Structured Review

Developmental Studies Hybridoma Bank anti isl1
Pallial boundaries during development . Micrographs of transverse sections through the telencephalon of Xenopus laevis at embryonic (A–D) , premetamorphic (E–G) and prometamorphic (H–N) stages. In each panel the developmental stage and the color code for the used markers are indicated. In the developing telencephalon, the combined immunohistochemical detection of Tbr1, expressed in the pallium, and <t>Isl1,</t> a subpallial marker, clearly allowed the identification of the boundary between both regions throughout the rostrocaudal extent (A–G,J,L–N) . The localization of Tbr1 (H) at rostral level, in comparison with GABA (I) , highlights the olfactory bulb, where GABA was very abundant, in contrast to the pallium, where the Tbr1 expression was observed (H,I) . Simultaneous labeling for GABA and Isl1 discerns the SPa from the pallium (K) . Scale bars = 50 μm (A–G) , 100 μm (H–N) . See list for abbreviations.
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Images

1) Product Images from "Pattern of Neurogenesis and Identification of Neuronal Progenitor Subtypes during Pallial Development in Xenopus laevis"

Article Title: Pattern of Neurogenesis and Identification of Neuronal Progenitor Subtypes during Pallial Development in Xenopus laevis

Journal: Frontiers in Neuroanatomy

doi: 10.3389/fnana.2017.00024

Pallial boundaries during development . Micrographs of transverse sections through the telencephalon of Xenopus laevis at embryonic (A–D) , premetamorphic (E–G) and prometamorphic (H–N) stages. In each panel the developmental stage and the color code for the used markers are indicated. In the developing telencephalon, the combined immunohistochemical detection of Tbr1, expressed in the pallium, and Isl1, a subpallial marker, clearly allowed the identification of the boundary between both regions throughout the rostrocaudal extent (A–G,J,L–N) . The localization of Tbr1 (H) at rostral level, in comparison with GABA (I) , highlights the olfactory bulb, where GABA was very abundant, in contrast to the pallium, where the Tbr1 expression was observed (H,I) . Simultaneous labeling for GABA and Isl1 discerns the SPa from the pallium (K) . Scale bars = 50 μm (A–G) , 100 μm (H–N) . See list for abbreviations.
Figure Legend Snippet: Pallial boundaries during development . Micrographs of transverse sections through the telencephalon of Xenopus laevis at embryonic (A–D) , premetamorphic (E–G) and prometamorphic (H–N) stages. In each panel the developmental stage and the color code for the used markers are indicated. In the developing telencephalon, the combined immunohistochemical detection of Tbr1, expressed in the pallium, and Isl1, a subpallial marker, clearly allowed the identification of the boundary between both regions throughout the rostrocaudal extent (A–G,J,L–N) . The localization of Tbr1 (H) at rostral level, in comparison with GABA (I) , highlights the olfactory bulb, where GABA was very abundant, in contrast to the pallium, where the Tbr1 expression was observed (H,I) . Simultaneous labeling for GABA and Isl1 discerns the SPa from the pallium (K) . Scale bars = 50 μm (A–G) , 100 μm (H–N) . See list for abbreviations.

Techniques Used: Immunohistochemistry, Marker, Expressing, Labeling

2) Product Images from "A Cre Transgenic Line for Studying V2 Neuronal Lineages and Functions in the Spinal Cord"

Article Title: A Cre Transgenic Line for Studying V2 Neuronal Lineages and Functions in the Spinal Cord

Journal: Genesis (New York, N.Y. : 2000)

doi: 10.1002/dvg.20669

Foxn4-expressing cells give rise to all interneurons of the three known V2 lineages. ( a-p ) Spinal cord sections from E14.5 (a-h) and E12.5 (i-p) Foxn4-Cre;R26R-YFP embryos were stained by double-immunofluorescence using the indicated antibodies. Sections in (a,e,i,m) were also weakly counterstained with DAPI. Note the colocalization between YFP and Chx10 in V2a neurons (a-d), between YFP and Gata2 in V2b neurons (e-h), and between YFP and Sox1 in V2c neurons (indicated by arrows) (i-l). YFP and Isl1/2 are not colocalized in motor neurons (m-p). The dashed ovals in (i) outline the regions where Sox1-immunoreactive V2c cells are located. CC, central canal; VLF, ventrolateral funiculus; VZ, ventricular zone. Scale bar equals 50 μm (a,e,i,m), 12.5 μm (b-d,f-h,j-l), and 12 μm (n-p).
Figure Legend Snippet: Foxn4-expressing cells give rise to all interneurons of the three known V2 lineages. ( a-p ) Spinal cord sections from E14.5 (a-h) and E12.5 (i-p) Foxn4-Cre;R26R-YFP embryos were stained by double-immunofluorescence using the indicated antibodies. Sections in (a,e,i,m) were also weakly counterstained with DAPI. Note the colocalization between YFP and Chx10 in V2a neurons (a-d), between YFP and Gata2 in V2b neurons (e-h), and between YFP and Sox1 in V2c neurons (indicated by arrows) (i-l). YFP and Isl1/2 are not colocalized in motor neurons (m-p). The dashed ovals in (i) outline the regions where Sox1-immunoreactive V2c cells are located. CC, central canal; VLF, ventrolateral funiculus; VZ, ventricular zone. Scale bar equals 50 μm (a,e,i,m), 12.5 μm (b-d,f-h,j-l), and 12 μm (n-p).

Techniques Used: Expressing, Staining, Immunofluorescence

Generation of Foxn4-Cre transgenic mice that express Cre recombinase in the p2/V2 domain of the spinal cord. ( a ) The Cre-loxP system for conditional activation of reporter YFP expression using Foxn4-Cre and R26R-YFP mice. To generate the Foxn4-Cre BAC, a BAC containing the Foxn4 locus was modified by recombineering to insert the Cre coding region (Cre-pA) at the Foxn4 translation initiation site (ATG) to generate the Foxn4-Cre BAC. The exons are indicated by vertical black bars and the estimated lengths of 5′ and 3′ flanking sequences are also indicated. The transgenic line was crossed with R26R-YFP mice to activate YFP expression in the spinal cord. ( b-g ) Spinal cord sections from E10.5 wild-type (b) and Foxn4-Cre (c-g) embryos were labeled by double-immunofluorescence using the indicated antibodies and weakly counterstained with nuclear DAPI. There is colocalization between Cre and Foxn4, Chx10 and Gata2 in cells of the p2/V2 domain but no colocalization between Cre and En1 or Isl1/2. Insets in (c-e) show corresponding outlined regions at a higher magnification. Scale bar equals 25 μm (b-g).
Figure Legend Snippet: Generation of Foxn4-Cre transgenic mice that express Cre recombinase in the p2/V2 domain of the spinal cord. ( a ) The Cre-loxP system for conditional activation of reporter YFP expression using Foxn4-Cre and R26R-YFP mice. To generate the Foxn4-Cre BAC, a BAC containing the Foxn4 locus was modified by recombineering to insert the Cre coding region (Cre-pA) at the Foxn4 translation initiation site (ATG) to generate the Foxn4-Cre BAC. The exons are indicated by vertical black bars and the estimated lengths of 5′ and 3′ flanking sequences are also indicated. The transgenic line was crossed with R26R-YFP mice to activate YFP expression in the spinal cord. ( b-g ) Spinal cord sections from E10.5 wild-type (b) and Foxn4-Cre (c-g) embryos were labeled by double-immunofluorescence using the indicated antibodies and weakly counterstained with nuclear DAPI. There is colocalization between Cre and Foxn4, Chx10 and Gata2 in cells of the p2/V2 domain but no colocalization between Cre and En1 or Isl1/2. Insets in (c-e) show corresponding outlined regions at a higher magnification. Scale bar equals 25 μm (b-g).

Techniques Used: Transgenic Assay, Mouse Assay, Activation Assay, Expressing, BAC Assay, Modification, Labeling, Immunofluorescence

3) Product Images from "Islet ?-, ?-, and ?-Cell Development Is Controlled by the Ldb1 Coregulator, Acting Primarily With the Islet-1 Transcription Factor"

Article Title: Islet ?-, ?-, and ?-Cell Development Is Controlled by the Ldb1 Coregulator, Acting Primarily With the Islet-1 Transcription Factor

Journal: Diabetes

doi: 10.2337/db12-0952

E18.5 Glut2 and Hb9 mRNA and protein expression is only compromised in Ldb1 mutant mice. Immunofluorescence analysis of Glut2 (red) and insulin (green) in the E18.5 control, mutant Ldb1 ( A ), and mutant Isl1 ( B ) pancreas. Insets show magnified insulin + cell clusters. C : ChIP-Seq pictograph demonstrating Isl1 occupancy at distal Glut2 Re1 (5′) and Re2 (3′) domains in βTC-3 cells. The red line denotes the Glut2-coding region, whereas ChIP-tested proximal 5′ promoter region is represented by the red box. D : βTC-3 ChIP analysis of Isl1 ( top panel ) and Ldb1 ( bottom panel ) occupancy of Glut2 Re1, Re2, and the proximal domain compared with the PEPCK control (from top to bottom , respectively). H 2 O serves as a negative PCR control. Results recapitulate observed Isl1 ChIP-Seq occupation of Glut2 Re1 and Re2, whereas Ldb1 also binds to the proximal domain. E : qPCR analysis of E18.5 Hb9 mRNA levels in pancreata from Ldb1- (blue bar) and Isl1-deficient (red bar) pancreata. Littermate control mRNA level was set at onefold (dashed line) ± SEM. F : E18.5 immunostaining analysis demonstrates that Hb9 protein (white) is maintained in the insulin + (red) nuclei of Pdx1-Cre ; Isl1 F/F pancreata as compared with littermate controls, as denoted by the white arrowheads. G : However, Hb9 is lost from most remaining insulin + cells in the Pax6-Cre ; Ldb1 F/F pancreata seen by comparing white arrowhead–labeled Hb9 + cells of control and mutant in F and G . The nuclear Hb9 signals are shown in the right panels . Yellow arrowheads in G illustrate autofluorescence from erythrocytes. * P
Figure Legend Snippet: E18.5 Glut2 and Hb9 mRNA and protein expression is only compromised in Ldb1 mutant mice. Immunofluorescence analysis of Glut2 (red) and insulin (green) in the E18.5 control, mutant Ldb1 ( A ), and mutant Isl1 ( B ) pancreas. Insets show magnified insulin + cell clusters. C : ChIP-Seq pictograph demonstrating Isl1 occupancy at distal Glut2 Re1 (5′) and Re2 (3′) domains in βTC-3 cells. The red line denotes the Glut2-coding region, whereas ChIP-tested proximal 5′ promoter region is represented by the red box. D : βTC-3 ChIP analysis of Isl1 ( top panel ) and Ldb1 ( bottom panel ) occupancy of Glut2 Re1, Re2, and the proximal domain compared with the PEPCK control (from top to bottom , respectively). H 2 O serves as a negative PCR control. Results recapitulate observed Isl1 ChIP-Seq occupation of Glut2 Re1 and Re2, whereas Ldb1 also binds to the proximal domain. E : qPCR analysis of E18.5 Hb9 mRNA levels in pancreata from Ldb1- (blue bar) and Isl1-deficient (red bar) pancreata. Littermate control mRNA level was set at onefold (dashed line) ± SEM. F : E18.5 immunostaining analysis demonstrates that Hb9 protein (white) is maintained in the insulin + (red) nuclei of Pdx1-Cre ; Isl1 F/F pancreata as compared with littermate controls, as denoted by the white arrowheads. G : However, Hb9 is lost from most remaining insulin + cells in the Pax6-Cre ; Ldb1 F/F pancreata seen by comparing white arrowhead–labeled Hb9 + cells of control and mutant in F and G . The nuclear Hb9 signals are shown in the right panels . Yellow arrowheads in G illustrate autofluorescence from erythrocytes. * P

Techniques Used: Expressing, Mutagenesis, Mouse Assay, Immunofluorescence, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Immunostaining, Labeling

Glp1r is a novel Ldb1- and Isl1-activated target gene. A : qPCR quantification of Isl1 ChIP-Seq candidates from Ldb1- (blue bars) and Isl1-deficient (red bars) E18.5 pancreata ( n = 4–6). Data are presented as fold of the littermate control, which was set at 1 (marked by the dashed line), ± SEM. B and C : Immunostaining of Glp1r (brown) and insulin (red) at P6 illustrates reduced Glp1r protein levels in insulin + cells lacking Ldb1 or Isl1. D : βTC-3 ChIP-Seq pictograph demonstrating the four distal 5′ peaks of Isl1 occupancy near Glp1r . The red line denotes the Glp1r locus. E : ChIP enrichment of peak 1 Glp1r 5′ DNA in Isl1 ( top panel ) and Ldb1 βTC-3 immunoprecipitates ( bottom panel ) as compared with IgG control-treated DNA. H 2 O serves as a negative control for the PCR. * P
Figure Legend Snippet: Glp1r is a novel Ldb1- and Isl1-activated target gene. A : qPCR quantification of Isl1 ChIP-Seq candidates from Ldb1- (blue bars) and Isl1-deficient (red bars) E18.5 pancreata ( n = 4–6). Data are presented as fold of the littermate control, which was set at 1 (marked by the dashed line), ± SEM. B and C : Immunostaining of Glp1r (brown) and insulin (red) at P6 illustrates reduced Glp1r protein levels in insulin + cells lacking Ldb1 or Isl1. D : βTC-3 ChIP-Seq pictograph demonstrating the four distal 5′ peaks of Isl1 occupancy near Glp1r . The red line denotes the Glp1r locus. E : ChIP enrichment of peak 1 Glp1r 5′ DNA in Isl1 ( top panel ) and Ldb1 βTC-3 immunoprecipitates ( bottom panel ) as compared with IgG control-treated DNA. H 2 O serves as a negative control for the PCR. * P

Techniques Used: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunostaining, Negative Control, Polymerase Chain Reaction

Ldb1 is enriched in islet and ductal cells. A : Ldb1 ( left ) and Ldb2 ( middle ) mRNA expression was visualized by RNA ISH under identical conditions in E15.5 tissue. A higher-magnification view of pancreatic Ldb1 and Ldb2 expression is shown on the right . B : qPCR was performed to measure Ldb1 , Ldb2 , and Isl1 mRNA levels in E15.5 total pancreas (black bars) and 3-month-old isolated islets (gray bars). Expression levels are displayed relative to TATA-binding protein (TBP), which is set as onefold. Error bars represent ± SEM ( n = 5). Ldb1 mRNA is significantly more abundant than Ldb2 in E15.5 and adult samples. C – K : Ldb1, Pdx1, Isl1, hormone (insulin, glucagon, and somatostatin), and ductal (DBA, CK-19) markers were visualized at E10.5, E18.5, P5, and P21 by coimmunofluorescence. Yellow dashed lines mark dorsal and ventral pancreas domains in C and D . Notably, only a few of the pancreatic Ldb1 + cells in D are copositive for Isl1 at this stage (some marked by white arrowheads). L : Immunohistochemical analysis illustrates enriched Ldb1 protein (brown) expression in adult human islet cells; the sample is eosin (pink) counterstained. * P
Figure Legend Snippet: Ldb1 is enriched in islet and ductal cells. A : Ldb1 ( left ) and Ldb2 ( middle ) mRNA expression was visualized by RNA ISH under identical conditions in E15.5 tissue. A higher-magnification view of pancreatic Ldb1 and Ldb2 expression is shown on the right . B : qPCR was performed to measure Ldb1 , Ldb2 , and Isl1 mRNA levels in E15.5 total pancreas (black bars) and 3-month-old isolated islets (gray bars). Expression levels are displayed relative to TATA-binding protein (TBP), which is set as onefold. Error bars represent ± SEM ( n = 5). Ldb1 mRNA is significantly more abundant than Ldb2 in E15.5 and adult samples. C – K : Ldb1, Pdx1, Isl1, hormone (insulin, glucagon, and somatostatin), and ductal (DBA, CK-19) markers were visualized at E10.5, E18.5, P5, and P21 by coimmunofluorescence. Yellow dashed lines mark dorsal and ventral pancreas domains in C and D . Notably, only a few of the pancreatic Ldb1 + cells in D are copositive for Isl1 at this stage (some marked by white arrowheads). L : Immunohistochemical analysis illustrates enriched Ldb1 protein (brown) expression in adult human islet cells; the sample is eosin (pink) counterstained. * P

Techniques Used: Expressing, In Situ Hybridization, Real-time Polymerase Chain Reaction, Isolation, Binding Assay, Immunohistochemistry

Isl1-regulated MafA and Arx expression is greatly reduced in the E18.5 Ldb1 mutant pancreas. A : mRNA levels of islet-enriched transcription factors in E18.5 littermate control (blue bars) and Pax6-Cre ; Ldb1 F/F (red bars) pancreas ( n = 4–6). Littermate control mRNA level was set at onefold ± SEM. Immunostaining levels of β-cell MafA (red) ( B ) and α-cell Arx (red) ( C ) were greatly reduced in E18.5 Pax6 -Cre; Ldb1 F/F pancreata. Arrowheads in C mark Arx + glucagon + (white, top ) or Arx − glucagon + cells (yellow, bottom ), with some magnified hormone + cell clusters shown. D : ChIP analysis of Ldb1 binding to MafA Region 3 ( top ) as well as Arx Re1 and Re2 ( bottom ). The PEPCK promoter served as the negative background control. Dilute input as well as Ldb1- and IgG-enriched DNA were analyzed by PCR using βTC-3 and αTC-6 chromatin isolated from whole-cell extract (WCE) and/or nuclear extract (NE). H 2 O control serves as a negative control for the PCR. E : Binding between endogenous Ldb1 and Isl1 were found in coimmunoprecipitation experiments using βTC-3 nuclear extracts, whereas Ldb1 and Isl1 did not bind to Pdx1, NeuroD1, Hnf1α, MafA, or Pax6. Diluted βTC-3 nuclear extract served as input positive control (1%), and immunoprecipitation (IP) results were compared with species-matched IgG treatments. F : Dominant-negative acting Ldb1ΔN significantly reduced MafA region 3-driven reporter expression in βTC-3 cells. Data are presented as mean fold reporter activity, with the empty pFox-Luc + CMV cotransfection set at onefold ± SEM; n = 3. * P
Figure Legend Snippet: Isl1-regulated MafA and Arx expression is greatly reduced in the E18.5 Ldb1 mutant pancreas. A : mRNA levels of islet-enriched transcription factors in E18.5 littermate control (blue bars) and Pax6-Cre ; Ldb1 F/F (red bars) pancreas ( n = 4–6). Littermate control mRNA level was set at onefold ± SEM. Immunostaining levels of β-cell MafA (red) ( B ) and α-cell Arx (red) ( C ) were greatly reduced in E18.5 Pax6 -Cre; Ldb1 F/F pancreata. Arrowheads in C mark Arx + glucagon + (white, top ) or Arx − glucagon + cells (yellow, bottom ), with some magnified hormone + cell clusters shown. D : ChIP analysis of Ldb1 binding to MafA Region 3 ( top ) as well as Arx Re1 and Re2 ( bottom ). The PEPCK promoter served as the negative background control. Dilute input as well as Ldb1- and IgG-enriched DNA were analyzed by PCR using βTC-3 and αTC-6 chromatin isolated from whole-cell extract (WCE) and/or nuclear extract (NE). H 2 O control serves as a negative control for the PCR. E : Binding between endogenous Ldb1 and Isl1 were found in coimmunoprecipitation experiments using βTC-3 nuclear extracts, whereas Ldb1 and Isl1 did not bind to Pdx1, NeuroD1, Hnf1α, MafA, or Pax6. Diluted βTC-3 nuclear extract served as input positive control (1%), and immunoprecipitation (IP) results were compared with species-matched IgG treatments. F : Dominant-negative acting Ldb1ΔN significantly reduced MafA region 3-driven reporter expression in βTC-3 cells. Data are presented as mean fold reporter activity, with the empty pFox-Luc + CMV cotransfection set at onefold ± SEM; n = 3. * P

Techniques Used: Expressing, Mutagenesis, Immunostaining, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Isolation, Negative Control, Positive Control, Immunoprecipitation, Dominant Negative Mutation, Activity Assay, Cotransfection

4) Product Images from "Dual role for neural crest cells during outflow tract septation in the neural crest-deficient mutant Splotch2H"

Article Title: Dual role for neural crest cells during outflow tract septation in the neural crest-deficient mutant Splotch2H

Journal: Journal of Anatomy

doi: 10.1111/j.1469-7580.2008.01028.x

Isl1 immunostaining of SHF cells and βgal expression by NCC in Sp 2H embryos at E10.5. (A–C) The dorsal wall of the aortic sac between the 4th and 6th arch arteries, which will fuse with the distal outflow cushions, is filled with Isl1-expressing
Figure Legend Snippet: Isl1 immunostaining of SHF cells and βgal expression by NCC in Sp 2H embryos at E10.5. (A–C) The dorsal wall of the aortic sac between the 4th and 6th arch arteries, which will fuse with the distal outflow cushions, is filled with Isl1-expressing

Techniques Used: Immunostaining, Expressing

5) Product Images from "Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement"

Article Title: Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

Journal: Nature Communications

doi: 10.1038/s41467-018-07307-6

Chromatin accessibility in CPCs is shaped by Isl1. a Number of differential chromatin accessibility peaks (log2(FC) > 2, false discovery rate [FDR]
Figure Legend Snippet: Chromatin accessibility in CPCs is shaped by Isl1. a Number of differential chromatin accessibility peaks (log2(FC) > 2, false discovery rate [FDR]

Techniques Used:

Inactivation of Isl1 prevents CPC fate bifurcation. a Schematic illustration depicting generation of Isl1 embryos and scRNA-seq. b t-SNE plots showing the predicted diffusion pseudotime of Isl1 knockout CPCs projected on Isl1 + cells (left), and clustering with Isl1 + cells (right). c Ratios of cycling and non-cycling Isl1 knockout and wild type Isl1 + CPCs. χ 2 test: p = 0.062. n indicates cells numbers. d Heatmap showing expression of deregulated genes in Isl1 + cells at E8.5 and E9.5 (cluster 1, 2, and 5) isolated from Isl1 knockout and control embryos. Source data are provided in the Source Data file
Figure Legend Snippet: Inactivation of Isl1 prevents CPC fate bifurcation. a Schematic illustration depicting generation of Isl1 embryos and scRNA-seq. b t-SNE plots showing the predicted diffusion pseudotime of Isl1 knockout CPCs projected on Isl1 + cells (left), and clustering with Isl1 + cells (right). c Ratios of cycling and non-cycling Isl1 knockout and wild type Isl1 + CPCs. χ 2 test: p = 0.062. n indicates cells numbers. d Heatmap showing expression of deregulated genes in Isl1 + cells at E8.5 and E9.5 (cluster 1, 2, and 5) isolated from Isl1 knockout and control embryos. Source data are provided in the Source Data file

Techniques Used: Diffusion-based Assay, Knock-Out, Expressing, Isolation

Chromatin accessibility of transcription factor binding sites. a t-SNE showing clustering of Z -scores of TF motif accessibility. Colors denote the same clusters as Fig. 7b . b Heatmap showing smoothened Z -scores of TF motif accessibility across defined clusters. Source data are provided in the Source Data file. c , d t-SNE visualization of highlighted single-cells progressing through the inferred ( c ) cardiomyocyte, ( d ) endothelial developmental trajectory (red dashed lines). Cells used for inference are colored by Z -scores of TF motif accessibility. All other cells are shown in gray. e Inferred model showing TF dynamics during Isl1 + CPC developmental bifurcation. f , g Smoothened heatmap showing dynamic RNA expression and motif accessibility of indicated TFs during cardiomyocyte ( f ), endothelial ( d ) pseudotime trajectories for gene-motif pairs (RNA:ATAC pairs). EC, endothelial cell. CM, cardiomyocyte
Figure Legend Snippet: Chromatin accessibility of transcription factor binding sites. a t-SNE showing clustering of Z -scores of TF motif accessibility. Colors denote the same clusters as Fig. 7b . b Heatmap showing smoothened Z -scores of TF motif accessibility across defined clusters. Source data are provided in the Source Data file. c , d t-SNE visualization of highlighted single-cells progressing through the inferred ( c ) cardiomyocyte, ( d ) endothelial developmental trajectory (red dashed lines). Cells used for inference are colored by Z -scores of TF motif accessibility. All other cells are shown in gray. e Inferred model showing TF dynamics during Isl1 + CPC developmental bifurcation. f , g Smoothened heatmap showing dynamic RNA expression and motif accessibility of indicated TFs during cardiomyocyte ( f ), endothelial ( d ) pseudotime trajectories for gene-motif pairs (RNA:ATAC pairs). EC, endothelial cell. CM, cardiomyocyte

Techniques Used: Binding Assay, RNA Expression

Single cell chromatin accessibility profiles of Isl1 + CPCs. a Representative genomic region showing ATAC-seq tracks of single, aggregate and bulk cells. b, c t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations based on chromatin accessibility. Colors denote corresponding clusters ( b ), and ( c ) development stages. d Gene ontology (GO) enrichment analyses of scATAC-seq clusters 1, 2, 5 of Isl1 + CPCs. Each bubble represents one of the top enriched GO terms. The relevant GO terms ( p
Figure Legend Snippet: Single cell chromatin accessibility profiles of Isl1 + CPCs. a Representative genomic region showing ATAC-seq tracks of single, aggregate and bulk cells. b, c t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations based on chromatin accessibility. Colors denote corresponding clusters ( b ), and ( c ) development stages. d Gene ontology (GO) enrichment analyses of scATAC-seq clusters 1, 2, 5 of Isl1 + CPCs. Each bubble represents one of the top enriched GO terms. The relevant GO terms ( p

Techniques Used:

Identification of CPC subpopulations by single-cell RNA-seq. a Schematic representation of the Nkx2-5-emGFP transgenic reporter and Isl1 nGFP/+ allele (top). Expression of Nkx2-5-emGFP and Isl1-nGFP at E8.5 in mouse embryonic hearts. (bottom). b Sampling time points for scRNA-seq, bulk RNA-seq, scATAC-seq, and bulk ATAC-seq. The table shows numbers of cells used for scRNA-seq. QC: quality control. c , d t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations. Colors denote corresponding clusters, and ( d ) development stages. Outlier cells are indicated by gray crosses. e Hierarchical clustering of expression heatmaps showing differentially expressed marker genes (AUROC > 0.8, FDR
Figure Legend Snippet: Identification of CPC subpopulations by single-cell RNA-seq. a Schematic representation of the Nkx2-5-emGFP transgenic reporter and Isl1 nGFP/+ allele (top). Expression of Nkx2-5-emGFP and Isl1-nGFP at E8.5 in mouse embryonic hearts. (bottom). b Sampling time points for scRNA-seq, bulk RNA-seq, scATAC-seq, and bulk ATAC-seq. The table shows numbers of cells used for scRNA-seq. QC: quality control. c , d t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations. Colors denote corresponding clusters, and ( d ) development stages. Outlier cells are indicated by gray crosses. e Hierarchical clustering of expression heatmaps showing differentially expressed marker genes (AUROC > 0.8, FDR

Techniques Used: RNA Sequencing Assay, Transgenic Assay, Expressing, Sampling, Marker

Reconstruction of trajectories and transition states of CPCs. a t-SNE plots showing diffusion pseudotimes (gray arrows) of Nkx2-5+ and b Isl1+ CPCs. Clusters and development stages of individual cells are color-coded as indicated. c Boxplots representing the distribution of I C (C) values from all marker genes for each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Lower and upper hinges correspond to the first and third quantile (25th and 75th percentile), while whiskers extend from the hinge to the smallest (largest) datum not further than 1.5 times the interquartile range. Outliers are plotted individually. d Violin plots showing the distribution of pairwise cell-to-cell distances across each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Inset boxplots show the median, lower and upper hinges as well as whiskers and outliers as in ( c ). e, f Expression levels of different transcription factors and key marker genes on the pseudotime axis in Nkx2-5 + ( e ) and Isl1 + ( f ) cells. Trend lines calculated by Loess regression are indicated in gray. Source data for ( c – f ) are provided in the Source Data file
Figure Legend Snippet: Reconstruction of trajectories and transition states of CPCs. a t-SNE plots showing diffusion pseudotimes (gray arrows) of Nkx2-5+ and b Isl1+ CPCs. Clusters and development stages of individual cells are color-coded as indicated. c Boxplots representing the distribution of I C (C) values from all marker genes for each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Lower and upper hinges correspond to the first and third quantile (25th and 75th percentile), while whiskers extend from the hinge to the smallest (largest) datum not further than 1.5 times the interquartile range. Outliers are plotted individually. d Violin plots showing the distribution of pairwise cell-to-cell distances across each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Inset boxplots show the median, lower and upper hinges as well as whiskers and outliers as in ( c ). e, f Expression levels of different transcription factors and key marker genes on the pseudotime axis in Nkx2-5 + ( e ) and Isl1 + ( f ) cells. Trend lines calculated by Loess regression are indicated in gray. Source data for ( c – f ) are provided in the Source Data file

Techniques Used: Diffusion-based Assay, Marker, Expressing

Spatial expression pattern of genes identified by scRNA-seq of CPCs. a Heatmap showing expression of selected genes in Isl1 + and Nkx2-5 + CPCs at E8.5. b – d In situ hybridization of sections from E8.5 embryos to reveal spatial expression profiles of genes identified by scRNA-seq. Scale bar: 100 μm for ( b ), 50 μm for ( c , d ). V: ventricle. PA: primitive atria. PhA: pharyngeal arches. OFT: outflow tract. Arrows indicate positive cells
Figure Legend Snippet: Spatial expression pattern of genes identified by scRNA-seq of CPCs. a Heatmap showing expression of selected genes in Isl1 + and Nkx2-5 + CPCs at E8.5. b – d In situ hybridization of sections from E8.5 embryos to reveal spatial expression profiles of genes identified by scRNA-seq. Scale bar: 100 μm for ( b ), 50 μm for ( c , d ). V: ventricle. PA: primitive atria. PhA: pharyngeal arches. OFT: outflow tract. Arrows indicate positive cells

Techniques Used: Expressing, In Situ Hybridization

Comparison of Isl1 + and Nkx2-5 + cardiac progenitor cells. a Confocal images showing nuclear-, cytoplasmic- and co-localization of GFP in CPCs FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos. Nuclei were stained with DAPI (blue). b Immunofluorescence-based quantification of ( a ). Isl1 + Nkx2-5 − , Isl1 + Nkx2-5 + and Isl1 − Nkx2-5 + cells were FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos at E8.5 and E9.5. Quantification of different cell populations was achieved by counting all immunostained cells in a multiwell dish. Mean ± s.d. are shown. Circles represent results from different biological replicates [ n = 3; Σ (cell number) of E8.5 = 225, 260, 100; Σ (cell number) of E9.5 = 175, 180, 100]. c Clustering of Isl1 and Nkx2-5 co-expressing cells in Nkx2-5 + and Isl1 + CPC subpopulations. Cells that are not double-positive are labeled in gray, and clusters are indicated by colored circles. d , e Plots showing the predicted diffusion pseudotime of Nkx2-5 + cells projected on t-SNE plots of Isl1 + cells, and the expression of Isl1 + ( d ) and Nkx2-5 + ( e ). Expression levels of Isl1 and Nkx2-5 in CPCs are represented by a color spectrum as indicated. EC, endothelial cell. CM, cardiomyocyte
Figure Legend Snippet: Comparison of Isl1 + and Nkx2-5 + cardiac progenitor cells. a Confocal images showing nuclear-, cytoplasmic- and co-localization of GFP in CPCs FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos. Nuclei were stained with DAPI (blue). b Immunofluorescence-based quantification of ( a ). Isl1 + Nkx2-5 − , Isl1 + Nkx2-5 + and Isl1 − Nkx2-5 + cells were FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos at E8.5 and E9.5. Quantification of different cell populations was achieved by counting all immunostained cells in a multiwell dish. Mean ± s.d. are shown. Circles represent results from different biological replicates [ n = 3; Σ (cell number) of E8.5 = 225, 260, 100; Σ (cell number) of E9.5 = 175, 180, 100]. c Clustering of Isl1 and Nkx2-5 co-expressing cells in Nkx2-5 + and Isl1 + CPC subpopulations. Cells that are not double-positive are labeled in gray, and clusters are indicated by colored circles. d , e Plots showing the predicted diffusion pseudotime of Nkx2-5 + cells projected on t-SNE plots of Isl1 + cells, and the expression of Isl1 + ( d ) and Nkx2-5 + ( e ). Expression levels of Isl1 and Nkx2-5 in CPCs are represented by a color spectrum as indicated. EC, endothelial cell. CM, cardiomyocyte

Techniques Used: FACS, Staining, Immunofluorescence, Expressing, Labeling, Diffusion-based Assay

6) Product Images from "The heart endocardium is derived from vascular endothelial progenitors"

Article Title: The heart endocardium is derived from vascular endothelial progenitors

Journal: Development (Cambridge, England)

doi: 10.1242/dev.061192

Lineage analysis of endocardial cells in the mouse. ( A-C ′) lacZ staining of Isl1Cre;R26R mouse embryos at different stages of development. ( D-F ′′) Immunostaining of sections of Isl1Cre;R26R E12.5 embryos for PECAM1 (red) and β-gal (green). White arrowheads indicate cells that co-express these markers, indicating that they are derivatives of the Isl1 lineage ( Isl1 + ); open arrowheads indicate PECAM1 pos cells that do not express β-gal ( Isl1 – ). ( G ) Quantification of Isl1 + and Isl1 – lineage-derived endothelial/endocardial cells. Error bars indicate s.d. All fluorescent images are counterstained with DAPI. rv, right ventricle; lv, left ventricle; ra, right atrium; la, left atrium; oft, outflow tract. Scale bars: 100 μm.
Figure Legend Snippet: Lineage analysis of endocardial cells in the mouse. ( A-C ′) lacZ staining of Isl1Cre;R26R mouse embryos at different stages of development. ( D-F ′′) Immunostaining of sections of Isl1Cre;R26R E12.5 embryos for PECAM1 (red) and β-gal (green). White arrowheads indicate cells that co-express these markers, indicating that they are derivatives of the Isl1 lineage ( Isl1 + ); open arrowheads indicate PECAM1 pos cells that do not express β-gal ( Isl1 – ). ( G ) Quantification of Isl1 + and Isl1 – lineage-derived endothelial/endocardial cells. Error bars indicate s.d. All fluorescent images are counterstained with DAPI. rv, right ventricle; lv, left ventricle; ra, right atrium; la, left atrium; oft, outflow tract. Scale bars: 100 μm.

Techniques Used: Staining, Immunostaining, Derivative Assay

Expression of cardiac and endothelial markers in the pharyngeal mesoderm. ( A , B , H ) Staining for endothelial (QH1, quail; PECAM1, mouse) and cardiac (ISL1) progenitors at the indicated stages of development. ( C ) Model depicting that endothelial and cardiac progenitors form distinct, non-overlapping populations. ( D ) Quantification of the results in E-G′ showing the percentage of FLK1 pos cells within the ISL1 pos mesoderm. ( E-G ′) Immunostaining in the mouse for ISL1 (green) and FLK1 (red) at (E) E7.5, (F,F′) E8 and (G-G′) E9.5. White arrowheads indicate cells co-expressing these markers; open arrowheads indicate FLK1 pos cells that do not express ISL1. All fluorescent images are counterstained with DAPI. nt, neural tube; ph, pharynx; pa2, pharyngeal arch 2; as, aortic sac; en, endocardium; myo, myocardium. Scale bars: 100 μm.
Figure Legend Snippet: Expression of cardiac and endothelial markers in the pharyngeal mesoderm. ( A , B , H ) Staining for endothelial (QH1, quail; PECAM1, mouse) and cardiac (ISL1) progenitors at the indicated stages of development. ( C ) Model depicting that endothelial and cardiac progenitors form distinct, non-overlapping populations. ( D ) Quantification of the results in E-G′ showing the percentage of FLK1 pos cells within the ISL1 pos mesoderm. ( E-G ′) Immunostaining in the mouse for ISL1 (green) and FLK1 (red) at (E) E7.5, (F,F′) E8 and (G-G′) E9.5. White arrowheads indicate cells co-expressing these markers; open arrowheads indicate FLK1 pos cells that do not express ISL1. All fluorescent images are counterstained with DAPI. nt, neural tube; ph, pharynx; pa2, pharyngeal arch 2; as, aortic sac; en, endocardium; myo, myocardium. Scale bars: 100 μm.

Techniques Used: Expressing, Staining, Immunostaining

The role of FLK1 in ISL1 lineage-derived progenitors in the mouse. ( A , E ) In situ hybridization for Pecam1 in E9.5 control and Isl1Cre;R26R;Flk1 null embryos. ( B-D , F-H ) Heart sections of control (B-D) and Isl1Cre;R26R;Flk1 (F-H) embryos at different stages of development, immunostained for MHC (green) and PECAM1 (red). ( I , J ) Immunostaining of sections for PECAM1 (red) and β-gal (green). White arrowheads indicate cells that co-express these markers and hence are Isl1 lineage derived ( Isl1 + ); open arrowheads indicate PECAM1 pos cells that do not express β-gal ( Isl1 – ). ( K ) Quantification of Isl1 versus non- Isl1 lineage-derived endothelial cells in mutant and control embryos. All fluorescent images are counterstained with DAPI. rv, right ventricle; lv, left ventricle; oft, outflow tract. Scale bars: 100 μm.
Figure Legend Snippet: The role of FLK1 in ISL1 lineage-derived progenitors in the mouse. ( A , E ) In situ hybridization for Pecam1 in E9.5 control and Isl1Cre;R26R;Flk1 null embryos. ( B-D , F-H ) Heart sections of control (B-D) and Isl1Cre;R26R;Flk1 (F-H) embryos at different stages of development, immunostained for MHC (green) and PECAM1 (red). ( I , J ) Immunostaining of sections for PECAM1 (red) and β-gal (green). White arrowheads indicate cells that co-express these markers and hence are Isl1 lineage derived ( Isl1 + ); open arrowheads indicate PECAM1 pos cells that do not express β-gal ( Isl1 – ). ( K ) Quantification of Isl1 versus non- Isl1 lineage-derived endothelial cells in mutant and control embryos. All fluorescent images are counterstained with DAPI. rv, right ventricle; lv, left ventricle; oft, outflow tract. Scale bars: 100 μm.

Techniques Used: Derivative Assay, In Situ Hybridization, Immunostaining, Mutagenesis

The heart endocardium is derived from vascular endothelial progenitors. ( A ) E9.5 mouse embryo showing that the heart endocardium is physically and functionally linked to the early endothelial network. ( B ) Cardiovascular lineage tree highlighting the hierarchy of MESP1, ISL1 and TIE2 cardiovascular progenitor populations and their respective contributions to the embryonic heart. ec, endothelial cells; ov, otic vesicle; pa1, pharyngeal arch 1; pa2, pharyngeal arch 2; som, somites; ph, pharynx; as, aortic sac; en, endocardium; myo, myocardium.
Figure Legend Snippet: The heart endocardium is derived from vascular endothelial progenitors. ( A ) E9.5 mouse embryo showing that the heart endocardium is physically and functionally linked to the early endothelial network. ( B ) Cardiovascular lineage tree highlighting the hierarchy of MESP1, ISL1 and TIE2 cardiovascular progenitor populations and their respective contributions to the embryonic heart. ec, endothelial cells; ov, otic vesicle; pa1, pharyngeal arch 1; pa2, pharyngeal arch 2; som, somites; ph, pharynx; as, aortic sac; en, endocardium; myo, myocardium.

Techniques Used: Derivative Assay

7) Product Images from "Energy metabolism and mitochondrial defects in X-linked Charcot-Marie-Tooth (CMTX6) iPSC-derived motor neurons with the p.R158H PDK3 mutation"

Article Title: Energy metabolism and mitochondrial defects in X-linked Charcot-Marie-Tooth (CMTX6) iPSC-derived motor neurons with the p.R158H PDK3 mutation

Journal: Scientific Reports

doi: 10.1038/s41598-020-66266-5

Differentiation of CMTX6 patient-derived and control iPSCs into motor neurons. ( A ) Timeline for spinal cord motor neuron differentiation from iPSC following a dual SMAD inhibition protocol. Diagram shows media used and factors added throughout the process. Cells were grown in suspension (3D) between days 18 and 26. Abbreviations: DM (Dorsomorphin); SB (SB431542); RA (Retinoic acid); SAG (Smoothened agonist). GDNF (Glial cell line-derived neurotrophic factor); CNTF (Human Ciliary neurotrophic factor); BDNF (Brain-derived neurotrophic factor); phosphorylated 7-ethyl-10-hydroxycamptothecin (SN38-P). ( B ) Differentiated motor neurons show a robust expression of ISL1, HB9 and NEFL markers at DIV 32. Motor neurons organise into clusters of cell bodies at longer maturation times (DIV 37, DIV 42) with connecting axons (TUBB3) occupying larger areas of the culture.
Figure Legend Snippet: Differentiation of CMTX6 patient-derived and control iPSCs into motor neurons. ( A ) Timeline for spinal cord motor neuron differentiation from iPSC following a dual SMAD inhibition protocol. Diagram shows media used and factors added throughout the process. Cells were grown in suspension (3D) between days 18 and 26. Abbreviations: DM (Dorsomorphin); SB (SB431542); RA (Retinoic acid); SAG (Smoothened agonist). GDNF (Glial cell line-derived neurotrophic factor); CNTF (Human Ciliary neurotrophic factor); BDNF (Brain-derived neurotrophic factor); phosphorylated 7-ethyl-10-hydroxycamptothecin (SN38-P). ( B ) Differentiated motor neurons show a robust expression of ISL1, HB9 and NEFL markers at DIV 32. Motor neurons organise into clusters of cell bodies at longer maturation times (DIV 37, DIV 42) with connecting axons (TUBB3) occupying larger areas of the culture.

Techniques Used: Derivative Assay, Inhibition, Expressing

8) Product Images from "Modelling the pathogenesis of X-linked distal hereditary motor neuropathy using patient-derived iPSCs"

Article Title: Modelling the pathogenesis of X-linked distal hereditary motor neuropathy using patient-derived iPSCs

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.041541

Differentiation of dHMNX patient-derived and control iPSCs into motor neurons. (A) Timeline for spinal cord motor neuron differentiation from iPSCs following a dual SMAD inhibition protocol. Diagram shows media used (see Materials and Methods for full description) and factors added throughout the process. Cells were grown in suspension (3D) between days 18 and 26. DM, dorsomorphin; SB, SB431542; RA, retinoic acid; SAG, Smoothened agonist; GDNF, glial-cell-line-derived neurotrophic factor; CNTF, human ciliary neurotrophic factor; BDNF, brain-derived neurotrophic factor; SN38-P, phosphorylated 7-ethyl-10-hydroxycamptothecin; hEB, human embryoid body medium. (B) Differentiated motor neurons show a robust expression of ISL1, HB9, βIII-tubulin (TUBB3) and NEFL markers at day 32. Maturation of motor neurons in suspension followed by incubation with SN38-P enriches for ISL1/HB9-expressing cells and reduces the proportion of PAX6 + cells. Mean fluorescence intensity of the indicated markers within all nuclei (DAPI) was determined and cells with values above 50 units (threshold empirically determined for each marker) were considered positive for ISL1, HB9 or PAX6, respectively. Violin plot shows full distribution of all data points acquired ( n > 3000 nuclei).
Figure Legend Snippet: Differentiation of dHMNX patient-derived and control iPSCs into motor neurons. (A) Timeline for spinal cord motor neuron differentiation from iPSCs following a dual SMAD inhibition protocol. Diagram shows media used (see Materials and Methods for full description) and factors added throughout the process. Cells were grown in suspension (3D) between days 18 and 26. DM, dorsomorphin; SB, SB431542; RA, retinoic acid; SAG, Smoothened agonist; GDNF, glial-cell-line-derived neurotrophic factor; CNTF, human ciliary neurotrophic factor; BDNF, brain-derived neurotrophic factor; SN38-P, phosphorylated 7-ethyl-10-hydroxycamptothecin; hEB, human embryoid body medium. (B) Differentiated motor neurons show a robust expression of ISL1, HB9, βIII-tubulin (TUBB3) and NEFL markers at day 32. Maturation of motor neurons in suspension followed by incubation with SN38-P enriches for ISL1/HB9-expressing cells and reduces the proportion of PAX6 + cells. Mean fluorescence intensity of the indicated markers within all nuclei (DAPI) was determined and cells with values above 50 units (threshold empirically determined for each marker) were considered positive for ISL1, HB9 or PAX6, respectively. Violin plot shows full distribution of all data points acquired ( n > 3000 nuclei).

Techniques Used: Derivative Assay, Inhibition, Expressing, Incubation, Fluorescence, Marker

9) Product Images from "Metabolic differentiation in retinal cells"

Article Title: Metabolic differentiation in retinal cells

Journal: Nature cell biology

doi: 10.1038/ncb2531

Cell differentiation can affect energy metabolism, while shifting energy metabolism to oxidative phosphorylation does not influence aspects of proliferation and differentiation. (a) Activation of Xath5GR (expressed in cyan cells) by dexamethasone (bottom panel) promotes cell cycle exit, migration to the basal layer where ganglion cells normally reside, and expression of isl1 , compared to non-expressing cells in the same retina, or to Xath5GR-expressing cells receiving ethanol solvent (top panel). (b) Sorted Xath5GR-positive cells lose ATP faster after NaN 3 addition compared to Xath5GR-negative cells in the same retina (n=4, p
Figure Legend Snippet: Cell differentiation can affect energy metabolism, while shifting energy metabolism to oxidative phosphorylation does not influence aspects of proliferation and differentiation. (a) Activation of Xath5GR (expressed in cyan cells) by dexamethasone (bottom panel) promotes cell cycle exit, migration to the basal layer where ganglion cells normally reside, and expression of isl1 , compared to non-expressing cells in the same retina, or to Xath5GR-expressing cells receiving ethanol solvent (top panel). (b) Sorted Xath5GR-positive cells lose ATP faster after NaN 3 addition compared to Xath5GR-negative cells in the same retina (n=4, p

Techniques Used: Cell Differentiation, Activation Assay, Migration, Expressing

10) Product Images from "Human axial progenitors generate trunk neural crest cells in vitro"

Article Title: Human axial progenitors generate trunk neural crest cells in vitro

Journal: eLife

doi: 10.7554/eLife.35786

Characterisation of hPSC- derived axial progenitor differentiation products. ( A ) Representative section of a chick embryo grafted with ZsGREEN+ human axial progenitor-derived trunk NC cells. The arrowhead indicates a migratory stream of donor human trunk NC cells emerging from the graft site on the dorsal neural tube. Nuclei were counterstained with DAPI. The asterisk marks debris. NT, neural tube; D, dorsal; V, ventral. ( B ) Immunofluorescence analysis of ZsGREEN and SOX10 expression in a section of a chick embryo grafted with ZsGREEN+ human axial progenitor-derived trunk NC cells. The image on the right is a magnification of the DRG region marked by the box. Note that the anti-SOX10 antibody used detects specifically the human SOX10 protein. V, ventral neural tube. Scale bar = 100 µm. ( C ) Immunofluorescence analysis of ZsGREEN and ISL1 expression in a section of a chick embryo grafted with ZsGREEN+ human axial progenitor-derived trunk NC cells. The image on the right is a magnification of the DRG region marked by the box. V, ventral neural tube. Scale bar = 100 µm. ( D ) Quantification of cells marked by different combinations of HOXC9 and SOX10 expression in day eight trunk NC cultures derived from axial progenitors cultured in the presence or absence of LDN during their induction from hPSCs. The data in the graph were obtained after scoring 8–10 random fields per experiment (three independent replicates). The error bars/standard deviation represent the variation across all fields and three experiments. Error bars = s.d. ( E ) Immunofluorescence analysis of MSGN1-VENUS expression in human axial progenitor-derived PXM cells. Scale bar = 100 µm. ( F ) qPCR expression analysis of indicated paraxial mesoderm (PXM) markers following differentiation of human axial progenitors toward PXM as indicated in Figure 3A . Error bars = s.d. (n = 2). ( G ) Representative purity check of sorted T-VENUS+ axial progenitors. 10.7554/eLife.35786.012 Raw data for Figure 3—figure supplement 2 .
Figure Legend Snippet: Characterisation of hPSC- derived axial progenitor differentiation products. ( A ) Representative section of a chick embryo grafted with ZsGREEN+ human axial progenitor-derived trunk NC cells. The arrowhead indicates a migratory stream of donor human trunk NC cells emerging from the graft site on the dorsal neural tube. Nuclei were counterstained with DAPI. The asterisk marks debris. NT, neural tube; D, dorsal; V, ventral. ( B ) Immunofluorescence analysis of ZsGREEN and SOX10 expression in a section of a chick embryo grafted with ZsGREEN+ human axial progenitor-derived trunk NC cells. The image on the right is a magnification of the DRG region marked by the box. Note that the anti-SOX10 antibody used detects specifically the human SOX10 protein. V, ventral neural tube. Scale bar = 100 µm. ( C ) Immunofluorescence analysis of ZsGREEN and ISL1 expression in a section of a chick embryo grafted with ZsGREEN+ human axial progenitor-derived trunk NC cells. The image on the right is a magnification of the DRG region marked by the box. V, ventral neural tube. Scale bar = 100 µm. ( D ) Quantification of cells marked by different combinations of HOXC9 and SOX10 expression in day eight trunk NC cultures derived from axial progenitors cultured in the presence or absence of LDN during their induction from hPSCs. The data in the graph were obtained after scoring 8–10 random fields per experiment (three independent replicates). The error bars/standard deviation represent the variation across all fields and three experiments. Error bars = s.d. ( E ) Immunofluorescence analysis of MSGN1-VENUS expression in human axial progenitor-derived PXM cells. Scale bar = 100 µm. ( F ) qPCR expression analysis of indicated paraxial mesoderm (PXM) markers following differentiation of human axial progenitors toward PXM as indicated in Figure 3A . Error bars = s.d. (n = 2). ( G ) Representative purity check of sorted T-VENUS+ axial progenitors. 10.7554/eLife.35786.012 Raw data for Figure 3—figure supplement 2 .

Techniques Used: Derivative Assay, Immunofluorescence, Expressing, Cell Culture, Standard Deviation, Real-time Polymerase Chain Reaction

11) Product Images from "Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo"

Article Title: Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1005328

DLL4 expressed from the Dll1 locus rescues DLL1 loss-of-function in the retina. (A) Dll1 null mutant retinas show epithelial disruption with formation of polarised rosettes in which the apical markers N-Cadherin (NCad, a) and ZO-1 (ZO1, b) are abnormally present at the central lumen. Ectopic proliferating progenitors, labelled with PHH3 (b, arrowheads), are located close to the apical lumen of these rosettes. (B) In contrast, the neuroepithelium of homozygous Dll1 Dll1ki and Dll1 Dll4ki embryos is correctly organised without rosettes, and N-Cadherin shows the normal apical localisation close to the retinal pigmented epithelium (a,b). Mitotic progenitors (PHH3+) are only detected at the apical region of the neuroepithelium (a,b arrowheads). A normal stratification of CHX10+ progenitors and P27+ differentiating neurons is also observed (c,d). (C, D) E13.5 homozygous Dll1 Dll1ki and Dll1 Dll4ki retinas show no significant difference in the number of ISL1+ RGCs (C) and CRABP+ amacrine cells (D). Cells immunopositive for Islet-1 and Crabp were counted and related to the total number of cells in the retina (DAPI+). Percentages are shown as mean ± SEM; ns, not significant. (E) Expression of DLL4 in homozygous Dll1 Dll1ki (a,c) and in homozygous Dll1 Dll4ki (b,d) E13.5 retinas as detected by an anti-DLL4 antibody. (c) and (d) are magnifications of (a) and (b), respectively. Endogenous plus transgenic DLL4 is expressed in more cells in Dll1 Dll4ki/Dll4ki as compared to endogenous DLL4 expression in Dll1 Dll1ki/Dll1ki while signal strength is similar. Scale bars are 50 μm in (A, B) and 100 μm in (E).
Figure Legend Snippet: DLL4 expressed from the Dll1 locus rescues DLL1 loss-of-function in the retina. (A) Dll1 null mutant retinas show epithelial disruption with formation of polarised rosettes in which the apical markers N-Cadherin (NCad, a) and ZO-1 (ZO1, b) are abnormally present at the central lumen. Ectopic proliferating progenitors, labelled with PHH3 (b, arrowheads), are located close to the apical lumen of these rosettes. (B) In contrast, the neuroepithelium of homozygous Dll1 Dll1ki and Dll1 Dll4ki embryos is correctly organised without rosettes, and N-Cadherin shows the normal apical localisation close to the retinal pigmented epithelium (a,b). Mitotic progenitors (PHH3+) are only detected at the apical region of the neuroepithelium (a,b arrowheads). A normal stratification of CHX10+ progenitors and P27+ differentiating neurons is also observed (c,d). (C, D) E13.5 homozygous Dll1 Dll1ki and Dll1 Dll4ki retinas show no significant difference in the number of ISL1+ RGCs (C) and CRABP+ amacrine cells (D). Cells immunopositive for Islet-1 and Crabp were counted and related to the total number of cells in the retina (DAPI+). Percentages are shown as mean ± SEM; ns, not significant. (E) Expression of DLL4 in homozygous Dll1 Dll1ki (a,c) and in homozygous Dll1 Dll4ki (b,d) E13.5 retinas as detected by an anti-DLL4 antibody. (c) and (d) are magnifications of (a) and (b), respectively. Endogenous plus transgenic DLL4 is expressed in more cells in Dll1 Dll4ki/Dll4ki as compared to endogenous DLL4 expression in Dll1 Dll1ki/Dll1ki while signal strength is similar. Scale bars are 50 μm in (A, B) and 100 μm in (E).

Techniques Used: Mutagenesis, Expressing, Transgenic Assay

12) Product Images from "Cardiac-specific developmental and epigenetic functions of Jarid2 during embryonic development"

Article Title: Cardiac-specific developmental and epigenetic functions of Jarid2 during embryonic development

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.002482

Identification of dysregulated genes in Jarid2 Nkx mice. qRT-PCR was performed on control or Jarid2 Nkx hearts at E13.5 ( A ) and E16.5 ( B ). The expression levels were normalized to the control, n = 3–5. C, Western blotting was performed on E13.5 and E15.5 control or Jarid2 Nkx hearts with phospho-Smad1/5/8 or Isl1 antibody. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) is a loading control. D, the graph shows the protein levels of phospho-Smad1/5/8 and Isl1 that were standardized to GAPDH and normalized to the control heart at E13.5, n = 4 (*, p ≤ 0.05; **, p ≤ 0.01).
Figure Legend Snippet: Identification of dysregulated genes in Jarid2 Nkx mice. qRT-PCR was performed on control or Jarid2 Nkx hearts at E13.5 ( A ) and E16.5 ( B ). The expression levels were normalized to the control, n = 3–5. C, Western blotting was performed on E13.5 and E15.5 control or Jarid2 Nkx hearts with phospho-Smad1/5/8 or Isl1 antibody. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) is a loading control. D, the graph shows the protein levels of phospho-Smad1/5/8 and Isl1 that were standardized to GAPDH and normalized to the control heart at E13.5, n = 4 (*, p ≤ 0.05; **, p ≤ 0.01).

Techniques Used: Mouse Assay, Quantitative RT-PCR, Expressing, Western Blot

Jarid2 occupies a specific region at the Isl1 locus. A, VISTA alignment was performed at the Isl1 genomic locus spanning about 70 kb for mouse, monkey, human, and rat to determine conserved regions. Here, the promoter region around the 10–kb region near the transcription start site ( arrow ) was analyzed. Gray bars indicate regions with greater than 75% conservation, and black bars indicate exons. Arrowheads indicate primer sites. B, Jarid2 occupancy at the conserved regions was measured by qChIP assays on Jarid2 Nkx or control hearts using Jarid2 antibody. The bars show enrichment compared with each input. C, qChIP assays were performed on control or Jarid2 Nkx hearts at the −0.5–kb region of Isl1 using Jarid2, Ezh2, H3K27me3, or H3K4me3 antibody, n = 3 (*, p ≤ 0.05; **, p ≤ 0.01).
Figure Legend Snippet: Jarid2 occupies a specific region at the Isl1 locus. A, VISTA alignment was performed at the Isl1 genomic locus spanning about 70 kb for mouse, monkey, human, and rat to determine conserved regions. Here, the promoter region around the 10–kb region near the transcription start site ( arrow ) was analyzed. Gray bars indicate regions with greater than 75% conservation, and black bars indicate exons. Arrowheads indicate primer sites. B, Jarid2 occupancy at the conserved regions was measured by qChIP assays on Jarid2 Nkx or control hearts using Jarid2 antibody. The bars show enrichment compared with each input. C, qChIP assays were performed on control or Jarid2 Nkx hearts at the −0.5–kb region of Isl1 using Jarid2, Ezh2, H3K27me3, or H3K4me3 antibody, n = 3 (*, p ≤ 0.05; **, p ≤ 0.01).

Techniques Used:

Jarid2 represses the Isl1 reporter gene. A, pGL3, pGL3-Isl1 including the −0.5–kb region (−0.9/+0.15 kb) or without the −0.5–kb region (−0.12/+0.15 kb) was transfected into 10T1/2 cells with increasing amounts of Jarid2 (μg). B, a schematic diagram shows Jarid2, and Jarid2 mutants (N-term, 1–528 aa; TR, 1–222 aa; C-term, 529–1234 aa; NLS/C-term, 1–130/529-1234 aa). Jarid2 (0.2 μg) was transfected into 10T1/2 cells with the pGL3-Isl1 (−0.9/+0.15 kb) reporter. TR , transcription repression domain; JN , Jumonji N domain; JC , Jumonji C domain; ARID , AT-rich interaction domain. C, pGL3-Isl1 (−0.9/+0.15 kb) reporter was transfected into 10T1/2 cells with Jarid2, EED, and/or EZH2 at a low dose (50 ng). D, the TR domain of Jarid2 (1–222 aa, 25 ng) was transfected into 10T1/2 cells with or without EED and/or EZH2 (50 ng) for luciferase activity assays of pGL3-Isl1 (−0.9/+0.15 kb). Luciferase activity was normalized to the reporter gene alone. Asterisks indicate a significant difference compared with the reporter gene alone (*, p ≤ 0.05; **, p ≤ 0.01, n = 3). A number sign indicates a significant difference between a combination of any two factors and all three factors together.
Figure Legend Snippet: Jarid2 represses the Isl1 reporter gene. A, pGL3, pGL3-Isl1 including the −0.5–kb region (−0.9/+0.15 kb) or without the −0.5–kb region (−0.12/+0.15 kb) was transfected into 10T1/2 cells with increasing amounts of Jarid2 (μg). B, a schematic diagram shows Jarid2, and Jarid2 mutants (N-term, 1–528 aa; TR, 1–222 aa; C-term, 529–1234 aa; NLS/C-term, 1–130/529-1234 aa). Jarid2 (0.2 μg) was transfected into 10T1/2 cells with the pGL3-Isl1 (−0.9/+0.15 kb) reporter. TR , transcription repression domain; JN , Jumonji N domain; JC , Jumonji C domain; ARID , AT-rich interaction domain. C, pGL3-Isl1 (−0.9/+0.15 kb) reporter was transfected into 10T1/2 cells with Jarid2, EED, and/or EZH2 at a low dose (50 ng). D, the TR domain of Jarid2 (1–222 aa, 25 ng) was transfected into 10T1/2 cells with or without EED and/or EZH2 (50 ng) for luciferase activity assays of pGL3-Isl1 (−0.9/+0.15 kb). Luciferase activity was normalized to the reporter gene alone. Asterisks indicate a significant difference compared with the reporter gene alone (*, p ≤ 0.05; **, p ≤ 0.01, n = 3). A number sign indicates a significant difference between a combination of any two factors and all three factors together.

Techniques Used: Transfection, Luciferase, Activity Assay

13) Product Images from "Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement"

Article Title: Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

Journal: Nature Communications

doi: 10.1038/s41467-018-07307-6

Chromatin accessibility in CPCs is shaped by Isl1. a Number of differential chromatin accessibility peaks (log2(FC) > 2, false discovery rate [FDR]
Figure Legend Snippet: Chromatin accessibility in CPCs is shaped by Isl1. a Number of differential chromatin accessibility peaks (log2(FC) > 2, false discovery rate [FDR]

Techniques Used:

Inactivation of Isl1 prevents CPC fate bifurcation. a Schematic illustration depicting generation of Isl1 embryos and scRNA-seq. b t-SNE plots showing the predicted diffusion pseudotime of Isl1 knockout CPCs projected on Isl1 + cells (left), and clustering with Isl1 + cells (right). c Ratios of cycling and non-cycling Isl1 knockout and wild type Isl1 + CPCs. χ 2 test: p = 0.062. n indicates cells numbers. d Heatmap showing expression of deregulated genes in Isl1 + cells at E8.5 and E9.5 (cluster 1, 2, and 5) isolated from Isl1 knockout and control embryos. Source data are provided in the Source Data file
Figure Legend Snippet: Inactivation of Isl1 prevents CPC fate bifurcation. a Schematic illustration depicting generation of Isl1 embryos and scRNA-seq. b t-SNE plots showing the predicted diffusion pseudotime of Isl1 knockout CPCs projected on Isl1 + cells (left), and clustering with Isl1 + cells (right). c Ratios of cycling and non-cycling Isl1 knockout and wild type Isl1 + CPCs. χ 2 test: p = 0.062. n indicates cells numbers. d Heatmap showing expression of deregulated genes in Isl1 + cells at E8.5 and E9.5 (cluster 1, 2, and 5) isolated from Isl1 knockout and control embryos. Source data are provided in the Source Data file

Techniques Used: Diffusion-based Assay, Knock-Out, Expressing, Isolation

Single cell chromatin accessibility profiles of Isl1 + CPCs. a Representative genomic region showing ATAC-seq tracks of single, aggregate and bulk cells. b, c t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations based on chromatin accessibility. Colors denote corresponding clusters ( b ), and ( c ) development stages. d Gene ontology (GO) enrichment analyses of scATAC-seq clusters 1, 2, 5 of Isl1 + CPCs. Each bubble represents one of the top enriched GO terms. The relevant GO terms ( p
Figure Legend Snippet: Single cell chromatin accessibility profiles of Isl1 + CPCs. a Representative genomic region showing ATAC-seq tracks of single, aggregate and bulk cells. b, c t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations based on chromatin accessibility. Colors denote corresponding clusters ( b ), and ( c ) development stages. d Gene ontology (GO) enrichment analyses of scATAC-seq clusters 1, 2, 5 of Isl1 + CPCs. Each bubble represents one of the top enriched GO terms. The relevant GO terms ( p

Techniques Used:

Identification of CPC subpopulations by single-cell RNA-seq. a Schematic representation of the Nkx2-5-emGFP transgenic reporter and Isl1 nGFP/+ allele (top). Expression of Nkx2-5-emGFP and Isl1-nGFP at E8.5 in mouse embryonic hearts. (bottom). b Sampling time points for scRNA-seq, bulk RNA-seq, scATAC-seq, and bulk ATAC-seq. The table shows numbers of cells used for scRNA-seq. QC: quality control. c , d t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations. Colors denote corresponding clusters, and ( d ) development stages. Outlier cells are indicated by gray crosses. e Hierarchical clustering of expression heatmaps showing differentially expressed marker genes (AUROC > 0.8, FDR
Figure Legend Snippet: Identification of CPC subpopulations by single-cell RNA-seq. a Schematic representation of the Nkx2-5-emGFP transgenic reporter and Isl1 nGFP/+ allele (top). Expression of Nkx2-5-emGFP and Isl1-nGFP at E8.5 in mouse embryonic hearts. (bottom). b Sampling time points for scRNA-seq, bulk RNA-seq, scATAC-seq, and bulk ATAC-seq. The table shows numbers of cells used for scRNA-seq. QC: quality control. c , d t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations. Colors denote corresponding clusters, and ( d ) development stages. Outlier cells are indicated by gray crosses. e Hierarchical clustering of expression heatmaps showing differentially expressed marker genes (AUROC > 0.8, FDR

Techniques Used: RNA Sequencing Assay, Transgenic Assay, Expressing, Sampling, Marker

Reconstruction of trajectories and transition states of CPCs. a t-SNE plots showing diffusion pseudotimes (gray arrows) of Nkx2-5+ and b Isl1+ CPCs. Clusters and development stages of individual cells are color-coded as indicated. c Boxplots representing the distribution of I C (C) values from all marker genes for each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Lower and upper hinges correspond to the first and third quantile (25th and 75th percentile), while whiskers extend from the hinge to the smallest (largest) datum not further than 1.5 times the interquartile range. Outliers are plotted individually. d Violin plots showing the distribution of pairwise cell-to-cell distances across each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Inset boxplots show the median, lower and upper hinges as well as whiskers and outliers as in ( c ). e, f Expression levels of different transcription factors and key marker genes on the pseudotime axis in Nkx2-5 + ( e ) and Isl1 + ( f ) cells. Trend lines calculated by Loess regression are indicated in gray. Source data for ( c – f ) are provided in the Source Data file
Figure Legend Snippet: Reconstruction of trajectories and transition states of CPCs. a t-SNE plots showing diffusion pseudotimes (gray arrows) of Nkx2-5+ and b Isl1+ CPCs. Clusters and development stages of individual cells are color-coded as indicated. c Boxplots representing the distribution of I C (C) values from all marker genes for each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Lower and upper hinges correspond to the first and third quantile (25th and 75th percentile), while whiskers extend from the hinge to the smallest (largest) datum not further than 1.5 times the interquartile range. Outliers are plotted individually. d Violin plots showing the distribution of pairwise cell-to-cell distances across each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Inset boxplots show the median, lower and upper hinges as well as whiskers and outliers as in ( c ). e, f Expression levels of different transcription factors and key marker genes on the pseudotime axis in Nkx2-5 + ( e ) and Isl1 + ( f ) cells. Trend lines calculated by Loess regression are indicated in gray. Source data for ( c – f ) are provided in the Source Data file

Techniques Used: Diffusion-based Assay, Marker, Expressing

Spatial expression pattern of genes identified by scRNA-seq of CPCs. a Heatmap showing expression of selected genes in Isl1 + and Nkx2-5 + CPCs at E8.5. b – d In situ hybridization of sections from E8.5 embryos to reveal spatial expression profiles of genes identified by scRNA-seq. Scale bar: 100 μm for ( b ), 50 μm for ( c , d ). V: ventricle. PA: primitive atria. PhA: pharyngeal arches. OFT: outflow tract. Arrows indicate positive cells
Figure Legend Snippet: Spatial expression pattern of genes identified by scRNA-seq of CPCs. a Heatmap showing expression of selected genes in Isl1 + and Nkx2-5 + CPCs at E8.5. b – d In situ hybridization of sections from E8.5 embryos to reveal spatial expression profiles of genes identified by scRNA-seq. Scale bar: 100 μm for ( b ), 50 μm for ( c , d ). V: ventricle. PA: primitive atria. PhA: pharyngeal arches. OFT: outflow tract. Arrows indicate positive cells

Techniques Used: Expressing, In Situ Hybridization

Comparison of Isl1 + and Nkx2-5 + cardiac progenitor cells. a Confocal images showing nuclear-, cytoplasmic- and co-localization of GFP in CPCs FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos. Nuclei were stained with DAPI (blue). b Immunofluorescence-based quantification of ( a ). Isl1 + Nkx2-5 − , Isl1 + Nkx2-5 + and Isl1 − Nkx2-5 + cells were FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos at E8.5 and E9.5. Quantification of different cell populations was achieved by counting all immunostained cells in a multiwell dish. Mean ± s.d. are shown. Circles represent results from different biological replicates [ n = 3; Σ (cell number) of E8.5 = 225, 260, 100; Σ (cell number) of E9.5 = 175, 180, 100]. c Clustering of Isl1 and Nkx2-5 co-expressing cells in Nkx2-5 + and Isl1 + CPC subpopulations. Cells that are not double-positive are labeled in gray, and clusters are indicated by colored circles. d , e Plots showing the predicted diffusion pseudotime of Nkx2-5 + cells projected on t-SNE plots of Isl1 + cells, and the expression of Isl1 + ( d ) and Nkx2-5 + ( e ). Expression levels of Isl1 and Nkx2-5 in CPCs are represented by a color spectrum as indicated. EC, endothelial cell. CM, cardiomyocyte
Figure Legend Snippet: Comparison of Isl1 + and Nkx2-5 + cardiac progenitor cells. a Confocal images showing nuclear-, cytoplasmic- and co-localization of GFP in CPCs FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos. Nuclei were stained with DAPI (blue). b Immunofluorescence-based quantification of ( a ). Isl1 + Nkx2-5 − , Isl1 + Nkx2-5 + and Isl1 − Nkx2-5 + cells were FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos at E8.5 and E9.5. Quantification of different cell populations was achieved by counting all immunostained cells in a multiwell dish. Mean ± s.d. are shown. Circles represent results from different biological replicates [ n = 3; Σ (cell number) of E8.5 = 225, 260, 100; Σ (cell number) of E9.5 = 175, 180, 100]. c Clustering of Isl1 and Nkx2-5 co-expressing cells in Nkx2-5 + and Isl1 + CPC subpopulations. Cells that are not double-positive are labeled in gray, and clusters are indicated by colored circles. d , e Plots showing the predicted diffusion pseudotime of Nkx2-5 + cells projected on t-SNE plots of Isl1 + cells, and the expression of Isl1 + ( d ) and Nkx2-5 + ( e ). Expression levels of Isl1 and Nkx2-5 in CPCs are represented by a color spectrum as indicated. EC, endothelial cell. CM, cardiomyocyte

Techniques Used: FACS, Staining, Immunofluorescence, Expressing, Labeling, Diffusion-based Assay

14) Product Images from "Lhx4 Deficiency: Increased Cyclin-Dependent Kinase Inhibitor Expression and Pituitary Hypoplasia"

Article Title: Lhx4 Deficiency: Increased Cyclin-Dependent Kinase Inhibitor Expression and Pituitary Hypoplasia

Journal: Molecular Endocrinology

doi: 10.1210/me.2014-1380

LIM-homeodomain genes Lhx3 and Isl1 do not compensate for Lhx4 deficiency. Midsagittal sections of wild-type and Lhx4 −/− embryos collected from E9.5 to E14.5 were immunostained for ISL1 and LHX3. Arrow indicates the presence of ISL1-immunopositive
Figure Legend Snippet: LIM-homeodomain genes Lhx3 and Isl1 do not compensate for Lhx4 deficiency. Midsagittal sections of wild-type and Lhx4 −/− embryos collected from E9.5 to E14.5 were immunostained for ISL1 and LHX3. Arrow indicates the presence of ISL1-immunopositive

Techniques Used:

15) Product Images from "Reprogramming amacrine and photoreceptor progenitors into retinal ganglion cells by replacing Neurod1 with Atoh7"

Article Title: Reprogramming amacrine and photoreceptor progenitors into retinal ganglion cells by replacing Neurod1 with Atoh7

Journal: Development (Cambridge, England)

doi: 10.1242/dev.085886

Activation of early expressed RGC genes in Atoh7 G/G :Neurod1 Atoh7HA/Atoh7HA retinas. ( A-G ) Immunostaining of retinas from E13 embryos with anti-Isl1 antibody (A-D) on (A) Atoh7 G/+ , (B) Neurod1 Atoh7HA/Atoh7HA , (C) Atoh7 G/G and (D) Atoh7 G/G :Neurod1 Atoh7HA/Atoh7HA retinal sections, and with anti-Pou4f2/Brn3b antibody (E-G) on (E) Atoh7 G/+ , (F) Atoh7 G/G and (G) Atoh7 G/G :Neurod1 Atoh7HA/Atoh7HA retinal sections. Scale bars: 100 μm.
Figure Legend Snippet: Activation of early expressed RGC genes in Atoh7 G/G :Neurod1 Atoh7HA/Atoh7HA retinas. ( A-G ) Immunostaining of retinas from E13 embryos with anti-Isl1 antibody (A-D) on (A) Atoh7 G/+ , (B) Neurod1 Atoh7HA/Atoh7HA , (C) Atoh7 G/G and (D) Atoh7 G/G :Neurod1 Atoh7HA/Atoh7HA retinal sections, and with anti-Pou4f2/Brn3b antibody (E-G) on (E) Atoh7 G/+ , (F) Atoh7 G/G and (G) Atoh7 G/G :Neurod1 Atoh7HA/Atoh7HA retinal sections. Scale bars: 100 μm.

Techniques Used: Activation Assay, Immunostaining

16) Product Images from "A Regulatory Pathway Involving Notch1/?-Catenin/Isl1 Determines Cardiac Progenitor Cell Fate"

Article Title: A Regulatory Pathway Involving Notch1/?-Catenin/Isl1 Determines Cardiac Progenitor Cell Fate

Journal: Nature cell biology

doi: 10.1038/ncb1906

Identification of genes affected by stabilized β-Catenin in cardiac progenitors. a, Lateral view of Rosa YFP ; Isl1 Cre ; β-catenin(ex3) loxP embryo at E9.0 showing YFP + cells in precardiac mesoderm (pm). b, Histograms of YFP + cell populations from control ( Isl1 Cre , left) and stabilized β-cat ( Isl1 Cre ; β-catenin(ex3) loxP , right) embryos. c, A heatmap of expression arrays showing significantly downregulated cardiac genes (green) in stabilized β-catenin pm cells. Color bar indicates fold change in log 2 scale. d, qPCR data of downregulated genes in FACS-purified cardiac progenitors with stabilized β-Catenin (Top). These genes were similarly affected in pm of Notch1 loss-of-function embryos (Bottom). Data are mean ± s. d.; n =3; * P
Figure Legend Snippet: Identification of genes affected by stabilized β-Catenin in cardiac progenitors. a, Lateral view of Rosa YFP ; Isl1 Cre ; β-catenin(ex3) loxP embryo at E9.0 showing YFP + cells in precardiac mesoderm (pm). b, Histograms of YFP + cell populations from control ( Isl1 Cre , left) and stabilized β-cat ( Isl1 Cre ; β-catenin(ex3) loxP , right) embryos. c, A heatmap of expression arrays showing significantly downregulated cardiac genes (green) in stabilized β-catenin pm cells. Color bar indicates fold change in log 2 scale. d, qPCR data of downregulated genes in FACS-purified cardiac progenitors with stabilized β-Catenin (Top). These genes were similarly affected in pm of Notch1 loss-of-function embryos (Bottom). Data are mean ± s. d.; n =3; * P

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

Isl1 targets Myocd and β-Catenin regulates Bhlhb2 to repress Smyd1 . a, Relative expression levels of Myocd and Smyd1 in FACS-purified control and Isl1 knockdown (KD) CPCs, determined by qPCR (mean ± s. d.; n =4; * P
Figure Legend Snippet: Isl1 targets Myocd and β-Catenin regulates Bhlhb2 to repress Smyd1 . a, Relative expression levels of Myocd and Smyd1 in FACS-purified control and Isl1 knockdown (KD) CPCs, determined by qPCR (mean ± s. d.; n =4; * P

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

Notch1 loss-of-function causes CPC expansion and increases free β-Catenin levels. a–f, Control embryos. g–l, Isl1 Cre , Notch1 flox/flox embryos (N1-KO). a , g , Lateral views of ED10.5 embryos. b, c, h, i, Lateral ( b , h ) or frontal ( c , i ) view of embryos focused on cardiac regions showing absence of right ventricle (rv) in mutants. d , e , j , k , Transverse sections (H E) of embryos ( d, j ) with enlargement of boxed areas ( e, k ) showing hyperplasia of precardiac progenitors (asterisk). f , l, Phosphohistone3 (Ph3, red) and Isl1 (green) immunostaining of transverse sections through the precardiac region. To compensate for the severe downregulation of Isl1 in Notch1 mutant embryos, Isl1 signals were amplified with the TSA system. DAPI (blue) was used to counterstain the nuclei. m , Percentage of ph3-positive cells in precardiac mesoderm region shown in e and k (mean ± s. d.; n =4; * P
Figure Legend Snippet: Notch1 loss-of-function causes CPC expansion and increases free β-Catenin levels. a–f, Control embryos. g–l, Isl1 Cre , Notch1 flox/flox embryos (N1-KO). a , g , Lateral views of ED10.5 embryos. b, c, h, i, Lateral ( b , h ) or frontal ( c , i ) view of embryos focused on cardiac regions showing absence of right ventricle (rv) in mutants. d , e , j , k , Transverse sections (H E) of embryos ( d, j ) with enlargement of boxed areas ( e, k ) showing hyperplasia of precardiac progenitors (asterisk). f , l, Phosphohistone3 (Ph3, red) and Isl1 (green) immunostaining of transverse sections through the precardiac region. To compensate for the severe downregulation of Isl1 in Notch1 mutant embryos, Isl1 signals were amplified with the TSA system. DAPI (blue) was used to counterstain the nuclei. m , Percentage of ph3-positive cells in precardiac mesoderm region shown in e and k (mean ± s. d.; n =4; * P

Techniques Used: Immunostaining, Mutagenesis, Amplification

Isl1 loss-of-function results in expansion of CPCs and suppression of their myocardial and smooth muscle lineages. a, YFP expression in control ( Rosa YFP , Isl1 Cre/+ , left) and Isl1-null ( Rosa YFP , Isl1 Cre/Cre , right) embryos at the 5-somite stage. Arrows indicate YFP + CPCs. Scale bars, 50 µm. b, Quantification of YFP + cells in indicated embryos at somite 5 (mean ± s. d.; n =3; * P
Figure Legend Snippet: Isl1 loss-of-function results in expansion of CPCs and suppression of their myocardial and smooth muscle lineages. a, YFP expression in control ( Rosa YFP , Isl1 Cre/+ , left) and Isl1-null ( Rosa YFP , Isl1 Cre/Cre , right) embryos at the 5-somite stage. Arrows indicate YFP + CPCs. Scale bars, 50 µm. b, Quantification of YFP + cells in indicated embryos at somite 5 (mean ± s. d.; n =3; * P

Techniques Used: Expressing

Increased levels of Isl1 promote myocardial differentiation. a, Schematic diagram of differentiation of Myh7-GFP ES cells with Isl1 overexpression. b, c, Relative expression levels of Isl1 on ED6 EBs ( b ), and endothelial ( Flk1 ), cardiac sarcomeric ( Actc1, Mlc2v, Myh7 ) and smooth muscle ( Sma ) genes on day 8 EBs ( c ), determined by qPCR. d, FACS analyses on ED 9 EBs to identify % of cells entering myocardial-lineage. Data are mean ± s. e. m.; n =3; * P
Figure Legend Snippet: Increased levels of Isl1 promote myocardial differentiation. a, Schematic diagram of differentiation of Myh7-GFP ES cells with Isl1 overexpression. b, c, Relative expression levels of Isl1 on ED6 EBs ( b ), and endothelial ( Flk1 ), cardiac sarcomeric ( Actc1, Mlc2v, Myh7 ) and smooth muscle ( Sma ) genes on day 8 EBs ( c ), determined by qPCR. d, FACS analyses on ED 9 EBs to identify % of cells entering myocardial-lineage. Data are mean ± s. e. m.; n =3; * P

Techniques Used: Over Expression, Expressing, Real-time Polymerase Chain Reaction, FACS

17) Product Images from "Impaired enteroendocrine development in intestinal-specific Islet1 mouse mutants causes impaired glucose homeostasis"

Article Title: Impaired enteroendocrine development in intestinal-specific Islet1 mouse mutants causes impaired glucose homeostasis

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

doi: 10.1152/ajpgi.00390.2013

Isl1 int nursing pups have fat malabsorption. Oil Red O staining for neutral fat in stool samples of P7 ( A ) and P28 ( B ) animals from control ( left ) and Isl1 int ( right ) littermates. Oil Red O staining of tissue samples in P7 ileum ( C ) and P7 colon ( D ) from
Figure Legend Snippet: Isl1 int nursing pups have fat malabsorption. Oil Red O staining for neutral fat in stool samples of P7 ( A ) and P28 ( B ) animals from control ( left ) and Isl1 int ( right ) littermates. Oil Red O staining of tissue samples in P7 ileum ( C ) and P7 colon ( D ) from

Techniques Used: Staining

Isl1 int female mice have reduced growth but normal histology. Representative images of immunohistochemistry for Isl1 expression in control ( A , top , arrows) and Isl1 int mice ( A , bottom ) demonstrating loss of epithelial expression although mesenchymal expression
Figure Legend Snippet: Isl1 int female mice have reduced growth but normal histology. Representative images of immunohistochemistry for Isl1 expression in control ( A , top , arrows) and Isl1 int mice ( A , bottom ) demonstrating loss of epithelial expression although mesenchymal expression

Techniques Used: Mouse Assay, Immunohistochemistry, Expressing

Isl1 is not expressed in tuft, Paneth, or goblet cells. Immunofluorescence for DCAMKL1, marker of tuft cells (red in A and C ), and lysozyme (Lys1; red in D and F ). The white arrows in A and C designate a DCAMKL1-positive cell. Immunohistochemistry for
Figure Legend Snippet: Isl1 is not expressed in tuft, Paneth, or goblet cells. Immunofluorescence for DCAMKL1, marker of tuft cells (red in A and C ), and lysozyme (Lys1; red in D and F ). The white arrows in A and C designate a DCAMKL1-positive cell. Immunohistochemistry for

Techniques Used: Immunofluorescence, Marker, Immunohistochemistry

Isl1 int mutant mice are hyperglycemic after oral glucose tolerance test (OGTT). Serum glucose measurements after glucose load in control (black bars) and Isl1 int (dotted line) mice during OGTT ( A and B ) or intraperitoneal glucose tolerance test (IPGTT;
Figure Legend Snippet: Isl1 int mutant mice are hyperglycemic after oral glucose tolerance test (OGTT). Serum glucose measurements after glucose load in control (black bars) and Isl1 int (dotted line) mice during OGTT ( A and B ) or intraperitoneal glucose tolerance test (IPGTT;

Techniques Used: Mutagenesis, Mouse Assay

Lineage map. Lineage map demonstrating role of Isl1 in determination of the enteroendocrine lineage.
Figure Legend Snippet: Lineage map. Lineage map demonstrating role of Isl1 in determination of the enteroendocrine lineage.

Techniques Used:

Islet1 (Isl1) is rarely expressed in chromogranin A (CGA) or serotonin (5-HT)-positive enteroendocrine cells. A–C : immunohistochemistry for Isl1 in duodenal crypts (arrows) and villi (asterisks) in postnatal day (P)7 ( A ) and 4-wk-old ( B ) and 4-mo-old
Figure Legend Snippet: Islet1 (Isl1) is rarely expressed in chromogranin A (CGA) or serotonin (5-HT)-positive enteroendocrine cells. A–C : immunohistochemistry for Isl1 in duodenal crypts (arrows) and villi (asterisks) in postnatal day (P)7 ( A ) and 4-wk-old ( B ) and 4-mo-old

Techniques Used: Immunohistochemistry

Isl1 is expressed in somatostatin, glucagon-like peptide-1 (GLP-1), ghrelin, glucose-dependent insulinotropic polypeptide (GIP), and CCK cells. Left : Isl1 expression (brown). Left middle : hormone expression with SST red in A , GLP1 green in B , ghrelin
Figure Legend Snippet: Isl1 is expressed in somatostatin, glucagon-like peptide-1 (GLP-1), ghrelin, glucose-dependent insulinotropic polypeptide (GIP), and CCK cells. Left : Isl1 expression (brown). Left middle : hormone expression with SST red in A , GLP1 green in B , ghrelin

Techniques Used: Expressing

Hormone subpopulation changes in Isl1 int mutant mice. The control images for each hormone are at left and Isl1 int mutants are at left middle . Graph at right middle is hormone-positive cells per mm 2 , and graph at right is mRNA expression level. Black bars
Figure Legend Snippet: Hormone subpopulation changes in Isl1 int mutant mice. The control images for each hormone are at left and Isl1 int mutants are at left middle . Graph at right middle is hormone-positive cells per mm 2 , and graph at right is mRNA expression level. Black bars

Techniques Used: Mutagenesis, Mouse Assay, Expressing

Isl1 int adult mice have normal body weight, body composition, food intake, and stool output. A : graph depicting body weight in 3- to 4-mo-old animals (initial weight) before high-fat diet (HFD) and after 14 wk on HFD. B : NMR fat (dark bars) and lean (white
Figure Legend Snippet: Isl1 int adult mice have normal body weight, body composition, food intake, and stool output. A : graph depicting body weight in 3- to 4-mo-old animals (initial weight) before high-fat diet (HFD) and after 14 wk on HFD. B : NMR fat (dark bars) and lean (white

Techniques Used: Mouse Assay, Nuclear Magnetic Resonance

18) Product Images from "Rbfox3-regulated alternative splicing of Numb promotes neuronal differentiation during development"

Article Title: Rbfox3-regulated alternative splicing of Numb promotes neuronal differentiation during development

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201206146

Effects of forced expression of Rbfox3-full on progenitors in the VZ. The expression constructs indicated at the top were transfected and sections of the neural tube were stained for markers indicated on the left and nuclei (DAPI). The right side in each panel is the transfected side, as demonstrated by GFP in A and B. A–L are E3 embryos, and M–R are E4 embryos. Arrowheads in E and F indicate some of the TUNEL + apoptotic cells. Arrowheads and an arrow in H and I indicate precocious expression of Isl1 in motor neuron progenitor and D2 neuron progenitor domains, respectively. Arrowheads in N and O indicate precocious expression of the interneuron marker Lim1/2 in the VZ. Expression of the postmitotic cell marker p27kip1 is not increased in the VZ (K, L, Q, and R). F, I, L, O, and R show only red fluorescence signals without DAPI, which is shown in E, H, K, N, and Q. Bars, 50 µm.
Figure Legend Snippet: Effects of forced expression of Rbfox3-full on progenitors in the VZ. The expression constructs indicated at the top were transfected and sections of the neural tube were stained for markers indicated on the left and nuclei (DAPI). The right side in each panel is the transfected side, as demonstrated by GFP in A and B. A–L are E3 embryos, and M–R are E4 embryos. Arrowheads in E and F indicate some of the TUNEL + apoptotic cells. Arrowheads and an arrow in H and I indicate precocious expression of Isl1 in motor neuron progenitor and D2 neuron progenitor domains, respectively. Arrowheads in N and O indicate precocious expression of the interneuron marker Lim1/2 in the VZ. Expression of the postmitotic cell marker p27kip1 is not increased in the VZ (K, L, Q, and R). F, I, L, O, and R show only red fluorescence signals without DAPI, which is shown in E, H, K, N, and Q. Bars, 50 µm.

Techniques Used: Expressing, Construct, Transfection, Staining, TUNEL Assay, Marker, Fluorescence

Impaired neuronal differentiation after Rbfox3-full knockdown. (A–J) Immunostaining of E4 neural tubes for Rbfox3 and neuronal markers. The right side of the neural tube in each panel has been transfected with siRNA indicated at the top and the GFP construct, as demonstrated by GFP in A and B. Sections were stained for proteins indicated on the left. Isl1 + cells indicated by open arrowheads in I are D2 interneurons. Bars, 50 µm. (K) Quantification of marker + cells. Each bar represents the marker + cell number in the transfected side divided by those in the contra-lateral untransfected side, except for Rbfox3 (mean ± SD, 5 embryos, 3 sections/embryo). In the case of Rbfox3, the fluorescence intensity is used instead of marker + cell number. Isl1 + cells in the D2 domain are not included for quantification. *, P
Figure Legend Snippet: Impaired neuronal differentiation after Rbfox3-full knockdown. (A–J) Immunostaining of E4 neural tubes for Rbfox3 and neuronal markers. The right side of the neural tube in each panel has been transfected with siRNA indicated at the top and the GFP construct, as demonstrated by GFP in A and B. Sections were stained for proteins indicated on the left. Isl1 + cells indicated by open arrowheads in I are D2 interneurons. Bars, 50 µm. (K) Quantification of marker + cells. Each bar represents the marker + cell number in the transfected side divided by those in the contra-lateral untransfected side, except for Rbfox3 (mean ± SD, 5 embryos, 3 sections/embryo). In the case of Rbfox3, the fluorescence intensity is used instead of marker + cell number. Isl1 + cells in the D2 domain are not included for quantification. *, P

Techniques Used: Immunostaining, Transfection, Construct, Staining, Marker, Fluorescence

Related Articles

Immunostaining:

Article Title: A Cre Transgenic Line for Studying V2 Neuronal Lineages and Functions in the Spinal Cord
Article Snippet: .. The antibodies used in the immunostaining analysis are: anti-GFP (for YFP) (goat, 1:1000, Abcam), anti-GFP (for YFP) (rabbit, 1:400, MBL International), anti-Foxn4 (rabbit, 1:50) , anti-Cre (mouse, 1:1000, Covance), anti-Cre (rabbit, 1:10000, EMD Chemicals), anti-Chx10 (sheep, 1:1600, Exalpha Biologicals), anti-Gata2 (rabbit, 1:200, Santa Cruz Biotechnology), anti-Gata2 (guinea pig, 1:2000) , anti-En1 [mouse, 1:25, Developmental Studies Hybridoma Bank (DSHB)], anti-Nkx2.2 (mouse, 1:50, DSHB), anti-Isl1 (mouse, 1:50, DSHB), and anti-Sox1 (guinea pig, 1:500) ( ). .. To quantify YFP+ cells and YFP+ cells colocalized with cell type-specific markers in the spinal cord, three comparable samples of each type were counted for positively labeled cells on serial semi-sections located at the thoracic region.

Concentration Assay:

Article Title: Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement
Article Snippet: .. The following antibodies with indicated concentration were used: anti-Nkx2-5 (ThermoScientific Cat# PA5-49431, 1:1,000) and anti-Isl1 (DSHB 39.4D5, 1:100). .. Purified Isl1+/GFP Nkx2-5-emGFP CPCs were stained using anti-GFP antibody (ThermoFisher Scientific Cat# A11120, 1:2,000).

Incubation:

Article Title: Islet ?-, ?-, and ?-Cell Development Is Controlled by the Ldb1 Coregulator, Acting Primarily With the Islet-1 Transcription Factor
Article Snippet: .. Chromatin was precleared with protein G-Sepharose (#101242; Invitrogen/Life Technologies, Carlsbad, CA) and then incubated with anti-Ldb1 (sc-11198X; Santa Cruz Biotechnology), anti-Isl1 (DSHB, 39.4D5), species-matched preimmune IgG (Santa Cruz Biotechnology), or without antibody. ..

IA:

Article Title: Pattern of Neurogenesis and Identification of Neuronal Progenitor Subtypes during Pallial Development in Xenopus laevis
Article Snippet: .. The anti-Isl1 (39.4D5), BrdU (G3G4), and Lhx2 (2C10) monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242. ..

Staining:

Article Title: Enhanced Aggregation of Androgen Receptor in Induced Pluripotent Stem Cell-derived Neurons from Spinal and Bulbar Muscular Atrophy *
Article Snippet: .. Immunofluorescence staining was performed using the following primary antibodies: anti-SSEA 3 (Abcam; 1:200), anti-SSEA 4 (Abcam; 1:200), anti-Tra-1–60 (Millipore; 1:200), anti-Tra-1–81 (Millipore; 1:200), anti-SSEA1 (Abcam; 1:200), anti-MAP-2 (Chemicon; 1:200), anti-tubulin βIII, clone 2G10 (Millipore 05-559; 1:200), anti-AR N-20 (Santa Cruz sc-816; 1:200), and anti-islet-1 (39.4D5-c; Developmental Studies Hybridoma Bank; 1:100). .. DAPI (Molecular Probes) was used for nuclear staining.

Immunofluorescence:

Article Title: Enhanced Aggregation of Androgen Receptor in Induced Pluripotent Stem Cell-derived Neurons from Spinal and Bulbar Muscular Atrophy *
Article Snippet: .. Immunofluorescence staining was performed using the following primary antibodies: anti-SSEA 3 (Abcam; 1:200), anti-SSEA 4 (Abcam; 1:200), anti-Tra-1–60 (Millipore; 1:200), anti-Tra-1–81 (Millipore; 1:200), anti-SSEA1 (Abcam; 1:200), anti-MAP-2 (Chemicon; 1:200), anti-tubulin βIII, clone 2G10 (Millipore 05-559; 1:200), anti-AR N-20 (Santa Cruz sc-816; 1:200), and anti-islet-1 (39.4D5-c; Developmental Studies Hybridoma Bank; 1:100). .. DAPI (Molecular Probes) was used for nuclear staining.

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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
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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
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    Developmental Studies Hybridoma Bank anti isl1 2
    Dorsal interneuron differentiation is normal in Nova and Dcc KOs. ( A ) Immunohistochemistry of <t>ISL1/2</t> and LHX5 in Nova WT, Nova dKO, Dcc WT, and Dcc KO spinal cords at E10.5. The markers are expressed by different subpopulations of interneurons in the dorsal spinal cord. ( B ) Quantification of ISL1/2+ neurons located in the dorsal half of the spinal cord. Data are normalized to WT and are represented as the mean ± SEM (Student’s t-test, ns, not significant). Scale bar, 50 μm. DOI: http://dx.doi.org/10.7554/eLife.14264.012
    Anti Isl1 2, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    Dorsal interneuron differentiation is normal in Nova and Dcc KOs. ( A ) Immunohistochemistry of ISL1/2 and LHX5 in Nova WT, Nova dKO, Dcc WT, and Dcc KO spinal cords at E10.5. The markers are expressed by different subpopulations of interneurons in the dorsal spinal cord. ( B ) Quantification of ISL1/2+ neurons located in the dorsal half of the spinal cord. Data are normalized to WT and are represented as the mean ± SEM (Student’s t-test, ns, not significant). Scale bar, 50 μm. DOI: http://dx.doi.org/10.7554/eLife.14264.012

    Journal: eLife

    Article Title: NOVA regulates Dcc alternative splicing during neuronal migration and axon guidance in the spinal cord

    doi: 10.7554/eLife.14264

    Figure Lengend Snippet: Dorsal interneuron differentiation is normal in Nova and Dcc KOs. ( A ) Immunohistochemistry of ISL1/2 and LHX5 in Nova WT, Nova dKO, Dcc WT, and Dcc KO spinal cords at E10.5. The markers are expressed by different subpopulations of interneurons in the dorsal spinal cord. ( B ) Quantification of ISL1/2+ neurons located in the dorsal half of the spinal cord. Data are normalized to WT and are represented as the mean ± SEM (Student’s t-test, ns, not significant). Scale bar, 50 μm. DOI: http://dx.doi.org/10.7554/eLife.14264.012

    Article Snippet: Antibodies used in the study include anti-PAX3/7 (PA1-107, Thermo Fisher, Waltham, MA, raised against PAX3 and cross reacts with PAX7), anti-BARHL2 (NBP2-32013, Novus Biologicals, Littleton, CO), anti-LHX5 (AF6290, R & D, Minneapolis, MN), anti-ISL1/2 (39.4D5, DSHB, Iowa City, IA), anti-ROBO3 (rabbit polyclonal, ), anti-TAG1 (4D7, DSHB), anti-pH3 (9701, CST, Danvers, MA), anti-Ki-67 (12202, CST), and anti-SOX2 (3728, CST).

    Techniques: Droplet Countercurrent Chromatography, Immunohistochemistry