Structured Review

Developmental Studies Hybridoma Bank mouse anti isl1
Hb9::Cre-derived INs do not overlap with the Shox2 non-V2a population. ( A ) Co-expression of YFP (green) and <t>Isl1</t> antibody (red) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Motor neurons are also labeled by Isl1 antibody (blue box). Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Isl1 (red) and Hb9::Cre-derived INs (green). Scale bars: 100 μm and 50 μm. ( B ) Co-expression of YFP (green), Shox2 antibody (red) and/or Chx10 antibody (blue) in the Hb9 :: Cre;Rosa26-YFP mouse ventral spinal cord at E11.5. Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Hb9::Cre-derived INs (green) and Shox2 + Chx10 − (red), Shox2 − Chx10 + (blue) or Shox2 + Chx10 + (pink). Scale bars: 100 μm and 50 μm. ( C ) Quantification of overlap in (A) and (B). Bar graph showing percent of overlap between Hb9::Cre-derived INs (YFP + ) and Shox2 V2a (Shox2 + Chx10 + , 4% ± 1%), Shox2 OFF V2a (Shox2 − Chx10 + , 2% ± 0.1%), Shox2 non-V2a (Shox2 + Chx10 − , 1.3% ± 0.2%), and Isl1 (Isl1 + , 6% ± 0.2%) INs. Error bars represent ± SEM. ( D ) Percent of the Shox2 non-V2a IN population (Shox2 + Chx10 − ) that overlaps with Hb9::Cre-derived INs (YFP + ) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Shox2 non-V2a INs rarely co-express YFP (Shox2 + YFP + , darker grey) (12% ± 2%). Error bars represent ± SEM.
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Images

1) Product Images from "Spinal Hb9::Cre-derived excitatory interneurons contribute to rhythm generation in the mouse"

Article Title: Spinal Hb9::Cre-derived excitatory interneurons contribute to rhythm generation in the mouse

Journal: Scientific Reports

doi: 10.1038/srep41369

Hb9::Cre-derived INs do not overlap with the Shox2 non-V2a population. ( A ) Co-expression of YFP (green) and Isl1 antibody (red) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Motor neurons are also labeled by Isl1 antibody (blue box). Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Isl1 (red) and Hb9::Cre-derived INs (green). Scale bars: 100 μm and 50 μm. ( B ) Co-expression of YFP (green), Shox2 antibody (red) and/or Chx10 antibody (blue) in the Hb9 :: Cre;Rosa26-YFP mouse ventral spinal cord at E11.5. Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Hb9::Cre-derived INs (green) and Shox2 + Chx10 − (red), Shox2 − Chx10 + (blue) or Shox2 + Chx10 + (pink). Scale bars: 100 μm and 50 μm. ( C ) Quantification of overlap in (A) and (B). Bar graph showing percent of overlap between Hb9::Cre-derived INs (YFP + ) and Shox2 V2a (Shox2 + Chx10 + , 4% ± 1%), Shox2 OFF V2a (Shox2 − Chx10 + , 2% ± 0.1%), Shox2 non-V2a (Shox2 + Chx10 − , 1.3% ± 0.2%), and Isl1 (Isl1 + , 6% ± 0.2%) INs. Error bars represent ± SEM. ( D ) Percent of the Shox2 non-V2a IN population (Shox2 + Chx10 − ) that overlaps with Hb9::Cre-derived INs (YFP + ) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Shox2 non-V2a INs rarely co-express YFP (Shox2 + YFP + , darker grey) (12% ± 2%). Error bars represent ± SEM.
Figure Legend Snippet: Hb9::Cre-derived INs do not overlap with the Shox2 non-V2a population. ( A ) Co-expression of YFP (green) and Isl1 antibody (red) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Motor neurons are also labeled by Isl1 antibody (blue box). Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Isl1 (red) and Hb9::Cre-derived INs (green). Scale bars: 100 μm and 50 μm. ( B ) Co-expression of YFP (green), Shox2 antibody (red) and/or Chx10 antibody (blue) in the Hb9 :: Cre;Rosa26-YFP mouse ventral spinal cord at E11.5. Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Hb9::Cre-derived INs (green) and Shox2 + Chx10 − (red), Shox2 − Chx10 + (blue) or Shox2 + Chx10 + (pink). Scale bars: 100 μm and 50 μm. ( C ) Quantification of overlap in (A) and (B). Bar graph showing percent of overlap between Hb9::Cre-derived INs (YFP + ) and Shox2 V2a (Shox2 + Chx10 + , 4% ± 1%), Shox2 OFF V2a (Shox2 − Chx10 + , 2% ± 0.1%), Shox2 non-V2a (Shox2 + Chx10 − , 1.3% ± 0.2%), and Isl1 (Isl1 + , 6% ± 0.2%) INs. Error bars represent ± SEM. ( D ) Percent of the Shox2 non-V2a IN population (Shox2 + Chx10 − ) that overlaps with Hb9::Cre-derived INs (YFP + ) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Shox2 non-V2a INs rarely co-express YFP (Shox2 + YFP + , darker grey) (12% ± 2%). Error bars represent ± SEM.

Techniques Used: Derivative Assay, Expressing, Labeling

2) Product Images from "Corepressors TLE1 and TLE3 Interact with HESX1 and PROP1"

Article Title: Corepressors TLE1 and TLE3 Interact with HESX1 and PROP1

Journal: Molecular Endocrinology

doi: 10.1210/me.2008-0359

Initiation of gonadotroph differentiation in double-transgenic embryos. The three highest expressing Tg ( Cga - Tle3 ), Tg ( Cga - Hesx1 ) double-transgenic embryos (designated αTLE3,αHESX1 double tgs) were selected based on ectopic immunohistochemical staining for TLE3 at e14.5 in the ventral cells of Rathke’s pouch in sagittal sections ( columns beginning with B, C, and D; gradation bar indicates decreasing transgene expression levels) compared with a nontransgenic littermate ( column beginning with A). SF1 (NR5A1) immunohistochemical staining detected both mature and pre-gonadotrophs (E–H, arrows ). Immunohistochemistry using antibodies against αGSU (CGA) (I–L) demonstrated a decrease in expression correlating with level of transgene expression (area of expression outlined ). There was no difference in protein levels of ISL1 (M–P) or PITX2 (Q–T) in transgenics and nontransgenic littermates.
Figure Legend Snippet: Initiation of gonadotroph differentiation in double-transgenic embryos. The three highest expressing Tg ( Cga - Tle3 ), Tg ( Cga - Hesx1 ) double-transgenic embryos (designated αTLE3,αHESX1 double tgs) were selected based on ectopic immunohistochemical staining for TLE3 at e14.5 in the ventral cells of Rathke’s pouch in sagittal sections ( columns beginning with B, C, and D; gradation bar indicates decreasing transgene expression levels) compared with a nontransgenic littermate ( column beginning with A). SF1 (NR5A1) immunohistochemical staining detected both mature and pre-gonadotrophs (E–H, arrows ). Immunohistochemistry using antibodies against αGSU (CGA) (I–L) demonstrated a decrease in expression correlating with level of transgene expression (area of expression outlined ). There was no difference in protein levels of ISL1 (M–P) or PITX2 (Q–T) in transgenics and nontransgenic littermates.

Techniques Used: Transgenic Assay, Expressing, Immunohistochemistry, Staining

3) Product Images from "Dynamic expression of ganglion cell markers in retinal progenitors during the terminal cell cycle"

Article Title: Dynamic expression of ganglion cell markers in retinal progenitors during the terminal cell cycle

Journal: Molecular and Cellular Neurosciences

doi: 10.1016/j.mcn.2012.05.002

The onset of Brn3b and Isl1 expression within individual cells is progressively delayed during retinal development. (A) Sections from E13.5 embryos co-stained for Brn3b, cyclinD1 (cycD1) and EdU following a 1 hr chase. In some cells, Brn3b is co-localized
Figure Legend Snippet: The onset of Brn3b and Isl1 expression within individual cells is progressively delayed during retinal development. (A) Sections from E13.5 embryos co-stained for Brn3b, cyclinD1 (cycD1) and EdU following a 1 hr chase. In some cells, Brn3b is co-localized

Techniques Used: Expressing, Staining

Co-expression of Brn3b and Isl1 during or shortly after the terminal cell cycle. (A–C) Sections from E11.5, E13.5, and E16.0 embryos co-stained for Brn3b, Isl1 and EdU following a 30 min chase. At E11.5 (A) and E13.5 (B), multiple Brn3b+ Isl1+
Figure Legend Snippet: Co-expression of Brn3b and Isl1 during or shortly after the terminal cell cycle. (A–C) Sections from E11.5, E13.5, and E16.0 embryos co-stained for Brn3b, Isl1 and EdU following a 30 min chase. At E11.5 (A) and E13.5 (B), multiple Brn3b+ Isl1+

Techniques Used: Expressing, Staining

4) Product Images from "Essential roles of mitochondrial biogenesis regulator Nrf1 in retinal development and homeostasis"

Article Title: Essential roles of mitochondrial biogenesis regulator Nrf1 in retinal development and homeostasis

Journal: Molecular Neurodegeneration

doi: 10.1186/s13024-018-0287-z

Delayed onset of RGC differentiation in Nrf1 f/f ;Six3-Cre retina. ( a–f ) Immunostaining of wildtype ( a , c , and e ) and Nrf1 f/f ;Six3-Cre ( b , d , and f ) retinas. ( a , b ) E12.5 retinal sections labeled with anti-Isl1 antibody. ( c , d ) E14.5 and ( e , f ) E16.5 retinal sections labeled with anti-Pou4f2 antibody. Arrowheads indicate clumped Pou4f2+ cells in the central area of Nrf1 f/f ;Six3-Cre retina. Scale bars: 50 μm in a–d , 100 μm in e and f . WT: wildtype
Figure Legend Snippet: Delayed onset of RGC differentiation in Nrf1 f/f ;Six3-Cre retina. ( a–f ) Immunostaining of wildtype ( a , c , and e ) and Nrf1 f/f ;Six3-Cre ( b , d , and f ) retinas. ( a , b ) E12.5 retinal sections labeled with anti-Isl1 antibody. ( c , d ) E14.5 and ( e , f ) E16.5 retinal sections labeled with anti-Pou4f2 antibody. Arrowheads indicate clumped Pou4f2+ cells in the central area of Nrf1 f/f ;Six3-Cre retina. Scale bars: 50 μm in a–d , 100 μm in e and f . WT: wildtype

Techniques Used: Immunostaining, Labeling

5) Product Images from "Schwann Cell Precursors Generate the Majority of Chromaffin Cells in Zuckerkandl Organ and Some Sympathetic Neurons in Paraganglia"

Article Title: Schwann Cell Precursors Generate the Majority of Chromaffin Cells in Zuckerkandl Organ and Some Sympathetic Neurons in Paraganglia

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2019.00006

Ablation of Schwann cell precursors or the preganglionic nerves that serve as their route toward the dorsal aorta results in a reduction of chromaffin cell numbers of the Zuckerkandl organ. (A,B) Immunofluorescence on cryosections against CHAT (choline acetyltransferase) (upper panels) at the level of the spinal cord shows the absence of CHAT + (cholinergic) preganglionic motorneurons in the gray matter, also seen as absence of ISL1 + motorneurons (lower panel). Scale bar = 100 μm. (C,D) Immunofluorescence against CART and TH at the level of the Zuckerkandl organ (ZO) shows a severely abnormal phenotype in the ZO and para-aortic ganglia (PAG) in Hb9 Cre/+ ; Isl2 DTA/+ E14.5 embryos in comparison to Isl2 DTA/+ control E14.5 embryos. Scale bar = 100 μm. (E) Quantification of TH + cells at E14.5 in control Isl2 DTA/+ and mutant Hb9 Cre/+ ; Isl2 DTA/+ embryos shows a decrease in the ZO and PAG, while the mesenteric ganglia (MG) remain unaffected. In Isl2 DTA/+ versus Hb9 Cre/+ ; Isl2 DTA/+ respectively: total TH + cells in ZO = 426.6 ± 52.66 vs. 201.9 ± 27.43 ( P = 0.0194), in MG = 178.4 ± 21.44 vs. 198.4 ± 15.34 ( P = 0.4889) and in PAG = 133.1 ± 9.57 vs. 83.22 ± 5.79 ( P = 0.0112), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (F) Schematic showing the experimental design for induction of the visceral nerve ablation using the Hb9 Cre ; Isl2 DTA strain. (G,H) Immunofluorescence against SOX10, TH and neurofilaments (NF) on cryosections from Sox10 CreERT2/+ and Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following tamoxifen (TAM) injection at E11.5 and E12.5 and analysis at E17.5 showing almost complete Schwann cell precursor (SCP)-ablation and significantly abnormal morphology in ZO, PAG and MG. Scale bar = 100 μm. (I) Quantification of SOX10 + cells in control Sox10 CreERT2/+ and SCP-ablated Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following TAM injection at E11.5 and E12.5 and analysis at E17.5. In Sox10 CreERT2/+ vs. Sox10 CreERT2/+ ; R26 DTA/DTA respectively: total SOX10 + cells in ZO = 205.47 ± 28.96 vs. 5.33 ± 4.43 ( P = 0.0024), in MG = 369.33 ± 27.56 vs. 5.55 ± 5.22 ( P = 0.0002) and in PAG = 88.11 ± 6.46 vs. 3.24 ± 2.58 ( P = 0.0002), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (J) Quantification of TH+ cells in control Sox10 CreERT2/+ and SCP-ablated Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following TAM injection at E11.5 and E12.5 and analysis at E17.5. In Sox10 CreERT2/+ vs. Sox10 CreERT2/+ ; R26 DTA/DTA respectively: total TH + cells in ZO = 588.08 ± 28.41 vs. 397.22 ± 23.69 ( P = 0.0067), in MG = 243.66 ± 22.98 vs. 226.55 ± 15.24 ( P = 0.5685) and in PAG = 103.00 ± 7.61 vs. 94.11 ± 8.04 ( P = 0.4672), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (K) Schematic outlining the TAM injection times and collection time point in the Schwann cell precursor (SCP)-ablation experiment using the Sox10 CreERT2 ; R26 DTA strain. SC, spinal cord; GM, gray matter; MG, mesenteric ganglion; PAG, para-aortic ganglion; MNs, motorneurons; DA, dorsal aorta; ZO, Zuckerkandl organ; ns, non-significant; ∗ P ≤ 0.05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001.
Figure Legend Snippet: Ablation of Schwann cell precursors or the preganglionic nerves that serve as their route toward the dorsal aorta results in a reduction of chromaffin cell numbers of the Zuckerkandl organ. (A,B) Immunofluorescence on cryosections against CHAT (choline acetyltransferase) (upper panels) at the level of the spinal cord shows the absence of CHAT + (cholinergic) preganglionic motorneurons in the gray matter, also seen as absence of ISL1 + motorneurons (lower panel). Scale bar = 100 μm. (C,D) Immunofluorescence against CART and TH at the level of the Zuckerkandl organ (ZO) shows a severely abnormal phenotype in the ZO and para-aortic ganglia (PAG) in Hb9 Cre/+ ; Isl2 DTA/+ E14.5 embryos in comparison to Isl2 DTA/+ control E14.5 embryos. Scale bar = 100 μm. (E) Quantification of TH + cells at E14.5 in control Isl2 DTA/+ and mutant Hb9 Cre/+ ; Isl2 DTA/+ embryos shows a decrease in the ZO and PAG, while the mesenteric ganglia (MG) remain unaffected. In Isl2 DTA/+ versus Hb9 Cre/+ ; Isl2 DTA/+ respectively: total TH + cells in ZO = 426.6 ± 52.66 vs. 201.9 ± 27.43 ( P = 0.0194), in MG = 178.4 ± 21.44 vs. 198.4 ± 15.34 ( P = 0.4889) and in PAG = 133.1 ± 9.57 vs. 83.22 ± 5.79 ( P = 0.0112), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (F) Schematic showing the experimental design for induction of the visceral nerve ablation using the Hb9 Cre ; Isl2 DTA strain. (G,H) Immunofluorescence against SOX10, TH and neurofilaments (NF) on cryosections from Sox10 CreERT2/+ and Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following tamoxifen (TAM) injection at E11.5 and E12.5 and analysis at E17.5 showing almost complete Schwann cell precursor (SCP)-ablation and significantly abnormal morphology in ZO, PAG and MG. Scale bar = 100 μm. (I) Quantification of SOX10 + cells in control Sox10 CreERT2/+ and SCP-ablated Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following TAM injection at E11.5 and E12.5 and analysis at E17.5. In Sox10 CreERT2/+ vs. Sox10 CreERT2/+ ; R26 DTA/DTA respectively: total SOX10 + cells in ZO = 205.47 ± 28.96 vs. 5.33 ± 4.43 ( P = 0.0024), in MG = 369.33 ± 27.56 vs. 5.55 ± 5.22 ( P = 0.0002) and in PAG = 88.11 ± 6.46 vs. 3.24 ± 2.58 ( P = 0.0002), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (J) Quantification of TH+ cells in control Sox10 CreERT2/+ and SCP-ablated Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following TAM injection at E11.5 and E12.5 and analysis at E17.5. In Sox10 CreERT2/+ vs. Sox10 CreERT2/+ ; R26 DTA/DTA respectively: total TH + cells in ZO = 588.08 ± 28.41 vs. 397.22 ± 23.69 ( P = 0.0067), in MG = 243.66 ± 22.98 vs. 226.55 ± 15.24 ( P = 0.5685) and in PAG = 103.00 ± 7.61 vs. 94.11 ± 8.04 ( P = 0.4672), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (K) Schematic outlining the TAM injection times and collection time point in the Schwann cell precursor (SCP)-ablation experiment using the Sox10 CreERT2 ; R26 DTA strain. SC, spinal cord; GM, gray matter; MG, mesenteric ganglion; PAG, para-aortic ganglion; MNs, motorneurons; DA, dorsal aorta; ZO, Zuckerkandl organ; ns, non-significant; ∗ P ≤ 0.05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001.

Techniques Used: Immunofluorescence, Mutagenesis, Two Tailed Test, Injection

The development and anatomy of the Zuckerkandl organ and associated paraganglia in relation to the dorsal aorta. (A) Schematic showing the molecular differences and similarities between sympathetic neurons (SNs) and chromaffin cells (ChCs) during development in relation to the expression of CART, (ISL1 and TH. (B) Side-view of whole-mount immunofluorescence against smooth-muscle-actin (SMA, showing the dorsal aorta -DA), TH and CART on an E12.5 wild type embryo. Note the presence of TH + cells at the dorsal part of the DA just posteriorly to its branching into the inferior mesenteric artery (shown by the white arrowhead). (C) Immunofluorescence on E12.5 wild type embryos against SOX10 and TH (left panel) and SOX10, CART and ISL1 (right panel) showing the close proximity of the developing CART - /ISL1 + /TH + Zuckerkandl organ (ZO) and CART + /ISL1 + /TH + mesenteric ganglion (MG). (D) Ventral-view of whole-mount immunofluorescence against NF200, TH and CD31 (showing the endothelium of the DA) on an E12.5 wild type embryo. Note the presence of the ZO below the inferior mesenteric artery (shown by the white arrowhead), which is surrounded by NF200 + axons. (E) Immunofluorescence on cryosection of an E13.5 wild type embryo against NF200, TH and CART. Note the innervation pattern around the DA and the TH+ cells of the ZO. (F) Ventral-view of whole-mount immunofluorescence against SMA, TH and CART on an E13.5 wild type embryo. Note the close proximity of the MG and ZO just posteriorly to the inferior mesenteric artery and the separation of the two structures based on the CART + /TH low immunoreactivity in the MG in contrast to the TH high immunoreactivity of the ZO (shown by the white arrowhead). Also note the presence of TH high chromaffin structures in close proximity to the MG and ZO (shown by yellow arrows). (G) Immunofluorescence against CART, TH and DAPI on cryosections of a wild type E17.5 embryo, clearly showing the composite nature of the ZO at this later developmental stage, with both TH + /CART - and TH + /CART + cells. Scale bar = 100 μm. SNs, sympathetic neurons; ChCs, chromaffin cells; SC, sympathetic chain; DA, dorsal aorta; PAG, para-aortic ganglion; ZO, Zuckerkandl organ; AM, adrenal medulla; MG, mesenteric ganglion.)
Figure Legend Snippet: The development and anatomy of the Zuckerkandl organ and associated paraganglia in relation to the dorsal aorta. (A) Schematic showing the molecular differences and similarities between sympathetic neurons (SNs) and chromaffin cells (ChCs) during development in relation to the expression of CART, (ISL1 and TH. (B) Side-view of whole-mount immunofluorescence against smooth-muscle-actin (SMA, showing the dorsal aorta -DA), TH and CART on an E12.5 wild type embryo. Note the presence of TH + cells at the dorsal part of the DA just posteriorly to its branching into the inferior mesenteric artery (shown by the white arrowhead). (C) Immunofluorescence on E12.5 wild type embryos against SOX10 and TH (left panel) and SOX10, CART and ISL1 (right panel) showing the close proximity of the developing CART - /ISL1 + /TH + Zuckerkandl organ (ZO) and CART + /ISL1 + /TH + mesenteric ganglion (MG). (D) Ventral-view of whole-mount immunofluorescence against NF200, TH and CD31 (showing the endothelium of the DA) on an E12.5 wild type embryo. Note the presence of the ZO below the inferior mesenteric artery (shown by the white arrowhead), which is surrounded by NF200 + axons. (E) Immunofluorescence on cryosection of an E13.5 wild type embryo against NF200, TH and CART. Note the innervation pattern around the DA and the TH+ cells of the ZO. (F) Ventral-view of whole-mount immunofluorescence against SMA, TH and CART on an E13.5 wild type embryo. Note the close proximity of the MG and ZO just posteriorly to the inferior mesenteric artery and the separation of the two structures based on the CART + /TH low immunoreactivity in the MG in contrast to the TH high immunoreactivity of the ZO (shown by the white arrowhead). Also note the presence of TH high chromaffin structures in close proximity to the MG and ZO (shown by yellow arrows). (G) Immunofluorescence against CART, TH and DAPI on cryosections of a wild type E17.5 embryo, clearly showing the composite nature of the ZO at this later developmental stage, with both TH + /CART - and TH + /CART + cells. Scale bar = 100 μm. SNs, sympathetic neurons; ChCs, chromaffin cells; SC, sympathetic chain; DA, dorsal aorta; PAG, para-aortic ganglion; ZO, Zuckerkandl organ; AM, adrenal medulla; MG, mesenteric ganglion.)

Techniques Used: Expressing, Immunofluorescence

The sympathetic ganglia at the level of the Zuckerkandl organ and the organ itself have distinct early-defined origin despite the intermingling anatomy. (A) Dorsal view of whole-mount immunofluorescence (left panel) against the sympathetic marker CART, the chromaffin and sympathetic marker TH and NF200 (showing the innervation on the trunk of an E15.5 wild type embryo and schematic (right panel) showing the sympathetic and chromaffin structures in relation to the dorsal aorta. Note that the mesenteric (MG) and suprarenal ganglion (SRG), as well as the sympathetic chain (SC), are CART + , while the Zuckerkandl organ (ZO) is composed mainly by TH + /CART - cells. Additionally note that the para-aortic ganglia (PAG) are the continuation of the sympathetic chain that extends along the anteroposterior axis of the embryo trunk just at the dorsal view of the dorsal aorta, at the level of the ZO and MG. (B,C) Immunofluorescence on cryosections against CART, Ret TOM and TH on tamoxifen-injected (TAM-injected) embryos at E10.5 and E11.5 respectively shows that Ret TOM specifically delineated the sympathetic compartment when analyzed at E15.5, with clear tracing of the MG and PAG, while only few Ret TOM+ cells can be seen in the ZO. Note the difference in CART immunofluorescence levels in the MG and PAG. (D) Immunofluorescence on cryosections against ISL1, Ret TOM and TH on TAM-injected embryos at E10.5 shows Ret TOM specific expression by the sympathetic ganglion (SG) and SRG when analyzed at E15.5, while almost no Ret TOM+ cells can be seen in the adrenal medulla (AM). (E) Ventral view of whole-mount immunofluorescence against CART, Ascl1 TOM and TH on embryos with TAM injection at E11.5 and analyzed at E13.5 shows tracing in the chromaffin cells of the ZO, while no tracing in the MG (shown by white arrowheads). Scale bar in (A–E) = 100 μm. A, anterior; P, posterior; AM, adrenal medulla; MG, mesenteric ganglion; ZO, Zuckerkandl organ; DA, dorsal aorta; SC, sympathetic chain; SRG, suprarenal ganglion; AG, adrenal gland; PAG, para-aortic ganglion; SG, sympathetic ganglion; ChCs, chromaffin cells; SNs, sympathetic neurons.)
Figure Legend Snippet: The sympathetic ganglia at the level of the Zuckerkandl organ and the organ itself have distinct early-defined origin despite the intermingling anatomy. (A) Dorsal view of whole-mount immunofluorescence (left panel) against the sympathetic marker CART, the chromaffin and sympathetic marker TH and NF200 (showing the innervation on the trunk of an E15.5 wild type embryo and schematic (right panel) showing the sympathetic and chromaffin structures in relation to the dorsal aorta. Note that the mesenteric (MG) and suprarenal ganglion (SRG), as well as the sympathetic chain (SC), are CART + , while the Zuckerkandl organ (ZO) is composed mainly by TH + /CART - cells. Additionally note that the para-aortic ganglia (PAG) are the continuation of the sympathetic chain that extends along the anteroposterior axis of the embryo trunk just at the dorsal view of the dorsal aorta, at the level of the ZO and MG. (B,C) Immunofluorescence on cryosections against CART, Ret TOM and TH on tamoxifen-injected (TAM-injected) embryos at E10.5 and E11.5 respectively shows that Ret TOM specifically delineated the sympathetic compartment when analyzed at E15.5, with clear tracing of the MG and PAG, while only few Ret TOM+ cells can be seen in the ZO. Note the difference in CART immunofluorescence levels in the MG and PAG. (D) Immunofluorescence on cryosections against ISL1, Ret TOM and TH on TAM-injected embryos at E10.5 shows Ret TOM specific expression by the sympathetic ganglion (SG) and SRG when analyzed at E15.5, while almost no Ret TOM+ cells can be seen in the adrenal medulla (AM). (E) Ventral view of whole-mount immunofluorescence against CART, Ascl1 TOM and TH on embryos with TAM injection at E11.5 and analyzed at E13.5 shows tracing in the chromaffin cells of the ZO, while no tracing in the MG (shown by white arrowheads). Scale bar in (A–E) = 100 μm. A, anterior; P, posterior; AM, adrenal medulla; MG, mesenteric ganglion; ZO, Zuckerkandl organ; DA, dorsal aorta; SC, sympathetic chain; SRG, suprarenal ganglion; AG, adrenal gland; PAG, para-aortic ganglion; SG, sympathetic ganglion; ChCs, chromaffin cells; SNs, sympathetic neurons.)

Techniques Used: Immunofluorescence, Marker, Injection, Expressing

6) Product Images from "Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA"

Article Title: Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Journal: Cell Research

doi: 10.1038/cr.2013.112

Endothelial differentiation of the human Isl1 + progenitors can be achieved by repeated transfections of the VEGF-A modRNA. (A) FACS result showing expression of mCherry in the day-7 purified human Isl1-cre eGFP + cells 24 h post transfection of the mCherry modRNA (1 μg/10 6 cells). (B) Quantification of mCherry expression by FACS showing percentage of mCherry + Isl1-cre eGFP + cells following 0-150 h post transfection. (C) ELISA analyses using VEGF-A-containing supernatant (refreshed 6 h before collection) cultured with the day-7 purified human Isl1-cre eGFP + cells following 0-72 h post transfection of different concentrations of the VEGF-A modRNA. (D) Trypan blue staining showing the survival percentage of the day-7 purified human Isl1-cre eGFP + cells following 24 h post transfection of different concentrations of the VEGF-A modRNA. (E) ELISA analyses showing the rate of VEGF-A secreted by the day-7 purified human Isl1-cre eGFP + cells (supernatant refreshed 6 h before collection) following two rounds of daily transfections of 1 μg/10 6 cells with VEGF-A modRNA. (F) FACS analyses showing the efficiency of CD144 + CD31 + EC differentiation by one transfection or repeated transfections (following medium change) of the VEGF-A modRNA compared to the use of VEGF-A protein 3 or 7 days post treatment.
Figure Legend Snippet: Endothelial differentiation of the human Isl1 + progenitors can be achieved by repeated transfections of the VEGF-A modRNA. (A) FACS result showing expression of mCherry in the day-7 purified human Isl1-cre eGFP + cells 24 h post transfection of the mCherry modRNA (1 μg/10 6 cells). (B) Quantification of mCherry expression by FACS showing percentage of mCherry + Isl1-cre eGFP + cells following 0-150 h post transfection. (C) ELISA analyses using VEGF-A-containing supernatant (refreshed 6 h before collection) cultured with the day-7 purified human Isl1-cre eGFP + cells following 0-72 h post transfection of different concentrations of the VEGF-A modRNA. (D) Trypan blue staining showing the survival percentage of the day-7 purified human Isl1-cre eGFP + cells following 24 h post transfection of different concentrations of the VEGF-A modRNA. (E) ELISA analyses showing the rate of VEGF-A secreted by the day-7 purified human Isl1-cre eGFP + cells (supernatant refreshed 6 h before collection) following two rounds of daily transfections of 1 μg/10 6 cells with VEGF-A modRNA. (F) FACS analyses showing the efficiency of CD144 + CD31 + EC differentiation by one transfection or repeated transfections (following medium change) of the VEGF-A modRNA compared to the use of VEGF-A protein 3 or 7 days post treatment.

Techniques Used: Transfection, FACS, Expressing, Purification, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining

VEGF-A drives differentiation of the human Isl1 + progenitors toward an EC lineage, but away from the SMC lineage in vivo . (A) Schematic diagram of the experimental design. (B - U) Frozen sections from matrigel plugs incubated with the day-7 purified human Isl1-cre eGFP + cells in the presence of (B - K) vehicle or (L - U) VEGF-A modRNA were stained for eGFP with smooth muscle- or endothelial cell-specific markers (scale bar in C = 100 μm, scale bar in M = 50 μm, scale bars in all the remaining panels = 25 μm). (V) Quantification analyzed by ImageJ showing number of double positive cells (eGFP + SMMHC + , eGFP + Vimentin + or eGFP + CD31 + ) per unit area of the matrigel plug following treatment with vehicle or VEGF-A modRNA.
Figure Legend Snippet: VEGF-A drives differentiation of the human Isl1 + progenitors toward an EC lineage, but away from the SMC lineage in vivo . (A) Schematic diagram of the experimental design. (B - U) Frozen sections from matrigel plugs incubated with the day-7 purified human Isl1-cre eGFP + cells in the presence of (B - K) vehicle or (L - U) VEGF-A modRNA were stained for eGFP with smooth muscle- or endothelial cell-specific markers (scale bar in C = 100 μm, scale bar in M = 50 μm, scale bars in all the remaining panels = 25 μm). (V) Quantification analyzed by ImageJ showing number of double positive cells (eGFP + SMMHC + , eGFP + Vimentin + or eGFP + CD31 + ) per unit area of the matrigel plug following treatment with vehicle or VEGF-A modRNA.

Techniques Used: In Vivo, Incubation, Purification, Staining

VEGF-A is the most abundantly expressed angiocrine factor in human fetal hearts and endothelial differentiation of the human Isl1 + progenitors is KDR dependent. (A) qPCR profiling showing expression levels of angiocrine factors derived from CD144 + CD31 + ECs purified from outflow tract of the 10-week human fetal hearts, expression levels were compared with those of the noncardiac, human cord blood-derived outgrowth endothelial cells (OECs, value on y axis = 1). (B) FACS analyses to determine the endothelial differentiation efficiency by factor X following treatment from day 7-12. (C) FACS analyses on the proliferation rate of the human Isl1-cre eGFP + cells following VEGF-A treatment from day 4-7, and the endothelial differentiation efficiency without VEGF-A, with VEGF-A or with VEGF-A+KDR inhibitor (SU5614) from day 7-14. (D) Clonal assay was performed by culturing the day-7 human Isl1-cre eGFP + cells on MEFs with VEGF-A ( n = 33) for 3 days. Result was normalized to cells without VEGF-A treatment. (E) qPCR data showing endothelial differentiation of the day-7 human Isl1-cre eGFP + cells in the presence of VEGF-A or VEGF-A+SU5614 for 7 days. Result was normalized to cells with VEGF-A alone (value on y axis =1).
Figure Legend Snippet: VEGF-A is the most abundantly expressed angiocrine factor in human fetal hearts and endothelial differentiation of the human Isl1 + progenitors is KDR dependent. (A) qPCR profiling showing expression levels of angiocrine factors derived from CD144 + CD31 + ECs purified from outflow tract of the 10-week human fetal hearts, expression levels were compared with those of the noncardiac, human cord blood-derived outgrowth endothelial cells (OECs, value on y axis = 1). (B) FACS analyses to determine the endothelial differentiation efficiency by factor X following treatment from day 7-12. (C) FACS analyses on the proliferation rate of the human Isl1-cre eGFP + cells following VEGF-A treatment from day 4-7, and the endothelial differentiation efficiency without VEGF-A, with VEGF-A or with VEGF-A+KDR inhibitor (SU5614) from day 7-14. (D) Clonal assay was performed by culturing the day-7 human Isl1-cre eGFP + cells on MEFs with VEGF-A ( n = 33) for 3 days. Result was normalized to cells without VEGF-A treatment. (E) qPCR data showing endothelial differentiation of the day-7 human Isl1-cre eGFP + cells in the presence of VEGF-A or VEGF-A+SU5614 for 7 days. Result was normalized to cells with VEGF-A alone (value on y axis =1).

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Purification, FACS, Clone Assay

VEGF-A-treated human Isl1 + progenitors express EC markers and secrete angiocrine factors in a similar pattern to the human outflow tract-derived ECs. (A) FACS analyses on the purified CD144 + CD31 + ECs from the human Isl1-cre eGFP + cells (Isl1-ECs) in the presence of VEGF-A from day 7-21. (B , C) Correlation of angiocrine gene expression between qPCR data obtained from the day-7 purified human Isl1-cre eGFP + cells (Isl1 progenitors) or Isl1-ECs in the presence of VEGF-A from day 7-21 and (B) the purified CD144 + CD31 + ECs from the outflow tract of human fetal hearts at 10 weeks of gestation (OFT-ECs) or (C) the noncardiac, human cord blood-derived outgrowth endothelial cells (OECs) ( n = 5).
Figure Legend Snippet: VEGF-A-treated human Isl1 + progenitors express EC markers and secrete angiocrine factors in a similar pattern to the human outflow tract-derived ECs. (A) FACS analyses on the purified CD144 + CD31 + ECs from the human Isl1-cre eGFP + cells (Isl1-ECs) in the presence of VEGF-A from day 7-21. (B , C) Correlation of angiocrine gene expression between qPCR data obtained from the day-7 purified human Isl1-cre eGFP + cells (Isl1 progenitors) or Isl1-ECs in the presence of VEGF-A from day 7-21 and (B) the purified CD144 + CD31 + ECs from the outflow tract of human fetal hearts at 10 weeks of gestation (OFT-ECs) or (C) the noncardiac, human cord blood-derived outgrowth endothelial cells (OECs) ( n = 5).

Techniques Used: Derivative Assay, FACS, Purification, Expressing, Real-time Polymerase Chain Reaction

Expression of VEGF receptors in the human Isl1 + progenitors. (A) Frozen sections from a human fetal heart at gestation week 9 were stained for DAPI (scale bar = 500 μm), Isl1, endothelial cell-specific markers: CD144, vWF, VEGF-A receptor 1 (Flt1) or 2 (KDR), or neurofilament (scale bars = 50 μm and 10 μm). Isl1 + cells are indicated by white asterisks (scale bar = 100 μm) and colocalization of Isl1 and EC markers are indicated by white arrows (scale bar = 10 μM). (B) Schematic diagram showing the Isl1 lineage-tracing construct in human ESCs. (C) Differentiation protocol used to derive the Isl1 + progenitors from human ESCs and to examine, which angiocrine factor (X) is responsible for endothelial differentiation of the progenitors. (D) FACS analyses showing expression of VEGFR1 or VEGFR2 in day-4 or day-7 human Isl1 + progenitors.
Figure Legend Snippet: Expression of VEGF receptors in the human Isl1 + progenitors. (A) Frozen sections from a human fetal heart at gestation week 9 were stained for DAPI (scale bar = 500 μm), Isl1, endothelial cell-specific markers: CD144, vWF, VEGF-A receptor 1 (Flt1) or 2 (KDR), or neurofilament (scale bars = 50 μm and 10 μm). Isl1 + cells are indicated by white asterisks (scale bar = 100 μm) and colocalization of Isl1 and EC markers are indicated by white arrows (scale bar = 10 μM). (B) Schematic diagram showing the Isl1 lineage-tracing construct in human ESCs. (C) Differentiation protocol used to derive the Isl1 + progenitors from human ESCs and to examine, which angiocrine factor (X) is responsible for endothelial differentiation of the progenitors. (D) FACS analyses showing expression of VEGFR1 or VEGFR2 in day-4 or day-7 human Isl1 + progenitors.

Techniques Used: Expressing, Staining, Construct, FACS

VEGF-A drives endothelial differentiation and promotes survival via increased proliferation and reduced apoptosis of the human Isl1 + progenitors in vivo . (A) FACS analyses to determine the percentage of CD144 + CD31 + ECs differentiated from the human Isl1-cre eGFP + cells isolated from matrigel plugs following incubation in the presence of vehicle or VEGF-A modRNA 2 weeks post s.c. implantation. (B , C) Cell counting to determine the percentage of proliferation (eGFP + Ki67 + by immunostaining), apoptosis (eGFP + Tunel + by immunostaining) or survival (total eGFP + cells by FACS) of the human Isl1-cre eGFP + cells in the vehicle- or VEGF-A modRNA-treated matrigel plugs two weeks post s.c. implantation. (D) Model for the proposed cell fate switch of the human Isl1 + progenitors following VEGF-A treatment.
Figure Legend Snippet: VEGF-A drives endothelial differentiation and promotes survival via increased proliferation and reduced apoptosis of the human Isl1 + progenitors in vivo . (A) FACS analyses to determine the percentage of CD144 + CD31 + ECs differentiated from the human Isl1-cre eGFP + cells isolated from matrigel plugs following incubation in the presence of vehicle or VEGF-A modRNA 2 weeks post s.c. implantation. (B , C) Cell counting to determine the percentage of proliferation (eGFP + Ki67 + by immunostaining), apoptosis (eGFP + Tunel + by immunostaining) or survival (total eGFP + cells by FACS) of the human Isl1-cre eGFP + cells in the vehicle- or VEGF-A modRNA-treated matrigel plugs two weeks post s.c. implantation. (D) Model for the proposed cell fate switch of the human Isl1 + progenitors following VEGF-A treatment.

Techniques Used: In Vivo, FACS, Isolation, Incubation, Cell Counting, Immunostaining, TUNEL Assay

7) Product Images from "Left-right locomotor circuitry depends upon RhoA-driven organization of the neuroepithelium in the developing spinal cord"

Article Title: Left-right locomotor circuitry depends upon RhoA-driven organization of the neuroepithelium in the developing spinal cord

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.6474-11.2012

Loss of RhoA does not affect the distribution of motor neurons. Immunostaining for Foxp1 (A, E), Isl1 (B, F), Lhx3 (C, G) and their merged images (D, H) of the lumbar spinal cord at E12.5. There were no obvious differences in the localization of motor
Figure Legend Snippet: Loss of RhoA does not affect the distribution of motor neurons. Immunostaining for Foxp1 (A, E), Isl1 (B, F), Lhx3 (C, G) and their merged images (D, H) of the lumbar spinal cord at E12.5. There were no obvious differences in the localization of motor

Techniques Used: Immunostaining

RhoA plays a role in the survival of motor neurons in the developing spinal cord. (A-D, I, J) The number of Isl1/2 + motor neurons in RhoA-Olig2-CKO embryos was not significantly different from that of control embryos at E10.5, but was significantly decreased
Figure Legend Snippet: RhoA plays a role in the survival of motor neurons in the developing spinal cord. (A-D, I, J) The number of Isl1/2 + motor neurons in RhoA-Olig2-CKO embryos was not significantly different from that of control embryos at E10.5, but was significantly decreased

Techniques Used:

8) Product Images from "Zebrafish Ext2 is necessary for Fgf and Wnt signaling, but not for Hh signaling"

Article Title: Zebrafish Ext2 is necessary for Fgf and Wnt signaling, but not for Hh signaling

Journal: BMC Developmental Biology

doi: 10.1186/1471-213X-11-53

Hh signaling in ext2 mutants elicits normal ptc1 expression and functions normally in differentiation of cells in the retina . Lateral view (A, C) and dorsal view (B, D) of ptc1 expression in 38 hpf WT embryos (A, B) and ext2 mutants (C-D). Asterisks label the developing limbs. The difference in somite staining between A-B and C-D is within the range of individual variation (also see additional figure 3A-B) (E-H) Confocal sections of the retina at 72 hpf, with anterior to the top. Detection of the Isl1 protein (E, G) and Zpr1 protein (F, H) in WT retinas (E-F) and ext2 mutant retinas (G-H) reveal normal Hh signaling in ext2 mutants during patterning of the zebrafish retina.
Figure Legend Snippet: Hh signaling in ext2 mutants elicits normal ptc1 expression and functions normally in differentiation of cells in the retina . Lateral view (A, C) and dorsal view (B, D) of ptc1 expression in 38 hpf WT embryos (A, B) and ext2 mutants (C-D). Asterisks label the developing limbs. The difference in somite staining between A-B and C-D is within the range of individual variation (also see additional figure 3A-B) (E-H) Confocal sections of the retina at 72 hpf, with anterior to the top. Detection of the Isl1 protein (E, G) and Zpr1 protein (F, H) in WT retinas (E-F) and ext2 mutant retinas (G-H) reveal normal Hh signaling in ext2 mutants during patterning of the zebrafish retina.

Techniques Used: Expressing, Staining, Mutagenesis

9) Product Images from "Necessity and Sufficiency of Ldb1 in the Generation, Differentiation and Maintenance of Non-photoreceptor Cell Types During Retinal Development"

Article Title: Necessity and Sufficiency of Ldb1 in the Generation, Differentiation and Maintenance of Non-photoreceptor Cell Types During Retinal Development

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00271

Conditional knockout of Ldb1 causes loss of all non-photoreceptor cell types. (A,A’) Ldb1 immunoreactivity was near completely abolished in the mutant retina. (B,B’) Immunoreactivity for Pax6, a cell marker for amacrine, horizontal and ganglion cells, was reduced in the mutant. (C,C) The Ldb1 binding cofactor Lmo4, usually present in nearly all major cell types in the INL and GCL, was decreased in the mutant. (D,D’) Syntaxin, a marker for all amacrine cells, was dramatically reduced in the mutant. (E,E’) GABA immunoreactive amacrine cells were diminished in the mutant. (F,F’) Calbindin is expressed in all horizontal cells and some amacrine cells in the wildtype retina. These cells were greatly reduced in the mutant. (G,G’) Chx10 + bipolar cells were vastly decreased in the mutant. (H,H’) Isl1 is present in ON-bipolar, cholinergic amacrine, and ganglion cells. These cells especially ganglion cells were drastically reduced in the mutant. (I,I’) Brn3b + ganglion cells nearly disappeared in the Ldb1 mutant. (J–L,J’–L’) Müller cells immunoreactive for Sox9, GS or Lhx2 were decreased in the mutant. (M) Quantification of some typical cell markers in control and Ldb1 mutant retinas. Each histogram represents the mean ± SD for three retinas. ∗ p
Figure Legend Snippet: Conditional knockout of Ldb1 causes loss of all non-photoreceptor cell types. (A,A’) Ldb1 immunoreactivity was near completely abolished in the mutant retina. (B,B’) Immunoreactivity for Pax6, a cell marker for amacrine, horizontal and ganglion cells, was reduced in the mutant. (C,C) The Ldb1 binding cofactor Lmo4, usually present in nearly all major cell types in the INL and GCL, was decreased in the mutant. (D,D’) Syntaxin, a marker for all amacrine cells, was dramatically reduced in the mutant. (E,E’) GABA immunoreactive amacrine cells were diminished in the mutant. (F,F’) Calbindin is expressed in all horizontal cells and some amacrine cells in the wildtype retina. These cells were greatly reduced in the mutant. (G,G’) Chx10 + bipolar cells were vastly decreased in the mutant. (H,H’) Isl1 is present in ON-bipolar, cholinergic amacrine, and ganglion cells. These cells especially ganglion cells were drastically reduced in the mutant. (I,I’) Brn3b + ganglion cells nearly disappeared in the Ldb1 mutant. (J–L,J’–L’) Müller cells immunoreactive for Sox9, GS or Lhx2 were decreased in the mutant. (M) Quantification of some typical cell markers in control and Ldb1 mutant retinas. Each histogram represents the mean ± SD for three retinas. ∗ p

Techniques Used: Knock-Out, Mutagenesis, Marker, Binding Assay

10) Product Images from "Insm1 promotes neurogenic proliferation in delaminated otic progenitors"

Article Title: Insm1 promotes neurogenic proliferation in delaminated otic progenitors

Journal: Mechanisms of development

doi: 10.1016/j.mod.2015.11.001

A change in apoptosis does not accompany the reduction of SVG neurons in Insm1 −/− Representative anatomically matched sections of E12.5 ears from Insm1 +/+ (A–B) and Insm1 −/− (C–D) littermate embryos. (A,C) Immunohistochemistry for DP marker Isl1 (magenta) and apoptosis marker ACC3 (green). Colabeled cells appear white. (B,D) Nuclear pattern for A and C, respectively. Dashed lines delineate the otic epithelia. Solid lines delineate SVG and do not include nearby facial ganglion which is also expresses Isl1. (E) There is no significant difference in the number of delaminated neurons undergoing apoptosis per section between Insm1 −/− and Insm1 +/+ at E12.5 (3.87;SEM=1.6 and 1.98;SEM=0.32, respectively. P =0.14) or E14.5 (3.59;SEM=0.83 and 5.37;SEM=1.06, respectively. P =0.11). Sample size in the number of ears for each condition is indicated by the number within the column representing that group. The number of mice in each sample is written in parentheses below the number of ears. Error bars indicate SEM. Scale bar: 200 μm. gg: geniculate ganglion, Ot: otocyst.
Figure Legend Snippet: A change in apoptosis does not accompany the reduction of SVG neurons in Insm1 −/− Representative anatomically matched sections of E12.5 ears from Insm1 +/+ (A–B) and Insm1 −/− (C–D) littermate embryos. (A,C) Immunohistochemistry for DP marker Isl1 (magenta) and apoptosis marker ACC3 (green). Colabeled cells appear white. (B,D) Nuclear pattern for A and C, respectively. Dashed lines delineate the otic epithelia. Solid lines delineate SVG and do not include nearby facial ganglion which is also expresses Isl1. (E) There is no significant difference in the number of delaminated neurons undergoing apoptosis per section between Insm1 −/− and Insm1 +/+ at E12.5 (3.87;SEM=1.6 and 1.98;SEM=0.32, respectively. P =0.14) or E14.5 (3.59;SEM=0.83 and 5.37;SEM=1.06, respectively. P =0.11). Sample size in the number of ears for each condition is indicated by the number within the column representing that group. The number of mice in each sample is written in parentheses below the number of ears. Error bars indicate SEM. Scale bar: 200 μm. gg: geniculate ganglion, Ot: otocyst.

Techniques Used: Immunohistochemistry, Marker, Mouse Assay

Reduction in proliferation of DPs precedes decrease of neurons in Insm1 −/− (A,C) Immunohistochemistry for DP marker Isl1 (magenta) and mitosis marker PH3 (green) in representative, anatomically matched, coronal sections of E10.5 Insm1 +/+ (A) and Insm1 −/− (C) heads. Colabeled cells appear white. (B,D) Nuclear pattern for (A) and (C), respectively. Dotted lines delineate the basal lamina of the otic epithelia. Solid lines delineate SVG and do not include nearby facial ganglion which is also expresses Isl1. (E) Average number of DPs in mitosis per cross section of Insm1 +/+ and Insm1 −/− otic ganglia at E10.5 and E12.5. There are approximately 30% fewer DPs in mitosis in Insm1 −/− than in Insm1 +/+ ganglia at E10.5 (13.63;SEM=2.33 and 20.32;SEM=2.37) and E12.5 (3.87;SEM=0.46 and 5.55;SEM=0.41). (F) Average number of DPs per cross section of Insm1 +/+ and Insm1 −/− otic ganglia at E10.5. There is no significant difference in the average number of DPs in Insm1 +/+ and Insm1 −/− SVGs at E10.5 (125.97;SEM=11.24 and 119.29;SEM=18.49, respectively. P =0.38). (G) Average percent of DPs that are in mitosis per cross section of Insm1 +/+ and Insm1 −/− otic ganglia at E10.5. Of the total number of Isl1 cells, approximately 30% fewer are in mitosis in Insm1 −/− than Insm1 +/+ ganglia at E10.5 (11.29%;SEM=0.28% and 16.06%;SEM=0.92%). (H) Average number of mitotic progenitors within the epithelium per cross section of Insm1 +/+ and Insm1 −/− otic ganglia at E10.5 and E12.5. There is no significant difference in the average number of epithelial mitotic progenitors in Insm1 +/+ and Insm1 −/− SVGs at E10.5 (19.25;SEM=2.01 and 20.83;SEM=2.39. P =0.31) and at E12.5 (10.05;SEM=1.54 and 11.53;SEM=1.53. P =0.25). Sample size in the number of ears for each condition is indicated by the number within the column representing that group. The number of mice in each sample is written in parentheses below the number of ears. Error bars indicate SEM. * P ≤0.05, ** P ≤0.01, **** P ≤0.0005. Scale bars: 100 μm. gg: geniculate ganglion, Ot: otocyst.
Figure Legend Snippet: Reduction in proliferation of DPs precedes decrease of neurons in Insm1 −/− (A,C) Immunohistochemistry for DP marker Isl1 (magenta) and mitosis marker PH3 (green) in representative, anatomically matched, coronal sections of E10.5 Insm1 +/+ (A) and Insm1 −/− (C) heads. Colabeled cells appear white. (B,D) Nuclear pattern for (A) and (C), respectively. Dotted lines delineate the basal lamina of the otic epithelia. Solid lines delineate SVG and do not include nearby facial ganglion which is also expresses Isl1. (E) Average number of DPs in mitosis per cross section of Insm1 +/+ and Insm1 −/− otic ganglia at E10.5 and E12.5. There are approximately 30% fewer DPs in mitosis in Insm1 −/− than in Insm1 +/+ ganglia at E10.5 (13.63;SEM=2.33 and 20.32;SEM=2.37) and E12.5 (3.87;SEM=0.46 and 5.55;SEM=0.41). (F) Average number of DPs per cross section of Insm1 +/+ and Insm1 −/− otic ganglia at E10.5. There is no significant difference in the average number of DPs in Insm1 +/+ and Insm1 −/− SVGs at E10.5 (125.97;SEM=11.24 and 119.29;SEM=18.49, respectively. P =0.38). (G) Average percent of DPs that are in mitosis per cross section of Insm1 +/+ and Insm1 −/− otic ganglia at E10.5. Of the total number of Isl1 cells, approximately 30% fewer are in mitosis in Insm1 −/− than Insm1 +/+ ganglia at E10.5 (11.29%;SEM=0.28% and 16.06%;SEM=0.92%). (H) Average number of mitotic progenitors within the epithelium per cross section of Insm1 +/+ and Insm1 −/− otic ganglia at E10.5 and E12.5. There is no significant difference in the average number of epithelial mitotic progenitors in Insm1 +/+ and Insm1 −/− SVGs at E10.5 (19.25;SEM=2.01 and 20.83;SEM=2.39. P =0.31) and at E12.5 (10.05;SEM=1.54 and 11.53;SEM=1.53. P =0.25). Sample size in the number of ears for each condition is indicated by the number within the column representing that group. The number of mice in each sample is written in parentheses below the number of ears. Error bars indicate SEM. * P ≤0.05, ** P ≤0.01, **** P ≤0.0005. Scale bars: 100 μm. gg: geniculate ganglion, Ot: otocyst.

Techniques Used: Immunohistochemistry, Marker, Mouse Assay

Insm1 is expressed in delaminating and delaminated neuronal progenitors that will produce the SVG Representative horizontal sections of (A–D) wild type and (E–L) Insm1 GFP.Cre/+ E10.5 embryos demonstrating the Insm1 expression pattern in the otocyst during delamination. (A) In situ hybridization with an Insm1 antisense probe on a section through the anterior portion of the otocyst. Insm1 mRNA is expressed in DPs within the anteroventral aspect of the otic epithelium and in the mesenchyme. (B) Nuclear pattern of panel (A). (C) Close up of boxed region in (A). Arrowhead indicates an Insm1 positive cell located more apically in the epithelium, presumably prior to delamination. (D) Control in situ hybridization with an Insm1 sense probe shows no signal. (E) Immunohistochemistry for GFP (green) representing Insm1 GFP.Cre expression in epithelial and delaminated DPs of the anteroventral otocyst. (F) Nuclear pattern corresponding to panel (E). (G) Immunohistochemistry for DP marker Isl1 (magenta) in the same section as (E,F). (H) Close up of merge of boxed region in panels (E) and (G) shows that the GFP and Isl1 patterns overlap (white), except for a few GFP positive cells not positive for Isl1 (arrows) and even fewer Isl1 positive cells not expressing GFP (open arrow). (I,J) Immunohistochemistry co-staining for Insm1 GFP.Cre expression (I, green) and PH3 (J, magenta) shows that Insm1 is not expressed in apically-dividing cells within the epithelium (examples indicated by open arrows), but is expressed by delaminated cells in mitosis (arrows). (K,L) Immunohistochemistry co-staining for Insm1 GFP.Cre expression (K, green) and Ki67 (L, magenta) shows that while some Insm1 expressing cells are proliferating (Ki67+; examples indicated by arrows), many are not (examples indicated by open arrows). Dotted lines in all panels delineate the basal lamina of the otocyst. Solid lines delineate the apical edge of the otic epithelium. Boxes indicate regions magnified in panels (C) and (H). (A,D,E) Scale bar: 200 μm. (C,H,I,K) Scale bar: 50 μm. Ot: otocyst.
Figure Legend Snippet: Insm1 is expressed in delaminating and delaminated neuronal progenitors that will produce the SVG Representative horizontal sections of (A–D) wild type and (E–L) Insm1 GFP.Cre/+ E10.5 embryos demonstrating the Insm1 expression pattern in the otocyst during delamination. (A) In situ hybridization with an Insm1 antisense probe on a section through the anterior portion of the otocyst. Insm1 mRNA is expressed in DPs within the anteroventral aspect of the otic epithelium and in the mesenchyme. (B) Nuclear pattern of panel (A). (C) Close up of boxed region in (A). Arrowhead indicates an Insm1 positive cell located more apically in the epithelium, presumably prior to delamination. (D) Control in situ hybridization with an Insm1 sense probe shows no signal. (E) Immunohistochemistry for GFP (green) representing Insm1 GFP.Cre expression in epithelial and delaminated DPs of the anteroventral otocyst. (F) Nuclear pattern corresponding to panel (E). (G) Immunohistochemistry for DP marker Isl1 (magenta) in the same section as (E,F). (H) Close up of merge of boxed region in panels (E) and (G) shows that the GFP and Isl1 patterns overlap (white), except for a few GFP positive cells not positive for Isl1 (arrows) and even fewer Isl1 positive cells not expressing GFP (open arrow). (I,J) Immunohistochemistry co-staining for Insm1 GFP.Cre expression (I, green) and PH3 (J, magenta) shows that Insm1 is not expressed in apically-dividing cells within the epithelium (examples indicated by open arrows), but is expressed by delaminated cells in mitosis (arrows). (K,L) Immunohistochemistry co-staining for Insm1 GFP.Cre expression (K, green) and Ki67 (L, magenta) shows that while some Insm1 expressing cells are proliferating (Ki67+; examples indicated by arrows), many are not (examples indicated by open arrows). Dotted lines in all panels delineate the basal lamina of the otocyst. Solid lines delineate the apical edge of the otic epithelium. Boxes indicate regions magnified in panels (C) and (H). (A,D,E) Scale bar: 200 μm. (C,H,I,K) Scale bar: 50 μm. Ot: otocyst.

Techniques Used: Expressing, In Situ Hybridization, Immunohistochemistry, Marker, Staining

11) Product Images from "TDP-43-Mediated Neuron Loss In Vivo Requires RNA-Binding Activity"

Article Title: TDP-43-Mediated Neuron Loss In Vivo Requires RNA-Binding Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0012247

RNA-binding activity is required for TDP-43-mediated motor neuron loss in chick. (A) Stable unilateral expression of human TDP-43 variants in chick ( Gallus gallus ) spinal cord. [i] Schematic of expression system mediating motor neuron-restricted expression upon unilateral transfection. [ii] Unilateral expression of human TDP-43 FFLL (red) in embryonic day 9 (E9) chick spinal cord (nuclei labeled with DAPI: blue): transversal section at lumbar levels. “−” and “+” respectively indicate control and transfected hemicords. Isl1/2 labels motor neuron nuclei (green). (B) [i] Examples of thoracic motor columns (Isl1/2 + motor neurons: green) upon TDP-43 variant expression (red). [ii] Quantification of motor neuron loss upon TDP-43 variant expression over all obtained sections (in “-” versus “+” hemicord). Differences relative to control (t-student's test) are indicated. (C) [i] Activated Caspase-3 (green) detected in E5 motor neurons upon TDP-43 WT expression (indicated by IRES-cherry bi-cistronic reporter: red). Compared to E5 motor neurons expressing TDP-43 WT , little activation of Caspase-3 was detected upon TDP-43 CTF expression. [ii] Quantification of Caspase-3 activation in motor neurons of transfected hemicords versus vector control. Significant differences are indicated (t-student's test - relative to control). *p
Figure Legend Snippet: RNA-binding activity is required for TDP-43-mediated motor neuron loss in chick. (A) Stable unilateral expression of human TDP-43 variants in chick ( Gallus gallus ) spinal cord. [i] Schematic of expression system mediating motor neuron-restricted expression upon unilateral transfection. [ii] Unilateral expression of human TDP-43 FFLL (red) in embryonic day 9 (E9) chick spinal cord (nuclei labeled with DAPI: blue): transversal section at lumbar levels. “−” and “+” respectively indicate control and transfected hemicords. Isl1/2 labels motor neuron nuclei (green). (B) [i] Examples of thoracic motor columns (Isl1/2 + motor neurons: green) upon TDP-43 variant expression (red). [ii] Quantification of motor neuron loss upon TDP-43 variant expression over all obtained sections (in “-” versus “+” hemicord). Differences relative to control (t-student's test) are indicated. (C) [i] Activated Caspase-3 (green) detected in E5 motor neurons upon TDP-43 WT expression (indicated by IRES-cherry bi-cistronic reporter: red). Compared to E5 motor neurons expressing TDP-43 WT , little activation of Caspase-3 was detected upon TDP-43 CTF expression. [ii] Quantification of Caspase-3 activation in motor neurons of transfected hemicords versus vector control. Significant differences are indicated (t-student's test - relative to control). *p

Techniques Used: RNA Binding Assay, Activity Assay, Expressing, Transfection, Labeling, Variant Assay, Activation Assay, Plasmid Preparation

12) Product Images from "A bi-modal function of Wnt signalling directs an FGF activity gradient to spatially regulate neuronal differentiation in the midbrain"

Article Title: A bi-modal function of Wnt signalling directs an FGF activity gradient to spatially regulate neuronal differentiation in the midbrain

Journal: Development (Cambridge, England)

doi: 10.1242/dev.099507

FGF, Wnt and Her5 dictate the number of MTN neurons that form in the midbrain. In situ hybridisation with probes for drg11 (A-E) and isl1 (F-I) reveals increased numbers of MTN neurons in zebrafish embryos exposed to 40 μM SU5402 (B) or 4 μM
Figure Legend Snippet: FGF, Wnt and Her5 dictate the number of MTN neurons that form in the midbrain. In situ hybridisation with probes for drg11 (A-E) and isl1 (F-I) reveals increased numbers of MTN neurons in zebrafish embryos exposed to 40 μM SU5402 (B) or 4 μM

Techniques Used: In Situ, Hybridization

13) Product Images from "ISL1 and BRN3B co-regulate the differentiation of murine retinal ganglion cells"

Article Title: ISL1 and BRN3B co-regulate the differentiation of murine retinal ganglion cells

Journal:

doi: 10.1242/dev.010751

ISL1 and BRN3B regulate the expression of a common set of RGC-specific genes. (A–H) Compared with controls (left panels), in situ hybridization shows that at E14.5, the expression of RGC specific genes Brn3a, Isl2, Olf1, Irx4, Ablim1, Gap43, L1cam,
Figure Legend Snippet: ISL1 and BRN3B regulate the expression of a common set of RGC-specific genes. (A–H) Compared with controls (left panels), in situ hybridization shows that at E14.5, the expression of RGC specific genes Brn3a, Isl2, Olf1, Irx4, Ablim1, Gap43, L1cam,

Techniques Used: Expressing, In Situ Hybridization

Functional mechanism of ISL1 and BRN3B in the development of RGCs. (A) Concurrent binding of ISL1 and BRN3B to RGC-specific promoters. Anti-BRN3B and anti-ISL1 antibodies co-precipitate with the promoters of Brn3b , Shh , Brn3a and Isl2 . Both antibodies
Figure Legend Snippet: Functional mechanism of ISL1 and BRN3B in the development of RGCs. (A) Concurrent binding of ISL1 and BRN3B to RGC-specific promoters. Anti-BRN3B and anti-ISL1 antibodies co-precipitate with the promoters of Brn3b , Shh , Brn3a and Isl2 . Both antibodies

Techniques Used: Functional Assay, Binding Assay

More severe RGC loss in Isl1 and Brn3b compound null mice. (A–H) Immunostaining of adult whole-mount retinas with anti-BRN3A (A–D) and SMI32 (E–H). Compared with control (A,E), Isl1 -null (B,F), and Brn3b -null (C,G), a more severe
Figure Legend Snippet: More severe RGC loss in Isl1 and Brn3b compound null mice. (A–H) Immunostaining of adult whole-mount retinas with anti-BRN3A (A–D) and SMI32 (E–H). Compared with control (A,E), Isl1 -null (B,F), and Brn3b -null (C,G), a more severe

Techniques Used: Mouse Assay, Immunostaining

Generation of Isl1 conditional knockout and Isl1-lacZ knock-in alleles. (A) Generation of Isl1 conditional allele. Isl1 genomic structure and restriction enzyme map is shown at the top. Open boxes are the non-coding exon sequences and filled boxes the
Figure Legend Snippet: Generation of Isl1 conditional knockout and Isl1-lacZ knock-in alleles. (A) Generation of Isl1 conditional allele. Isl1 genomic structure and restriction enzyme map is shown at the top. Open boxes are the non-coding exon sequences and filled boxes the

Techniques Used: Knock-Out, Knock-In

Targeted disruption of Isl1 results in the developmental loss of RGCs. (A–H) Immunostaining of retina sections with anti-BRN3B shows that in Isl1- null retina, BRN3B+ RGCs are generated and positioned properly at E13.5 (A,E) and E15.5 (B,F). However,
Figure Legend Snippet: Targeted disruption of Isl1 results in the developmental loss of RGCs. (A–H) Immunostaining of retina sections with anti-BRN3B shows that in Isl1- null retina, BRN3B+ RGCs are generated and positioned properly at E13.5 (A,E) and E15.5 (B,F). However,

Techniques Used: Immunostaining, Generated

Loss of RGCs in adult Isl1 -null retina. (A–G) Immunostaining of whole-mount retina with anti-BRN3A (A,B), BRN3B (C,D) and SMI32 (F,G) antibodies reveals the reduction of RGCs in Isl1 - null retina. (E) Quantification of BRN3A+ and BRN3B+ cells in
Figure Legend Snippet: Loss of RGCs in adult Isl1 -null retina. (A–G) Immunostaining of whole-mount retina with anti-BRN3A (A,B), BRN3B (C,D) and SMI32 (F,G) antibodies reveals the reduction of RGCs in Isl1 - null retina. (E) Quantification of BRN3A+ and BRN3B+ cells in

Techniques Used: Immunostaining

Axon growth defects in mice deficient for Isl1 or Brn3b . (A–F) After DiI was placed at the right optic nerve head, brains were dissected to expose the optic pathways at the ventral diencephalons. RGC axons of wild type pass the midline (dot line),
Figure Legend Snippet: Axon growth defects in mice deficient for Isl1 or Brn3b . (A–F) After DiI was placed at the right optic nerve head, brains were dissected to expose the optic pathways at the ventral diencephalons. RGC axons of wild type pass the midline (dot line),

Techniques Used: Mouse Assay

Expression of ISL1 in developing mouse retina. (A–F) Horizontal sections of retinas at E11.5 (A–C) and E13.5 (D–F) were immunolabeled with anti-ISL1 (red) and anti-BRN3B (green). The expression of ISL1 is detected from E11.5 and
Figure Legend Snippet: Expression of ISL1 in developing mouse retina. (A–F) Horizontal sections of retinas at E11.5 (A–C) and E13.5 (D–F) were immunolabeled with anti-ISL1 (red) and anti-BRN3B (green). The expression of ISL1 is detected from E11.5 and

Techniques Used: Expressing, Immunolabeling

14) Product Images from "Slit and semaphorin signaling governed by Islet transcription factors positions motor neuron somata within the neural tube"

Article Title: Slit and semaphorin signaling governed by Islet transcription factors positions motor neuron somata within the neural tube

Journal: Experimental neurology

doi: 10.1016/j.expneurol.2015.03.024

Reduced expression of Neuropilin1 and Slit2 in the absence of Isl1 and Isl2
Figure Legend Snippet: Reduced expression of Neuropilin1 and Slit2 in the absence of Isl1 and Isl2

Techniques Used: Expressing

Ectopic motor neurons in the ventral roots of Isl1 hypo ( Isl1 h/h ) and Isl1 hypo; Isl2 ( Isl1 h/h ; Isl2 KO) null mice
Figure Legend Snippet: Ectopic motor neurons in the ventral roots of Isl1 hypo ( Isl1 h/h ) and Isl1 hypo; Isl2 ( Isl1 h/h ; Isl2 KO) null mice

Techniques Used: Mouse Assay

15) Product Images from "Distinct temporal requirements for Sonic hedgehog signaling in development of the tuberal hypothalamus"

Article Title: Distinct temporal requirements for Sonic hedgehog signaling in development of the tuberal hypothalamus

Journal: Development (Cambridge, England)

doi: 10.1242/dev.167379

Shh signaling is required for subtype identity of VMH and DMH neurons. Coronal sections through the tuberal hypothalamus of control and cSmo embryos at E14.5 that received tamoxifen at E10.5. (A-C) The number of cells expressing Isl1 in the DMH is reduced
Figure Legend Snippet: Shh signaling is required for subtype identity of VMH and DMH neurons. Coronal sections through the tuberal hypothalamus of control and cSmo embryos at E14.5 that received tamoxifen at E10.5. (A-C) The number of cells expressing Isl1 in the DMH is reduced

Techniques Used: Expressing

16) Product Images from "Hair Cell Overexpression of Islet1 Reduces Age-Related and Noise-Induced Hearing Loss"

Article Title: Hair Cell Overexpression of Islet1 Reduces Age-Related and Noise-Induced Hearing Loss

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1489-13.2013

Construction and characterization of Isl1-TG mice. A , A schematic diagram depicting the construction of the Pou4f3 promoter driven Isl1 expression, with an IRES signal that links an EGFP ORF as a separate marker. The total size of the construct is ∼14 kb. Positions of genotyping primers are also shown. B , C , In 20-month-old Is1l-TG mouse cochlea, GFP signal was detected in all OHCs ( B ) and inner hair cells ( B , IHC) and was colocalized with MYO7A-labeled hair cells ( C ). D , E , In P1 Isl1-TG cochlea, Isl1 transgene expression was detected in all hair cells ( D ), whereas in the WT control cochlea, endogenous Isl1 was no longer detectable in hair cells ( E ). F , qRT-PCR of 3-month-old adult showed overexpression of Isl1 in the Isl1-TG compared with WT control inner ear tissues. Expression levels were normalized with control gene Pgk1 . Ut, Utricle; Coch, cochlea. Isl1-TG , n = 2; WT, n = 3. Error bars indcate mean (±SEM). Scale bar, 10 μm.
Figure Legend Snippet: Construction and characterization of Isl1-TG mice. A , A schematic diagram depicting the construction of the Pou4f3 promoter driven Isl1 expression, with an IRES signal that links an EGFP ORF as a separate marker. The total size of the construct is ∼14 kb. Positions of genotyping primers are also shown. B , C , In 20-month-old Is1l-TG mouse cochlea, GFP signal was detected in all OHCs ( B ) and inner hair cells ( B , IHC) and was colocalized with MYO7A-labeled hair cells ( C ). D , E , In P1 Isl1-TG cochlea, Isl1 transgene expression was detected in all hair cells ( D ), whereas in the WT control cochlea, endogenous Isl1 was no longer detectable in hair cells ( E ). F , qRT-PCR of 3-month-old adult showed overexpression of Isl1 in the Isl1-TG compared with WT control inner ear tissues. Expression levels were normalized with control gene Pgk1 . Ut, Utricle; Coch, cochlea. Isl1-TG , n = 2; WT, n = 3. Error bars indcate mean (±SEM). Scale bar, 10 μm.

Techniques Used: Mouse Assay, Expressing, Marker, Construct, Immunohistochemistry, Labeling, Quantitative RT-PCR, Over Expression

Survival of hair cells and preservation of presynaptic ribbons in aged Isl1-TG cochleas. A , B , Confocal images from the 32 kHz region in 19-month-old cochleas from an Isl1-TG and a WT. Hair cells are immunostained with antibodies to myosin VIIA (cyan); ribbons are stained with antibodies to CtBP2 (red). Scale bar, 10 μm. C , H E staining of a 17-month-old WT cochlea section to show OHC loss. D , H E staining of a 17-month-old Isl1-TG cochlea section to show preservation of OHCs and the overall structure. E , F , H E staining of 15-month-old WT and Isl1-TG mid-turn sections to show similar ganglion survival. G , Hair cell counts from 19-month-old animals of both genotypes show significant enhancement of survival in the basal half of the cochlea in the Isl1-TG . Data are group means (±SEM). Group sizes were as follows: n = 8 for TG; n = 7 for WT. H , At 19 months, the number of synaptic ribbons per hair cell was significantly reduced in the basal half of the cochlea in WT mice but preserved in the Isl1-TG s. I , J , At 3 months of age, the numbers of hair cells or ribbons were not significantly different in WT versus Isl1-TG mice. K , By genomic PCR, a fragment of 194 bp for the Cdh23 ahl allele was amplified. Upon PstI digestion, both Isl1-TG and WT showed a 170 bp fragment corresponding to the mutant Cdh23 ahl allele, whereas the 194 bp fragment remained in CBA/CaJ. n = 4 in each comparison. ANOVA analysis: G , F = 25.07, *** p
Figure Legend Snippet: Survival of hair cells and preservation of presynaptic ribbons in aged Isl1-TG cochleas. A , B , Confocal images from the 32 kHz region in 19-month-old cochleas from an Isl1-TG and a WT. Hair cells are immunostained with antibodies to myosin VIIA (cyan); ribbons are stained with antibodies to CtBP2 (red). Scale bar, 10 μm. C , H E staining of a 17-month-old WT cochlea section to show OHC loss. D , H E staining of a 17-month-old Isl1-TG cochlea section to show preservation of OHCs and the overall structure. E , F , H E staining of 15-month-old WT and Isl1-TG mid-turn sections to show similar ganglion survival. G , Hair cell counts from 19-month-old animals of both genotypes show significant enhancement of survival in the basal half of the cochlea in the Isl1-TG . Data are group means (±SEM). Group sizes were as follows: n = 8 for TG; n = 7 for WT. H , At 19 months, the number of synaptic ribbons per hair cell was significantly reduced in the basal half of the cochlea in WT mice but preserved in the Isl1-TG s. I , J , At 3 months of age, the numbers of hair cells or ribbons were not significantly different in WT versus Isl1-TG mice. K , By genomic PCR, a fragment of 194 bp for the Cdh23 ahl allele was amplified. Upon PstI digestion, both Isl1-TG and WT showed a 170 bp fragment corresponding to the mutant Cdh23 ahl allele, whereas the 194 bp fragment remained in CBA/CaJ. n = 4 in each comparison. ANOVA analysis: G , F = 25.07, *** p

Techniques Used: Preserving, Staining, Mouse Assay, Polymerase Chain Reaction, Amplification, Mutagenesis, Crocin Bleaching Assay

Noise-induced threshold shift is reduced in Isl1-TG mice compared with WT 2 weeks after noise exposure, as measured by either ABRs ( A ) or DPOAEs ( B ). Data are group means (±SEM). Group sizes were as follows: n = 19 for control, n = 30 for Isl1-TG . The up arrow indicates a lack of response in some animals at the highest SPLs tested, which could lead to an underestimated threshold shift. ANOVA analysis: ABR: F = 4.718, * p = 0.035; DPOAE: F = 1.205, p = 0.278.
Figure Legend Snippet: Noise-induced threshold shift is reduced in Isl1-TG mice compared with WT 2 weeks after noise exposure, as measured by either ABRs ( A ) or DPOAEs ( B ). Data are group means (±SEM). Group sizes were as follows: n = 19 for control, n = 30 for Isl1-TG . The up arrow indicates a lack of response in some animals at the highest SPLs tested, which could lead to an underestimated threshold shift. ANOVA analysis: ABR: F = 4.718, * p = 0.035; DPOAE: F = 1.205, p = 0.278.

Techniques Used: Mouse Assay

Reduced age-related threshold shift. A–H , Age-related threshold shift is reduced in Isl1-TG mice, as measured either by ABRs ( A , C , E , G ) or DPOAEs ( B , D , F , H ). Thresholds were compared with 3-month-old WT and Isl1-TG mice, which had indistinguishable ABR and DPOAE at this age. Data are group means (±SEM). Group sizes were as follows: 3 months: n = 4 for control, n = 10 for Isl1-TG ; 6 months: n = 4 for control, n = 10 for Isl1-TG ; for 12 months: n = 4 for control, n = 9 for Isl1-TG ; for 17 months: n = 4 for control, n = 11 for Isl1-TG . The up arrows indicate that at the highest SPLs tested some animals showed no response, which will cause an underestimation of threshold shift. For ANOVA analysis: 6 months: ABR: F = 11.31, ** p = 0.0012; DPOAE: F = 0.560; p = 0.457; 12 months: ABR: F = 7.636, * p = 0.018; DPOAE: F = 1.773, p = 0.210; 17 months: ABR: F = 11.184; ** p = 0.006; DPOAE: F = 11.554, ** p = 0.005.
Figure Legend Snippet: Reduced age-related threshold shift. A–H , Age-related threshold shift is reduced in Isl1-TG mice, as measured either by ABRs ( A , C , E , G ) or DPOAEs ( B , D , F , H ). Thresholds were compared with 3-month-old WT and Isl1-TG mice, which had indistinguishable ABR and DPOAE at this age. Data are group means (±SEM). Group sizes were as follows: 3 months: n = 4 for control, n = 10 for Isl1-TG ; 6 months: n = 4 for control, n = 10 for Isl1-TG ; for 12 months: n = 4 for control, n = 9 for Isl1-TG ; for 17 months: n = 4 for control, n = 11 for Isl1-TG . The up arrows indicate that at the highest SPLs tested some animals showed no response, which will cause an underestimation of threshold shift. For ANOVA analysis: 6 months: ABR: F = 11.31, ** p = 0.0012; DPOAE: F = 0.560; p = 0.457; 12 months: ABR: F = 7.636, * p = 0.018; DPOAE: F = 1.773, p = 0.210; 17 months: ABR: F = 11.184; ** p = 0.006; DPOAE: F = 11.554, ** p = 0.005.

Techniques Used: Mouse Assay

Normal differentiation of Isl1-TG inner ear. A–J , There is no significant difference in the key protein distribution between Isl1-TG and WT control inner ear and hair cells that include PTPRQ for hair bundles ( A , B ), acetylated tubulin ( A , B , Tub), and neurofilament ( G , H , NF) for nerve fibers; prestin for OHC, and PROX1 for supporting cells ( C , D ); PCNA for proliferating cells and GFP only in transgenic hair cells ( E , F ), p27KIP1 for supporting cells ( G , H ), SOX2 for supporting cells, and MYO7A for hair cells ( I , J ). K , L , FM1-43 uptake showed colocalization between GFP fluorescence and hair bundles in P2 Isl1-TG cochlea ( K ) and utricle ( L ). M , A diagram depicting signal intensity of genes in hair cells and supporting cells between Is1l-TG and WT cochleas. Only Isl1 and GFP showed significant increase in the Isl1-TG versus WT control hair cells. * p
Figure Legend Snippet: Normal differentiation of Isl1-TG inner ear. A–J , There is no significant difference in the key protein distribution between Isl1-TG and WT control inner ear and hair cells that include PTPRQ for hair bundles ( A , B ), acetylated tubulin ( A , B , Tub), and neurofilament ( G , H , NF) for nerve fibers; prestin for OHC, and PROX1 for supporting cells ( C , D ); PCNA for proliferating cells and GFP only in transgenic hair cells ( E , F ), p27KIP1 for supporting cells ( G , H ), SOX2 for supporting cells, and MYO7A for hair cells ( I , J ). K , L , FM1-43 uptake showed colocalization between GFP fluorescence and hair bundles in P2 Isl1-TG cochlea ( K ) and utricle ( L ). M , A diagram depicting signal intensity of genes in hair cells and supporting cells between Is1l-TG and WT cochleas. Only Isl1 and GFP showed significant increase in the Isl1-TG versus WT control hair cells. * p

Techniques Used: Transgenic Assay, Fluorescence

Hair cell survival is enhanced after 116 dB noise in Isl1-TG mice. A , B , No significant difference in hair cell survival or ribbon counts was seen between Isl1-TG and WT mice 1 month after 100 dB exposure. C , After the 116 dB exposure, the OHC loss was significantly larger in some regions in WTs than in Isl1-TG s. ANOVA, F = 55.20, *** p
Figure Legend Snippet: Hair cell survival is enhanced after 116 dB noise in Isl1-TG mice. A , B , No significant difference in hair cell survival or ribbon counts was seen between Isl1-TG and WT mice 1 month after 100 dB exposure. C , After the 116 dB exposure, the OHC loss was significantly larger in some regions in WTs than in Isl1-TG s. ANOVA, F = 55.20, *** p

Techniques Used: Mouse Assay

Hair cell survival in Isl1-TG after intense noise exposure. A , B , One month after 100 dB exposure, in both WT and Isl1-TG mice, most OHCs survived with a few missing OHCs. The number of ribbon counts was also similar (CtBP2 labeling). C , D , 2 months after 116 dB exposure, WT mice exhibited major OHC loss, whereas in Isl1-TG mice most OHCs survived. E , Twenty-one hours after 116 dB exposure, many WT OHCs were labeled with cleaved Casp9 (arrow), and many condensed nuclei (arrowheads) were seen in the basal half of the cochlea. F , In Isl1-TG cochlea from the similar region, no Casp9-positive hair cells were detected. Scale bar, 10 μm.
Figure Legend Snippet: Hair cell survival in Isl1-TG after intense noise exposure. A , B , One month after 100 dB exposure, in both WT and Isl1-TG mice, most OHCs survived with a few missing OHCs. The number of ribbon counts was also similar (CtBP2 labeling). C , D , 2 months after 116 dB exposure, WT mice exhibited major OHC loss, whereas in Isl1-TG mice most OHCs survived. E , Twenty-one hours after 116 dB exposure, many WT OHCs were labeled with cleaved Casp9 (arrow), and many condensed nuclei (arrowheads) were seen in the basal half of the cochlea. F , In Isl1-TG cochlea from the similar region, no Casp9-positive hair cells were detected. Scale bar, 10 μm.

Techniques Used: Mouse Assay, Labeling

17) Product Images from "The Lineage Contribution and Role of Gbx2 in Spinal Cord Development"

Article Title: The Lineage Contribution and Role of Gbx2 in Spinal Cord Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020940

Gbx2 loss affects spinal cord progenitor patterning. Hemi-transverse sections from E10.5 wildtype (A–H) and Gbx2 CreER-ires-eGFP/CreER-ires-eGFP mutant embryos (A′–H′) triple immunolabeled with indicated markers. (A, A′) Broader Brn3a domain dorsally (*1) and Brn3a expressing cells in the ventral domain (*2) in Gbx2 mutants. (B, B′) Brn3a+/Isl1/2+ neurons (arrows) and depletion of the medial-ventral domain of Isl1/2+ neurons (medial motor column,*) in Gbx2 mutants. Qualitatively, some ventral Isl1/2+ neurons inappropriately expressed Brn3a (yellow overlap). (C,C′) Ectopic Brn3a expressing cells in close proximity to the ventral Nkx2.2 population (*2). (D,D′) Medial-lateral expansion of early differentiating neurons in Gbx2 mutant embryos (*1, brackets). In addition, ectopic Brn3a+/Lim1/2+ neurons were seen ventral to their normal position (arrow). (E,E′) Pax3/Pax2 showing that the Pax3 domain is unchanged. (F,F′) Immunolabeling for phosphorylated-Histone H3 (pH3) showing fewer mitotic dorsally in mutant littermates. Wnt1 expression in the roofplate (RP) was subtly expanded (G,G′,*) while Shh expression in the floor plate (FP) and notochord (NC) was unaffected in mutants (H,H′).
Figure Legend Snippet: Gbx2 loss affects spinal cord progenitor patterning. Hemi-transverse sections from E10.5 wildtype (A–H) and Gbx2 CreER-ires-eGFP/CreER-ires-eGFP mutant embryos (A′–H′) triple immunolabeled with indicated markers. (A, A′) Broader Brn3a domain dorsally (*1) and Brn3a expressing cells in the ventral domain (*2) in Gbx2 mutants. (B, B′) Brn3a+/Isl1/2+ neurons (arrows) and depletion of the medial-ventral domain of Isl1/2+ neurons (medial motor column,*) in Gbx2 mutants. Qualitatively, some ventral Isl1/2+ neurons inappropriately expressed Brn3a (yellow overlap). (C,C′) Ectopic Brn3a expressing cells in close proximity to the ventral Nkx2.2 population (*2). (D,D′) Medial-lateral expansion of early differentiating neurons in Gbx2 mutant embryos (*1, brackets). In addition, ectopic Brn3a+/Lim1/2+ neurons were seen ventral to their normal position (arrow). (E,E′) Pax3/Pax2 showing that the Pax3 domain is unchanged. (F,F′) Immunolabeling for phosphorylated-Histone H3 (pH3) showing fewer mitotic dorsally in mutant littermates. Wnt1 expression in the roofplate (RP) was subtly expanded (G,G′,*) while Shh expression in the floor plate (FP) and notochord (NC) was unaffected in mutants (H,H′).

Techniques Used: Mutagenesis, Immunolabeling, Expressing

Molecular identity of the Gbx2 lineage in E12.5 spinal cord. (A) Sagittal section of E12.5 embryo with nuclear staining (blue) showing regions analyzed. (B) Pax2 expression in dorsal spinal cord (indicated by bracket) in a hemi-transverse section. The box indicates the dorsolateral area of high magnification sampled for panels D–F, M–O (C) Isl1/2 expression in hemi-transverse sections of ventral spinal cord at the upper limb level. Isl1/2 is expressed in all developing motor neurons (MN) and dorsal root ganglia (DRG). Marker analysis of upper (D–L) and lower (M–U) limb levels at E12.5. The Gbx2 lineage (ß-gal+, red) marked at E8.5, E9.5 or E10.5 gave rise to Pax2+ neurons (green) at both upper (D–F) and lower (M–O) limb levels; insets highlight colocalization. (G–I) The Gbx2 lineage (ß-gal+, red) marked at E8.5, but not E9.5 or E10.5, contributed to ventral MNs (Isl1/2+, green) at upper limb level. (P–R) MNs (Is1/2+, green) at lower limb level were derived from the Gbx2 lineage (ß-gal+, red) marked at E8.5 and E9.5, but not E10.5. (J–L) Neurons in upper limb DRG (Isl1/2+, green) were derived from the Gbx2 lineage at E8.5 but not at later stages. (S–U) Caudal DRG (Isl1/2+, green) were derived from the Gbx2 lineage at E8.5 and E9.5.
Figure Legend Snippet: Molecular identity of the Gbx2 lineage in E12.5 spinal cord. (A) Sagittal section of E12.5 embryo with nuclear staining (blue) showing regions analyzed. (B) Pax2 expression in dorsal spinal cord (indicated by bracket) in a hemi-transverse section. The box indicates the dorsolateral area of high magnification sampled for panels D–F, M–O (C) Isl1/2 expression in hemi-transverse sections of ventral spinal cord at the upper limb level. Isl1/2 is expressed in all developing motor neurons (MN) and dorsal root ganglia (DRG). Marker analysis of upper (D–L) and lower (M–U) limb levels at E12.5. The Gbx2 lineage (ß-gal+, red) marked at E8.5, E9.5 or E10.5 gave rise to Pax2+ neurons (green) at both upper (D–F) and lower (M–O) limb levels; insets highlight colocalization. (G–I) The Gbx2 lineage (ß-gal+, red) marked at E8.5, but not E9.5 or E10.5, contributed to ventral MNs (Isl1/2+, green) at upper limb level. (P–R) MNs (Is1/2+, green) at lower limb level were derived from the Gbx2 lineage (ß-gal+, red) marked at E8.5 and E9.5, but not E10.5. (J–L) Neurons in upper limb DRG (Isl1/2+, green) were derived from the Gbx2 lineage at E8.5 but not at later stages. (S–U) Caudal DRG (Isl1/2+, green) were derived from the Gbx2 lineage at E8.5 and E9.5.

Techniques Used: Staining, Expressing, Marker, Derivative Assay

Gbx2 mutant lineage at E12.5. Control Gbx2 CreER-ires-eGFP/+ (A) and mutant Gbx2 CreER-ires-eGFP/CreER-ires-eGFP (B) embryos at E12.5; mutants have reduced r1 (r1*). (C,C′) GFP immunolabeling on level-matched hemi-transverse sections of E12.5 heterozygous control (C) versus Gbx2 mutant embryos (C′). Ectopic clusters of Gbx2 mutant (GFP+) cells (*) in the ventricular zone. (D,D′) Isl1/2 immunolabeling on transverse sections of E12.5 heterozygous (D) versus Gbx2 mutant embryos (D′) showing loss of medial motor neurons in Gbx2 mutant embryos (*). (E, E′) Immunolabeling for GFP and Pax2 showing ectopically located ventral Gbx2 (GFP)-mutant/Pax2+ interneurons (*) in mutant embryos; arrows indicate regions shown in insets. (F–G′) GIFM of thoracic sections from wildtype control (F,G) versus mutant (F′,G′) spinal cord. (F) ß-gal and GFP immunolabeling showing the wildtype Gbx2 lineage (ß-gal+, red) marked at E9.5 and Gbx2 -expressing neurons at E12.5. (F′) The Gbx2 -mutant lineage marked at E9.5 (ß-gal+, red) and Gbx2 -mutants cells (GFP+, green) analyzed at E12.5. (G, G′) ß-gal expression resulting from lineage marking at E9.5 versus Pax2 expression in E12.5 control (G) versus Gbx2 mutant embryos (G′). Note that some ventral Pax2+ cells are disorganized (*1, *2), and others are ectopically located (*3). (H–H′) Cells of the Gbx2 mutant lineage marked at E9.5 reside in ectopic locations (arrows) that are ventral to the lineage boundary seen in controls (arrowheads).
Figure Legend Snippet: Gbx2 mutant lineage at E12.5. Control Gbx2 CreER-ires-eGFP/+ (A) and mutant Gbx2 CreER-ires-eGFP/CreER-ires-eGFP (B) embryos at E12.5; mutants have reduced r1 (r1*). (C,C′) GFP immunolabeling on level-matched hemi-transverse sections of E12.5 heterozygous control (C) versus Gbx2 mutant embryos (C′). Ectopic clusters of Gbx2 mutant (GFP+) cells (*) in the ventricular zone. (D,D′) Isl1/2 immunolabeling on transverse sections of E12.5 heterozygous (D) versus Gbx2 mutant embryos (D′) showing loss of medial motor neurons in Gbx2 mutant embryos (*). (E, E′) Immunolabeling for GFP and Pax2 showing ectopically located ventral Gbx2 (GFP)-mutant/Pax2+ interneurons (*) in mutant embryos; arrows indicate regions shown in insets. (F–G′) GIFM of thoracic sections from wildtype control (F,G) versus mutant (F′,G′) spinal cord. (F) ß-gal and GFP immunolabeling showing the wildtype Gbx2 lineage (ß-gal+, red) marked at E9.5 and Gbx2 -expressing neurons at E12.5. (F′) The Gbx2 -mutant lineage marked at E9.5 (ß-gal+, red) and Gbx2 -mutants cells (GFP+, green) analyzed at E12.5. (G, G′) ß-gal expression resulting from lineage marking at E9.5 versus Pax2 expression in E12.5 control (G) versus Gbx2 mutant embryos (G′). Note that some ventral Pax2+ cells are disorganized (*1, *2), and others are ectopically located (*3). (H–H′) Cells of the Gbx2 mutant lineage marked at E9.5 reside in ectopic locations (arrows) that are ventral to the lineage boundary seen in controls (arrowheads).

Techniques Used: Mutagenesis, Immunolabeling, Expressing

Quantitative assessment of aberrantly distributed spinal cord progenitors in Gbx2 mutant embryos. Quantitative spatial analysis of control Gbx2 CreER-ires-eGFP/+ (A–F) and mutant Gbx2 CreER-ires-eGFP/CreER-ires-eGFP (G–L) spinal cords at E10.5. The average number of progenitors was assessed by counting cells with expressing the indicated markers in two sections at the upper limb level from control embryos (n = 3) and mutant embryos (n = 4). To facilitate a clear comparison of the spatial distribution across samples, we a Cartesian coordinate system where ML 1 -DV 1 represented the most medial-dorsal quadrant, ML 1 -DV 10 the most medial-ventral quadrant, ML 4 -DV 1 the most lateral-dorsal quadrant, and ML 4 -DV 10 the most lateral-ventral quadrant (M–R). The yellow boxes in panels M–R are shown at higher magnification with white dots used to track counted cells. The yellow boxes also correlate with the domains that were highlighted in the graphs with a yellow arrow. Quantitative spatial mapping revealed the distribution of Brn3a+ cells (A,G,M), Isl1/2+ (B,H,N), Lim1+ (C,I,O), Brn3a+/Isl1/2+ (D,J,P), Brn3a+/Lim1+ (E,K,Q), and pHH3 (F,L,R).
Figure Legend Snippet: Quantitative assessment of aberrantly distributed spinal cord progenitors in Gbx2 mutant embryos. Quantitative spatial analysis of control Gbx2 CreER-ires-eGFP/+ (A–F) and mutant Gbx2 CreER-ires-eGFP/CreER-ires-eGFP (G–L) spinal cords at E10.5. The average number of progenitors was assessed by counting cells with expressing the indicated markers in two sections at the upper limb level from control embryos (n = 3) and mutant embryos (n = 4). To facilitate a clear comparison of the spatial distribution across samples, we a Cartesian coordinate system where ML 1 -DV 1 represented the most medial-dorsal quadrant, ML 1 -DV 10 the most medial-ventral quadrant, ML 4 -DV 1 the most lateral-dorsal quadrant, and ML 4 -DV 10 the most lateral-ventral quadrant (M–R). The yellow boxes in panels M–R are shown at higher magnification with white dots used to track counted cells. The yellow boxes also correlate with the domains that were highlighted in the graphs with a yellow arrow. Quantitative spatial mapping revealed the distribution of Brn3a+ cells (A,G,M), Isl1/2+ (B,H,N), Lim1+ (C,I,O), Brn3a+/Isl1/2+ (D,J,P), Brn3a+/Lim1+ (E,K,Q), and pHH3 (F,L,R).

Techniques Used: Mutagenesis, Expressing

Dynamic expression of Gbx2 in the developing spinal cord. Gbx2 (GFP) expression detected in whole mount embryo (A). GFP immunolabeling (B, top row) and adjacent sections processed for Gbx2 in situ hybridization (B, bottom row) from E8.5 Gbx2 CreER-ires-eGFP embryo; inset in “A” shows wildtype littermate. (C) Gbx2 (GFP) expression in lateral view of an E9.5 embryo. (D–E) GFP and Pax7 immunolabeling on E9.5 Gbx2 CreER-ires-eGFP/+ sections. (F–G) Lateral (F) and (G) dorsal views of EGFP fluorescence in E10.5 Gbx2 CreER-ires-eGFP/+ embryo. (H–J) Antibody labeling of GFP and indicated markers on sagittal sections of E10.5 spinal cord; Note restricted ventral strip of Gbx2 (GFP) expression (J, arrows). (K–S) Antibody labeling of GFP and indicated D-V markers on transverse hemi-sections of E10.5 spinal cord at the upper limb level. The insets show a high magnification view of the region indicated by the arrow. (T–U) EGFP fluorescence of E12.5 Gbx2 CreER-ires-eGFP/+ embryo showing lateral (T) and dorsal (U) view. (V) GFP antibody labeling on sagittal sections of E12.5 spinal cord. GFP/Pax2 (W–W″) and GFP/Isl1/2 (X–X″) immunolabeling on transverse E12.5 hemi-sections of spinal cord at the upper limb (rostral) level. Abbreviations: mesencephalon (mes), rhombomere 1 (r1), intermediate (int) and posterior (post) neural tube, neuroepithelium (ne), blood vessel (bv), prosencephalon (pros), thalamus (thal), spinal cord (sc).
Figure Legend Snippet: Dynamic expression of Gbx2 in the developing spinal cord. Gbx2 (GFP) expression detected in whole mount embryo (A). GFP immunolabeling (B, top row) and adjacent sections processed for Gbx2 in situ hybridization (B, bottom row) from E8.5 Gbx2 CreER-ires-eGFP embryo; inset in “A” shows wildtype littermate. (C) Gbx2 (GFP) expression in lateral view of an E9.5 embryo. (D–E) GFP and Pax7 immunolabeling on E9.5 Gbx2 CreER-ires-eGFP/+ sections. (F–G) Lateral (F) and (G) dorsal views of EGFP fluorescence in E10.5 Gbx2 CreER-ires-eGFP/+ embryo. (H–J) Antibody labeling of GFP and indicated markers on sagittal sections of E10.5 spinal cord; Note restricted ventral strip of Gbx2 (GFP) expression (J, arrows). (K–S) Antibody labeling of GFP and indicated D-V markers on transverse hemi-sections of E10.5 spinal cord at the upper limb level. The insets show a high magnification view of the region indicated by the arrow. (T–U) EGFP fluorescence of E12.5 Gbx2 CreER-ires-eGFP/+ embryo showing lateral (T) and dorsal (U) view. (V) GFP antibody labeling on sagittal sections of E12.5 spinal cord. GFP/Pax2 (W–W″) and GFP/Isl1/2 (X–X″) immunolabeling on transverse E12.5 hemi-sections of spinal cord at the upper limb (rostral) level. Abbreviations: mesencephalon (mes), rhombomere 1 (r1), intermediate (int) and posterior (post) neural tube, neuroepithelium (ne), blood vessel (bv), prosencephalon (pros), thalamus (thal), spinal cord (sc).

Techniques Used: Expressing, Immunolabeling, In Situ Hybridization, Fluorescence, Antibody Labeling, Stripping Membranes

18) Product Images from "Transient inactivation of Notch signaling synchronizes differentiation of neural progenitor cells"

Article Title: Transient inactivation of Notch signaling synchronizes differentiation of neural progenitor cells

Journal: Developmental biology

doi: 10.1016/j.ydbio.2007.01.001

Notch signaling inactivation promotes stage appropriate neuronal differentiation (A–F) DAPT treatment of E12.5 mouse retina increased ganglion cell differentiation, visualized by Tuj1 and Islet1 (Isl1) immunolabeling of sections (A, B and D, E) and wholemounts (C) and (F) respectively: asterisk in (C) and (F) mark DAPT-treated explant. (G–J) DAPT treatment at E12.5 also increased cone photoreceptor differentiation visualized by Trß2 immunolabeling (G, H), but not later-born rod photoreceptors visualized by lack of Rhodopsin immunoreactivity in both control and DAPT-treated explants (I, J respectively). (K–R) DAPT treatment at P1 had no effect on earlier-born Tuj1+ neurons (K, L), but produced a clear increase in both Recoverin and Rhodopsin immunolabeling of rod photoreceptors in the apical region of the outer nuclear layer (M–P); also note the numerous newly generated rods in the more basal region of the outer nuclear layer (arrowheads, N, P). DAPT treatment also produced a clear decrease in the differentiation of Muller glia cells, which have just begun to differentiate as visualized by CRALBP and Cyclin D3 immunolabeling (Q, R).
Figure Legend Snippet: Notch signaling inactivation promotes stage appropriate neuronal differentiation (A–F) DAPT treatment of E12.5 mouse retina increased ganglion cell differentiation, visualized by Tuj1 and Islet1 (Isl1) immunolabeling of sections (A, B and D, E) and wholemounts (C) and (F) respectively: asterisk in (C) and (F) mark DAPT-treated explant. (G–J) DAPT treatment at E12.5 also increased cone photoreceptor differentiation visualized by Trß2 immunolabeling (G, H), but not later-born rod photoreceptors visualized by lack of Rhodopsin immunoreactivity in both control and DAPT-treated explants (I, J respectively). (K–R) DAPT treatment at P1 had no effect on earlier-born Tuj1+ neurons (K, L), but produced a clear increase in both Recoverin and Rhodopsin immunolabeling of rod photoreceptors in the apical region of the outer nuclear layer (M–P); also note the numerous newly generated rods in the more basal region of the outer nuclear layer (arrowheads, N, P). DAPT treatment also produced a clear decrease in the differentiation of Muller glia cells, which have just begun to differentiate as visualized by CRALBP and Cyclin D3 immunolabeling (Q, R).

Techniques Used: Cell Differentiation, Immunolabeling, Produced, Generated

19) Product Images from "Compensatory Response by Late Embryonic Tubular Epithelium to the Reduction in Pancreatic Progenitors"

Article Title: Compensatory Response by Late Embryonic Tubular Epithelium to the Reduction in Pancreatic Progenitors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0142286

Endocrine differentiation of pancreas is impaired in Pdx1 tTA/+ ;tetO MafA pancreas. At E17.5 Isl1 (green, AB ), Pax6 (green, CD ) and Hb9 (green, EF ), transcription factors implicated in endocrine differentiation and maturation, have severely reduced expression in Pdx1 tTA/+ ;tetO MafA pancreas ( ACE ) compared to tetO MafA pancreas ( BDF ). This finding is consistent with their reduced number of insulin + cells (red, A-F ), and suggests that misexpression of the MafA transgene inhibits the entire endocrine differentiation program. DAPI (blue). Bar: 50 μm.
Figure Legend Snippet: Endocrine differentiation of pancreas is impaired in Pdx1 tTA/+ ;tetO MafA pancreas. At E17.5 Isl1 (green, AB ), Pax6 (green, CD ) and Hb9 (green, EF ), transcription factors implicated in endocrine differentiation and maturation, have severely reduced expression in Pdx1 tTA/+ ;tetO MafA pancreas ( ACE ) compared to tetO MafA pancreas ( BDF ). This finding is consistent with their reduced number of insulin + cells (red, A-F ), and suggests that misexpression of the MafA transgene inhibits the entire endocrine differentiation program. DAPI (blue). Bar: 50 μm.

Techniques Used: Expressing

20) Product Images from "Gata6 restricts Isl1 to the posterior of nascent hindlimb buds through Isl1 cis-regulatory modules"

Article Title: Gata6 restricts Isl1 to the posterior of nascent hindlimb buds through Isl1 cis-regulatory modules

Journal: Developmental biology

doi: 10.1016/j.ydbio.2017.11.013

GATA6 synergizes with Zfpm2 to repress transcription in vitro (A) Schematic presentation of the luciferase reporter construct with thymidine kinase minimum promoter (TK mini.) and the CR1-CR2 sequence. The 1.3 kb sequence contains both CR1, CR2 and the sequence between them. (B) Luciferase reporter assay with indicated factors. GATA6 + Zfpn2 repressed the Isl1 -luciferase. * p
Figure Legend Snippet: GATA6 synergizes with Zfpm2 to repress transcription in vitro (A) Schematic presentation of the luciferase reporter construct with thymidine kinase minimum promoter (TK mini.) and the CR1-CR2 sequence. The 1.3 kb sequence contains both CR1, CR2 and the sequence between them. (B) Luciferase reporter assay with indicated factors. GATA6 + Zfpn2 repressed the Isl1 -luciferase. * p

Techniques Used: In Vitro, Luciferase, Construct, Sequencing, Reporter Assay

Expression patterns of Isl1 and Gata genes (A–F) Expression pattern of Gata1 – Gata6 at the 25/26 somite stage. The inset in A shows Gata1 signals in putative hematopoietic cells. Shown are dorsal views of posterior part of the body. Arrows in C point to Gata3 expression in the intermediate mesoderm. Arrowheads in F point to Gata6 signals in the flank and the anterior hindlimb forming region. (G, H) Immunofluorescence of GATA1 and TER-119 (G) and GATA1 and SOX2 (H). Dotted rectangles are shown as close-up of the lateral plate mesoderm. Single channel images are shown in black/white for better contrast. GATA1 positive signals and TER-119 positive signals overlap (arrowheads in G). GATA1 positive signals do not overlap with SOX2 signals (arrowheads in H). scale bar = 100 μm. (I–M) Expression pattern of Gata2 (I, J), Gata3 (K), Gata4 (L) and Gata5 (M) at E9.5 (I, K–M) and E10.5 (J). (I, J) Blue arrows and blue arrowheads point to Gata2 signals in the lateral nasal prominence and medial nasal prominence, respectively. (K) Red arrowheads point to Gata3 signals in the 3 rd pharyngeal pouch. (L) Green arrowheads point to Gata4 signals in the heart. (M) Yellow arrowheads point to Gata5 signals in the heart. Abbreviations: fl: forelimb bud, h: heart, hy: hyoid arch, lnp: lateral nasal prominence, ma: mandibular arch, mnp: medial nasal prominence, nt: neural tube, oft: outflow tract, p3: 3 rd pharyngeal pouch, v: ventricle,
Figure Legend Snippet: Expression patterns of Isl1 and Gata genes (A–F) Expression pattern of Gata1 – Gata6 at the 25/26 somite stage. The inset in A shows Gata1 signals in putative hematopoietic cells. Shown are dorsal views of posterior part of the body. Arrows in C point to Gata3 expression in the intermediate mesoderm. Arrowheads in F point to Gata6 signals in the flank and the anterior hindlimb forming region. (G, H) Immunofluorescence of GATA1 and TER-119 (G) and GATA1 and SOX2 (H). Dotted rectangles are shown as close-up of the lateral plate mesoderm. Single channel images are shown in black/white for better contrast. GATA1 positive signals and TER-119 positive signals overlap (arrowheads in G). GATA1 positive signals do not overlap with SOX2 signals (arrowheads in H). scale bar = 100 μm. (I–M) Expression pattern of Gata2 (I, J), Gata3 (K), Gata4 (L) and Gata5 (M) at E9.5 (I, K–M) and E10.5 (J). (I, J) Blue arrows and blue arrowheads point to Gata2 signals in the lateral nasal prominence and medial nasal prominence, respectively. (K) Red arrowheads point to Gata3 signals in the 3 rd pharyngeal pouch. (L) Green arrowheads point to Gata4 signals in the heart. (M) Yellow arrowheads point to Gata5 signals in the heart. Abbreviations: fl: forelimb bud, h: heart, hy: hyoid arch, lnp: lateral nasal prominence, ma: mandibular arch, mnp: medial nasal prominence, nt: neural tube, oft: outflow tract, p3: 3 rd pharyngeal pouch, v: ventricle,

Techniques Used: Expressing, Immunofluorescence

Comparison of expression patterns of Gata6 and Isl1 (A–D) Dorsal views Gata6 expression pattern in the hindlimb-forming region or the hindlimb bud at indicated stages. Arrowheads point to Gata6 signals. (E–H) Dorsal views Isl1 expression pattern in the hindlimb-forming region or the hindlimb bud at indicated stages. Arrowheads point to Isl1 signals.
Figure Legend Snippet: Comparison of expression patterns of Gata6 and Isl1 (A–D) Dorsal views Gata6 expression pattern in the hindlimb-forming region or the hindlimb bud at indicated stages. Arrowheads point to Gata6 signals. (E–H) Dorsal views Isl1 expression pattern in the hindlimb-forming region or the hindlimb bud at indicated stages. Arrowheads point to Isl1 signals.

Techniques Used: Expressing

Immunofluorescence analysis of ISL1 signals in the nascent hindlimb bud (A–H) Confocal images of the nascent hindlimb bud of the coronal section at the 28 somite stage. B, C, F and G are shown in rainbow color. Dotted areas in C, D, G and H show nascent hindlimb buds, where line scanning for ISL1 and GATA6 were performed. Scale bar: 200 μm. (I) Relative fluorescent intensity of GATA6 (magenta) and ISL1 (green) along the anterior-posterior axis of the nascent hindlimb bud in wild type embryos. The highest signal intensity of ISL1 and GATA6 in the section is set as 1.0 intensity, and other signal intensities are relative to the highest signal intensity. (J) Overlay of ISL1 signal intensity analysis along the anterior-posterior axis of the nascent hindlimb bud of wild type (green) and Tcre; Gata6 fl/fl (green with black dotted line) embryos at 28 somite stage. The highest ISL1 signals in wild type or Tcre; Gata6 fl/fl hindlimb are set at 1.0 intensity. Highest ISL1 signals are detected in more anterior regions in of Tcre; Gata6 fl/fl hindlimb buds, compared to wild type. Note that this panel does not compare the ISL1 signal intensity between wild type sections and Tcre; Gata6 fl/fl sections. Brackets denote highest levels of ISL1 signals in wild type (†) and Tcre; Gata6 fl/fl (††) embryos.
Figure Legend Snippet: Immunofluorescence analysis of ISL1 signals in the nascent hindlimb bud (A–H) Confocal images of the nascent hindlimb bud of the coronal section at the 28 somite stage. B, C, F and G are shown in rainbow color. Dotted areas in C, D, G and H show nascent hindlimb buds, where line scanning for ISL1 and GATA6 were performed. Scale bar: 200 μm. (I) Relative fluorescent intensity of GATA6 (magenta) and ISL1 (green) along the anterior-posterior axis of the nascent hindlimb bud in wild type embryos. The highest signal intensity of ISL1 and GATA6 in the section is set as 1.0 intensity, and other signal intensities are relative to the highest signal intensity. (J) Overlay of ISL1 signal intensity analysis along the anterior-posterior axis of the nascent hindlimb bud of wild type (green) and Tcre; Gata6 fl/fl (green with black dotted line) embryos at 28 somite stage. The highest ISL1 signals in wild type or Tcre; Gata6 fl/fl hindlimb are set at 1.0 intensity. Highest ISL1 signals are detected in more anterior regions in of Tcre; Gata6 fl/fl hindlimb buds, compared to wild type. Note that this panel does not compare the ISL1 signal intensity between wild type sections and Tcre; Gata6 fl/fl sections. Brackets denote highest levels of ISL1 signals in wild type (†) and Tcre; Gata6 fl/fl (††) embryos.

Techniques Used: Immunofluorescence

Isl1 enhancer drives reporter expression in the hindlimb progenitors ) analysis shows conservation of human sequence (hg19) in mouse (mm10), chicken (galGal3) and frog (xenTro2), but not zebrafish (Zv9) in the 3′ region from the ISL1 gene. The CR1, CR2 and CR3 sequences are indicated. Purple and light blue peaks denote coding and 3′ UTR sequences, respectively, in the Isl1 exon 6. (B) Schematic presentation of the transgenic construct with the 1.3 kb region containing the CR1 and CR2 sequences. The 1.3 kb sequence contains the sequence between CR1 and CR2. (C) Isl1 expression pattern in a wild type embryo at E9.5. (D, E) LacZ-stained embryo after injection of the transgenic construct. E shows close up of the hindlimb-forming region in D. The bracket shows the hindlimb-forming region. ma: mandibular arch, mn: motor neuron.
Figure Legend Snippet: Isl1 enhancer drives reporter expression in the hindlimb progenitors ) analysis shows conservation of human sequence (hg19) in mouse (mm10), chicken (galGal3) and frog (xenTro2), but not zebrafish (Zv9) in the 3′ region from the ISL1 gene. The CR1, CR2 and CR3 sequences are indicated. Purple and light blue peaks denote coding and 3′ UTR sequences, respectively, in the Isl1 exon 6. (B) Schematic presentation of the transgenic construct with the 1.3 kb region containing the CR1 and CR2 sequences. The 1.3 kb sequence contains the sequence between CR1 and CR2. (C) Isl1 expression pattern in a wild type embryo at E9.5. (D, E) LacZ-stained embryo after injection of the transgenic construct. E shows close up of the hindlimb-forming region in D. The bracket shows the hindlimb-forming region. ma: mandibular arch, mn: motor neuron.

Techniques Used: Expressing, Sequencing, Transgenic Assay, Construct, Staining, Injection

21) Product Images from "The transcriptional regulator PRDM12 is critical for Pomc expression in the mouse hypothalamus and controlling food intake, adiposity, and body weight"

Article Title: The transcriptional regulator PRDM12 is critical for Pomc expression in the mouse hypothalamus and controlling food intake, adiposity, and body weight

Journal: Molecular Metabolism

doi: 10.1016/j.molmet.2020.01.007

Overlapping expression patterns of PRDM12, ISL1, and POMC in the developing hypothalamus. Immunofluorescence using anti-PRDM12 (red) and anti-ISL1 (blue) antibodies in sagittal cryosections of E10.5 (left) and E12.5 (right) Pomc -EGFP mouse embryos. Confocal images showing superimposed PRDM12 and ISL1 immunopositive neurons (middle lane) and triple merged with Pomc -EGFP-immunoreactive neurons (bottom).
Figure Legend Snippet: Overlapping expression patterns of PRDM12, ISL1, and POMC in the developing hypothalamus. Immunofluorescence using anti-PRDM12 (red) and anti-ISL1 (blue) antibodies in sagittal cryosections of E10.5 (left) and E12.5 (right) Pomc -EGFP mouse embryos. Confocal images showing superimposed PRDM12 and ISL1 immunopositive neurons (middle lane) and triple merged with Pomc -EGFP-immunoreactive neurons (bottom).

Techniques Used: Expressing, Immunofluorescence

Specific deletion of Prdm12 from ISL1 neurons impairs the onset of Pomc expression. Immunofluorescence performed in sagittal cryosections of E12.5 embryos using an anti-ACTH antibody in the hypothalamic mantle zone. ISL1 Prdm12 KO embryos show highly reduced numbers of POMC neurons compared with Prdm12 loxP/loxP controls. Bottom, magnified views of the hypothalamic areas boxed in the figures above.
Figure Legend Snippet: Specific deletion of Prdm12 from ISL1 neurons impairs the onset of Pomc expression. Immunofluorescence performed in sagittal cryosections of E12.5 embryos using an anti-ACTH antibody in the hypothalamic mantle zone. ISL1 Prdm12 KO embryos show highly reduced numbers of POMC neurons compared with Prdm12 loxP/loxP controls. Bottom, magnified views of the hypothalamic areas boxed in the figures above.

Techniques Used: Expressing, Immunofluorescence

Hypothalamic Pomc expression is impaired in Prdm12 knockout mice. (A) Immunofluorescence analysis using anti-PRDM12 (red) and anti-ACTH (green) antibodies in sagittal cryosections of wild-type , heterozygous Prdm12 +/− , and homozygous Prdm12 −/− E12.5 mouse embryos reveal that the absence of PRDM12 prevents Pomc expression in the developing hypothalamus. ISL1 immunofluorescence (magenta) labels the neurons present in the mantle zone where Pomc is normally expressed. Insets are magnified views of the indicated boxes. (B) At E15.5, hypothalamic POMC immunolabeled neurons continued to be negligible in Prdm12 knockout mice. Bottom, magnified views of the hypothalamic areas embedded in the figures above.
Figure Legend Snippet: Hypothalamic Pomc expression is impaired in Prdm12 knockout mice. (A) Immunofluorescence analysis using anti-PRDM12 (red) and anti-ACTH (green) antibodies in sagittal cryosections of wild-type , heterozygous Prdm12 +/− , and homozygous Prdm12 −/− E12.5 mouse embryos reveal that the absence of PRDM12 prevents Pomc expression in the developing hypothalamus. ISL1 immunofluorescence (magenta) labels the neurons present in the mantle zone where Pomc is normally expressed. Insets are magnified views of the indicated boxes. (B) At E15.5, hypothalamic POMC immunolabeled neurons continued to be negligible in Prdm12 knockout mice. Bottom, magnified views of the hypothalamic areas embedded in the figures above.

Techniques Used: Expressing, Knock-Out, Mouse Assay, Immunofluorescence, Immunolabeling

22) Product Images from "Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the β-catenin pathway"

Article Title: Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the β-catenin pathway

Journal: Developmental biology

doi: 10.1016/j.ydbio.2014.01.001

Isl1 is essential for nuclear β-catenin accumulation and Fgf8 expression in BA1 epithelium
Figure Legend Snippet: Isl1 is essential for nuclear β-catenin accumulation and Fgf8 expression in BA1 epithelium

Techniques Used: Expressing

Inactivation of β-catenin in the Isl1 -lineage causes cell death in the mesenchyme not in the epithelium of BA1
Figure Legend Snippet: Inactivation of β-catenin in the Isl1 -lineage causes cell death in the mesenchyme not in the epithelium of BA1

Techniques Used:

Expression of Isl1 in the epithelium of the branchial arches
Figure Legend Snippet: Expression of Isl1 in the epithelium of the branchial arches

Techniques Used: Expressing

23) Product Images from "Positional differences of axon growth rates between sensory neurons encoded by runx3"

Article Title: Positional differences of axon growth rates between sensory neurons encoded by runx3

Journal: The EMBO Journal

doi: 10.1038/emboj.2012.228

Axial differences in axon growth behaviour established by Runx3 in the mouse. ( A ) Immunohistochemistry for Runx3 in E11.5 brachial ( B ) compared to thoracic (T) DRG neurons. ( B ) Quantification of Runx3 and Isl1 at single cell level of ( A ) ( n =2 embryos).
Figure Legend Snippet: Axial differences in axon growth behaviour established by Runx3 in the mouse. ( A ) Immunohistochemistry for Runx3 in E11.5 brachial ( B ) compared to thoracic (T) DRG neurons. ( B ) Quantification of Runx3 and Isl1 at single cell level of ( A ) ( n =2 embryos).

Techniques Used: Immunohistochemistry

24) Product Images from "The temporal and spatial expression pattern of ABCG2 in the developing human heart"

Article Title: The temporal and spatial expression pattern of ABCG2 in the developing human heart

Journal: International journal of cardiology

doi: 10.1016/j.ijcard.2010.10.025

Islet-1 is expressed during development of the human heart. (A) RT-PCR results for islet1 mRNA expression at CS18 (6 weeks pc) and F3 (11 weeks pc). Immunohistochemical analysis for islet-1 expressing cells, large numbers of isl+ cells are present in
Figure Legend Snippet: Islet-1 is expressed during development of the human heart. (A) RT-PCR results for islet1 mRNA expression at CS18 (6 weeks pc) and F3 (11 weeks pc). Immunohistochemical analysis for islet-1 expressing cells, large numbers of isl+ cells are present in

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry

Representative results for co-expression analysis for ABCG2 and other stem, progenitor endothelial and cardiac markers. No cells expressed islet −1 and ABCG2 in the OFT (A) or RA (B) at CS23 (8 weeks pc). Rare cells that expressed both CD31 and
Figure Legend Snippet: Representative results for co-expression analysis for ABCG2 and other stem, progenitor endothelial and cardiac markers. No cells expressed islet −1 and ABCG2 in the OFT (A) or RA (B) at CS23 (8 weeks pc). Rare cells that expressed both CD31 and

Techniques Used: Expressing

25) Product Images from "Identification and Functional Characterization of Cardiac Pacemaker Cells in Zebrafish"

Article Title: Identification and Functional Characterization of Cardiac Pacemaker Cells in Zebrafish

Journal: PLoS ONE

doi: 10.1371/journal.pone.0047644

Isl1 cells have pacemaker activity. ( A–C ) Optical mapping on an explanted, contracting adult zebrafish tg(isl1BAC:GalFF; UAS:GFP) heart. Arrow indicates the sinus venosus in all panels. (A) Explanted adult zebrafish heart. (B) GFP-fluorescent cells reporting Isl1 expression are situated at the sinus venosus. (C) The activation pattern measured by di-4-ANEPPS fluorescence shows that the GFP+ myocytes are situated in the area of earliest activation. ( D ) Typical action potentials of freshly isolated GFP + and GFP − myocytes. The GFP − cell was stimulated at 3 Hz. The inset displays a representative example of a GFP+ myocyte.
Figure Legend Snippet: Isl1 cells have pacemaker activity. ( A–C ) Optical mapping on an explanted, contracting adult zebrafish tg(isl1BAC:GalFF; UAS:GFP) heart. Arrow indicates the sinus venosus in all panels. (A) Explanted adult zebrafish heart. (B) GFP-fluorescent cells reporting Isl1 expression are situated at the sinus venosus. (C) The activation pattern measured by di-4-ANEPPS fluorescence shows that the GFP+ myocytes are situated in the area of earliest activation. ( D ) Typical action potentials of freshly isolated GFP + and GFP − myocytes. The GFP − cell was stimulated at 3 Hz. The inset displays a representative example of a GFP+ myocyte.

Techniques Used: Activity Assay, Expressing, Activation Assay, Fluorescence, Isolation

Characterization of the embryonic Isl1 −/− cardiac phenotype in vivo . ( A ) Zebrafish embryonic heart at 2 dpf. The embryonic heart is highlighted in black dotted contour; white dotted lines through the atrium (A) and the ventricle (V) are placed at kymograph positions. ( B ) Atrial (A) and ventricular (V) kymographs from 2 dpf embryonic hearts spanning approximately 2.8 s. Note the much longer period of the Isl −/− heart when compared to the sibling and the irregularity of the period (double arrow and white dotted vertical lines). Movies are available as Movies S1 and S2 , respectively. ( C ) Box-whisker plots representation of 20 successive heartbeats of 2 dpf Isl1 −/− and sibling embryos. ( D ) Kymograph recorded at 3 dpf covering a period of about 16 s of absent heart contractions. For all panels a: atrium; v: ventricle.
Figure Legend Snippet: Characterization of the embryonic Isl1 −/− cardiac phenotype in vivo . ( A ) Zebrafish embryonic heart at 2 dpf. The embryonic heart is highlighted in black dotted contour; white dotted lines through the atrium (A) and the ventricle (V) are placed at kymograph positions. ( B ) Atrial (A) and ventricular (V) kymographs from 2 dpf embryonic hearts spanning approximately 2.8 s. Note the much longer period of the Isl −/− heart when compared to the sibling and the irregularity of the period (double arrow and white dotted vertical lines). Movies are available as Movies S1 and S2 , respectively. ( C ) Box-whisker plots representation of 20 successive heartbeats of 2 dpf Isl1 −/− and sibling embryos. ( D ) Kymograph recorded at 3 dpf covering a period of about 16 s of absent heart contractions. For all panels a: atrium; v: ventricle.

Techniques Used: In Vivo, Whisker Assay

Isl1 expression in the embryonic and adult zebrafish heart. Single confocal scans of a fluorescent antibody labeling of Isl1 and eGFP in embryonic (2 dpf) ( A–D ) and adult ( E–H ) zebrafish expressing Tg(myl7:eGFP) in all cardiomyocytes. GFP + cardiomyocytes are displayed in grey (B, F) and in green in (D, H). Isl1 is shown in grey (C, G) and in red (D, H). Arrowheads indicate Isl + /GFP + cells. Illustrations of a lateral view of a 2 dpf (A) and adult (E) zebrafish heart indicate the location of Isl1 + cells (red). The box in panel E represents the area shown in (F–H). ( B–D ) Fluorescent immunolabeling of Isl1 and eGFP in a 2 dpf embryo (sagittal section 100 µm). At this time point Isl1 + /GFP + cells were only found in the IFT of the heart. ( F–H ) Fluorescent immunolabeling of Isl1 and eGFP in an adult zebrafish heart (sagittal section 100 µm). Isl1 + /GFP + cells are located at the junction of the sinus venosus and atrium in the inflow region of the heart (arrowheads). Isl1 + cells showed low expression of myl7 . v, ventricle; a, atrium; oft, outflow tract; ift, inflow tract; ba, bulbus arteriosus; sv, sinus venosus; a, anterior; p, posterior; d, dorsal; v, ventral. Scale bars represent 50 µm.
Figure Legend Snippet: Isl1 expression in the embryonic and adult zebrafish heart. Single confocal scans of a fluorescent antibody labeling of Isl1 and eGFP in embryonic (2 dpf) ( A–D ) and adult ( E–H ) zebrafish expressing Tg(myl7:eGFP) in all cardiomyocytes. GFP + cardiomyocytes are displayed in grey (B, F) and in green in (D, H). Isl1 is shown in grey (C, G) and in red (D, H). Arrowheads indicate Isl + /GFP + cells. Illustrations of a lateral view of a 2 dpf (A) and adult (E) zebrafish heart indicate the location of Isl1 + cells (red). The box in panel E represents the area shown in (F–H). ( B–D ) Fluorescent immunolabeling of Isl1 and eGFP in a 2 dpf embryo (sagittal section 100 µm). At this time point Isl1 + /GFP + cells were only found in the IFT of the heart. ( F–H ) Fluorescent immunolabeling of Isl1 and eGFP in an adult zebrafish heart (sagittal section 100 µm). Isl1 + /GFP + cells are located at the junction of the sinus venosus and atrium in the inflow region of the heart (arrowheads). Isl1 + cells showed low expression of myl7 . v, ventricle; a, atrium; oft, outflow tract; ift, inflow tract; ba, bulbus arteriosus; sv, sinus venosus; a, anterior; p, posterior; d, dorsal; v, ventral. Scale bars represent 50 µm.

Techniques Used: Expressing, Antibody Labeling, Immunolabeling

Molecular characterization of the adult isl1 expression domain. ( A–E,G,H ) Section in-situ hybridizations on adult wild-type zebrafish hearts. Probes are indicated in the panels. (A) 4-chamber view of a sagittal section through zebrafish heart labeled with the myocardial marker myl7. The box indicates the region shown enlarged in panels B–E,G,H. Demarcated areas (C) indicate isl1 expression in the tbx2b+ hcn4+ nppa- myocardium at the base of the valves surrounding the sinoatrial junction. BMP4 signal is pointed at in the valves by arrowheads in (H). ( F ) 3D reconstruction of the sinoatrial junction. isl1 (yellow) is expressed around the entire sinoatrial junction, forming a ring-like structure. ( I–L ) Reconstruction of a confocal scan through a sagittal section of the inflow region of adult Tg(isl1BAC:GalFF; UAS:GFP) transgenic zebrafish heart. Fluorescent antibody labeling for GFP (shown in grey (I, I', L) or green (K, K')) and for Isl1 (shown in grey (J, J') or red (K, K')). GFP+/Isl1+ cells were found in bilateral populations at the sinoatrial junction (arrows). (I'–K') Enlargement of the GFP+/Isl1+ cells at the dorsal rim of the inflow tract (indicated with dashed box in (I)). Nuclei of GFP+ cells were positive for Isl1 (arrowheads). (L) Enlargement of GFP+ cells (transverse section) shows a string of contiguous cells. a, atrium; avc, atrioventricular canal; ba, bulbus arteriosus; sv, sinus venosus; v, ventricle; l, left; r, right; a, anterior; p, posterior; d, dorsal; v, ventral. Scale bars represent 50 µm (A–G, I–K) or 10 µm (I'–K',L).
Figure Legend Snippet: Molecular characterization of the adult isl1 expression domain. ( A–E,G,H ) Section in-situ hybridizations on adult wild-type zebrafish hearts. Probes are indicated in the panels. (A) 4-chamber view of a sagittal section through zebrafish heart labeled with the myocardial marker myl7. The box indicates the region shown enlarged in panels B–E,G,H. Demarcated areas (C) indicate isl1 expression in the tbx2b+ hcn4+ nppa- myocardium at the base of the valves surrounding the sinoatrial junction. BMP4 signal is pointed at in the valves by arrowheads in (H). ( F ) 3D reconstruction of the sinoatrial junction. isl1 (yellow) is expressed around the entire sinoatrial junction, forming a ring-like structure. ( I–L ) Reconstruction of a confocal scan through a sagittal section of the inflow region of adult Tg(isl1BAC:GalFF; UAS:GFP) transgenic zebrafish heart. Fluorescent antibody labeling for GFP (shown in grey (I, I', L) or green (K, K')) and for Isl1 (shown in grey (J, J') or red (K, K')). GFP+/Isl1+ cells were found in bilateral populations at the sinoatrial junction (arrows). (I'–K') Enlargement of the GFP+/Isl1+ cells at the dorsal rim of the inflow tract (indicated with dashed box in (I)). Nuclei of GFP+ cells were positive for Isl1 (arrowheads). (L) Enlargement of GFP+ cells (transverse section) shows a string of contiguous cells. a, atrium; avc, atrioventricular canal; ba, bulbus arteriosus; sv, sinus venosus; v, ventricle; l, left; r, right; a, anterior; p, posterior; d, dorsal; v, ventral. Scale bars represent 50 µm (A–G, I–K) or 10 µm (I'–K',L).

Techniques Used: Expressing, In Situ, Labeling, Marker, Transgenic Assay, Antibody Labeling

26) Product Images from "TCF4 deficiency expands ventral diencephalon signaling and increases induction of pituitary progenitors"

Article Title: TCF4 deficiency expands ventral diencephalon signaling and increases induction of pituitary progenitors

Journal:

doi: 10.1016/j.ydbio.2007.08.046

Differentiation of ventral Rathke’s pouch cells is delayed in Tcf4 −/− mice. Immunohistochemistry for ISL1 at e11.5 (A, B) and e12.5 (C, D) as well as FOXL2 at e11.5 (E, F) and e12.5 (G, H) were used to determine the onset of cell
Figure Legend Snippet: Differentiation of ventral Rathke’s pouch cells is delayed in Tcf4 −/− mice. Immunohistochemistry for ISL1 at e11.5 (A, B) and e12.5 (C, D) as well as FOXL2 at e11.5 (E, F) and e12.5 (G, H) were used to determine the onset of cell

Techniques Used: Mouse Assay, Immunohistochemistry

27) Product Images from "Ebf Factors Are Required for Specifying Multiple Retinal Cell Types and Subtypes from Postmitotic Precursors"

Article Title: Ebf Factors Are Required for Specifying Multiple Retinal Cell Types and Subtypes from Postmitotic Precursors

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.2187-10.2010

Expression of Ebfs in multiple retinal neuronal types and subtypes. A-X , Sections from P21 mouse retinas were double-immunolabeled with a pan-Ebf antibody and those against the indicated cell type-specific markers. Sections in ( B,C,F,H-N,P-R ) were also weakly counterstained with DAPI. There is colocalization between Ebfs and Pax6, Barhl2, calretinin, syntaxin, GLYT1, or GlyRa1/2 in ACs and/or RGCs ( A-F ) but no colocalization with Dab1, GABA, GAD65, TH, ChAT or Isl1 in ACs ( G-L ). Ebfs are co-expressed with Isl1 or Brn3b in RGCs ( L,M ), with Chx10, Bhlhb5 or recoverin but not with PKC in bipolar cells ( O-Q,S-U ), with Lim1 or calbindin in horizontal cells ( R,V-X ); however, they are not co-expressed with glutamine synthase (GS) in Müller cells ( N ). Arrows point to representative colocalized cells and insets show corresponding outlined regions at a higher magnification. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar: A,D,E,G,O , 19 μm; B,C,F,H-N,P-X , 16.7 μm.
Figure Legend Snippet: Expression of Ebfs in multiple retinal neuronal types and subtypes. A-X , Sections from P21 mouse retinas were double-immunolabeled with a pan-Ebf antibody and those against the indicated cell type-specific markers. Sections in ( B,C,F,H-N,P-R ) were also weakly counterstained with DAPI. There is colocalization between Ebfs and Pax6, Barhl2, calretinin, syntaxin, GLYT1, or GlyRa1/2 in ACs and/or RGCs ( A-F ) but no colocalization with Dab1, GABA, GAD65, TH, ChAT or Isl1 in ACs ( G-L ). Ebfs are co-expressed with Isl1 or Brn3b in RGCs ( L,M ), with Chx10, Bhlhb5 or recoverin but not with PKC in bipolar cells ( O-Q,S-U ), with Lim1 or calbindin in horizontal cells ( R,V-X ); however, they are not co-expressed with glutamine synthase (GS) in Müller cells ( N ). Arrows point to representative colocalized cells and insets show corresponding outlined regions at a higher magnification. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar: A,D,E,G,O , 19 μm; B,C,F,H-N,P-X , 16.7 μm.

Techniques Used: Expressing, Immunolabeling

28) Product Images from "Brn3a/Pou4f1 Regulates Dorsal Root Ganglion Sensory Neuron Specification and Axonal Projection into the Spinal Cord"

Article Title: Brn3a/Pou4f1 Regulates Dorsal Root Ganglion Sensory Neuron Specification and Axonal Projection into the Spinal Cord

Journal: Developmental Biology

doi: 10.1016/j.ydbio.2012.01.021

The expression of TrkA/TrkB and TrkA/TrkC in DRG sensory neurons are not properly segregated in Brn3a mutant mice at E12.5. (A–H) Forelimb-level transverse DRG sections from E12.5 Brn3a +/− and Brn3a −/− mice were immunostained with indicated antibodies. Sections in (A, B, E, and F) were counter-labeled with Topro-3. In Brn3a −/− DRGs, TrkB + and TrkC + cells significantly increase (A, B, E, and F), so do the smaller-sized TrkC + cells in particular (E and F). There is also a significant increase in the number of TrkA + /TrkB + and TrkA + /TrkC + co-expressing cells in Brn3a −/− DRGs (C, D, G, and H). Arrow heads in D and H point to representative co-expressing cells and those in F point to representative smaller-sized TrkC + cells. (I and J) Quantification of sensory neuron subtypes in control and Brn3a mutant DRGs. Total neuron number is not changed in Brn3a mutant DRGs at E11.5, E12.5 and E18.5, as measured by unchanged Isl1 + cells (J). The numbers of TrkB + , TrkC + , TrkA + /B + , and TrkA + /C + neurons are all increased but TrkA + cells remain unchanged in E12.5 Brn3a −/− DRGs (I). (K and L) Distribution of cell cross-sectional areas of TrkB + and TrkC + neurons in E12.5 control and Brn3a −/− DRGs. Areas were measured by the NIH Image J software and data were plotted using Microsoft Excel. In Brn3a −/− DRGs, the peak of TrkC + neuron sizes has shifted to the left compared to the control (L). There is no obvious change in the distribution of TrkB + neuron sizes in Brn3a −/− DRGs (K). Control includes both Brn3a +/+ and Brn3a +/− DRGs, which are indistinguishable in neuron number and size. Data are shown as mean±s.d.; **p
Figure Legend Snippet: The expression of TrkA/TrkB and TrkA/TrkC in DRG sensory neurons are not properly segregated in Brn3a mutant mice at E12.5. (A–H) Forelimb-level transverse DRG sections from E12.5 Brn3a +/− and Brn3a −/− mice were immunostained with indicated antibodies. Sections in (A, B, E, and F) were counter-labeled with Topro-3. In Brn3a −/− DRGs, TrkB + and TrkC + cells significantly increase (A, B, E, and F), so do the smaller-sized TrkC + cells in particular (E and F). There is also a significant increase in the number of TrkA + /TrkB + and TrkA + /TrkC + co-expressing cells in Brn3a −/− DRGs (C, D, G, and H). Arrow heads in D and H point to representative co-expressing cells and those in F point to representative smaller-sized TrkC + cells. (I and J) Quantification of sensory neuron subtypes in control and Brn3a mutant DRGs. Total neuron number is not changed in Brn3a mutant DRGs at E11.5, E12.5 and E18.5, as measured by unchanged Isl1 + cells (J). The numbers of TrkB + , TrkC + , TrkA + /B + , and TrkA + /C + neurons are all increased but TrkA + cells remain unchanged in E12.5 Brn3a −/− DRGs (I). (K and L) Distribution of cell cross-sectional areas of TrkB + and TrkC + neurons in E12.5 control and Brn3a −/− DRGs. Areas were measured by the NIH Image J software and data were plotted using Microsoft Excel. In Brn3a −/− DRGs, the peak of TrkC + neuron sizes has shifted to the left compared to the control (L). There is no obvious change in the distribution of TrkB + neuron sizes in Brn3a −/− DRGs (K). Control includes both Brn3a +/+ and Brn3a +/− DRGs, which are indistinguishable in neuron number and size. Data are shown as mean±s.d.; **p

Techniques Used: Expressing, Mutagenesis, Mouse Assay, Labeling, Software

Expression patterns of Brn3 proteins during mouse DRG sensory neuron development. (A–F) Transverse DRG sections from wild-type mouse embryos at indicated developmental stages were co-immunostained with antibodies against Isl1 (A–F) and Brn3a (A and B), Brn3b (C and D), or Brn3c (E and F). They were also counter-labeled with DAPI (blue). Isl1 was used as a pan-sensory maker for the DRG. Brn3a is strongly expressed in almost all Isl1-labeled cells at E10.5 and E11.5, whereas Brn3b and Brn3c are expressed weakly only in some of the Isl1-immunoreactive cells at these stages. Insets show Brn3b- (C and D) and Brn3c- (E and F) immunoreactive cells in corresponding outlined regions at a higher magnification and arrows point to representative Brn3b- or Brn3c-immunoreactive cells. (G–N) Co-expression of Brn3b or Brn3c with Brn3a in DRG sensory neurons and the regulation of their expression by Brn3a at E12.5 and E15.5. Brn3b is expressed in a subset of Brn3a-immunoreactive cells at E12.5 but in the majority of them at E15.5 (G and I). Brn3c is expressed in a subset of Brn3a-exrpessing cells at both E12.5 and E15.5 (K and M). In Brn3a −/− mice, the expression of Brn3b is abolished in the DRG at E12.5 and E15.5 (H and J), but not in the spinal cord (H). The expression of Brn3c is largely unchanged in Brn3a −/− DRGs at E12.5 (L), but becomes abolished at E15.5 (N). (O–Q) Transverse sections from E10.5 (O and P) and E11.5 (Q) embryos pulse-labeled with EdU were double-stained for Brn3a and EdU. None of the Brn3a-immunoreactive cells were co-labeled by EdU in the spinal cord (O and P) and only a few were colabeled in the DRG (P and Q). Arrows in P and Q point to representative co-localized cells. (R–T) Double-immunostaining between Brn3a and TrkA, TrkB or TrkC showed co-localization of Brn3a and these Trk receptors in E11.5 DRG neurons. Insets show corresponding outlined regions at a higher magnification. Abbreviations: drg, dorsal root ganglion; sc, spinal cord. Scale bar: A–F, P–T, 50 µm; G–O, 100 µm.
Figure Legend Snippet: Expression patterns of Brn3 proteins during mouse DRG sensory neuron development. (A–F) Transverse DRG sections from wild-type mouse embryos at indicated developmental stages were co-immunostained with antibodies against Isl1 (A–F) and Brn3a (A and B), Brn3b (C and D), or Brn3c (E and F). They were also counter-labeled with DAPI (blue). Isl1 was used as a pan-sensory maker for the DRG. Brn3a is strongly expressed in almost all Isl1-labeled cells at E10.5 and E11.5, whereas Brn3b and Brn3c are expressed weakly only in some of the Isl1-immunoreactive cells at these stages. Insets show Brn3b- (C and D) and Brn3c- (E and F) immunoreactive cells in corresponding outlined regions at a higher magnification and arrows point to representative Brn3b- or Brn3c-immunoreactive cells. (G–N) Co-expression of Brn3b or Brn3c with Brn3a in DRG sensory neurons and the regulation of their expression by Brn3a at E12.5 and E15.5. Brn3b is expressed in a subset of Brn3a-immunoreactive cells at E12.5 but in the majority of them at E15.5 (G and I). Brn3c is expressed in a subset of Brn3a-exrpessing cells at both E12.5 and E15.5 (K and M). In Brn3a −/− mice, the expression of Brn3b is abolished in the DRG at E12.5 and E15.5 (H and J), but not in the spinal cord (H). The expression of Brn3c is largely unchanged in Brn3a −/− DRGs at E12.5 (L), but becomes abolished at E15.5 (N). (O–Q) Transverse sections from E10.5 (O and P) and E11.5 (Q) embryos pulse-labeled with EdU were double-stained for Brn3a and EdU. None of the Brn3a-immunoreactive cells were co-labeled by EdU in the spinal cord (O and P) and only a few were colabeled in the DRG (P and Q). Arrows in P and Q point to representative co-localized cells. (R–T) Double-immunostaining between Brn3a and TrkA, TrkB or TrkC showed co-localization of Brn3a and these Trk receptors in E11.5 DRG neurons. Insets show corresponding outlined regions at a higher magnification. Abbreviations: drg, dorsal root ganglion; sc, spinal cord. Scale bar: A–F, P–T, 50 µm; G–O, 100 µm.

Techniques Used: Expressing, Labeling, Mouse Assay, Staining, Double Immunostaining

29) Product Images from "Mechanisms Underlying Pituitary Hypoplasia and Failed Cell Specification in Lhx3 Deficient Mice"

Article Title: Mechanisms Underlying Pituitary Hypoplasia and Failed Cell Specification in Lhx3 Deficient Mice

Journal: Developmental biology

doi: 10.1016/j.ydbio.2007.10.006

Lhx3 plays an important role in pituitary development A) While Lhx4 ), absence of Lhx3 causes a transient loss of ISL1 expression at this age, demonstrating that Lhx3 and Lhx4 can have very different effects on gene expression in the pituitary. B) Tpit is required to suppress SF1 expression in the dorsal aspect of the pituitary gland. Without Tpit , SF1 is expressed in the intermediate lobe and complete gonadotrope differentiation occurs as evidenced by CGA and LHβ expression. Prop1 df/df mutants have no obvious abnormalities in TPIT expression, but SF1 is expressed ectopically in the dorsal aspect of the gland. SF1 is not, however, sufficient to complete the gonadotrope differentiation program in this case. NOTCH2 expression fails completely in Prop1 df/df mutants. Lhx3 nulls lack both TPIT and NOTCH2 and exhibit a loss of gonadotrope differentiation that appears to correlate with the lack of NOTCH2 expression. Ectopic expression of gonadotrope marker, SF1, occurs, but no gonadotropin expression ensues. This suggests that Lhx3 is required to complete the gonadotrope differentiation program. C) Lhx3 is important for cell survival of pre-corticotropes. Lhx3 deficiency results in a severe reduction in pre-corticotropes as evidenced by reduced expression of NEUROD1 and TPIT. A reduction in differentiated corticotropes, as well as melanotropes, is demonstrated by a reduction in POMC expression. Gonadotropes are able to proceed with aspects of their differentiation process without Lhx3 ). Lhx3 is required for NOTCH2 expression, which may be important for appropriate dorsal-ventral cell specification and/or suppression of precocious differentiation of dorsal cell types. Finally, Lhx3 is required for expression of Pit1 and the Pit1 ). Together, these studies demonstrate that Lhx3 is a critical factor in the complex process of pituitary patterning and cell specification. Transcription factors are shown in green and hormones and other genes are shown in blue. Proteins studied using immunohistochemistry are represented in all capital letters and genes studied using in situ hybridization are represented in italics.
Figure Legend Snippet: Lhx3 plays an important role in pituitary development A) While Lhx4 ), absence of Lhx3 causes a transient loss of ISL1 expression at this age, demonstrating that Lhx3 and Lhx4 can have very different effects on gene expression in the pituitary. B) Tpit is required to suppress SF1 expression in the dorsal aspect of the pituitary gland. Without Tpit , SF1 is expressed in the intermediate lobe and complete gonadotrope differentiation occurs as evidenced by CGA and LHβ expression. Prop1 df/df mutants have no obvious abnormalities in TPIT expression, but SF1 is expressed ectopically in the dorsal aspect of the gland. SF1 is not, however, sufficient to complete the gonadotrope differentiation program in this case. NOTCH2 expression fails completely in Prop1 df/df mutants. Lhx3 nulls lack both TPIT and NOTCH2 and exhibit a loss of gonadotrope differentiation that appears to correlate with the lack of NOTCH2 expression. Ectopic expression of gonadotrope marker, SF1, occurs, but no gonadotropin expression ensues. This suggests that Lhx3 is required to complete the gonadotrope differentiation program. C) Lhx3 is important for cell survival of pre-corticotropes. Lhx3 deficiency results in a severe reduction in pre-corticotropes as evidenced by reduced expression of NEUROD1 and TPIT. A reduction in differentiated corticotropes, as well as melanotropes, is demonstrated by a reduction in POMC expression. Gonadotropes are able to proceed with aspects of their differentiation process without Lhx3 ). Lhx3 is required for NOTCH2 expression, which may be important for appropriate dorsal-ventral cell specification and/or suppression of precocious differentiation of dorsal cell types. Finally, Lhx3 is required for expression of Pit1 and the Pit1 ). Together, these studies demonstrate that Lhx3 is a critical factor in the complex process of pituitary patterning and cell specification. Transcription factors are shown in green and hormones and other genes are shown in blue. Proteins studied using immunohistochemistry are represented in all capital letters and genes studied using in situ hybridization are represented in italics.

Techniques Used: Expressing, Marker, Immunohistochemistry, In Situ Hybridization

ISL1 and SF1 are expressed ectopically in the dorsal region of Lhx3 mutant pituitaries SF1 (A–D), LHβ (E–F) and ISL1 (G–L) were detected by immunohistochemistry and labeled with fluorescein (green). Cell nuclei are labeled with DAPI (blue). SF1 and ISL1 are expressed in some ventral cells as expected, however they also exhibit ectopic expression at e18.5 in the dorsal aspect of Lhx3 ), LHβ is not detected in the mutant pituitary at e18.5 (E–F) or e16.5 (data not shown). A–L) The anterior lobe is identified with white brackets and null pituitaries are outlined with dotted lines.
Figure Legend Snippet: ISL1 and SF1 are expressed ectopically in the dorsal region of Lhx3 mutant pituitaries SF1 (A–D), LHβ (E–F) and ISL1 (G–L) were detected by immunohistochemistry and labeled with fluorescein (green). Cell nuclei are labeled with DAPI (blue). SF1 and ISL1 are expressed in some ventral cells as expected, however they also exhibit ectopic expression at e18.5 in the dorsal aspect of Lhx3 ), LHβ is not detected in the mutant pituitary at e18.5 (E–F) or e16.5 (data not shown). A–L) The anterior lobe is identified with white brackets and null pituitaries are outlined with dotted lines.

Techniques Used: Mutagenesis, Immunohistochemistry, Labeling, Expressing

30) Product Images from "Establishment of Motor Neuron-V3 Interneuron Progenitor Domain Boundary in Ventral Spinal Cord Requires Groucho-Mediated Transcriptional Corepression"

Article Title: Establishment of Motor Neuron-V3 Interneuron Progenitor Domain Boundary in Ventral Spinal Cord Requires Groucho-Mediated Transcriptional Corepression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0031176

TLE expression in the embryonic chick spinal cord. (A) Schematic representation of the five progenitor cell (p) domains of the ventral spinal cord, termed p0, p1, p2, pMN and p3 from dorsal to ventral positions, respectively. These domains are defined by the specific expression of combinations of HD and bHLH transcription factors. Refinement and maintenance of these progenitor domains is achieved through cross-repressive interactions between pairs of transcription factors, for example between Pax6 and Nkx2.2 at the pMN/p3 boundary. In turn, each progenitor domain generates different neuronal populations, V0, V1 and V2 INs, somatic MNs and V3 INs, respectively. Like the progenitor domains, separate populations of postmitotic neurons can be defined by the expression of specific transcription factors, such as HB9 and Isl1 in MNs derived from the pMN domain or other factors in other cell types, as indicated in the right-hand column. (B–E) Sections through the spinal cord of HH stage 18 chick embryos were subjected to double-labeling immunohistochemical analysis using a panTLE antibody together with antibodies against the indicated proteins. Panels in the right-hand column show high-magnification views of the boxed areas in the adjacent panels. Arrows point to examples of double-labeled cells. Arrowheads point to examples of cells expressing only TLE. TLE expression was observed in most ventral spinal cord cells, including domains p0–p2 (region of high Pax6 immunoreactivity dorsal to the Olig2+ domain), pMN (region expressing Nkx6.1, Olig2 and low levels of Pax6) and p3 (region expressing Nkx6.1 and Nkx2.2) of the ventral area. Notice in particular how virtually all Nkx2.2+ cells also express TLE.
Figure Legend Snippet: TLE expression in the embryonic chick spinal cord. (A) Schematic representation of the five progenitor cell (p) domains of the ventral spinal cord, termed p0, p1, p2, pMN and p3 from dorsal to ventral positions, respectively. These domains are defined by the specific expression of combinations of HD and bHLH transcription factors. Refinement and maintenance of these progenitor domains is achieved through cross-repressive interactions between pairs of transcription factors, for example between Pax6 and Nkx2.2 at the pMN/p3 boundary. In turn, each progenitor domain generates different neuronal populations, V0, V1 and V2 INs, somatic MNs and V3 INs, respectively. Like the progenitor domains, separate populations of postmitotic neurons can be defined by the expression of specific transcription factors, such as HB9 and Isl1 in MNs derived from the pMN domain or other factors in other cell types, as indicated in the right-hand column. (B–E) Sections through the spinal cord of HH stage 18 chick embryos were subjected to double-labeling immunohistochemical analysis using a panTLE antibody together with antibodies against the indicated proteins. Panels in the right-hand column show high-magnification views of the boxed areas in the adjacent panels. Arrows point to examples of double-labeled cells. Arrowheads point to examples of cells expressing only TLE. TLE expression was observed in most ventral spinal cord cells, including domains p0–p2 (region of high Pax6 immunoreactivity dorsal to the Olig2+ domain), pMN (region expressing Nkx6.1, Olig2 and low levels of Pax6) and p3 (region expressing Nkx6.1 and Nkx2.2) of the ventral area. Notice in particular how virtually all Nkx2.2+ cells also express TLE.

Techniques Used: Expressing, Derivative Assay, Labeling, Immunohistochemistry

Effect of TLE1ΔQ expression on ventral spinal cord Pax6+ and Nkx2.2+ progenitor populations and neuronal fate acquisition. (A) Schematic representation of TLE1ΔQ, compared to TLE1 and AES, depicting the lack of the Q domain but retention of the WDR domain in TLE1ΔQ. (B) Coimmunoprecipitation experiments performed using lysates from chick embryo spinal cords electroporated with plasmid encoding FLAG-TLE1ΔQ. Immunoprecipitation (IP) was performed using anti-FLAG antibody, followed by Western blotting (WB) analysis of input lysate (10%) and immunoprecipitated material using a panTLE antibody that recognizes all full-length TLE proteins and also TLE1ΔQ because it is directed against the WDR domain [19] . Endogenous TLE did not coimmunoprecipitate with exogenous TLE1ΔQ. (C) Quantification of the number of GFP+ cells expressing Nkx2.2 [in either the ventricular zone (VZ) or marginal zone (MZ)], Pax6, Hb9, or Isl1 in chick embryos electroporated with GFP alone or together with TLE1 or TLE1ΔQ. Expression of TLE1ΔQ resulted in an increase in the number of Pax6+ progenitor cells as well as Hb9+ and Isl1+ MNs compared to the control conditions. These effects were opposite to the effects of TLE1. See Figure S5 for double-labeling immunohistochemical analysis of electroporated embryos. (D) Quantification of the number of GFP+ cells expressing Pax6 in chick embryo spinal cord electroporated with GFP alone or together with TLE1, TLE1ΔQ, or TLE1 and TLE1ΔQ together, as indicated. Data in (C and D) are expressed as mean ± SEM (* p
Figure Legend Snippet: Effect of TLE1ΔQ expression on ventral spinal cord Pax6+ and Nkx2.2+ progenitor populations and neuronal fate acquisition. (A) Schematic representation of TLE1ΔQ, compared to TLE1 and AES, depicting the lack of the Q domain but retention of the WDR domain in TLE1ΔQ. (B) Coimmunoprecipitation experiments performed using lysates from chick embryo spinal cords electroporated with plasmid encoding FLAG-TLE1ΔQ. Immunoprecipitation (IP) was performed using anti-FLAG antibody, followed by Western blotting (WB) analysis of input lysate (10%) and immunoprecipitated material using a panTLE antibody that recognizes all full-length TLE proteins and also TLE1ΔQ because it is directed against the WDR domain [19] . Endogenous TLE did not coimmunoprecipitate with exogenous TLE1ΔQ. (C) Quantification of the number of GFP+ cells expressing Nkx2.2 [in either the ventricular zone (VZ) or marginal zone (MZ)], Pax6, Hb9, or Isl1 in chick embryos electroporated with GFP alone or together with TLE1 or TLE1ΔQ. Expression of TLE1ΔQ resulted in an increase in the number of Pax6+ progenitor cells as well as Hb9+ and Isl1+ MNs compared to the control conditions. These effects were opposite to the effects of TLE1. See Figure S5 for double-labeling immunohistochemical analysis of electroporated embryos. (D) Quantification of the number of GFP+ cells expressing Pax6 in chick embryo spinal cord electroporated with GFP alone or together with TLE1, TLE1ΔQ, or TLE1 and TLE1ΔQ together, as indicated. Data in (C and D) are expressed as mean ± SEM (* p

Techniques Used: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Labeling, Immunohistochemistry

Effect of TLE1 overexpression in the developing chick ventral spinal cord. (A) Schematic representation of the TLE domain structure. Notice the Q domain involved in oligomerization and transcriptional repression and the WDR domain important for protein-protein interactions [8] . Nkx2.2 binds to the TLE WDR domain using an Eh-1 motif. (B) Western blotting analysis of lysates from chick embryo spinal cords electroporated with plasmids encoding GFP alone or together with FLAG epitope-tagged TLE1 demonstrating the expression of exogenous TLE1 using anti-FLAG antibody. “n.s.” indicates a non-specific band detected by this antibody. (C) Double-labeling analysis of the expression of GFP and the indicated proteins in embryos electroporated with GFP alone (control) or GFP+TLE1 (TLE1). Nkx2.2+ cells were observed in both the ventricular zone (VZ) and mantle zone (MZ). Arrows in the two right-hand columns point to examples of double-labeled cells coexpressing GFP and either Hb9 or Isl1. (D and E) Quantification of the numbers of electroporated cells (GFP+) expressing Nkx2.2 [in either the VZ (D) or the MZ (E)], Pax6, Nkx6.1, Hb9, or Isl1, as indicated. TLE1 overexpression caused an increase in the number of Nkx2.2+ cells in the VZ, with a concomitant decrease in Pax6+ cells. The number of cells expressing Nkx6.1 was not altered. These changes were associated with an increase in Nkx2.2+ cells in the MZ and a decrease in the number of electroporated cells expressing the MN markers Hb9 and Isl1. Data are expressed as mean ± SEM (* p
Figure Legend Snippet: Effect of TLE1 overexpression in the developing chick ventral spinal cord. (A) Schematic representation of the TLE domain structure. Notice the Q domain involved in oligomerization and transcriptional repression and the WDR domain important for protein-protein interactions [8] . Nkx2.2 binds to the TLE WDR domain using an Eh-1 motif. (B) Western blotting analysis of lysates from chick embryo spinal cords electroporated with plasmids encoding GFP alone or together with FLAG epitope-tagged TLE1 demonstrating the expression of exogenous TLE1 using anti-FLAG antibody. “n.s.” indicates a non-specific band detected by this antibody. (C) Double-labeling analysis of the expression of GFP and the indicated proteins in embryos electroporated with GFP alone (control) or GFP+TLE1 (TLE1). Nkx2.2+ cells were observed in both the ventricular zone (VZ) and mantle zone (MZ). Arrows in the two right-hand columns point to examples of double-labeled cells coexpressing GFP and either Hb9 or Isl1. (D and E) Quantification of the numbers of electroporated cells (GFP+) expressing Nkx2.2 [in either the VZ (D) or the MZ (E)], Pax6, Nkx6.1, Hb9, or Isl1, as indicated. TLE1 overexpression caused an increase in the number of Nkx2.2+ cells in the VZ, with a concomitant decrease in Pax6+ cells. The number of cells expressing Nkx6.1 was not altered. These changes were associated with an increase in Nkx2.2+ cells in the MZ and a decrease in the number of electroporated cells expressing the MN markers Hb9 and Isl1. Data are expressed as mean ± SEM (* p

Techniques Used: Over Expression, Western Blot, FLAG-tag, Expressing, Labeling

31) Product Images from "SOX9 is required for maintenance of the pancreatic progenitor cell pool"

Article Title: SOX9 is required for maintenance of the pancreatic progenitor cell pool

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0609217104

SOX9 expression is restricted to uncommitted pancreatic progenitor cells. At E9.0, SOX9 colocalizes with PDX1 in the prepancreatic endoderm ( A ), persists in the PDX1 + pancreatic progenitors at E12.5 ( C ), and by E15.5 becomes restricted to a core subset of PDX1 + epithelial cords ( D ). At E15.5, ≈40% of SOX9 + cells incorporate the mitotic marker BrdU ( E , arrowheads) and coexpress HES1 ( F , arrowheads). Committed NGN3 + endocrine progenitors are intercalated within the SOX9 + epithelial cords but rarely express SOX9 ( G , arrowheads). SOX9 rarely colocalizes with the endocrine differentiation factors NKX2.2 ( H , arrowhead), NKX6.1 ( I , arrowheads), and MAFB ( J , arrowhead), and it is similarly excluded from differentiated endocrine cells expressing ISL1 ( K ), insulin or glucagon ( B and L ), differentiated amylase + acinar cells ( M ), and MUC1 + ductal cells ( N ). In the adult, SOX9 expression is restricted to a subset of ductal epithelial and centroacinar (arrows in O and P ) cells in both mouse ( O ) and human ( P ). VP, ventral; DP, dorsal prepancreatic endoderm; BRDU, bromodeoxyuridine; GLU, glucagon; INS, insulin; AMY, amylase; MUC1, mucin-1; e, embryonic day. [Scale bar, 50 μm ( A , B , D–P ); 100 μm ( C ).]
Figure Legend Snippet: SOX9 expression is restricted to uncommitted pancreatic progenitor cells. At E9.0, SOX9 colocalizes with PDX1 in the prepancreatic endoderm ( A ), persists in the PDX1 + pancreatic progenitors at E12.5 ( C ), and by E15.5 becomes restricted to a core subset of PDX1 + epithelial cords ( D ). At E15.5, ≈40% of SOX9 + cells incorporate the mitotic marker BrdU ( E , arrowheads) and coexpress HES1 ( F , arrowheads). Committed NGN3 + endocrine progenitors are intercalated within the SOX9 + epithelial cords but rarely express SOX9 ( G , arrowheads). SOX9 rarely colocalizes with the endocrine differentiation factors NKX2.2 ( H , arrowhead), NKX6.1 ( I , arrowheads), and MAFB ( J , arrowhead), and it is similarly excluded from differentiated endocrine cells expressing ISL1 ( K ), insulin or glucagon ( B and L ), differentiated amylase + acinar cells ( M ), and MUC1 + ductal cells ( N ). In the adult, SOX9 expression is restricted to a subset of ductal epithelial and centroacinar (arrows in O and P ) cells in both mouse ( O ) and human ( P ). VP, ventral; DP, dorsal prepancreatic endoderm; BRDU, bromodeoxyuridine; GLU, glucagon; INS, insulin; AMY, amylase; MUC1, mucin-1; e, embryonic day. [Scale bar, 50 μm ( A , B , D–P ); 100 μm ( C ).]

Techniques Used: Expressing, Marker

32) Product Images from "The Homeodomain Transcription Factor NKX2.1 Is Essential for the Early Specification of Melanocortin Neuron Identity and Activates Pomc Expression in the Developing Hypothalamus"

Article Title: The Homeodomain Transcription Factor NKX2.1 Is Essential for the Early Specification of Melanocortin Neuron Identity and Activates Pomc Expression in the Developing Hypothalamus

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2924-18.2019

Hypothalamic NPY neurons in conditional Nkx2.1 mutant mice. a–f , The number of NPY + neurons is normal in Nkx2.1 KO@E9.5 ( b ), Isl1 Nkx2.1 KO ( d ), and Pomc Nkx2.1 KO ( f ) E12.5 mouse embryos compared with their corresponding control littermates ( a , c , e ), as determined by immunofluorescence using anti-NPY (green) antibody on sagittal cryosections. g–j , The signal density of NPY + cells in Pomc Nkx2.1 KO mice is also normal at E15.5 ( g , h ) and in the adult arcuate nucleus ( i , j ).
Figure Legend Snippet: Hypothalamic NPY neurons in conditional Nkx2.1 mutant mice. a–f , The number of NPY + neurons is normal in Nkx2.1 KO@E9.5 ( b ), Isl1 Nkx2.1 KO ( d ), and Pomc Nkx2.1 KO ( f ) E12.5 mouse embryos compared with their corresponding control littermates ( a , c , e ), as determined by immunofluorescence using anti-NPY (green) antibody on sagittal cryosections. g–j , The signal density of NPY + cells in Pomc Nkx2.1 KO mice is also normal at E15.5 ( g , h ) and in the adult arcuate nucleus ( i , j ).

Techniques Used: Mutagenesis, Mouse Assay, Immunofluorescence

Hypothalamic Pomc expression is impaired in Nkx2.1 conditional knock-out mice. a–h , Immunofluorescence analysis using anti-NKX2.1 (red) and anti-ACTH (green) antibodies in sagittal cryosections of E12.5 mouse embryos injected with tamoxifen at E8.5 ( a–d ) or E9.5 ( e–h ). Cre + and Cre − embryos are littermates. i–n , Immunofluorescence analysis of E12.5 mouse embryos injected with tamoxifen at E9.5 using antibodies against ISL1 ( i , l ) or ACTH ( j , m ); closed-up merged pictures are also shown ( k , n ).
Figure Legend Snippet: Hypothalamic Pomc expression is impaired in Nkx2.1 conditional knock-out mice. a–h , Immunofluorescence analysis using anti-NKX2.1 (red) and anti-ACTH (green) antibodies in sagittal cryosections of E12.5 mouse embryos injected with tamoxifen at E8.5 ( a–d ) or E9.5 ( e–h ). Cre + and Cre − embryos are littermates. i–n , Immunofluorescence analysis of E12.5 mouse embryos injected with tamoxifen at E9.5 using antibodies against ISL1 ( i , l ) or ACTH ( j , m ); closed-up merged pictures are also shown ( k , n ).

Techniques Used: Expressing, Knock-Out, Mouse Assay, Immunofluorescence, Injection

Specific deletion of Nkx2.1 from ISL1 + neurons impairs the onset of Pomc expression. a–l , Immunofluorescence analysis in sagittal cryosections of E11.5 embryos using anti-ACTH ( a , b ), anti-NKX2.1 ( c , d ), anti-ISL1 ( e , f ), anti-ASCL1 ( i , j ), and anti-NGN3 ( k , l ) antibodies. g , h , Loss of Nkx2.1 in ISL1 + neurons is evident by double labeling. Arrows point to the mantle zone of the mediobasal hypothalamus detailed in the insets above. m–p , E12.5 Isl1 Nkx2.1 KO embryos show reduced numbers of ACTH + neurons compared with controls ( m , n ) and loss of NKX2.1 in the mantle zone ( o , p ). DAPI is shown in blue.
Figure Legend Snippet: Specific deletion of Nkx2.1 from ISL1 + neurons impairs the onset of Pomc expression. a–l , Immunofluorescence analysis in sagittal cryosections of E11.5 embryos using anti-ACTH ( a , b ), anti-NKX2.1 ( c , d ), anti-ISL1 ( e , f ), anti-ASCL1 ( i , j ), and anti-NGN3 ( k , l ) antibodies. g , h , Loss of Nkx2.1 in ISL1 + neurons is evident by double labeling. Arrows point to the mantle zone of the mediobasal hypothalamus detailed in the insets above. m–p , E12.5 Isl1 Nkx2.1 KO embryos show reduced numbers of ACTH + neurons compared with controls ( m , n ) and loss of NKX2.1 in the mantle zone ( o , p ). DAPI is shown in blue.

Techniques Used: Expressing, Immunofluorescence, Labeling

Overlapping expression patterns of Nkx2.1 , Isl1 , and Pomc in the developing hypothalamus. a–j , Immunofluorescence analysis using anti-NKX2.1 (blue) and anti-ISL1 (red) antibodies in sagittal cryosections of E10.5 ( a–e ) and E12.5 ( f–j ) Pomc -EGFP mouse embryos. Confocal images showing superimposed NKX2.1- and ISL1-immunopositive neurons ( a , f ) and triple merged with Pomc -EGFP-immunoreactive neurons ( e , j ). Arrows denote examples of neurons coexpressing NKX2.1, ISL1, and EGFP.
Figure Legend Snippet: Overlapping expression patterns of Nkx2.1 , Isl1 , and Pomc in the developing hypothalamus. a–j , Immunofluorescence analysis using anti-NKX2.1 (blue) and anti-ISL1 (red) antibodies in sagittal cryosections of E10.5 ( a–e ) and E12.5 ( f–j ) Pomc -EGFP mouse embryos. Confocal images showing superimposed NKX2.1- and ISL1-immunopositive neurons ( a , f ) and triple merged with Pomc -EGFP-immunoreactive neurons ( e , j ). Arrows denote examples of neurons coexpressing NKX2.1, ISL1, and EGFP.

Techniques Used: Expressing, Immunofluorescence

33) Product Images from "Dynamic regulation of Pdx1 enhancers by Foxa1 and Foxa2 is essential for pancreas development"

Article Title: Dynamic regulation of Pdx1 enhancers by Foxa1 and Foxa2 is essential for pancreas development

Journal: Genes & Development

doi: 10.1101/gad.1752608

The impact of Foxa1 and Foxa2 ablation on the pancreatic transcription factor network. E18.5 pancreas sections from control and compound mutant mice were stained with Foxa1/2, Nkx6.1, and Pdx1 ( A–H ); Pax6, Nkx2.2, and Foxa1/2 ( I–P ); or Isl1 and Foxa1/2 ( Q–T ) antibodies by indirect immunofluorescence and visualized by confocal microscopy. Arrows, in compound mutant sections, point to remaining Foxa1/2-positive cells that coexpress additional pancreatic transcription factors. Asterisks in G, H, R , and S mark the ducts. H, P , and Q–T are bright-field images or merged images including bright-field images. Bars: R , T , 17.5 μm; all others, 30 μm.
Figure Legend Snippet: The impact of Foxa1 and Foxa2 ablation on the pancreatic transcription factor network. E18.5 pancreas sections from control and compound mutant mice were stained with Foxa1/2, Nkx6.1, and Pdx1 ( A–H ); Pax6, Nkx2.2, and Foxa1/2 ( I–P ); or Isl1 and Foxa1/2 ( Q–T ) antibodies by indirect immunofluorescence and visualized by confocal microscopy. Arrows, in compound mutant sections, point to remaining Foxa1/2-positive cells that coexpress additional pancreatic transcription factors. Asterisks in G, H, R , and S mark the ducts. H, P , and Q–T are bright-field images or merged images including bright-field images. Bars: R , T , 17.5 μm; all others, 30 μm.

Techniques Used: Mutagenesis, Mouse Assay, Staining, Immunofluorescence, Confocal Microscopy

34) Product Images from "The transcription factor Hmx1 and growth factor receptor activities control sympathetic neurons diversification"

Article Title: The transcription factor Hmx1 and growth factor receptor activities control sympathetic neurons diversification

Journal: The EMBO Journal

doi: 10.1038/emboj.2013.85

A failure of Hmx1 repression and development of cholinergic neurons in mice lacking TrkC . ( A ) Triple immunostaining for TRKC, Hmx1 -βgal and ISL1 on SG section of E15.5 Hmx1 LcZ/+ embryos. Note βgal + /TRKC + neurons
Figure Legend Snippet: A failure of Hmx1 repression and development of cholinergic neurons in mice lacking TrkC . ( A ) Triple immunostaining for TRKC, Hmx1 -βgal and ISL1 on SG section of E15.5 Hmx1 LcZ/+ embryos. Note βgal + /TRKC + neurons

Techniques Used: Mouse Assay, Triple Immunostaining

35) Product Images from "Necessity and Sufficiency of Ldb1 in the Generation, Differentiation and Maintenance of Non-photoreceptor Cell Types During Retinal Development"

Article Title: Necessity and Sufficiency of Ldb1 in the Generation, Differentiation and Maintenance of Non-photoreceptor Cell Types During Retinal Development

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00271

Conditional knockout of Ldb1 causes loss of all non-photoreceptor cell types. (A,A’) Ldb1 immunoreactivity was near completely abolished in the mutant retina. (B,B’) Immunoreactivity for Pax6, a cell marker for amacrine, horizontal and ganglion cells, was reduced in the mutant. (C,C) The Ldb1 binding cofactor Lmo4, usually present in nearly all major cell types in the INL and GCL, was decreased in the mutant. (D,D’) Syntaxin, a marker for all amacrine cells, was dramatically reduced in the mutant. (E,E’) GABA immunoreactive amacrine cells were diminished in the mutant. (F,F’) Calbindin is expressed in all horizontal cells and some amacrine cells in the wildtype retina. These cells were greatly reduced in the mutant. (G,G’) Chx10 + bipolar cells were vastly decreased in the mutant. (H,H’) Isl1 is present in ON-bipolar, cholinergic amacrine, and ganglion cells. These cells especially ganglion cells were drastically reduced in the mutant. (I,I’) Brn3b + ganglion cells nearly disappeared in the Ldb1 mutant. (J–L,J’–L’) Müller cells immunoreactive for Sox9, GS or Lhx2 were decreased in the mutant. (M) Quantification of some typical cell markers in control and Ldb1 mutant retinas. Each histogram represents the mean ± SD for three retinas. ∗ p
Figure Legend Snippet: Conditional knockout of Ldb1 causes loss of all non-photoreceptor cell types. (A,A’) Ldb1 immunoreactivity was near completely abolished in the mutant retina. (B,B’) Immunoreactivity for Pax6, a cell marker for amacrine, horizontal and ganglion cells, was reduced in the mutant. (C,C) The Ldb1 binding cofactor Lmo4, usually present in nearly all major cell types in the INL and GCL, was decreased in the mutant. (D,D’) Syntaxin, a marker for all amacrine cells, was dramatically reduced in the mutant. (E,E’) GABA immunoreactive amacrine cells were diminished in the mutant. (F,F’) Calbindin is expressed in all horizontal cells and some amacrine cells in the wildtype retina. These cells were greatly reduced in the mutant. (G,G’) Chx10 + bipolar cells were vastly decreased in the mutant. (H,H’) Isl1 is present in ON-bipolar, cholinergic amacrine, and ganglion cells. These cells especially ganglion cells were drastically reduced in the mutant. (I,I’) Brn3b + ganglion cells nearly disappeared in the Ldb1 mutant. (J–L,J’–L’) Müller cells immunoreactive for Sox9, GS or Lhx2 were decreased in the mutant. (M) Quantification of some typical cell markers in control and Ldb1 mutant retinas. Each histogram represents the mean ± SD for three retinas. ∗ p

Techniques Used: Knock-Out, Mutagenesis, Marker, Binding Assay

36) Product Images from "Lungfishes, Like Tetrapods, Possess a Vomeronasal System"

Article Title: Lungfishes, Like Tetrapods, Possess a Vomeronasal System

Journal: Frontiers in Neuroanatomy

doi: 10.3389/fnana.2010.00130

Characterization of the MeA . (A,B) The MeA can be identified by its specific OTP-immunoreactivity. (C,D) The presence of ISL1-immunoreactivity and the virtual lack of NKX2.1 labeling characterize the MeA. (E,F) The distinct distribution of NOS and SP immunoreactive structures identifies the MeA. (G) Retrogradely labeled cells in the MeA after DiI application to the lateral hypothalamus. All photographs correspond to transverse sections and in (E–G) only the left side is shown. Abbreviations: ac, anterior commissure; MeA, medial amygdala; PO, preoptic area. Scale bars = 200 μm.
Figure Legend Snippet: Characterization of the MeA . (A,B) The MeA can be identified by its specific OTP-immunoreactivity. (C,D) The presence of ISL1-immunoreactivity and the virtual lack of NKX2.1 labeling characterize the MeA. (E,F) The distinct distribution of NOS and SP immunoreactive structures identifies the MeA. (G) Retrogradely labeled cells in the MeA after DiI application to the lateral hypothalamus. All photographs correspond to transverse sections and in (E–G) only the left side is shown. Abbreviations: ac, anterior commissure; MeA, medial amygdala; PO, preoptic area. Scale bars = 200 μm.

Techniques Used: Microelectrode Array, Labeling

37) Product Images from "Ablation of CNTN2+ Pyramidal Neurons During Development Results in Defects in Neocortical Size and Axonal Tract Formation"

Article Title: Ablation of CNTN2+ Pyramidal Neurons During Development Results in Defects in Neocortical Size and Axonal Tract Formation

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2019.00454

Emx1 Cre -mediated recombination in Tag-1 loxP − EGFP − loxP − DTA embryos results in EGFP removal and neocortical cell death during stages E11.5 to E13.5. (A) Immunofluorescence against EGFP and TAG-1 in in Tag-1 EGFP and Emx1 Cre ;Tag-1 DTA mice at E11.5 and E13.5. Note the lack of recombination at the ventral telencephalon (vT). (B) Whole mount immunofluorescence against EGFP, neurofilaments and ISL1 in control and double transgenic mice at E12.5 shows the selective EGFP switch-off in the dorsal telencephalon (see white arrowheads). Note that sensory ganglia (ISL1+, shown by asterisks) remain unaffected upon Emx1 Cre -mediated recombination. (C–J) Immunofluorescence against cleaved caspase-3 (reflecting cell death) in in Tag-1 EGFP and Emx1 Cre ;Tag-1 DTA mice at E11.5 to P0. RMTW, rostromedial telencephalic wall, vT, ventral telencephalon, mb, midbrain, m, medulla, p, pons, str, striatum.
Figure Legend Snippet: Emx1 Cre -mediated recombination in Tag-1 loxP − EGFP − loxP − DTA embryos results in EGFP removal and neocortical cell death during stages E11.5 to E13.5. (A) Immunofluorescence against EGFP and TAG-1 in in Tag-1 EGFP and Emx1 Cre ;Tag-1 DTA mice at E11.5 and E13.5. Note the lack of recombination at the ventral telencephalon (vT). (B) Whole mount immunofluorescence against EGFP, neurofilaments and ISL1 in control and double transgenic mice at E12.5 shows the selective EGFP switch-off in the dorsal telencephalon (see white arrowheads). Note that sensory ganglia (ISL1+, shown by asterisks) remain unaffected upon Emx1 Cre -mediated recombination. (C–J) Immunofluorescence against cleaved caspase-3 (reflecting cell death) in in Tag-1 EGFP and Emx1 Cre ;Tag-1 DTA mice at E11.5 to P0. RMTW, rostromedial telencephalic wall, vT, ventral telencephalon, mb, midbrain, m, medulla, p, pons, str, striatum.

Techniques Used: Immunofluorescence, Mouse Assay, Transgenic Assay

38) Product Images from "The Homeodomain Transcription Factor NKX2.1 Is Essential for the Early Specification of Melanocortin Neuron Identity and Activates Pomc Expression in the Developing Hypothalamus"

Article Title: The Homeodomain Transcription Factor NKX2.1 Is Essential for the Early Specification of Melanocortin Neuron Identity and Activates Pomc Expression in the Developing Hypothalamus

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2924-18.2019

Hypothalamic NPY neurons in conditional Nkx2.1 mutant mice. a–f , The number of NPY + neurons is normal in Nkx2.1 KO@E9.5 ( b ), Isl1 Nkx2.1 KO ( d ), and Pomc Nkx2.1 KO ( f ) E12.5 mouse embryos compared with their corresponding control littermates ( a , c , e ), as determined by immunofluorescence using anti-NPY (green) antibody on sagittal cryosections. g–j , The signal density of NPY + cells in Pomc Nkx2.1 KO mice is also normal at E15.5 ( g , h ) and in the adult arcuate nucleus ( i , j ).
Figure Legend Snippet: Hypothalamic NPY neurons in conditional Nkx2.1 mutant mice. a–f , The number of NPY + neurons is normal in Nkx2.1 KO@E9.5 ( b ), Isl1 Nkx2.1 KO ( d ), and Pomc Nkx2.1 KO ( f ) E12.5 mouse embryos compared with their corresponding control littermates ( a , c , e ), as determined by immunofluorescence using anti-NPY (green) antibody on sagittal cryosections. g–j , The signal density of NPY + cells in Pomc Nkx2.1 KO mice is also normal at E15.5 ( g , h ) and in the adult arcuate nucleus ( i , j ).

Techniques Used: Mutagenesis, Mouse Assay, Immunofluorescence

Hypothalamic Pomc expression is impaired in Nkx2.1 conditional knock-out mice. a–h , Immunofluorescence analysis using anti-NKX2.1 (red) and anti-ACTH (green) antibodies in sagittal cryosections of E12.5 mouse embryos injected with tamoxifen at E8.5 ( a–d ) or E9.5 ( e–h ). Cre + and Cre − embryos are littermates. i–n , Immunofluorescence analysis of E12.5 mouse embryos injected with tamoxifen at E9.5 using antibodies against ISL1 ( i , l ) or ACTH ( j , m ); closed-up merged pictures are also shown ( k , n ).
Figure Legend Snippet: Hypothalamic Pomc expression is impaired in Nkx2.1 conditional knock-out mice. a–h , Immunofluorescence analysis using anti-NKX2.1 (red) and anti-ACTH (green) antibodies in sagittal cryosections of E12.5 mouse embryos injected with tamoxifen at E8.5 ( a–d ) or E9.5 ( e–h ). Cre + and Cre − embryos are littermates. i–n , Immunofluorescence analysis of E12.5 mouse embryos injected with tamoxifen at E9.5 using antibodies against ISL1 ( i , l ) or ACTH ( j , m ); closed-up merged pictures are also shown ( k , n ).

Techniques Used: Expressing, Knock-Out, Mouse Assay, Immunofluorescence, Injection

Specific deletion of Nkx2.1 from ISL1 + neurons impairs the onset of Pomc expression. a–l , Immunofluorescence analysis in sagittal cryosections of E11.5 embryos using anti-ACTH ( a , b ), anti-NKX2.1 ( c , d ), anti-ISL1 ( e , f ), anti-ASCL1 ( i , j ), and anti-NGN3 ( k , l ) antibodies. g , h , Loss of Nkx2.1 in ISL1 + neurons is evident by double labeling. Arrows point to the mantle zone of the mediobasal hypothalamus detailed in the insets above. m–p , E12.5 Isl1 Nkx2.1 KO embryos show reduced numbers of ACTH + neurons compared with controls ( m , n ) and loss of NKX2.1 in the mantle zone ( o , p ). DAPI is shown in blue.
Figure Legend Snippet: Specific deletion of Nkx2.1 from ISL1 + neurons impairs the onset of Pomc expression. a–l , Immunofluorescence analysis in sagittal cryosections of E11.5 embryos using anti-ACTH ( a , b ), anti-NKX2.1 ( c , d ), anti-ISL1 ( e , f ), anti-ASCL1 ( i , j ), and anti-NGN3 ( k , l ) antibodies. g , h , Loss of Nkx2.1 in ISL1 + neurons is evident by double labeling. Arrows point to the mantle zone of the mediobasal hypothalamus detailed in the insets above. m–p , E12.5 Isl1 Nkx2.1 KO embryos show reduced numbers of ACTH + neurons compared with controls ( m , n ) and loss of NKX2.1 in the mantle zone ( o , p ). DAPI is shown in blue.

Techniques Used: Expressing, Immunofluorescence, Labeling

Overlapping expression patterns of Nkx2.1 , Isl1 , and Pomc in the developing hypothalamus. a–j , Immunofluorescence analysis using anti-NKX2.1 (blue) and anti-ISL1 (red) antibodies in sagittal cryosections of E10.5 ( a–e ) and E12.5 ( f–j ) Pomc -EGFP mouse embryos. Confocal images showing superimposed NKX2.1- and ISL1-immunopositive neurons ( a , f ) and triple merged with Pomc -EGFP-immunoreactive neurons ( e , j ). Arrows denote examples of neurons coexpressing NKX2.1, ISL1, and EGFP.
Figure Legend Snippet: Overlapping expression patterns of Nkx2.1 , Isl1 , and Pomc in the developing hypothalamus. a–j , Immunofluorescence analysis using anti-NKX2.1 (blue) and anti-ISL1 (red) antibodies in sagittal cryosections of E10.5 ( a–e ) and E12.5 ( f–j ) Pomc -EGFP mouse embryos. Confocal images showing superimposed NKX2.1- and ISL1-immunopositive neurons ( a , f ) and triple merged with Pomc -EGFP-immunoreactive neurons ( e , j ). Arrows denote examples of neurons coexpressing NKX2.1, ISL1, and EGFP.

Techniques Used: Expressing, Immunofluorescence

39) Product Images from "Schwann Cell Precursors Generate the Majority of Chromaffin Cells in Zuckerkandl Organ and Some Sympathetic Neurons in Paraganglia"

Article Title: Schwann Cell Precursors Generate the Majority of Chromaffin Cells in Zuckerkandl Organ and Some Sympathetic Neurons in Paraganglia

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2019.00006

Ablation of Schwann cell precursors or the preganglionic nerves that serve as their route toward the dorsal aorta results in a reduction of chromaffin cell numbers of the Zuckerkandl organ. (A,B) Immunofluorescence on cryosections against CHAT (choline acetyltransferase) (upper panels) at the level of the spinal cord shows the absence of CHAT + (cholinergic) preganglionic motorneurons in the gray matter, also seen as absence of ISL1 + motorneurons (lower panel). Scale bar = 100 μm. (C,D) Immunofluorescence against CART and TH at the level of the Zuckerkandl organ (ZO) shows a severely abnormal phenotype in the ZO and para-aortic ganglia (PAG) in Hb9 Cre/+ ; Isl2 DTA/+ E14.5 embryos in comparison to Isl2 DTA/+ control E14.5 embryos. Scale bar = 100 μm. (E) Quantification of TH + cells at E14.5 in control Isl2 DTA/+ and mutant Hb9 Cre/+ ; Isl2 DTA/+ embryos shows a decrease in the ZO and PAG, while the mesenteric ganglia (MG) remain unaffected. In Isl2 DTA/+ versus Hb9 Cre/+ ; Isl2 DTA/+ respectively: total TH + cells in ZO = 426.6 ± 52.66 vs. 201.9 ± 27.43 ( P = 0.0194), in MG = 178.4 ± 21.44 vs. 198.4 ± 15.34 ( P = 0.4889) and in PAG = 133.1 ± 9.57 vs. 83.22 ± 5.79 ( P = 0.0112), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (F) Schematic showing the experimental design for induction of the visceral nerve ablation using the Hb9 Cre ; Isl2 DTA strain. (G,H) Immunofluorescence against SOX10, TH and neurofilaments (NF) on cryosections from Sox10 CreERT2/+ and Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following tamoxifen (TAM) injection at E11.5 and E12.5 and analysis at E17.5 showing almost complete Schwann cell precursor (SCP)-ablation and significantly abnormal morphology in ZO, PAG and MG. Scale bar = 100 μm. (I) Quantification of SOX10 + cells in control Sox10 CreERT2/+ and SCP-ablated Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following TAM injection at E11.5 and E12.5 and analysis at E17.5. In Sox10 CreERT2/+ vs. Sox10 CreERT2/+ ; R26 DTA/DTA respectively: total SOX10 + cells in ZO = 205.47 ± 28.96 vs. 5.33 ± 4.43 ( P = 0.0024), in MG = 369.33 ± 27.56 vs. 5.55 ± 5.22 ( P = 0.0002) and in PAG = 88.11 ± 6.46 vs. 3.24 ± 2.58 ( P = 0.0002), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (J) Quantification of TH+ cells in control Sox10 CreERT2/+ and SCP-ablated Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following TAM injection at E11.5 and E12.5 and analysis at E17.5. In Sox10 CreERT2/+ vs. Sox10 CreERT2/+ ; R26 DTA/DTA respectively: total TH + cells in ZO = 588.08 ± 28.41 vs. 397.22 ± 23.69 ( P = 0.0067), in MG = 243.66 ± 22.98 vs. 226.55 ± 15.24 ( P = 0.5685) and in PAG = 103.00 ± 7.61 vs. 94.11 ± 8.04 ( P = 0.4672), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (K) Schematic outlining the TAM injection times and collection time point in the Schwann cell precursor (SCP)-ablation experiment using the Sox10 CreERT2 ; R26 DTA strain. SC, spinal cord; GM, gray matter; MG, mesenteric ganglion; PAG, para-aortic ganglion; MNs, motorneurons; DA, dorsal aorta; ZO, Zuckerkandl organ; ns, non-significant; ∗ P ≤ 0.05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001.
Figure Legend Snippet: Ablation of Schwann cell precursors or the preganglionic nerves that serve as their route toward the dorsal aorta results in a reduction of chromaffin cell numbers of the Zuckerkandl organ. (A,B) Immunofluorescence on cryosections against CHAT (choline acetyltransferase) (upper panels) at the level of the spinal cord shows the absence of CHAT + (cholinergic) preganglionic motorneurons in the gray matter, also seen as absence of ISL1 + motorneurons (lower panel). Scale bar = 100 μm. (C,D) Immunofluorescence against CART and TH at the level of the Zuckerkandl organ (ZO) shows a severely abnormal phenotype in the ZO and para-aortic ganglia (PAG) in Hb9 Cre/+ ; Isl2 DTA/+ E14.5 embryos in comparison to Isl2 DTA/+ control E14.5 embryos. Scale bar = 100 μm. (E) Quantification of TH + cells at E14.5 in control Isl2 DTA/+ and mutant Hb9 Cre/+ ; Isl2 DTA/+ embryos shows a decrease in the ZO and PAG, while the mesenteric ganglia (MG) remain unaffected. In Isl2 DTA/+ versus Hb9 Cre/+ ; Isl2 DTA/+ respectively: total TH + cells in ZO = 426.6 ± 52.66 vs. 201.9 ± 27.43 ( P = 0.0194), in MG = 178.4 ± 21.44 vs. 198.4 ± 15.34 ( P = 0.4889) and in PAG = 133.1 ± 9.57 vs. 83.22 ± 5.79 ( P = 0.0112), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (F) Schematic showing the experimental design for induction of the visceral nerve ablation using the Hb9 Cre ; Isl2 DTA strain. (G,H) Immunofluorescence against SOX10, TH and neurofilaments (NF) on cryosections from Sox10 CreERT2/+ and Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following tamoxifen (TAM) injection at E11.5 and E12.5 and analysis at E17.5 showing almost complete Schwann cell precursor (SCP)-ablation and significantly abnormal morphology in ZO, PAG and MG. Scale bar = 100 μm. (I) Quantification of SOX10 + cells in control Sox10 CreERT2/+ and SCP-ablated Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following TAM injection at E11.5 and E12.5 and analysis at E17.5. In Sox10 CreERT2/+ vs. Sox10 CreERT2/+ ; R26 DTA/DTA respectively: total SOX10 + cells in ZO = 205.47 ± 28.96 vs. 5.33 ± 4.43 ( P = 0.0024), in MG = 369.33 ± 27.56 vs. 5.55 ± 5.22 ( P = 0.0002) and in PAG = 88.11 ± 6.46 vs. 3.24 ± 2.58 ( P = 0.0002), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (J) Quantification of TH+ cells in control Sox10 CreERT2/+ and SCP-ablated Sox10 CreERT2/+ ; R26 DTA/DTA E17.5 embryos following TAM injection at E11.5 and E12.5 and analysis at E17.5. In Sox10 CreERT2/+ vs. Sox10 CreERT2/+ ; R26 DTA/DTA respectively: total TH + cells in ZO = 588.08 ± 28.41 vs. 397.22 ± 23.69 ( P = 0.0067), in MG = 243.66 ± 22.98 vs. 226.55 ± 15.24 ( P = 0.5685) and in PAG = 103.00 ± 7.61 vs. 94.11 ± 8.04 ( P = 0.4672), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t -test. (K) Schematic outlining the TAM injection times and collection time point in the Schwann cell precursor (SCP)-ablation experiment using the Sox10 CreERT2 ; R26 DTA strain. SC, spinal cord; GM, gray matter; MG, mesenteric ganglion; PAG, para-aortic ganglion; MNs, motorneurons; DA, dorsal aorta; ZO, Zuckerkandl organ; ns, non-significant; ∗ P ≤ 0.05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001.

Techniques Used: Immunofluorescence, Mutagenesis, Two Tailed Test, Injection

The development and anatomy of the Zuckerkandl organ and associated paraganglia in relation to the dorsal aorta. (A) Schematic showing the molecular differences and similarities between sympathetic neurons (SNs) and chromaffin cells (ChCs) during development in relation to the expression of CART, (ISL1 and TH. (B) Side-view of whole-mount immunofluorescence against smooth-muscle-actin (SMA, showing the dorsal aorta -DA), TH and CART on an E12.5 wild type embryo. Note the presence of TH + cells at the dorsal part of the DA just posteriorly to its branching into the inferior mesenteric artery (shown by the white arrowhead). (C) Immunofluorescence on E12.5 wild type embryos against SOX10 and TH (left panel) and SOX10, CART and ISL1 (right panel) showing the close proximity of the developing CART - /ISL1 + /TH + Zuckerkandl organ (ZO) and CART + /ISL1 + /TH + mesenteric ganglion (MG). (D) Ventral-view of whole-mount immunofluorescence against NF200, TH and CD31 (showing the endothelium of the DA) on an E12.5 wild type embryo. Note the presence of the ZO below the inferior mesenteric artery (shown by the white arrowhead), which is surrounded by NF200 + axons. (E) Immunofluorescence on cryosection of an E13.5 wild type embryo against NF200, TH and CART. Note the innervation pattern around the DA and the TH+ cells of the ZO. (F) Ventral-view of whole-mount immunofluorescence against SMA, TH and CART on an E13.5 wild type embryo. Note the close proximity of the MG and ZO just posteriorly to the inferior mesenteric artery and the separation of the two structures based on the CART + /TH low immunoreactivity in the MG in contrast to the TH high immunoreactivity of the ZO (shown by the white arrowhead). Also note the presence of TH high chromaffin structures in close proximity to the MG and ZO (shown by yellow arrows). (G) Immunofluorescence against CART, TH and DAPI on cryosections of a wild type E17.5 embryo, clearly showing the composite nature of the ZO at this later developmental stage, with both TH + /CART - and TH + /CART + cells. Scale bar = 100 μm. SNs, sympathetic neurons; ChCs, chromaffin cells; SC, sympathetic chain; DA, dorsal aorta; PAG, para-aortic ganglion; ZO, Zuckerkandl organ; AM, adrenal medulla; MG, mesenteric ganglion.)
Figure Legend Snippet: The development and anatomy of the Zuckerkandl organ and associated paraganglia in relation to the dorsal aorta. (A) Schematic showing the molecular differences and similarities between sympathetic neurons (SNs) and chromaffin cells (ChCs) during development in relation to the expression of CART, (ISL1 and TH. (B) Side-view of whole-mount immunofluorescence against smooth-muscle-actin (SMA, showing the dorsal aorta -DA), TH and CART on an E12.5 wild type embryo. Note the presence of TH + cells at the dorsal part of the DA just posteriorly to its branching into the inferior mesenteric artery (shown by the white arrowhead). (C) Immunofluorescence on E12.5 wild type embryos against SOX10 and TH (left panel) and SOX10, CART and ISL1 (right panel) showing the close proximity of the developing CART - /ISL1 + /TH + Zuckerkandl organ (ZO) and CART + /ISL1 + /TH + mesenteric ganglion (MG). (D) Ventral-view of whole-mount immunofluorescence against NF200, TH and CD31 (showing the endothelium of the DA) on an E12.5 wild type embryo. Note the presence of the ZO below the inferior mesenteric artery (shown by the white arrowhead), which is surrounded by NF200 + axons. (E) Immunofluorescence on cryosection of an E13.5 wild type embryo against NF200, TH and CART. Note the innervation pattern around the DA and the TH+ cells of the ZO. (F) Ventral-view of whole-mount immunofluorescence against SMA, TH and CART on an E13.5 wild type embryo. Note the close proximity of the MG and ZO just posteriorly to the inferior mesenteric artery and the separation of the two structures based on the CART + /TH low immunoreactivity in the MG in contrast to the TH high immunoreactivity of the ZO (shown by the white arrowhead). Also note the presence of TH high chromaffin structures in close proximity to the MG and ZO (shown by yellow arrows). (G) Immunofluorescence against CART, TH and DAPI on cryosections of a wild type E17.5 embryo, clearly showing the composite nature of the ZO at this later developmental stage, with both TH + /CART - and TH + /CART + cells. Scale bar = 100 μm. SNs, sympathetic neurons; ChCs, chromaffin cells; SC, sympathetic chain; DA, dorsal aorta; PAG, para-aortic ganglion; ZO, Zuckerkandl organ; AM, adrenal medulla; MG, mesenteric ganglion.)

Techniques Used: Expressing, Immunofluorescence

The sympathetic ganglia at the level of the Zuckerkandl organ and the organ itself have distinct early-defined origin despite the intermingling anatomy. (A) Dorsal view of whole-mount immunofluorescence (left panel) against the sympathetic marker CART, the chromaffin and sympathetic marker TH and NF200 (showing the innervation on the trunk of an E15.5 wild type embryo and schematic (right panel) showing the sympathetic and chromaffin structures in relation to the dorsal aorta. Note that the mesenteric (MG) and suprarenal ganglion (SRG), as well as the sympathetic chain (SC), are CART + , while the Zuckerkandl organ (ZO) is composed mainly by TH + /CART - cells. Additionally note that the para-aortic ganglia (PAG) are the continuation of the sympathetic chain that extends along the anteroposterior axis of the embryo trunk just at the dorsal view of the dorsal aorta, at the level of the ZO and MG. (B,C) Immunofluorescence on cryosections against CART, Ret TOM and TH on tamoxifen-injected (TAM-injected) embryos at E10.5 and E11.5 respectively shows that Ret TOM specifically delineated the sympathetic compartment when analyzed at E15.5, with clear tracing of the MG and PAG, while only few Ret TOM+ cells can be seen in the ZO. Note the difference in CART immunofluorescence levels in the MG and PAG. (D) Immunofluorescence on cryosections against ISL1, Ret TOM and TH on TAM-injected embryos at E10.5 shows Ret TOM specific expression by the sympathetic ganglion (SG) and SRG when analyzed at E15.5, while almost no Ret TOM+ cells can be seen in the adrenal medulla (AM). (E) Ventral view of whole-mount immunofluorescence against CART, Ascl1 TOM and TH on embryos with TAM injection at E11.5 and analyzed at E13.5 shows tracing in the chromaffin cells of the ZO, while no tracing in the MG (shown by white arrowheads). Scale bar in (A–E) = 100 μm. A, anterior; P, posterior; AM, adrenal medulla; MG, mesenteric ganglion; ZO, Zuckerkandl organ; DA, dorsal aorta; SC, sympathetic chain; SRG, suprarenal ganglion; AG, adrenal gland; PAG, para-aortic ganglion; SG, sympathetic ganglion; ChCs, chromaffin cells; SNs, sympathetic neurons.)
Figure Legend Snippet: The sympathetic ganglia at the level of the Zuckerkandl organ and the organ itself have distinct early-defined origin despite the intermingling anatomy. (A) Dorsal view of whole-mount immunofluorescence (left panel) against the sympathetic marker CART, the chromaffin and sympathetic marker TH and NF200 (showing the innervation on the trunk of an E15.5 wild type embryo and schematic (right panel) showing the sympathetic and chromaffin structures in relation to the dorsal aorta. Note that the mesenteric (MG) and suprarenal ganglion (SRG), as well as the sympathetic chain (SC), are CART + , while the Zuckerkandl organ (ZO) is composed mainly by TH + /CART - cells. Additionally note that the para-aortic ganglia (PAG) are the continuation of the sympathetic chain that extends along the anteroposterior axis of the embryo trunk just at the dorsal view of the dorsal aorta, at the level of the ZO and MG. (B,C) Immunofluorescence on cryosections against CART, Ret TOM and TH on tamoxifen-injected (TAM-injected) embryos at E10.5 and E11.5 respectively shows that Ret TOM specifically delineated the sympathetic compartment when analyzed at E15.5, with clear tracing of the MG and PAG, while only few Ret TOM+ cells can be seen in the ZO. Note the difference in CART immunofluorescence levels in the MG and PAG. (D) Immunofluorescence on cryosections against ISL1, Ret TOM and TH on TAM-injected embryos at E10.5 shows Ret TOM specific expression by the sympathetic ganglion (SG) and SRG when analyzed at E15.5, while almost no Ret TOM+ cells can be seen in the adrenal medulla (AM). (E) Ventral view of whole-mount immunofluorescence against CART, Ascl1 TOM and TH on embryos with TAM injection at E11.5 and analyzed at E13.5 shows tracing in the chromaffin cells of the ZO, while no tracing in the MG (shown by white arrowheads). Scale bar in (A–E) = 100 μm. A, anterior; P, posterior; AM, adrenal medulla; MG, mesenteric ganglion; ZO, Zuckerkandl organ; DA, dorsal aorta; SC, sympathetic chain; SRG, suprarenal ganglion; AG, adrenal gland; PAG, para-aortic ganglion; SG, sympathetic ganglion; ChCs, chromaffin cells; SNs, sympathetic neurons.)

Techniques Used: Immunofluorescence, Marker, Injection, Expressing

40) Product Images from "ISL1 and BRN3B co-regulate the differentiation of murine retinal ganglion cells"

Article Title: ISL1 and BRN3B co-regulate the differentiation of murine retinal ganglion cells

Journal:

doi: 10.1242/dev.010751

ISL1 and BRN3B regulate the expression of a common set of RGC-specific genes. (A–H) Compared with controls (left panels), in situ hybridization shows that at E14.5, the expression of RGC specific genes Brn3a, Isl2, Olf1, Irx4, Ablim1, Gap43, L1cam,
Figure Legend Snippet: ISL1 and BRN3B regulate the expression of a common set of RGC-specific genes. (A–H) Compared with controls (left panels), in situ hybridization shows that at E14.5, the expression of RGC specific genes Brn3a, Isl2, Olf1, Irx4, Ablim1, Gap43, L1cam,

Techniques Used: Expressing, In Situ Hybridization

Functional mechanism of ISL1 and BRN3B in the development of RGCs. (A) Concurrent binding of ISL1 and BRN3B to RGC-specific promoters. Anti-BRN3B and anti-ISL1 antibodies co-precipitate with the promoters of Brn3b , Shh , Brn3a and Isl2 . Both antibodies
Figure Legend Snippet: Functional mechanism of ISL1 and BRN3B in the development of RGCs. (A) Concurrent binding of ISL1 and BRN3B to RGC-specific promoters. Anti-BRN3B and anti-ISL1 antibodies co-precipitate with the promoters of Brn3b , Shh , Brn3a and Isl2 . Both antibodies

Techniques Used: Functional Assay, Binding Assay

More severe RGC loss in Isl1 and Brn3b compound null mice. (A–H) Immunostaining of adult whole-mount retinas with anti-BRN3A (A–D) and SMI32 (E–H). Compared with control (A,E), Isl1 -null (B,F), and Brn3b -null (C,G), a more severe
Figure Legend Snippet: More severe RGC loss in Isl1 and Brn3b compound null mice. (A–H) Immunostaining of adult whole-mount retinas with anti-BRN3A (A–D) and SMI32 (E–H). Compared with control (A,E), Isl1 -null (B,F), and Brn3b -null (C,G), a more severe

Techniques Used: Mouse Assay, Immunostaining

Generation of Isl1 conditional knockout and Isl1-lacZ knock-in alleles. (A) Generation of Isl1 conditional allele. Isl1 genomic structure and restriction enzyme map is shown at the top. Open boxes are the non-coding exon sequences and filled boxes the
Figure Legend Snippet: Generation of Isl1 conditional knockout and Isl1-lacZ knock-in alleles. (A) Generation of Isl1 conditional allele. Isl1 genomic structure and restriction enzyme map is shown at the top. Open boxes are the non-coding exon sequences and filled boxes the

Techniques Used: Knock-Out, Knock-In

Targeted disruption of Isl1 results in the developmental loss of RGCs. (A–H) Immunostaining of retina sections with anti-BRN3B shows that in Isl1- null retina, BRN3B+ RGCs are generated and positioned properly at E13.5 (A,E) and E15.5 (B,F). However,
Figure Legend Snippet: Targeted disruption of Isl1 results in the developmental loss of RGCs. (A–H) Immunostaining of retina sections with anti-BRN3B shows that in Isl1- null retina, BRN3B+ RGCs are generated and positioned properly at E13.5 (A,E) and E15.5 (B,F). However,

Techniques Used: Immunostaining, Generated

Loss of RGCs in adult Isl1 -null retina. (A–G) Immunostaining of whole-mount retina with anti-BRN3A (A,B), BRN3B (C,D) and SMI32 (F,G) antibodies reveals the reduction of RGCs in Isl1 - null retina. (E) Quantification of BRN3A+ and BRN3B+ cells in
Figure Legend Snippet: Loss of RGCs in adult Isl1 -null retina. (A–G) Immunostaining of whole-mount retina with anti-BRN3A (A,B), BRN3B (C,D) and SMI32 (F,G) antibodies reveals the reduction of RGCs in Isl1 - null retina. (E) Quantification of BRN3A+ and BRN3B+ cells in

Techniques Used: Immunostaining

Axon growth defects in mice deficient for Isl1 or Brn3b . (A–F) After DiI was placed at the right optic nerve head, brains were dissected to expose the optic pathways at the ventral diencephalons. RGC axons of wild type pass the midline (dot line),
Figure Legend Snippet: Axon growth defects in mice deficient for Isl1 or Brn3b . (A–F) After DiI was placed at the right optic nerve head, brains were dissected to expose the optic pathways at the ventral diencephalons. RGC axons of wild type pass the midline (dot line),

Techniques Used: Mouse Assay

Expression of ISL1 in developing mouse retina. (A–F) Horizontal sections of retinas at E11.5 (A–C) and E13.5 (D–F) were immunolabeled with anti-ISL1 (red) and anti-BRN3B (green). The expression of ISL1 is detected from E11.5 and
Figure Legend Snippet: Expression of ISL1 in developing mouse retina. (A–F) Horizontal sections of retinas at E11.5 (A–C) and E13.5 (D–F) were immunolabeled with anti-ISL1 (red) and anti-BRN3B (green). The expression of ISL1 is detected from E11.5 and

Techniques Used: Expressing, Immunolabeling

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Article Title: Spinal Hb9::Cre-derived excitatory interneurons contribute to rhythm generation in the mouse
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Mass Spectrometry:

Article Title: Essential roles of mitochondrial biogenesis regulator Nrf1 in retinal development and homeostasis
Article Snippet: .. The primary antibody was applied to the sections for 1–3 days at 4o C. The primary antibodies used were mouse anti-Nrf1 (1:300, catalog #PCRP-NFR1-3D4; DSHB, The University of Iowa, Iowa City, IA), mouse anti-Isl1 (1:200, catalog# 37.3F7; DSHB), goat anti-Brn3/Pou4f2 (1:150, catalog #sc-6026; Santa Cruz Biotechnology, Dallas, TX), mouse anti-Pax6 (1:200, catalog #MAB5552; Chemicon, Burlington, MA), sheep anti-Chx10 (1:300, catalog #X1180P; Exalpha, Shirley, MA), rabbit anti-cleaved caspase-3 (1:300, catalog #9579; Cell Signaling, Danvers, MA), mouse anti-BrdU (1:10, catalog #05–633; Millipore, Burlington, MA), mouse anti-Phospho-Histone H3/PH3 (1:700, catalog #9706; Cell Signaling), rabbit anti-Cyclin D1 (1:300, catalog #MA1–39546; Thermo Fisher Scientific, Waltham, MA), mouse anti-rhodopsin (1:20, catalog #MS-1233-R7; Thermo Fisher Scientific), rabbit anti-cone arrestin (1:2000, catalog #AB16282; Millipore), and rabbit anti-Tfam (1:500, catalog #ab131607; Abcam, Cambridge, MA). .. Secondary antibodies conjugated with Alexa-488, 555 or 633 (Thermo Fisher Scientific) were used in 1:800 dilution.

IA:

Article Title: Essential roles of mitochondrial biogenesis regulator Nrf1 in retinal development and homeostasis
Article Snippet: .. The primary antibody was applied to the sections for 1–3 days at 4o C. The primary antibodies used were mouse anti-Nrf1 (1:300, catalog #PCRP-NFR1-3D4; DSHB, The University of Iowa, Iowa City, IA), mouse anti-Isl1 (1:200, catalog# 37.3F7; DSHB), goat anti-Brn3/Pou4f2 (1:150, catalog #sc-6026; Santa Cruz Biotechnology, Dallas, TX), mouse anti-Pax6 (1:200, catalog #MAB5552; Chemicon, Burlington, MA), sheep anti-Chx10 (1:300, catalog #X1180P; Exalpha, Shirley, MA), rabbit anti-cleaved caspase-3 (1:300, catalog #9579; Cell Signaling, Danvers, MA), mouse anti-BrdU (1:10, catalog #05–633; Millipore, Burlington, MA), mouse anti-Phospho-Histone H3/PH3 (1:700, catalog #9706; Cell Signaling), rabbit anti-Cyclin D1 (1:300, catalog #MA1–39546; Thermo Fisher Scientific, Waltham, MA), mouse anti-rhodopsin (1:20, catalog #MS-1233-R7; Thermo Fisher Scientific), rabbit anti-cone arrestin (1:2000, catalog #AB16282; Millipore), and rabbit anti-Tfam (1:500, catalog #ab131607; Abcam, Cambridge, MA). .. Secondary antibodies conjugated with Alexa-488, 555 or 633 (Thermo Fisher Scientific) were used in 1:800 dilution.

Article Title: Cell-Autonomous Inhibition of α7-Containing Nicotinic Acetylcholine Receptors Prevents Death of Parasympathetic Neurons during Development
Article Snippet: .. Primary antibodies and the dilutions in blocking buffer at which they were used were as follows: anti-mouse AMV-3C2 (Developmental Studies Hybridoma Bank, Iowa City, IA), which recognizes a viral p19 gag expressed by avian sarcoma and leukemia viruses , at 1:10; mouse anti-Islet-1, which recognizes a transcription factor expressed in ciliary ganglion neurons , at 1:100 dilution of the culture supernatant (prepared in the Nishi laboratory from clone 39.4D5; Developmental Studies Hybridoma Bank); mouse anti-Hu C/D (Invitrogen), which recognizes a neuron-specific RNA-binding protein ( ; ), at 1:250 dilution of the culture supernatant; rabbit anti-p27gag (SPAFAS, Norwich, CT), which recognizes avian sarcoma gag p27 , at 1:1000; rat anti-somatostatin (product number YMC1020; Accurate Chemical and Scientific Corporation, Westbury, NY) diluted 1:100; and rabbit anti- α as ). .. Secondary antibodies were as follows: biotinylated anti-mouse (Vector Laboratories, Burlingame, CA) at 1:250; biotinylated anti-rabbit (Vector Laboratories) at 1:250; goat anti-mouse Cy3 (Jackson ImmunoResearch, West Grove, PA) at 1:750; goat anti-rabbit Alexa 488 (Invitrogen) at 1:750; and goat anti-rat Cy3 (Jackson ImmunoResearch) at 1:750.

RNA Binding Assay:

Article Title: Cell-Autonomous Inhibition of α7-Containing Nicotinic Acetylcholine Receptors Prevents Death of Parasympathetic Neurons during Development
Article Snippet: .. Primary antibodies and the dilutions in blocking buffer at which they were used were as follows: anti-mouse AMV-3C2 (Developmental Studies Hybridoma Bank, Iowa City, IA), which recognizes a viral p19 gag expressed by avian sarcoma and leukemia viruses , at 1:10; mouse anti-Islet-1, which recognizes a transcription factor expressed in ciliary ganglion neurons , at 1:100 dilution of the culture supernatant (prepared in the Nishi laboratory from clone 39.4D5; Developmental Studies Hybridoma Bank); mouse anti-Hu C/D (Invitrogen), which recognizes a neuron-specific RNA-binding protein ( ; ), at 1:250 dilution of the culture supernatant; rabbit anti-p27gag (SPAFAS, Norwich, CT), which recognizes avian sarcoma gag p27 , at 1:1000; rat anti-somatostatin (product number YMC1020; Accurate Chemical and Scientific Corporation, Westbury, NY) diluted 1:100; and rabbit anti- α as ). .. Secondary antibodies were as follows: biotinylated anti-mouse (Vector Laboratories, Burlingame, CA) at 1:250; biotinylated anti-rabbit (Vector Laboratories) at 1:250; goat anti-mouse Cy3 (Jackson ImmunoResearch, West Grove, PA) at 1:750; goat anti-rabbit Alexa 488 (Invitrogen) at 1:750; and goat anti-rat Cy3 (Jackson ImmunoResearch) at 1:750.

In Situ Hybridization:

Article Title: Identification of retinal homeobox (rax) gene-dependent genes by a microarray approach: the DNA endoglycosylase neil3 is a major downstream component of the rax genetic pathway
Article Snippet: .. Primary antibodies were used at the following dilutions: mouse anti-rhodopsin (RetP1; Biomeda, Foster City, CA) 1:50; mouse anti-islet 1, 1:50 (39.4D5; Developmental Studies Hybridoma Bank [DSHB], University of Iowa)._Whole mount in situ hybridization was performed as described previously ( )._Section in situ hybridization was performed on 8 μM sections as described previously ( ). ..

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    Developmental Studies Hybridoma Bank mouse anti isl1 2
    Trnp1 marks ON type bipolar cells. Immunostaining of developing and adult retinas with Trnp1 ( green ) and cell type-specific markers. ( A – E ) Trnp1 costaining with Otx2 ( red ) and Pax6 ( gray ) at multiple ages. Otx2 is cropped and Pax6 shown only in the insets for clarity. At P0 ( A ) and P5 ( B ), no Trnp1 immunostaining is detected in the retina. ( C ) Starting at P7, Trnp1 nuclear staining is seen in the INL, where it overlaps completely with Otx2 ( arrows , insets ). The same pattern of Trnp1 expression is seen at P10 ( D ) and in adult ( E ) sections. Pax6+ amacrine cells in the ONL ( arrowheads , insets ) do not coexpress Trnp1 at any age. ( F – K ) Adult sections stained with Trnp1 and bipolar subtype specific markers ( red / gray ). ( F ) Cells that are Trnp1+ coexpress <t>Isl1/2</t> ( red , arrows , insets ), which marks ON type bipolar cells in the retina. Starburst amacrines labeled by Isl1/2 ( arrowheads ) do not express Trnp1. ( G – G'' ) A section showing Trnp1, Scgn ( gray ) and PKCα ( red ) costaining. A subset of Trnp1+ cells coexpresses Scgn ( arrowheads , insets ) or PKCα ( arrows , insets ). Nearly all of the PKCα+ rod bipolar cells express Trnp1 (G''), but only a fraction of Scgn+ cone bipolars are Trnp1+ ( G' ). ( H ) Type 2 cone OFF bipolar cells marked by Bhlhb5 staining ( arrowheads , insets ) do not coexpress Trnp1. Bhlhb5+ amacrine cells are marked with an “a”. ( I ) Calsenilin-positive type 4 cone OFF bipolar cells ( arrowheads , insets ) do not coexpress Trnp1. Scale bars : ( A – E , G – H ) 25 μm for panels and 10 μm for insets; ( F ) 100 μm and 10 μm for insets; ( I ) 50 μm and 10 μm for the insets. ( J ) Quantification of Trnp1 staining in the adult wild-type retina. The left panel shows the fraction of Trnp1+ cells that coexpress a cell-type specific marker. The right panel shows what percentage of a given population of cells expresses Trnp1. Error bars represent SD.
    Mouse Anti Isl1 2, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 89/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti isl1 2/product/Developmental Studies Hybridoma Bank
    Average 89 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
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    90
    Developmental Studies Hybridoma Bank mouse anti isl1
    Hb9::Cre-derived INs do not overlap with the Shox2 non-V2a population. ( A ) Co-expression of YFP (green) and <t>Isl1</t> antibody (red) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Motor neurons are also labeled by Isl1 antibody (blue box). Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Isl1 (red) and Hb9::Cre-derived INs (green). Scale bars: 100 μm and 50 μm. ( B ) Co-expression of YFP (green), Shox2 antibody (red) and/or Chx10 antibody (blue) in the Hb9 :: Cre;Rosa26-YFP mouse ventral spinal cord at E11.5. Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Hb9::Cre-derived INs (green) and Shox2 + Chx10 − (red), Shox2 − Chx10 + (blue) or Shox2 + Chx10 + (pink). Scale bars: 100 μm and 50 μm. ( C ) Quantification of overlap in (A) and (B). Bar graph showing percent of overlap between Hb9::Cre-derived INs (YFP + ) and Shox2 V2a (Shox2 + Chx10 + , 4% ± 1%), Shox2 OFF V2a (Shox2 − Chx10 + , 2% ± 0.1%), Shox2 non-V2a (Shox2 + Chx10 − , 1.3% ± 0.2%), and Isl1 (Isl1 + , 6% ± 0.2%) INs. Error bars represent ± SEM. ( D ) Percent of the Shox2 non-V2a IN population (Shox2 + Chx10 − ) that overlaps with Hb9::Cre-derived INs (YFP + ) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Shox2 non-V2a INs rarely co-express YFP (Shox2 + YFP + , darker grey) (12% ± 2%). Error bars represent ± SEM.
    Mouse Anti Isl1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 90/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti isl1/product/Developmental Studies Hybridoma Bank
    Average 90 stars, based on 39 article reviews
    Price from $9.99 to $1999.99
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    Trnp1 marks ON type bipolar cells. Immunostaining of developing and adult retinas with Trnp1 ( green ) and cell type-specific markers. ( A – E ) Trnp1 costaining with Otx2 ( red ) and Pax6 ( gray ) at multiple ages. Otx2 is cropped and Pax6 shown only in the insets for clarity. At P0 ( A ) and P5 ( B ), no Trnp1 immunostaining is detected in the retina. ( C ) Starting at P7, Trnp1 nuclear staining is seen in the INL, where it overlaps completely with Otx2 ( arrows , insets ). The same pattern of Trnp1 expression is seen at P10 ( D ) and in adult ( E ) sections. Pax6+ amacrine cells in the ONL ( arrowheads , insets ) do not coexpress Trnp1 at any age. ( F – K ) Adult sections stained with Trnp1 and bipolar subtype specific markers ( red / gray ). ( F ) Cells that are Trnp1+ coexpress Isl1/2 ( red , arrows , insets ), which marks ON type bipolar cells in the retina. Starburst amacrines labeled by Isl1/2 ( arrowheads ) do not express Trnp1. ( G – G'' ) A section showing Trnp1, Scgn ( gray ) and PKCα ( red ) costaining. A subset of Trnp1+ cells coexpresses Scgn ( arrowheads , insets ) or PKCα ( arrows , insets ). Nearly all of the PKCα+ rod bipolar cells express Trnp1 (G''), but only a fraction of Scgn+ cone bipolars are Trnp1+ ( G' ). ( H ) Type 2 cone OFF bipolar cells marked by Bhlhb5 staining ( arrowheads , insets ) do not coexpress Trnp1. Bhlhb5+ amacrine cells are marked with an “a”. ( I ) Calsenilin-positive type 4 cone OFF bipolar cells ( arrowheads , insets ) do not coexpress Trnp1. Scale bars : ( A – E , G – H ) 25 μm for panels and 10 μm for insets; ( F ) 100 μm and 10 μm for insets; ( I ) 50 μm and 10 μm for the insets. ( J ) Quantification of Trnp1 staining in the adult wild-type retina. The left panel shows the fraction of Trnp1+ cells that coexpress a cell-type specific marker. The right panel shows what percentage of a given population of cells expresses Trnp1. Error bars represent SD.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Gsg1, Trnp1, and Tmem215 Mark Subpopulations of Bipolar Interneurons in the Mouse Retina

    doi: 10.1167/iovs.16-19767

    Figure Lengend Snippet: Trnp1 marks ON type bipolar cells. Immunostaining of developing and adult retinas with Trnp1 ( green ) and cell type-specific markers. ( A – E ) Trnp1 costaining with Otx2 ( red ) and Pax6 ( gray ) at multiple ages. Otx2 is cropped and Pax6 shown only in the insets for clarity. At P0 ( A ) and P5 ( B ), no Trnp1 immunostaining is detected in the retina. ( C ) Starting at P7, Trnp1 nuclear staining is seen in the INL, where it overlaps completely with Otx2 ( arrows , insets ). The same pattern of Trnp1 expression is seen at P10 ( D ) and in adult ( E ) sections. Pax6+ amacrine cells in the ONL ( arrowheads , insets ) do not coexpress Trnp1 at any age. ( F – K ) Adult sections stained with Trnp1 and bipolar subtype specific markers ( red / gray ). ( F ) Cells that are Trnp1+ coexpress Isl1/2 ( red , arrows , insets ), which marks ON type bipolar cells in the retina. Starburst amacrines labeled by Isl1/2 ( arrowheads ) do not express Trnp1. ( G – G'' ) A section showing Trnp1, Scgn ( gray ) and PKCα ( red ) costaining. A subset of Trnp1+ cells coexpresses Scgn ( arrowheads , insets ) or PKCα ( arrows , insets ). Nearly all of the PKCα+ rod bipolar cells express Trnp1 (G''), but only a fraction of Scgn+ cone bipolars are Trnp1+ ( G' ). ( H ) Type 2 cone OFF bipolar cells marked by Bhlhb5 staining ( arrowheads , insets ) do not coexpress Trnp1. Bhlhb5+ amacrine cells are marked with an “a”. ( I ) Calsenilin-positive type 4 cone OFF bipolar cells ( arrowheads , insets ) do not coexpress Trnp1. Scale bars : ( A – E , G – H ) 25 μm for panels and 10 μm for insets; ( F ) 100 μm and 10 μm for insets; ( I ) 50 μm and 10 μm for the insets. ( J ) Quantification of Trnp1 staining in the adult wild-type retina. The left panel shows the fraction of Trnp1+ cells that coexpress a cell-type specific marker. The right panel shows what percentage of a given population of cells expresses Trnp1. Error bars represent SD.

    Article Snippet: Primary antibodies used were: mouse anti-Ap2α (1:250, clone 5E4; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); chicken anti–β-galactosidase (β-gal; 1:2000, AB9361; Abcam, Cambridge, MA, USA); goat anti-Bhlhb5 (1:1000, sc-6045; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); mouse anti-Cabp5 (1:10, a gift from F. Haeseleer, University of Washington) ; mouse anti-Calretinin (1:750) (MAB1568, Milipore, Billerica, MA, USA); mouse anti-Calsenilin (1:2000, 05-756; Milipore); rabbit anti-GAD65/67 (1:500, AB1511; Milipore); goat anti-GlyT1 (1:2000, AB1770; Milipore); rabbit anti-HCN4 (1:500, APC-052; Alomone Labs Ltd., Jerusalem, Israel); mouse anti-Isl1/2 (1:250, clone 39.4D5; Developmental Studies Hybridoma Bank); goat anti-Otx2 (1:200, BAF1979; R & D Systems, Minneapolis, MN, USA); rabbit anti-Pax6 (1:500, 901301; BioLegend, Inc., San Diego, CA, USA); mouse anti-PKARIIβ (1:3000, 610625; BD Biosciences, San Jose, CA, USA); mouse anti-PKCα (1:250, P5704; Sigma-Aldrich Corp., St. Louis, MO, USA); rabbit anti-Scgn (1:5000, RD181120100; Biovendor LLC, Ashville, NC, USA); goat anti-Sox2 (1:100, sc17320; Santa Cruz Biotechnology); guinea pig anti-Trnp1 (1:200, a gift from M. Götz, Helmholtz Zentrum Muenchen) ; and rabbit anti-Vsx1 (1:250, a gift from E. Levine, Vanderbilt University).

    Techniques: Immunostaining, Staining, Expressing, Labeling, Marker

    Tmem215 marks subsets of bipolar and amacrine cells. Adult Tmem215-LacZ heterozygous mice stained for β-gal ( green ) and cell-type specific markers ( red / gray ). ( A – A' ) Section stained with Scgn ( gray ) and PKCα ( red ). A large fraction of β-gal+ cells coexpress Scgn ( arrows , insets ), but none overlap with PKCα ( arrowheads , insets ). ( B ) Costaining with Scgn ( gray ) and Isl1/2 ( red ) to mark ON bipolar cells. A subset of β-gal+ cells coexpress Isl1/2 ( arrows , blue insets ). Other β-gal+ cells coexpress only Scgn ( arrowheads ), marking them as cone OFF bipolars. Thus, Tmem215-LacZ marks both ON and OFF cone bipolar cells. ( C – C' ) Costaining with Isl1/2 ( gray ) and Vsx1 ( red ), which mark types 1, 2, and 7 cone bipolars. A subset of β-gal+ cells coexpress Vsx1 and Isl1/2 ( arrows , insets ), marking them as type 7 cone ON bipolars. However, not all type 7 cone bipolars were β-gal+ ( magenta arrowheads , insets ). Some β-gal+ cells expressed Isl1/2, but not Vsx1 ( arrowheads , insets ). Of the β-gal+ cells that did not express Isl1/2, none coexpressed Vsx1 ( asterisks ). This argues that types 1 and 2 cone bipolars are not Tmem215+. Isl1/2+ amacrine cells do not coexpress β-gal. ( D ) A subset of β-gal+ cells coexpress Cabp5 ( red , arrows , insets ), which marks types 3 and 5 cone bipolars. ( E ) β-gal costaining with HCN4 to mark type 3a cone OFF bipolars. Most HCN4+ bipolar cells coexpress β-gal ( arrows , insets ), though some HCN4+ cells in the inner INL lack β-gal staining ( arrowheads ). ( F ) β-gal expression ( arrowheads , insets ) does not overlap with PKARIIβ ( red ), a marker of type 3b cone bipolars. ( G ) Type 2 cone OFF bipolars marked with Bhlhb5 did not express β-gal ( arrowheads , insets ). We did not see β-gal overlap with the type 4 cone OFF bipolar marker Csen (data not shown). ( H – H' ) Retinas stained with the amacrine markers GlyT1 ( red , glycinergic) and GAD65/67 ( gray , GABAergic). Roughly equal fractions coexpress GlyT1 ( arrows , insets ) and GAD65/67 ( arrowheads , insets ). There are no β-gal+ displaced amacrine cells seen. ( I ) A subset of the β-gal+ amacrine cells ( arrowheads , insets ) coexpress Ap2α ( red ) ( arrows , insets ). Scale bars : 50 μm for panels and 10 μm for insets. ( J ) Quantification of β-gal+ cells. The left panel shows the percentage of β-gal+ cells that coexpress a cell type–specific marker. There are approximately 9 β-gal+ bipolar cells (Otx2+) for every amacrine cell (Pax6+). The right panel shows the fraction of a cell type–specific marker population that coexpresses β-gal+. Error bars show SD.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Gsg1, Trnp1, and Tmem215 Mark Subpopulations of Bipolar Interneurons in the Mouse Retina

    doi: 10.1167/iovs.16-19767

    Figure Lengend Snippet: Tmem215 marks subsets of bipolar and amacrine cells. Adult Tmem215-LacZ heterozygous mice stained for β-gal ( green ) and cell-type specific markers ( red / gray ). ( A – A' ) Section stained with Scgn ( gray ) and PKCα ( red ). A large fraction of β-gal+ cells coexpress Scgn ( arrows , insets ), but none overlap with PKCα ( arrowheads , insets ). ( B ) Costaining with Scgn ( gray ) and Isl1/2 ( red ) to mark ON bipolar cells. A subset of β-gal+ cells coexpress Isl1/2 ( arrows , blue insets ). Other β-gal+ cells coexpress only Scgn ( arrowheads ), marking them as cone OFF bipolars. Thus, Tmem215-LacZ marks both ON and OFF cone bipolar cells. ( C – C' ) Costaining with Isl1/2 ( gray ) and Vsx1 ( red ), which mark types 1, 2, and 7 cone bipolars. A subset of β-gal+ cells coexpress Vsx1 and Isl1/2 ( arrows , insets ), marking them as type 7 cone ON bipolars. However, not all type 7 cone bipolars were β-gal+ ( magenta arrowheads , insets ). Some β-gal+ cells expressed Isl1/2, but not Vsx1 ( arrowheads , insets ). Of the β-gal+ cells that did not express Isl1/2, none coexpressed Vsx1 ( asterisks ). This argues that types 1 and 2 cone bipolars are not Tmem215+. Isl1/2+ amacrine cells do not coexpress β-gal. ( D ) A subset of β-gal+ cells coexpress Cabp5 ( red , arrows , insets ), which marks types 3 and 5 cone bipolars. ( E ) β-gal costaining with HCN4 to mark type 3a cone OFF bipolars. Most HCN4+ bipolar cells coexpress β-gal ( arrows , insets ), though some HCN4+ cells in the inner INL lack β-gal staining ( arrowheads ). ( F ) β-gal expression ( arrowheads , insets ) does not overlap with PKARIIβ ( red ), a marker of type 3b cone bipolars. ( G ) Type 2 cone OFF bipolars marked with Bhlhb5 did not express β-gal ( arrowheads , insets ). We did not see β-gal overlap with the type 4 cone OFF bipolar marker Csen (data not shown). ( H – H' ) Retinas stained with the amacrine markers GlyT1 ( red , glycinergic) and GAD65/67 ( gray , GABAergic). Roughly equal fractions coexpress GlyT1 ( arrows , insets ) and GAD65/67 ( arrowheads , insets ). There are no β-gal+ displaced amacrine cells seen. ( I ) A subset of the β-gal+ amacrine cells ( arrowheads , insets ) coexpress Ap2α ( red ) ( arrows , insets ). Scale bars : 50 μm for panels and 10 μm for insets. ( J ) Quantification of β-gal+ cells. The left panel shows the percentage of β-gal+ cells that coexpress a cell type–specific marker. There are approximately 9 β-gal+ bipolar cells (Otx2+) for every amacrine cell (Pax6+). The right panel shows the fraction of a cell type–specific marker population that coexpresses β-gal+. Error bars show SD.

    Article Snippet: Primary antibodies used were: mouse anti-Ap2α (1:250, clone 5E4; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); chicken anti–β-galactosidase (β-gal; 1:2000, AB9361; Abcam, Cambridge, MA, USA); goat anti-Bhlhb5 (1:1000, sc-6045; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); mouse anti-Cabp5 (1:10, a gift from F. Haeseleer, University of Washington) ; mouse anti-Calretinin (1:750) (MAB1568, Milipore, Billerica, MA, USA); mouse anti-Calsenilin (1:2000, 05-756; Milipore); rabbit anti-GAD65/67 (1:500, AB1511; Milipore); goat anti-GlyT1 (1:2000, AB1770; Milipore); rabbit anti-HCN4 (1:500, APC-052; Alomone Labs Ltd., Jerusalem, Israel); mouse anti-Isl1/2 (1:250, clone 39.4D5; Developmental Studies Hybridoma Bank); goat anti-Otx2 (1:200, BAF1979; R & D Systems, Minneapolis, MN, USA); rabbit anti-Pax6 (1:500, 901301; BioLegend, Inc., San Diego, CA, USA); mouse anti-PKARIIβ (1:3000, 610625; BD Biosciences, San Jose, CA, USA); mouse anti-PKCα (1:250, P5704; Sigma-Aldrich Corp., St. Louis, MO, USA); rabbit anti-Scgn (1:5000, RD181120100; Biovendor LLC, Ashville, NC, USA); goat anti-Sox2 (1:100, sc17320; Santa Cruz Biotechnology); guinea pig anti-Trnp1 (1:200, a gift from M. Götz, Helmholtz Zentrum Muenchen) ; and rabbit anti-Vsx1 (1:250, a gift from E. Levine, Vanderbilt University).

    Techniques: Mouse Assay, Staining, Expressing, Marker

    Hb9::Cre-derived INs do not overlap with the Shox2 non-V2a population. ( A ) Co-expression of YFP (green) and Isl1 antibody (red) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Motor neurons are also labeled by Isl1 antibody (blue box). Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Isl1 (red) and Hb9::Cre-derived INs (green). Scale bars: 100 μm and 50 μm. ( B ) Co-expression of YFP (green), Shox2 antibody (red) and/or Chx10 antibody (blue) in the Hb9 :: Cre;Rosa26-YFP mouse ventral spinal cord at E11.5. Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Hb9::Cre-derived INs (green) and Shox2 + Chx10 − (red), Shox2 − Chx10 + (blue) or Shox2 + Chx10 + (pink). Scale bars: 100 μm and 50 μm. ( C ) Quantification of overlap in (A) and (B). Bar graph showing percent of overlap between Hb9::Cre-derived INs (YFP + ) and Shox2 V2a (Shox2 + Chx10 + , 4% ± 1%), Shox2 OFF V2a (Shox2 − Chx10 + , 2% ± 0.1%), Shox2 non-V2a (Shox2 + Chx10 − , 1.3% ± 0.2%), and Isl1 (Isl1 + , 6% ± 0.2%) INs. Error bars represent ± SEM. ( D ) Percent of the Shox2 non-V2a IN population (Shox2 + Chx10 − ) that overlaps with Hb9::Cre-derived INs (YFP + ) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Shox2 non-V2a INs rarely co-express YFP (Shox2 + YFP + , darker grey) (12% ± 2%). Error bars represent ± SEM.

    Journal: Scientific Reports

    Article Title: Spinal Hb9::Cre-derived excitatory interneurons contribute to rhythm generation in the mouse

    doi: 10.1038/srep41369

    Figure Lengend Snippet: Hb9::Cre-derived INs do not overlap with the Shox2 non-V2a population. ( A ) Co-expression of YFP (green) and Isl1 antibody (red) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Motor neurons are also labeled by Isl1 antibody (blue box). Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Isl1 (red) and Hb9::Cre-derived INs (green). Scale bars: 100 μm and 50 μm. ( B ) Co-expression of YFP (green), Shox2 antibody (red) and/or Chx10 antibody (blue) in the Hb9 :: Cre;Rosa26-YFP mouse ventral spinal cord at E11.5. Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Hb9::Cre-derived INs (green) and Shox2 + Chx10 − (red), Shox2 − Chx10 + (blue) or Shox2 + Chx10 + (pink). Scale bars: 100 μm and 50 μm. ( C ) Quantification of overlap in (A) and (B). Bar graph showing percent of overlap between Hb9::Cre-derived INs (YFP + ) and Shox2 V2a (Shox2 + Chx10 + , 4% ± 1%), Shox2 OFF V2a (Shox2 − Chx10 + , 2% ± 0.1%), Shox2 non-V2a (Shox2 + Chx10 − , 1.3% ± 0.2%), and Isl1 (Isl1 + , 6% ± 0.2%) INs. Error bars represent ± SEM. ( D ) Percent of the Shox2 non-V2a IN population (Shox2 + Chx10 − ) that overlaps with Hb9::Cre-derived INs (YFP + ) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Shox2 non-V2a INs rarely co-express YFP (Shox2 + YFP + , darker grey) (12% ± 2%). Error bars represent ± SEM.

    Article Snippet: Sections were incubated for 24 hours with one or several of the following primary antibodies: rabbit anti-Shox2 #860 (1:32,000, generated against the peptide CKTTSKNSSIADLR), sheep anti-Chx10 (1:400, Chemicon), mouse anti-Isl1 (40.2D6 and 39.4D5) (1:250, DSHB), rabbit anti-Hb9 (1:16000, DSHB).

    Techniques: Derivative Assay, Expressing, Labeling

    neil3 knockdown embryos exhibit deficits in retinal development. A – C. Knockdown of neil3 results in a disorganized retina. Histological staining of retinas from uninjected (A) or injected (B,C) sides of embryos injected with neil3 antisense morpholino oligonucleotide (ASMO), sectioned, and stained with hemotoxylin/eosin. D – F. Knockdown of neil3 results in aberrant retinal cell differentiation. Embryos injected with neil3 ASMO were fixed and subjected to immunohistochemistry using antibodies against Islet-1 (D,E) or rhodopsin (F,G). Panels D and F show uninjected side and E and G show injected sides. R – retinal pigmented epithelium (RPE); P – photoreceptor layer; I – inner nuclear layer (INL), G – ganglion cell layer; L – lens. Arrows indicate examples of putative photoreceptor rosettes.

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: Identification of retinal homeobox (rax) gene-dependent genes by a microarray approach: the DNA endoglycosylase neil3 is a major downstream component of the rax genetic pathway

    doi: 10.1002/dvdy.24679

    Figure Lengend Snippet: neil3 knockdown embryos exhibit deficits in retinal development. A – C. Knockdown of neil3 results in a disorganized retina. Histological staining of retinas from uninjected (A) or injected (B,C) sides of embryos injected with neil3 antisense morpholino oligonucleotide (ASMO), sectioned, and stained with hemotoxylin/eosin. D – F. Knockdown of neil3 results in aberrant retinal cell differentiation. Embryos injected with neil3 ASMO were fixed and subjected to immunohistochemistry using antibodies against Islet-1 (D,E) or rhodopsin (F,G). Panels D and F show uninjected side and E and G show injected sides. R – retinal pigmented epithelium (RPE); P – photoreceptor layer; I – inner nuclear layer (INL), G – ganglion cell layer; L – lens. Arrows indicate examples of putative photoreceptor rosettes.

    Article Snippet: Primary antibodies were used at the following dilutions: mouse anti-rhodopsin (RetP1; Biomeda, Foster City, CA) 1:50; mouse anti-islet 1, 1:50 (39.4D5; Developmental Studies Hybridoma Bank [DSHB], University of Iowa)._Whole mount in situ hybridization was performed as described previously ( )._Section in situ hybridization was performed on 8 μM sections as described previously ( ).

    Techniques: Staining, Injection, Cell Differentiation, Immunohistochemistry

    Initiation of gonadotroph differentiation in double-transgenic embryos. The three highest expressing Tg ( Cga - Tle3 ), Tg ( Cga - Hesx1 ) double-transgenic embryos (designated αTLE3,αHESX1 double tgs) were selected based on ectopic immunohistochemical staining for TLE3 at e14.5 in the ventral cells of Rathke’s pouch in sagittal sections ( columns beginning with B, C, and D; gradation bar indicates decreasing transgene expression levels) compared with a nontransgenic littermate ( column beginning with A). SF1 (NR5A1) immunohistochemical staining detected both mature and pre-gonadotrophs (E–H, arrows ). Immunohistochemistry using antibodies against αGSU (CGA) (I–L) demonstrated a decrease in expression correlating with level of transgene expression (area of expression outlined ). There was no difference in protein levels of ISL1 (M–P) or PITX2 (Q–T) in transgenics and nontransgenic littermates.

    Journal: Molecular Endocrinology

    Article Title: Corepressors TLE1 and TLE3 Interact with HESX1 and PROP1

    doi: 10.1210/me.2008-0359

    Figure Lengend Snippet: Initiation of gonadotroph differentiation in double-transgenic embryos. The three highest expressing Tg ( Cga - Tle3 ), Tg ( Cga - Hesx1 ) double-transgenic embryos (designated αTLE3,αHESX1 double tgs) were selected based on ectopic immunohistochemical staining for TLE3 at e14.5 in the ventral cells of Rathke’s pouch in sagittal sections ( columns beginning with B, C, and D; gradation bar indicates decreasing transgene expression levels) compared with a nontransgenic littermate ( column beginning with A). SF1 (NR5A1) immunohistochemical staining detected both mature and pre-gonadotrophs (E–H, arrows ). Immunohistochemistry using antibodies against αGSU (CGA) (I–L) demonstrated a decrease in expression correlating with level of transgene expression (area of expression outlined ). There was no difference in protein levels of ISL1 (M–P) or PITX2 (Q–T) in transgenics and nontransgenic littermates.

    Article Snippet: Rabbit anti-PITX2 (1:400; Dr. Tord Hjalt, Lund University, Sweden), mouse anti-ISL1 (1:600; Developmental Studies Hybridoma Band, University of Iowa), and rabbit anti-TLE3 (1:150; Millipore, Billerica, MA) were incubated overnight at 4 C. The following day, the sections were washed and then incubated with either a biotinylated antimouse IgG (1:200, Vectastain Mouse on Mouse kit; Vector Laboratories, Burlingame, CA) or a biotinylated antirabbit IgG (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

    Techniques: Transgenic Assay, Expressing, Immunohistochemistry, Staining