anti islet 1 Search Results


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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 384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Developmental Studies Hybridoma Bank mouse anti islet1
    Quantitative PCR analysis of mRNA expression of the LIM homeobox genes and their cofactors. The Lhx1 , Isl1 , Isl2 , Lmx1a , Lmx1b , Ldb1 and Ldb2 mRNAs have been detected in mouse small intestine. Lhx1 , <t>Islet1</t> , Ldb1 and Ldb2 mRNAs were expressed at high level. Lhx2 , Lhx3 , Lhx4 , Lhx5 , Lhx6 and Lhx8 mRNAs were not detected. All values are in log2 scale and were normalized to the level of Lmx1b mRNA which is considered 1. At least three independent experiments were performed for each gene. The error bars show standard deviations.
    Mouse Anti Islet1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 91/100, based on 464 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Developmental Studies Hybridoma Bank isl1
    Loss of Nkx2-5 , but not <t>Isl1</t> , leads to malformation of proepicardium (A) Wholemount figure of Nkx2-5 Cre/Cre ( Nkx2-5 KO ) and littermate control Nkx2-5 +/+ . Black arrow indicates PE in control embryo, and corresponding region of the mutant embryo. (B) Immunostaining of Wt1 (red) and Nkx2-5 (green) shows decreased Wt1 expression and no Nkx2-5 expression in mutants. Arrow indicates Wt1+ cells in PE of control embryo, and corresponding area deficient in Wt1 expression in mutant embryo. Arrowhead indicates preserved Wt1 expression outside of the heart-forming regions of the mutant embryo. (C) Wholemount figure of Isl1 Cre/Cre ( Isl1 KO ) and littermate control Isl1 +/+ . Black arrow indicates PE in control and mutant embryos. (D) Immunostaining of Wt1 (red) and cardiac marker Desmin (green) shows existence of proepicardium. White arrow indicates Wt1-expressing proepicardium. A,C bar = 500 µm; B,D bar = 50 µm.
    Isl1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 777 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Developmental Studies Hybridoma Bank anti islet1
    Pre- and postsynaptic components of in vitro NMJ. ( A ) Expression of the myogenic markers MYH, MEF2C, and AChR. ( B ) Expression of the MN-specific markers HB9, <t>Islet1,</t> and choline acetyltransferase (ChAT) at day 30. ( C ) Expression of the Schwann cell marker S100 (green). Scale bars: 10 μm ( A – C ).
    Anti Islet1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 89/100, based on 266 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti islet 1
    Effect of fluorofenidone on <t>Islet-1</t> expression in murine retinas
    Anti Islet 1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    isl1  (Abcam)
    93
    Abcam isl1
    MED17.11 express the early neuronal markers FOX3 (NeuN), <t>Isl1</t> and Tuj1, and can be transfected with GFP. Immunolabelling of MED17.11 cells cultured in permissive conditions for large T antigen expression. GFP transgene expression in MED17.11. Scale bar is 100 μm.
    Isl1, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Abcam rabbit anti isl1
    <t>ISL1</t> directly regulates a number of genes required for normal pacemaker function in mice and human.
    Rabbit Anti Isl1, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Developmental Studies Hybridoma Bank anti islet1 2
    Effect of RCASBP(B)-RFP-GluA2 RNAi infection on chicken embryos. A–B ) Expression of red fluorescence protein (RFP) transgene in the lumbar spinal cord of E6 ( A ) and E11 ( B ) chicken embryos following retroviral infection with an RCASBP(B)-RFP-GluA2 RNAi construct. Embryos infected with the RCASBP(B)-RFP-GluA2 RNAi construct show strong fluorescent labeling throughout the whole spinal cord cross section. cc = central canal, nc = notochord. C ) Averaged number of labeled neurons for the RCASBP(B) viral protein p27 gag as a percent of the total number of neuron labeled with the motoneuron marker <t>Islet1/2</t> in chicken embryos infected with an RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP- GluA2 RNAi construct. Lumbar ventral neurons were isolated from E6, E8 or E11 chicken embryos and immunolabeled with p27 gag and Islet1/2 in order to assess the extent of viral infection of spinal motoneurons. Notice that ≥60% of infected cells also tested positive for the motoneuron marker Islet1/2 at all ages tested. D ) Representative example of Western Blot data collected from the E6 and E11 ventral spinal cords from control chicken embryos (non-infected) or embryos infected with an RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi constructs. The anti-GluA2 antibody detected a band with a relative molecular weight of ∼102 kD. To normalize for changes in protein loading in each well, membranes were reprobed for β-actin (∼42 kD). Infection of chicken embryos with an RCASBP(B)-RFP- GluA2 RNAi construct causes a significant reduction in GluA2 expression at E11 as determined by immunoblot analysis. E ) Expression of GluA2 protein as a function of β-actin in chicken ventral spinal cords. The age-dependent increase in GluA2 protein expression between E6 and E11 chicken spinal cords was reversed by infection of chicken embryos with an RCASBP(B)-RFP- GluA2 RNAi construct. In these experiments, RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi viral particles were injected into the developing neural tube at E2 (approximately 36 hr after incubation). Embryos were allowed to develop up to E11 before tissue isolation and processing.
    Anti Islet1 2, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 90/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam anti islet 1 antibody ep4182
    Loss of SMN reactivates the cell cycle. a Ki67 and <t>ISL1</t> immunostaining analysis of wild-type (BJ iPS and 18a), SMA Type I (1-38 G), and SMA Type II (1-51 N) motor neuron cultures at day 28. The percentages of ISL1 + Ki67 + cells amongst all ISL1 + motor neurons are shown. b Knockdown of SMN in wild-type cell line (BJ-iPS) increased the percentage of ISL1 + motor neurons co-expressing Ki67. c Co-staining of ISL1 (red) and Ki67 (green) showing increased Ki67 + cells upon SMN knockdown in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Ki67 and cCASP3 immunostaining analysis of wild-type motor neurons demonstrated higher cCASP3 expression in Ki67 + motor neurons than Ki67 − motor neurons. *** p
    Anti Islet 1 Antibody Ep4182, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam islet1
    TDP-43 localization in patient iPSC-derived motor neurons (A) iPSC-MN stained for TDP-43 shows TDP-43 is ubiquitously expressed and nuclear in all cells including in <t>ISLET1-positive</t> neurons from control (left panel) and ALS (right panel) patients. ISLET1-positive cells from sporadic ALS iPSC-MN have nuclear staining as well as nuclear aggregates that stain with higher intensity for TDP-43 (arrowheads, right panel) but aggregates are not present in healthy controls (left panel). Fibroblasts from healthy control and sALS patients do not show nuclear aggregates. Scale bar: 30 µm. (B) Sporadic ALS patient-derived iPSC-MN cells stained with nuclear envelop marker LAMIN-A (green) and TDP-43 (red) shows TDP-43 aggregates are inside the nuclear envelope; scale bar: 20µm. (C) Quantification of healthy control IPRN.0013 and sALS IPRN.0048 clone 1 iPSC-MN cultures shows TDP-43 aggregation is present in higher fraction of ISLET1 or HB9-positive cells compared to negative cells. (30.7% of ISLET1/HB9-positive motor neurons as compared to 16.2% of ISLET1/HB9-negative cells in sALS iPSC-MN cultures. Bars are standard deviation 9% and 8% respectively; P
    Islet1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam mouse anti isl1
    Figure 6. Structure of cardiomyocyte colonies grown in the primary culture of rat neonatal myocardial cells. ( A–C ) Different stages of development of the colonies stemming from <t>Isl1</t> + CSCs. ( A ) Cell division, DIV 2. Isl1 + (FITC, green), GATA-4 (phycoerythrin, red). ( B ) Colony consisting of approximately 8 cells, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( C ) Large Isl1 + colony, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( D ) The optical sections of colonies formed by Isl1 + , c-kit + , and Sca1 + CSCs on the 11th DIV. Isl1 + CSCs (Alexa 405, blue), Z = 12. c-kit + CSCs (FITC, green), Z = 12. Sca1 + CSCs (Alexa 405, blue), Z = 11. Actin was stained using rhodamine-phalloidin (red). ( E ) Differentiation of c-kit + CSCs inside the colony on the 13th DIV. Overlaid optical section of transmitted light and fluorescent images in 2 emitting wavelengths: 488 nm (FITC) and 543 nm (Alexa) in the bottom (Z = 5), in the middle (Z = 10), and the top (Z = 20) parts of the colony. c-kit + expression was revealed by FITC-conjugated antibodies (green), and α-sarcomeric actinin was revealed by Alexa-conjugated antibodies (red). Confocal microscope, Leica TCS SP5 (Germany), objective ×63, oil.
    Mouse Anti Isl1, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    Quantitative PCR analysis of mRNA expression of the LIM homeobox genes and their cofactors. The Lhx1 , Isl1 , Isl2 , Lmx1a , Lmx1b , Ldb1 and Ldb2 mRNAs have been detected in mouse small intestine. Lhx1 , Islet1 , Ldb1 and Ldb2 mRNAs were expressed at high level. Lhx2 , Lhx3 , Lhx4 , Lhx5 , Lhx6 and Lhx8 mRNAs were not detected. All values are in log2 scale and were normalized to the level of Lmx1b mRNA which is considered 1. At least three independent experiments were performed for each gene. The error bars show standard deviations.

    Journal: PLoS ONE

    Article Title: Islet1 and Its Co-Factor Ldb1 Are Expressed in Quiescent Cells of Mouse Intestinal Epithelium

    doi: 10.1371/journal.pone.0095256

    Figure Lengend Snippet: Quantitative PCR analysis of mRNA expression of the LIM homeobox genes and their cofactors. The Lhx1 , Isl1 , Isl2 , Lmx1a , Lmx1b , Ldb1 and Ldb2 mRNAs have been detected in mouse small intestine. Lhx1 , Islet1 , Ldb1 and Ldb2 mRNAs were expressed at high level. Lhx2 , Lhx3 , Lhx4 , Lhx5 , Lhx6 and Lhx8 mRNAs were not detected. All values are in log2 scale and were normalized to the level of Lmx1b mRNA which is considered 1. At least three independent experiments were performed for each gene. The error bars show standard deviations.

    Article Snippet: Li and P. Love, NICHD, NIH), mouse anti-Islet1 at 1∶100 (clone 40.2D6, Developmental Studies Hybridoma Bank), rabbit anti-Islet1 at 1∶4000 (ab26122, Abcam), rat anti-CD45 (clone 30-F11, BD Pharmingen), rabbit anti-CD3 at 1∶200 (VP-RM01, Vector Laboratories), mouse anti-Multi-cytokeratine at 1∶50 (VP-C419,Vector Laboratories), mouse anti-BrdU at 1∶50 (clone Bu20a, Daco), rabbit anti-Chromogranin A at 1∶1000 (ab15160, Abcam), rat anti-Ki-67 at 1∶50 (clone TEC-3, Daco), rabbit anti-Phospho Histone H3 at 1∶100 (9701, Cell Signaling), and rabbit anti-Cleaved Caspase3 at 1∶500 (9661, Cell Signaling).

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Skin-derived NT3 does not regulate DRG neuron Trk expression. A–D , Immunostaining for NF200, TrkB, and TrkC in T7 DRG sections from P0 control littermate and K14; Ntf3 CKO mice. Asterisks indicate double-labeled cells. E , Numbers of Islet1/2 + , NF200 + , TrkB + , TrkC + , and TrkB + TrkC + neurons in P0 T7 DRGs ( n = 6 mice/genotype). Error bars represent the SEM, and asterisks indicate statistically significant differences between genotypes. *** p

    Journal: The Journal of Neuroscience

    Article Title: Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes

    doi: 10.1523/JNEUROSCI.3781-15.2016

    Figure Lengend Snippet: Skin-derived NT3 does not regulate DRG neuron Trk expression. A–D , Immunostaining for NF200, TrkB, and TrkC in T7 DRG sections from P0 control littermate and K14; Ntf3 CKO mice. Asterisks indicate double-labeled cells. E , Numbers of Islet1/2 + , NF200 + , TrkB + , TrkC + , and TrkB + TrkC + neurons in P0 T7 DRGs ( n = 6 mice/genotype). Error bars represent the SEM, and asterisks indicate statistically significant differences between genotypes. *** p

    Article Snippet: The primary antibodies used were mouse anti-Islet1/2 (1:50; catalog #39.4D5, Developmental Studies Hybridoma Bank), rabbit anti-NF200 (1:1000; N4142, Sigma-Aldrich), goat anti-TrkB (1:200; AF1494, R & D Systems), goat anti-TrkC [AF1404 (1:100) and BAF1404 (1:20), R & D Systems], rabbit anti-Ret (1:50; catalog #18121, Immuno-Biological Laboratories), rabbit anti-parvalbumin (PV; 1:1000; PV25, Swant), rabbit anti-CGRP (1:1000; T-4032, Peninsula Laboratories), rabbit anti-phospho-SMAD1/5/8 (1:250; catalog 9511, Cell Signaling Technology), goat anti-TBX3 (1:100; sc-31656, Santa Cruz Biotechnology), rat anti-Keratin8 (1:20, TROMA-1, Developmental Studies Hybridoma Bank), guinea pig anti-Asic1 (1:250; Alomone Labs), rabbit anti-δ opioid receptor (DOR; 1:500; Alomone Labs), and rabbit anti-γENaC (1:500; StressMarq Biosciences).

    Techniques: Derivative Assay, Expressing, Immunostaining, Mouse Assay, Labeling

    Loss of TrkC expression but not neurons in DRGs of P0 K14; Atoh1 CKO mice is consistent across spinal levels. A–D , Immunostaining for TrkC in L2 and L4/5 DRG sections from P0 control littermate and K14; Atoh1 CKO mice. E , Numbers of Islet1/2 + neurons in P0 L2 and L4/5 DRGs from control and K14; Atoh1 CKO mice ( n ≥ 7 mice/genotype at each level). F , Percentages (numbers ± SEM shown in bars on graph) of Islet1/2 + L2 ( n = 7 mice/genotype) and L4/5 ( n = 15–18 mice/genotype) DRG neurons that were TrkC + in P0 control and K14; Atoh1 CKO mice. In graphs, error bars represent the SEM, and asterisks indicate statistically significant differences between genotypes. * p

    Journal: The Journal of Neuroscience

    Article Title: Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes

    doi: 10.1523/JNEUROSCI.3781-15.2016

    Figure Lengend Snippet: Loss of TrkC expression but not neurons in DRGs of P0 K14; Atoh1 CKO mice is consistent across spinal levels. A–D , Immunostaining for TrkC in L2 and L4/5 DRG sections from P0 control littermate and K14; Atoh1 CKO mice. E , Numbers of Islet1/2 + neurons in P0 L2 and L4/5 DRGs from control and K14; Atoh1 CKO mice ( n ≥ 7 mice/genotype at each level). F , Percentages (numbers ± SEM shown in bars on graph) of Islet1/2 + L2 ( n = 7 mice/genotype) and L4/5 ( n = 15–18 mice/genotype) DRG neurons that were TrkC + in P0 control and K14; Atoh1 CKO mice. In graphs, error bars represent the SEM, and asterisks indicate statistically significant differences between genotypes. * p

    Article Snippet: The primary antibodies used were mouse anti-Islet1/2 (1:50; catalog #39.4D5, Developmental Studies Hybridoma Bank), rabbit anti-NF200 (1:1000; N4142, Sigma-Aldrich), goat anti-TrkB (1:200; AF1494, R & D Systems), goat anti-TrkC [AF1404 (1:100) and BAF1404 (1:20), R & D Systems], rabbit anti-Ret (1:50; catalog #18121, Immuno-Biological Laboratories), rabbit anti-parvalbumin (PV; 1:1000; PV25, Swant), rabbit anti-CGRP (1:1000; T-4032, Peninsula Laboratories), rabbit anti-phospho-SMAD1/5/8 (1:250; catalog 9511, Cell Signaling Technology), goat anti-TBX3 (1:100; sc-31656, Santa Cruz Biotechnology), rat anti-Keratin8 (1:20, TROMA-1, Developmental Studies Hybridoma Bank), guinea pig anti-Asic1 (1:250; Alomone Labs), rabbit anti-δ opioid receptor (DOR; 1:500; Alomone Labs), and rabbit anti-γENaC (1:500; StressMarq Biosciences).

    Techniques: Expressing, Mouse Assay, Immunostaining

    Merkel cell deletion alters the expression of SAI ion channel components. A , qPCR analysis of ion channels in P21 thoracic DRGs ( n ≥ 8 mice/genotype at each age). B–G , Immunostaining for NF200, γENaC, Asic1, and DOR in T7 DRG sections from P21 control littermate and K14; Atoh1 CKO mice. Asterisks indicate double-positive cells. H , Percentages of Islet1/2 + T7 DRG neurons that were NF200 + γENaC + , NF200 + Asic1, and NF200 + DOR + in P21 control and K14; Atoh1 CKO mice ( n ≥ 3 mice/genotype). Error bars in graphs represent the SEM, and asterisks indicate statistically significant differences between genotypes. * p

    Journal: The Journal of Neuroscience

    Article Title: Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes

    doi: 10.1523/JNEUROSCI.3781-15.2016

    Figure Lengend Snippet: Merkel cell deletion alters the expression of SAI ion channel components. A , qPCR analysis of ion channels in P21 thoracic DRGs ( n ≥ 8 mice/genotype at each age). B–G , Immunostaining for NF200, γENaC, Asic1, and DOR in T7 DRG sections from P21 control littermate and K14; Atoh1 CKO mice. Asterisks indicate double-positive cells. H , Percentages of Islet1/2 + T7 DRG neurons that were NF200 + γENaC + , NF200 + Asic1, and NF200 + DOR + in P21 control and K14; Atoh1 CKO mice ( n ≥ 3 mice/genotype). Error bars in graphs represent the SEM, and asterisks indicate statistically significant differences between genotypes. * p

    Article Snippet: The primary antibodies used were mouse anti-Islet1/2 (1:50; catalog #39.4D5, Developmental Studies Hybridoma Bank), rabbit anti-NF200 (1:1000; N4142, Sigma-Aldrich), goat anti-TrkB (1:200; AF1494, R & D Systems), goat anti-TrkC [AF1404 (1:100) and BAF1404 (1:20), R & D Systems], rabbit anti-Ret (1:50; catalog #18121, Immuno-Biological Laboratories), rabbit anti-parvalbumin (PV; 1:1000; PV25, Swant), rabbit anti-CGRP (1:1000; T-4032, Peninsula Laboratories), rabbit anti-phospho-SMAD1/5/8 (1:250; catalog 9511, Cell Signaling Technology), goat anti-TBX3 (1:100; sc-31656, Santa Cruz Biotechnology), rat anti-Keratin8 (1:20, TROMA-1, Developmental Studies Hybridoma Bank), guinea pig anti-Asic1 (1:250; Alomone Labs), rabbit anti-δ opioid receptor (DOR; 1:500; Alomone Labs), and rabbit anti-γENaC (1:500; StressMarq Biosciences).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Immunostaining

    Merkel cell deletion reduces BDNF-stimulated signaling in SAI neurons and alters transcription factor expression. A–H , Immunostaining for NF200 and phospho-SMAD1/5/8 ( A–D ) or Tbx3 ( E–H ) in T7 DRG sections from E13.5 and E16.5 control littermate and K14; Atoh1 CKO mice. Asterisks indicate double-positive cells. I , J , The mean percentage of Islet1/2 + T7 DRG neurons that were NF200 + pSMAD1/5/8 + ( I ) and NF200 + Tbx3 + ( J ) in E13.5 and E16.5 control and K14; Atoh1 CKO mice ( n ≥ 3 mice/genotype at each age). K , Percentage of pSMAD1/5/8 + NF200 + or Tbx3 + NF200 + neurons in E13.5 and E16.5 control littermate and K14; Atoh1 CKO mice. L , qPCR analysis of genes regulated by BDNF signaling ( n ≥ 8 mice/genotype). In photographs, asterisks indicate double-labeled cells. In graphs, error bars represent the SEM, and asterisks indicate statistically significant differences between genotypes. * p

    Journal: The Journal of Neuroscience

    Article Title: Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes

    doi: 10.1523/JNEUROSCI.3781-15.2016

    Figure Lengend Snippet: Merkel cell deletion reduces BDNF-stimulated signaling in SAI neurons and alters transcription factor expression. A–H , Immunostaining for NF200 and phospho-SMAD1/5/8 ( A–D ) or Tbx3 ( E–H ) in T7 DRG sections from E13.5 and E16.5 control littermate and K14; Atoh1 CKO mice. Asterisks indicate double-positive cells. I , J , The mean percentage of Islet1/2 + T7 DRG neurons that were NF200 + pSMAD1/5/8 + ( I ) and NF200 + Tbx3 + ( J ) in E13.5 and E16.5 control and K14; Atoh1 CKO mice ( n ≥ 3 mice/genotype at each age). K , Percentage of pSMAD1/5/8 + NF200 + or Tbx3 + NF200 + neurons in E13.5 and E16.5 control littermate and K14; Atoh1 CKO mice. L , qPCR analysis of genes regulated by BDNF signaling ( n ≥ 8 mice/genotype). In photographs, asterisks indicate double-labeled cells. In graphs, error bars represent the SEM, and asterisks indicate statistically significant differences between genotypes. * p

    Article Snippet: The primary antibodies used were mouse anti-Islet1/2 (1:50; catalog #39.4D5, Developmental Studies Hybridoma Bank), rabbit anti-NF200 (1:1000; N4142, Sigma-Aldrich), goat anti-TrkB (1:200; AF1494, R & D Systems), goat anti-TrkC [AF1404 (1:100) and BAF1404 (1:20), R & D Systems], rabbit anti-Ret (1:50; catalog #18121, Immuno-Biological Laboratories), rabbit anti-parvalbumin (PV; 1:1000; PV25, Swant), rabbit anti-CGRP (1:1000; T-4032, Peninsula Laboratories), rabbit anti-phospho-SMAD1/5/8 (1:250; catalog 9511, Cell Signaling Technology), goat anti-TBX3 (1:100; sc-31656, Santa Cruz Biotechnology), rat anti-Keratin8 (1:20, TROMA-1, Developmental Studies Hybridoma Bank), guinea pig anti-Asic1 (1:250; Alomone Labs), rabbit anti-δ opioid receptor (DOR; 1:500; Alomone Labs), and rabbit anti-γENaC (1:500; StressMarq Biosciences).

    Techniques: Expressing, Immunostaining, Mouse Assay, Real-time Polymerase Chain Reaction, Labeling

    Lineage reconstruction and cell type identification. ( A ) Snapshots of a time-lapse video showing a rat retinal progenitor cell (RPC) undergoing a P/D division ( t =20:30) and a terminal D/D division ( t =58:11) giving rise to a three-cell clone composed of an amacrine (Am), a bipolar (Bi) and a rod photoreceptor (RPh or ph) cell. Time is given in hours:minutes. ( B-D ) Retinal cell type identification in the clone recorded in A. In this clone, cell 4 stained for Islet1 and cell 5 stained for Pax6, identifying them as Bi and Am, respectively. The remaining cell was negative for both markers and displayed typical RPh cell morphology with a small round cell body and simple processes and showed characteristic Hoechst staining. ( E ) Based on the staining data in B-D and the videomicroscopy in A, the lineage tree of this clone was reconstructed. ( F-I ) A clone containing two RPh cells and one Müller glial cell (Mu; Pax6 negative and Islet1 negative) with distinct glial morphology, large nucleus, absence of neurites and lack of expression of neuronal markers. Scale bars: 20 μm in A; 8 μm in B-D; 20 μm in F-I.

    Journal: Development (Cambridge, England)

    Article Title: Reconstruction of rat retinal progenitor cell lineages in vitro reveals a surprising degree of stochasticity in cell fate decisions

    doi: 10.1242/dev.059683

    Figure Lengend Snippet: Lineage reconstruction and cell type identification. ( A ) Snapshots of a time-lapse video showing a rat retinal progenitor cell (RPC) undergoing a P/D division ( t =20:30) and a terminal D/D division ( t =58:11) giving rise to a three-cell clone composed of an amacrine (Am), a bipolar (Bi) and a rod photoreceptor (RPh or ph) cell. Time is given in hours:minutes. ( B-D ) Retinal cell type identification in the clone recorded in A. In this clone, cell 4 stained for Islet1 and cell 5 stained for Pax6, identifying them as Bi and Am, respectively. The remaining cell was negative for both markers and displayed typical RPh cell morphology with a small round cell body and simple processes and showed characteristic Hoechst staining. ( E ) Based on the staining data in B-D and the videomicroscopy in A, the lineage tree of this clone was reconstructed. ( F-I ) A clone containing two RPh cells and one Müller glial cell (Mu; Pax6 negative and Islet1 negative) with distinct glial morphology, large nucleus, absence of neurites and lack of expression of neuronal markers. Scale bars: 20 μm in A; 8 μm in B-D; 20 μm in F-I.

    Article Snippet: After the recording, the cells were fixed with 4% paraformaldehyde for 10 minutes and the following antibodies were used for immunostaining: monoclonal mouse anti-Islet1 (1:2000; produced by T. Jessell and obtained from the Developmental Studies Hybridoma Bank) and rabbit anti-Pax6 (1:10,000; Santa Cruz Biotech).

    Techniques: Staining, Expressing

    Loss of Nkx2-5 , but not Isl1 , leads to malformation of proepicardium (A) Wholemount figure of Nkx2-5 Cre/Cre ( Nkx2-5 KO ) and littermate control Nkx2-5 +/+ . Black arrow indicates PE in control embryo, and corresponding region of the mutant embryo. (B) Immunostaining of Wt1 (red) and Nkx2-5 (green) shows decreased Wt1 expression and no Nkx2-5 expression in mutants. Arrow indicates Wt1+ cells in PE of control embryo, and corresponding area deficient in Wt1 expression in mutant embryo. Arrowhead indicates preserved Wt1 expression outside of the heart-forming regions of the mutant embryo. (C) Wholemount figure of Isl1 Cre/Cre ( Isl1 KO ) and littermate control Isl1 +/+ . Black arrow indicates PE in control and mutant embryos. (D) Immunostaining of Wt1 (red) and cardiac marker Desmin (green) shows existence of proepicardium. White arrow indicates Wt1-expressing proepicardium. A,C bar = 500 µm; B,D bar = 50 µm.

    Journal: Biochemical and biophysical research communications

    Article Title: Nkx2-5- and Isl1-expressing cardiac progenitors contribute to proepicardium

    doi: 10.1016/j.bbrc.2008.08.044

    Figure Lengend Snippet: Loss of Nkx2-5 , but not Isl1 , leads to malformation of proepicardium (A) Wholemount figure of Nkx2-5 Cre/Cre ( Nkx2-5 KO ) and littermate control Nkx2-5 +/+ . Black arrow indicates PE in control embryo, and corresponding region of the mutant embryo. (B) Immunostaining of Wt1 (red) and Nkx2-5 (green) shows decreased Wt1 expression and no Nkx2-5 expression in mutants. Arrow indicates Wt1+ cells in PE of control embryo, and corresponding area deficient in Wt1 expression in mutant embryo. Arrowhead indicates preserved Wt1 expression outside of the heart-forming regions of the mutant embryo. (C) Wholemount figure of Isl1 Cre/Cre ( Isl1 KO ) and littermate control Isl1 +/+ . Black arrow indicates PE in control and mutant embryos. (D) Immunostaining of Wt1 (red) and cardiac marker Desmin (green) shows existence of proepicardium. White arrow indicates Wt1-expressing proepicardium. A,C bar = 500 µm; B,D bar = 50 µm.

    Article Snippet: The primary antibodies Wt1 (1:100, Santa Cruz), Tbx18 (1:100, Santa Cruz), Nkx2-5 (1:100, Santa Cruz), β-gal (1:5000, MP biomedicals), Isl1 (1:100, Iowa Developmental Studies Hybridoma Bank) were incubated for 1.5 hours at room temperature.

    Techniques: Mutagenesis, Immunostaining, Expressing, Marker

    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.

    Journal: Frontiers in Neuroanatomy

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

    doi: 10.3389/fnana.2017.00024

    Figure Lengend 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.

    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.

    Techniques: Immunohistochemistry, Marker, Expressing, Labeling

    Tbx1 regulates the Mef2c AHF enhancer in vivo . (A-E) Immunofluorescence of transverse sections of E9.5 (20 somites) embryos with the genotype indicated, using anti-Cre (green) and anti-Isl1 (red) antibodies. The first row of sections (A) corresponds to a level immediately below the outflow tract. The second row (B) is just anteriorly to the inflow tract, while the third fourth and fifth rows (C-E) correspond to the inflow tract. Note that the Cre signals in A and B are comparable in the three genotypes, while in C-E, reduced dosage of Tbx1 shows Tbx1 gene expression in similar sections. A, atrium; DPW, dorsal pericardial wall (line arrowhead); IFT, inflow tract; NT, neural tube; OFT, outflow tract; Ph, pharynx. Scale bar: 100 μm.

    Journal: Disease Models & Mechanisms

    Article Title: Tbx1 represses Mef2c gene expression and is correlated with histone 3 deacetylation of the anterior heart field enhancer

    doi: 10.1242/dmm.029967

    Figure Lengend Snippet: Tbx1 regulates the Mef2c AHF enhancer in vivo . (A-E) Immunofluorescence of transverse sections of E9.5 (20 somites) embryos with the genotype indicated, using anti-Cre (green) and anti-Isl1 (red) antibodies. The first row of sections (A) corresponds to a level immediately below the outflow tract. The second row (B) is just anteriorly to the inflow tract, while the third fourth and fifth rows (C-E) correspond to the inflow tract. Note that the Cre signals in A and B are comparable in the three genotypes, while in C-E, reduced dosage of Tbx1 shows Tbx1 gene expression in similar sections. A, atrium; DPW, dorsal pericardial wall (line arrowhead); IFT, inflow tract; NT, neural tube; OFT, outflow tract; Ph, pharynx. Scale bar: 100 μm.

    Article Snippet: Immunofluorescence on paraffin-embedded E9.5 embryo sections was performed using an anti-Cre antibody (69050-3, Novagen, Madison, WI, USA; 1:1000), and an anti-Isl1 antibody (39.4D5-s, Developmental Studies Hybridoma Bank, Iowa City, IA, USA; 1:50). β-galactosidase assays were performed on whole-mount embryos using salmon-gal (6-chloro-3-indolyl-beta-D-galactopyranoside, , Alfa Aesar, Haverhill, MA, USA).

    Techniques: In Vivo, Immunofluorescence, Expressing

    VEGF expression is regulated by HIF1α. ( a – c ) Representative images of SCs at the developmental stages indicated, showing HIF1α staining in post-mitotic MNs (Isl1/2 + ). ( d – i ) Higher magnifications of insets in a – c showing co-localization of HIF1α and Isl1/2 + ( d – f ) or just HIF1α staining ( g – i ). Note the changes of HIF1α nuclear localization during development. ( j – m ) qRT-PCR analysis of changes in expression levels of Vegf ( j ) and other prototypical HIF target genes ( Bnip3 ( k ) , Pdk1 ( l ) and Egln3 ( m )), when explants from mouse E11.5 MN columns are cultured under normoxia (20% O 2 ) (green bars) or hypoxia conditions (1% O 2 ) (red bars), with or without chetomin. Data are represented as mean±s.e.m. n =2 individual experiments done in triplicates. ( j ) * P =0.0161, ** P =0.0086 (normoxia versus hypoxia), ** P =0.0082 (hypoxia versus hypoxia+chetomin); ( k ) * P =0.0169, ** P =0.0056 (normoxia versus hypoxia), ** P =0.0018 (hypoxia versus hypoxia+chetomin); ( l ) *** P =0.0007, **** P

    Journal: Nature Communications

    Article Title: Motor neurons control blood vessel patterning in the developing spinal cord

    doi: 10.1038/ncomms14583

    Figure Lengend Snippet: VEGF expression is regulated by HIF1α. ( a – c ) Representative images of SCs at the developmental stages indicated, showing HIF1α staining in post-mitotic MNs (Isl1/2 + ). ( d – i ) Higher magnifications of insets in a – c showing co-localization of HIF1α and Isl1/2 + ( d – f ) or just HIF1α staining ( g – i ). Note the changes of HIF1α nuclear localization during development. ( j – m ) qRT-PCR analysis of changes in expression levels of Vegf ( j ) and other prototypical HIF target genes ( Bnip3 ( k ) , Pdk1 ( l ) and Egln3 ( m )), when explants from mouse E11.5 MN columns are cultured under normoxia (20% O 2 ) (green bars) or hypoxia conditions (1% O 2 ) (red bars), with or without chetomin. Data are represented as mean±s.e.m. n =2 individual experiments done in triplicates. ( j ) * P =0.0161, ** P =0.0086 (normoxia versus hypoxia), ** P =0.0082 (hypoxia versus hypoxia+chetomin); ( k ) * P =0.0169, ** P =0.0056 (normoxia versus hypoxia), ** P =0.0018 (hypoxia versus hypoxia+chetomin); ( l ) *** P =0.0007, **** P

    Article Snippet: Histology and immunohistochemistry For immunohistochemistry the following primary antibodies were used at the indicated dilutions: Isl1 (40.2D6, 1:100, DSHB), Isl1/2 (39.4D5, 1:200, DSHB), Nkx2.2 (74.5A5, 1:100, DSHB), Olig2 (AB9610, 1:100, Millipore), Pax7 (1:10, DSHB), anti-h/m/r Hif-1a (AF1935, 1:100, R & D Systems), pAb anti-Carbonic Anhydrase IX/CA9 (NB100-417, 1:100, Novus Biologicals), TER-119 (MAB1125, 1:100, R & D Systems), rabbit anti-FoxP1 (ab16645, 1:1.000, Abcam), mouse anti-neurofilament-M (RMO 270, 1:1.500, ThermoFischer), anti-mouse Flt1 (103-M31, 1:100, ReliaTech GmbH), En-1 (4G11, 1:50, DSHB).

    Techniques: Expressing, Staining, Quantitative RT-PCR, Cell Culture

    mFlt1 and sFlt1 are expressed in MN columns. ( a , b , e , f , i , j ) Representative images of immunostaining for Flt1 (mFlt1+sFlt1) combined with blood vessel staining (IB4 + ) at E10.5 ( a , b ), E11.5 ( e , f ) and E12.5 ( i , j ). ( c , d , g , h , k , l ). Representative images of immunostaining for Flt1 (mFlt1+sFlt1) and for Isl1/2 + (post-mitotic MNs) at E10.5 ( c , d ), E11.5 ( g , h ) and E12.5 ( k , l ). Note Flt1 can be detected in blood vessels (white arrowheads), radial glia (blue arrowheads, identified by its typical morphology), as well as in MN columns (yellow dotted outlines). ( m – p ) Representative images of RNAscope Multiplex Fluorescent Assay using specific probes to detect mFlt1 and sFlt1 combined with staining for nuclei (DAPI + ) and either staining for blood vessels (IB4 + ) ( m , o ) or immunostaining for MNs (Isl1/2 + ) ( n , p ) at E11.5 ( m , n ) and E12.5 ( o , p ). Scale bars 100 μm. Right panels: higher magnifications of insets. Scale bars of insets 10 μm.

    Journal: Nature Communications

    Article Title: Motor neurons control blood vessel patterning in the developing spinal cord

    doi: 10.1038/ncomms14583

    Figure Lengend Snippet: mFlt1 and sFlt1 are expressed in MN columns. ( a , b , e , f , i , j ) Representative images of immunostaining for Flt1 (mFlt1+sFlt1) combined with blood vessel staining (IB4 + ) at E10.5 ( a , b ), E11.5 ( e , f ) and E12.5 ( i , j ). ( c , d , g , h , k , l ). Representative images of immunostaining for Flt1 (mFlt1+sFlt1) and for Isl1/2 + (post-mitotic MNs) at E10.5 ( c , d ), E11.5 ( g , h ) and E12.5 ( k , l ). Note Flt1 can be detected in blood vessels (white arrowheads), radial glia (blue arrowheads, identified by its typical morphology), as well as in MN columns (yellow dotted outlines). ( m – p ) Representative images of RNAscope Multiplex Fluorescent Assay using specific probes to detect mFlt1 and sFlt1 combined with staining for nuclei (DAPI + ) and either staining for blood vessels (IB4 + ) ( m , o ) or immunostaining for MNs (Isl1/2 + ) ( n , p ) at E11.5 ( m , n ) and E12.5 ( o , p ). Scale bars 100 μm. Right panels: higher magnifications of insets. Scale bars of insets 10 μm.

    Article Snippet: Histology and immunohistochemistry For immunohistochemistry the following primary antibodies were used at the indicated dilutions: Isl1 (40.2D6, 1:100, DSHB), Isl1/2 (39.4D5, 1:200, DSHB), Nkx2.2 (74.5A5, 1:100, DSHB), Olig2 (AB9610, 1:100, Millipore), Pax7 (1:10, DSHB), anti-h/m/r Hif-1a (AF1935, 1:100, R & D Systems), pAb anti-Carbonic Anhydrase IX/CA9 (NB100-417, 1:100, Novus Biologicals), TER-119 (MAB1125, 1:100, R & D Systems), rabbit anti-FoxP1 (ab16645, 1:1.000, Abcam), mouse anti-neurofilament-M (RMO 270, 1:1.500, ThermoFischer), anti-mouse Flt1 (103-M31, 1:100, ReliaTech GmbH), En-1 (4G11, 1:50, DSHB).

    Techniques: Immunostaining, Staining, Multiplex Assay, Fluorescence

    MN-specific deletion of sFlt1 leads to premature ingression of blood vessels into MN columns. ( a , e ) Representative images of Hb9-EGFP-miFlt1#1 ( a ) or Hb9-EGFP-misFlt1#1 ( e ) electroporated chicken embryos showing blood vessel ingression (blood vessels (BV) labelled by ISH for Vegfr2 are traced in purple) into MN columns in the electroporated side of the SC (yellow arrowheads) but not in the non-electroporated one. ( b , f ) qRT-PCR analysis confirms downregulation of Flt1 ( mFlt1 + sFlt1) ( b ) or sFlt1 ( f ) levels. n =4, ** P =0.0022 for ( b ) and n =4, ** P =0.0042 for ( f ). ( c , g ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated side and non-electroporated one. n =8, ** P =0.0012 for ( c ) and n =8, * P =0.0366 for ( g ). ( d , h ) Quantification of the angle of ingression showing a significant increase in blood vessel sprouts ingressing into MN columns in the electroporated side but not in the non-electroporated one. n =11, * P =0.0147 for ( d ) and n =12, * P =0.0408 for ( h ). ( i ) Representative image of sFlt1 rescue experiment showing no blood vessel ingression into MN columns in the electroporated side of the SC as well as in the non-electroporated one. ( j ) qRT-PCR analysis confirms downregulation of chicken sFlt1 levels in MN columns in the electroporated side. n =6, **** P

    Journal: Nature Communications

    Article Title: Motor neurons control blood vessel patterning in the developing spinal cord

    doi: 10.1038/ncomms14583

    Figure Lengend Snippet: MN-specific deletion of sFlt1 leads to premature ingression of blood vessels into MN columns. ( a , e ) Representative images of Hb9-EGFP-miFlt1#1 ( a ) or Hb9-EGFP-misFlt1#1 ( e ) electroporated chicken embryos showing blood vessel ingression (blood vessels (BV) labelled by ISH for Vegfr2 are traced in purple) into MN columns in the electroporated side of the SC (yellow arrowheads) but not in the non-electroporated one. ( b , f ) qRT-PCR analysis confirms downregulation of Flt1 ( mFlt1 + sFlt1) ( b ) or sFlt1 ( f ) levels. n =4, ** P =0.0022 for ( b ) and n =4, ** P =0.0042 for ( f ). ( c , g ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated side and non-electroporated one. n =8, ** P =0.0012 for ( c ) and n =8, * P =0.0366 for ( g ). ( d , h ) Quantification of the angle of ingression showing a significant increase in blood vessel sprouts ingressing into MN columns in the electroporated side but not in the non-electroporated one. n =11, * P =0.0147 for ( d ) and n =12, * P =0.0408 for ( h ). ( i ) Representative image of sFlt1 rescue experiment showing no blood vessel ingression into MN columns in the electroporated side of the SC as well as in the non-electroporated one. ( j ) qRT-PCR analysis confirms downregulation of chicken sFlt1 levels in MN columns in the electroporated side. n =6, **** P

    Article Snippet: Histology and immunohistochemistry For immunohistochemistry the following primary antibodies were used at the indicated dilutions: Isl1 (40.2D6, 1:100, DSHB), Isl1/2 (39.4D5, 1:200, DSHB), Nkx2.2 (74.5A5, 1:100, DSHB), Olig2 (AB9610, 1:100, Millipore), Pax7 (1:10, DSHB), anti-h/m/r Hif-1a (AF1935, 1:100, R & D Systems), pAb anti-Carbonic Anhydrase IX/CA9 (NB100-417, 1:100, Novus Biologicals), TER-119 (MAB1125, 1:100, R & D Systems), rabbit anti-FoxP1 (ab16645, 1:1.000, Abcam), mouse anti-neurofilament-M (RMO 270, 1:1.500, ThermoFischer), anti-mouse Flt1 (103-M31, 1:100, ReliaTech GmbH), En-1 (4G11, 1:50, DSHB).

    Techniques: In Situ Hybridization, Quantitative RT-PCR

    Blood vessel sprouts invade the developing mouse spinal cord at specific locations and grow in a stereotypical pattern. ( a ) Scheme of SC vascularization during mouse development (E9.5 till E12.5), showing blood vessels (BV, red), the floor plate (FP, orange) and the MN columns (green). ( b – e ) Representative images of SCs at the developmental stages indicated, showing labelled endothelial cells (IB4 + ) and post-mitotic MNs (Isl1/2 + ). ( f – i ) Higher magnifications of insets in b – e . Note blood vessels stay outside the Isl1/2 + domain till E12.5. ( j – m ) Representative images of blood vessel staining (IB4 + ) in the SC combined with ISH for Vegf from E9.5 till E12.5 in mice. At E9.5 ( j ), Vegf is uniformly expressed in the entire SC (white arrowhead: PNVP). From E10.5 till E12.5 ( k – m ) Vegf expression becomes restricted to specific neuronal domains (yellow dotted lines: MN columns; orange arrowheads: FP; blue arrowhead: neuronal progenitors). ( n – q ) Representative images of ISH for Vegf combined with immunostaining for MNs (Isl1/2 + ) confirming that Vegf expression is highly localized and increased in MN columns from E10.5 onwards. Insets show higher magnifications of MN columns. ( r – u ) Representative images of ISH for Vegf combined with immunostaining for the p3 neuronal progenitor domain (Nkx2.2 + ) revealing expression of Vegf in the p3 domain and in the FP (orange arrowheads: FP (domain Nkx2.2 − , below Nkx2.2 + )). Insets show higher magnifications of the FP region (white dotted outlines: FP). Scale bars 100 μm.

    Journal: Nature Communications

    Article Title: Motor neurons control blood vessel patterning in the developing spinal cord

    doi: 10.1038/ncomms14583

    Figure Lengend Snippet: Blood vessel sprouts invade the developing mouse spinal cord at specific locations and grow in a stereotypical pattern. ( a ) Scheme of SC vascularization during mouse development (E9.5 till E12.5), showing blood vessels (BV, red), the floor plate (FP, orange) and the MN columns (green). ( b – e ) Representative images of SCs at the developmental stages indicated, showing labelled endothelial cells (IB4 + ) and post-mitotic MNs (Isl1/2 + ). ( f – i ) Higher magnifications of insets in b – e . Note blood vessels stay outside the Isl1/2 + domain till E12.5. ( j – m ) Representative images of blood vessel staining (IB4 + ) in the SC combined with ISH for Vegf from E9.5 till E12.5 in mice. At E9.5 ( j ), Vegf is uniformly expressed in the entire SC (white arrowhead: PNVP). From E10.5 till E12.5 ( k – m ) Vegf expression becomes restricted to specific neuronal domains (yellow dotted lines: MN columns; orange arrowheads: FP; blue arrowhead: neuronal progenitors). ( n – q ) Representative images of ISH for Vegf combined with immunostaining for MNs (Isl1/2 + ) confirming that Vegf expression is highly localized and increased in MN columns from E10.5 onwards. Insets show higher magnifications of MN columns. ( r – u ) Representative images of ISH for Vegf combined with immunostaining for the p3 neuronal progenitor domain (Nkx2.2 + ) revealing expression of Vegf in the p3 domain and in the FP (orange arrowheads: FP (domain Nkx2.2 − , below Nkx2.2 + )). Insets show higher magnifications of the FP region (white dotted outlines: FP). Scale bars 100 μm.

    Article Snippet: Histology and immunohistochemistry For immunohistochemistry the following primary antibodies were used at the indicated dilutions: Isl1 (40.2D6, 1:100, DSHB), Isl1/2 (39.4D5, 1:200, DSHB), Nkx2.2 (74.5A5, 1:100, DSHB), Olig2 (AB9610, 1:100, Millipore), Pax7 (1:10, DSHB), anti-h/m/r Hif-1a (AF1935, 1:100, R & D Systems), pAb anti-Carbonic Anhydrase IX/CA9 (NB100-417, 1:100, Novus Biologicals), TER-119 (MAB1125, 1:100, R & D Systems), rabbit anti-FoxP1 (ab16645, 1:1.000, Abcam), mouse anti-neurofilament-M (RMO 270, 1:1.500, ThermoFischer), anti-mouse Flt1 (103-M31, 1:100, ReliaTech GmbH), En-1 (4G11, 1:50, DSHB).

    Techniques: Staining, In Situ Hybridization, Mouse Assay, Expressing, Immunostaining

    NRP1 regulates sFlt1 expression and as a consequence blood vessel guidance. ( a – d ) Representative images of ISH for Nrp1 in mouse SC sections at the developmental stages indicated. Note the changes in Nrp1 expression levels and in its expression pattern during development. ( e ) qRT-PCR analysis of Nrp1 in microdissected MN columns at E10.5, E11.5 and E12.5. Data are represented as mean±s.e.m. n =4 individual experiments done in triplicates. ( f – k ) Representative images of ISH for Nrp1 in chicken SC sections at the developmental stages indicated. ( l ) qRT-PCR analysis showing downregulation of Nrp1 , Flt1 ( mFlt1+sFlt1 ), sFlt1 and mFlt1 levels in MN columns of chicken embryos electroporated with Hb9-EGFP-miNRP1#1. Vegf mRNA levels are unaffected. n =7, * P =0.0437 ( Nrp1 ), * P =0.0306 ( Flt1 ), ** P =0.0081 ( sFlt1 ), ** P =0.0055 ( mFlt1 ) and P =ns ( Vegf ). ( m ) Representative image of Hb9-EGFP-miNRP1#1 electroporated chicken embryos showing blood vessel ingression into MN columns in the electroporated side of the SC (yellow arrowhead) but not in the non-electroporated one. ( n ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated and non-electroporated side in Hb9-EGFP-miNRP1#1 embryos. n =8, * P =0.0274. ( o ) Representative image of rescue experiment, in which Hb9-EGFP-miNRP1#1 is co-electroporated with a mouse sFlt1-HA plasmid. ( p ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated and non-electroporated side. n =6, * P =0.0349. Scale bars 100 μm.

    Journal: Nature Communications

    Article Title: Motor neurons control blood vessel patterning in the developing spinal cord

    doi: 10.1038/ncomms14583

    Figure Lengend Snippet: NRP1 regulates sFlt1 expression and as a consequence blood vessel guidance. ( a – d ) Representative images of ISH for Nrp1 in mouse SC sections at the developmental stages indicated. Note the changes in Nrp1 expression levels and in its expression pattern during development. ( e ) qRT-PCR analysis of Nrp1 in microdissected MN columns at E10.5, E11.5 and E12.5. Data are represented as mean±s.e.m. n =4 individual experiments done in triplicates. ( f – k ) Representative images of ISH for Nrp1 in chicken SC sections at the developmental stages indicated. ( l ) qRT-PCR analysis showing downregulation of Nrp1 , Flt1 ( mFlt1+sFlt1 ), sFlt1 and mFlt1 levels in MN columns of chicken embryos electroporated with Hb9-EGFP-miNRP1#1. Vegf mRNA levels are unaffected. n =7, * P =0.0437 ( Nrp1 ), * P =0.0306 ( Flt1 ), ** P =0.0081 ( sFlt1 ), ** P =0.0055 ( mFlt1 ) and P =ns ( Vegf ). ( m ) Representative image of Hb9-EGFP-miNRP1#1 electroporated chicken embryos showing blood vessel ingression into MN columns in the electroporated side of the SC (yellow arrowhead) but not in the non-electroporated one. ( n ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated and non-electroporated side in Hb9-EGFP-miNRP1#1 embryos. n =8, * P =0.0274. ( o ) Representative image of rescue experiment, in which Hb9-EGFP-miNRP1#1 is co-electroporated with a mouse sFlt1-HA plasmid. ( p ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated and non-electroporated side. n =6, * P =0.0349. Scale bars 100 μm.

    Article Snippet: Histology and immunohistochemistry For immunohistochemistry the following primary antibodies were used at the indicated dilutions: Isl1 (40.2D6, 1:100, DSHB), Isl1/2 (39.4D5, 1:200, DSHB), Nkx2.2 (74.5A5, 1:100, DSHB), Olig2 (AB9610, 1:100, Millipore), Pax7 (1:10, DSHB), anti-h/m/r Hif-1a (AF1935, 1:100, R & D Systems), pAb anti-Carbonic Anhydrase IX/CA9 (NB100-417, 1:100, Novus Biologicals), TER-119 (MAB1125, 1:100, R & D Systems), rabbit anti-FoxP1 (ab16645, 1:1.000, Abcam), mouse anti-neurofilament-M (RMO 270, 1:1.500, ThermoFischer), anti-mouse Flt1 (103-M31, 1:100, ReliaTech GmbH), En-1 (4G11, 1:50, DSHB).

    Techniques: Expressing, In Situ Hybridization, Quantitative RT-PCR, Plasmid Preparation

    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

    Pre- and postsynaptic components of in vitro NMJ. ( A ) Expression of the myogenic markers MYH, MEF2C, and AChR. ( B ) Expression of the MN-specific markers HB9, Islet1, and choline acetyltransferase (ChAT) at day 30. ( C ) Expression of the Schwann cell marker S100 (green). Scale bars: 10 μm ( A – C ).

    Journal: JCI Insight

    Article Title: iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases

    doi: 10.1172/jci.insight.124299

    Figure Lengend Snippet: Pre- and postsynaptic components of in vitro NMJ. ( A ) Expression of the myogenic markers MYH, MEF2C, and AChR. ( B ) Expression of the MN-specific markers HB9, Islet1, and choline acetyltransferase (ChAT) at day 30. ( C ) Expression of the Schwann cell marker S100 (green). Scale bars: 10 μm ( A – C ).

    Article Snippet: The primary antibodies used were antineurofilament (MilliporeSigma MAB5254, 1:500), anti–synaptic vesicle protein 2 (DSHB SV2, 1:50), anti-AChRγ (antibodies-online, ABIN926968, 1:200), anti-AChRε (Santa Cruz Biotechnology Inc., sc-376826, 1:200), anti-MYH (MilliporeSigma A4.1025, 1:1000), anti-Islet1 (DSHB 40.2D6, 1:100), anti-HB9 (DSHB 81.5C10, 1:100), anti-ChAT (MilliporeSigma AB144P, 1:100), anti-S100 (Abcam ab14849, 1:300), and anti-Tuj1 (Covance MMS435P, 1:1000). α-Bungarotoxin, Alexa Fluor™ 647 conjugate (Thermo Fisher Scientific) (Molecular Probes, B3545, 0.5 ug/ml) was used to detect AChR.

    Techniques: In Vitro, Expressing, Marker

    Hair cell generation only from Lgr5-expressing neurospheres (A) Neurospheres formed by the Sox2 pos and Sox2 neg fractions incorporated BrdU. (B) Sorted Lgr5 pos and Lgr5 neg cells formed neurospheres that incorporated BrdU. (C) Neurospheres formed by the Sox2 pos and Sox2 neg fractions did not contain hair cells ( Myosin VIIa ). (D) Neurospheres made from sorted Lgr5 pos and Lgr5 neg cells showed no labeling for myosin VIIa. (E) Myosin VIIa-positive cells were observed upon differentiation of spheres made from the Sox2 pos cells but not the Sox2 neg cells. (F) After differentiation, neurospheres made from Lgr5 pos cells generated myosin VIIa-positive cells. No hair cells were observed upon differentiation of neurospheres made from the Lgr5 neg cells. (G) Differentiated Lgr5 pos neurospheres retained Lgr5 and Sox2 surrounding the myosin VIIa-positive cells that lost expression of Lgr5. (H) Myosin VIIa-staining cells from Lgr5 pos spheres co-stained for parvalbumin 3. (I) Cells surrounding the myosin VIIa- positive cells expressed islet1. (J) Neuropheres made from Atoh1 (negatively-sorted), or Sox2 or Lgr5 (positively-sorted) cells had similar rates of cell division. (K) Increasing capacity for hair cell differentiation was found in neurospheres made from Atoh1, Sox2 or Lgr5-sorted cells. Means ± S.E.M. are shown, n=3. Scale bars, 10 μm. (L) Secondary spheres made by sorting Lgr5 pos cells differentiated into myosin VIIa-positive hair cells. No hair cells were observed after differentiation of secondary spheres from Lgr5 neg cells. (M) Clonal spheres from single Lgr5 pos but not from single Lgr5 neg cells from cochlear sensory epithelium gave rise to hair cells upon differentiation.

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

    Article Title: Wnt Responsive Lgr5-Expressing Stem Cells Are Hair Cell Progenitors in the Cochlea

    doi: 10.1523/JNEUROSCI.1064-12.2012

    Figure Lengend Snippet: Hair cell generation only from Lgr5-expressing neurospheres (A) Neurospheres formed by the Sox2 pos and Sox2 neg fractions incorporated BrdU. (B) Sorted Lgr5 pos and Lgr5 neg cells formed neurospheres that incorporated BrdU. (C) Neurospheres formed by the Sox2 pos and Sox2 neg fractions did not contain hair cells ( Myosin VIIa ). (D) Neurospheres made from sorted Lgr5 pos and Lgr5 neg cells showed no labeling for myosin VIIa. (E) Myosin VIIa-positive cells were observed upon differentiation of spheres made from the Sox2 pos cells but not the Sox2 neg cells. (F) After differentiation, neurospheres made from Lgr5 pos cells generated myosin VIIa-positive cells. No hair cells were observed upon differentiation of neurospheres made from the Lgr5 neg cells. (G) Differentiated Lgr5 pos neurospheres retained Lgr5 and Sox2 surrounding the myosin VIIa-positive cells that lost expression of Lgr5. (H) Myosin VIIa-staining cells from Lgr5 pos spheres co-stained for parvalbumin 3. (I) Cells surrounding the myosin VIIa- positive cells expressed islet1. (J) Neuropheres made from Atoh1 (negatively-sorted), or Sox2 or Lgr5 (positively-sorted) cells had similar rates of cell division. (K) Increasing capacity for hair cell differentiation was found in neurospheres made from Atoh1, Sox2 or Lgr5-sorted cells. Means ± S.E.M. are shown, n=3. Scale bars, 10 μm. (L) Secondary spheres made by sorting Lgr5 pos cells differentiated into myosin VIIa-positive hair cells. No hair cells were observed after differentiation of secondary spheres from Lgr5 neg cells. (M) Clonal spheres from single Lgr5 pos but not from single Lgr5 neg cells from cochlear sensory epithelium gave rise to hair cells upon differentiation.

    Article Snippet: Antibodies used in this study were myosin VIIa (1:800, Proteus), myosin VIIa (1:100; DSHB), Sox2 (1:500; Santa Cruz), espin (1:200; Transduction Labs), parvabumin 3 (1:1,000; courtesy of Stefan Heller), Prox1 (1:200; Chemicon), p27kip1 (1:200, NeoMarkers), islet1 (1:100; DSHB), BrdU (1:100; ABD Serotec), GFP (1:1,000; Invitrogen), β-III-tubulin (1:500, Covance) collagen type II (1:100, Chemicon), nestin (1:100, DSHB), CD105 (1:500, BD Pharmingen) and Sca1 (1:500, BD Pharmingen).

    Techniques: Expressing, Labeling, Generated, Staining, Cell Differentiation

    OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the anti-islet-1 mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.

    Journal: The Journal of Neuroscience

    Article Title: Implication of OTX2 in Pigment Epithelium Determination and Neural Retina Differentiation

    doi: 10.1523/JNEUROSCI.17-11-04243.1997

    Figure Lengend Snippet: OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the anti-islet-1 mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.

    Article Snippet: The mAb anti-Islet-1, developed by Prof. T. M. Jessell, was obtained by the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University, School of Medicine (Baltimore, MD), and the Department of Biological Sciences, University of Iowa (Iowa City, IA), under contract N01-HD-2-3144 from the National Institute of Child Health and Human Development.

    Techniques: Staining, Expressing

    Immunohistochemistry of the chick retina at P10. A , cOpn5L2 IR cells in the inner nuclear layer (inl) and ganglion cell layer (gcl) (green, arrowheads). Parvalbumin is visualized in magenta to reveal subsets of amacrine cells in the inner nuclear layer (inl) and sublamina I and V in the inner plexiform layer (ipl) [47] . B , A representative cOpn5L2 IR cell in the INL. C , A representative cOpn5L2 IR cell in the GCL. D , A cOpn5L2 IR cell in the INL after two-color ABC immunostaining. E , A cOpn5m IR cell in the INL of the same retinal section as shown in (D). F-F″ , The cOpn5L2 IR cell (green in F , F″ ) in the GCL is not positive for ChAT (magenta in F′, F″). G-G″ , The cOpn5L2 IR cell (arrow) in the GCL (G, G″) is positive for Islet1 (G′, G″). H-H″ , cOpn5L2 IR cells in the INL (H, H″) are positive for Meis (H′, H″). I-I″ , The cOpn5L2 IR cell in the INL (I, I″) is positive for GAD65/67 (I′, I″). J , J′ , cOpn5L2 IR cells in the INL (green, arrowheads in J) are not positive for ChAT. Some cOpn5L2 IR cells are adjacent to ChAT IR cells (J), and others are separate from the ChAT IR cells (J′). K , One cOpn5L2 IR cell in the vicinity of a serotonin IR cell, while the other is located apart from it. L , cOpn5L2 IR cells are not positive for VIP. M , A cOpn5L2 IR cell adjacent to a TH IR cell. For all images, DAPI is blue. Scale bars, 50 µm (A), 10 µm (B–E, G-I″, K–M), and 20 µm (F-F″, J, J′).

    Journal: PLoS ONE

    Article Title: A Non-Mammalian Type Opsin 5 Functions Dually in the Photoreceptive and Non-Photoreceptive Organs of Birds

    doi: 10.1371/journal.pone.0031534

    Figure Lengend Snippet: Immunohistochemistry of the chick retina at P10. A , cOpn5L2 IR cells in the inner nuclear layer (inl) and ganglion cell layer (gcl) (green, arrowheads). Parvalbumin is visualized in magenta to reveal subsets of amacrine cells in the inner nuclear layer (inl) and sublamina I and V in the inner plexiform layer (ipl) [47] . B , A representative cOpn5L2 IR cell in the INL. C , A representative cOpn5L2 IR cell in the GCL. D , A cOpn5L2 IR cell in the INL after two-color ABC immunostaining. E , A cOpn5m IR cell in the INL of the same retinal section as shown in (D). F-F″ , The cOpn5L2 IR cell (green in F , F″ ) in the GCL is not positive for ChAT (magenta in F′, F″). G-G″ , The cOpn5L2 IR cell (arrow) in the GCL (G, G″) is positive for Islet1 (G′, G″). H-H″ , cOpn5L2 IR cells in the INL (H, H″) are positive for Meis (H′, H″). I-I″ , The cOpn5L2 IR cell in the INL (I, I″) is positive for GAD65/67 (I′, I″). J , J′ , cOpn5L2 IR cells in the INL (green, arrowheads in J) are not positive for ChAT. Some cOpn5L2 IR cells are adjacent to ChAT IR cells (J), and others are separate from the ChAT IR cells (J′). K , One cOpn5L2 IR cell in the vicinity of a serotonin IR cell, while the other is located apart from it. L , cOpn5L2 IR cells are not positive for VIP. M , A cOpn5L2 IR cell adjacent to a TH IR cell. For all images, DAPI is blue. Scale bars, 50 µm (A), 10 µm (B–E, G-I″, K–M), and 20 µm (F-F″, J, J′).

    Article Snippet: The anti-Islet1 antibody (40.2D6) developed by Jessell T. M. and Brenner-Morton S. was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.

    Techniques: Immunohistochemistry, Immunostaining

    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

    Effect of fluorofenidone on Islet-1 expression in murine retinas

    Journal: International Journal of Ophthalmology

    Article Title: Effect of pyridone agent on blood-retinal barrier in diabetic mice

    doi: 10.18240/ijo.2017.06.09

    Figure Lengend Snippet: Effect of fluorofenidone on Islet-1 expression in murine retinas

    Article Snippet: Total protein of murine retinas were collected and resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gel, then it was transferred onto a nitrocellulose membrane and incubated with anti-Islet-1 (Abcam, UK), anti-VEGF (Abcam, UK), anti-Albumin(Abcam, UK), anti-occludin (Abcam, UK) and anti-β-actin antibodies (Sigma, USA).

    Techniques: Expressing

    Sox13 overexpression partly antagonizes Phox2b activity. (a-m

    Journal: Neural Development

    Article Title: Identification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors

    doi: 10.1186/1749-8104-3-14

    Figure Lengend Snippet: Sox13 overexpression partly antagonizes Phox2b activity. (a-m") Chicken neural tubes electroporated at HH 12–13 either separately with pCAGGS::Phox2b-IRES-EGFP (a-a", d-d", g-g", j-j") or pCAGGS::Sox13-IRES-EGFP (b-b", e-e", h-h", k-k") or with pCAGGS::Phox2b-IRES-EGFP plus pCAGGS::Sox13-IRES-EGFP (c-c", f-f", i-i", l-l", m-m") were analyzed 48 h after electroporation. Transverse sections were stained with anti-GFP antibodies in combination with anti-NeuN (a-c"), anti-Sox2 (g-i"), anti-Islet1/2 (j-l") or anti-Lhx1,5 immunohistochemistry (m-m") or in combination with anti-BrdU antibodies (d-f") after BrdU injection into the amniotic cavity 2 h before fixation. Phox2b induces and Sox13 represses NeuN (asterisks in (a', a", b')) in the ML. When co-expressed with Sox13 , Phox2b is still capable of inducing NeuN, which, however, is now also switched on in the VZ (c', c"). Cells electroporated with Phox2b are BrdU-negative whether Sox13 was co-transfected (f) or not (d). Cells electroporated with Sox13 whether co-transfected with Phox2b (i) or not (h) express Sox2, while cells electroporated with Phox2b alone are always Sox2-negative (g). Islet1,2 induction by Phox2b (j) is abolished by co-transfection of Sox13 (l). Repression of Lhx1,5 by Phox2b is not prevented by co-expressing Sox13 (m). Co-transfection of Sox13 together with Phox2b inhibits relocation to the ML (c, f, i, l, m) implemented by expression of Phox2b alone (a, d, g, j).

    Article Snippet: The following antibodies were used: rabbit anti-axonin-1 [ ], mouse monoclonal anti-BrdU (1/100; Sigma), mouse monoclonal (Roche, Basel, Switzerland) and rabbit (Chemicon, Temecula, CA, USA) anti-GFP (1/400), mouse anti-Islet1,2 (1/100) [ ], rabbit anti-Islet1 (1/500; Abcam, Paris, France), mouse anti-Lhx1,5 (Developmental Studies Hybridoma Bank), mouse anti-NeuN (1/500), rabbit anti-Sox2 (1/1,000; Abcam), rabbit anti-Sox13 [ ] and adequate fluorescent secondary antibodies.

    Techniques: Over Expression, Activity Assay, Electroporation, Staining, Immunohistochemistry, Injection, Transfection, Cotransfection, Expressing

    Sfrp1 partially restores a commissural axonal phenotype. (a-c) Chicken neural tubes electroporated at HH12-13 with pCAGGS::Sfrp1-IRES-EGFP (a), pCAGGS::Phox2b-IRES-EGFP (b) or pCAGGS::Sfrp1-IRES-EGFP plus pCAGGS::Phox2b-IRES-EGFP (c) were analyzed by anti-GFP immunohistochemistry. Expression of Sfrp1 did not grossly alter the morphology of neuroepithelial cells, neither did it prevent Phox2b from promoting relocation to the ML. However, Sfrp1 prevented Phox2b from repressing the growth of commissural axons (arrowheads). (d-d

    Journal: Neural Development

    Article Title: Identification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors

    doi: 10.1186/1749-8104-3-14

    Figure Lengend Snippet: Sfrp1 partially restores a commissural axonal phenotype. (a-c) Chicken neural tubes electroporated at HH12-13 with pCAGGS::Sfrp1-IRES-EGFP (a), pCAGGS::Phox2b-IRES-EGFP (b) or pCAGGS::Sfrp1-IRES-EGFP plus pCAGGS::Phox2b-IRES-EGFP (c) were analyzed by anti-GFP immunohistochemistry. Expression of Sfrp1 did not grossly alter the morphology of neuroepithelial cells, neither did it prevent Phox2b from promoting relocation to the ML. However, Sfrp1 prevented Phox2b from repressing the growth of commissural axons (arrowheads). (d-d") Chicken neural tubes electroporated with pCAGGS::Sfrp1-IRES-EGFP and a Phox2b expression vector were analyzed by anti-GFP and anti-Phox2b immunohistochemistry as indicated. Virtually all the transfected cells express both GFP and Phox2b. (e-e") Chicken neural tubes electroporated with pCAGGS::Sfrp1-IRES-EGFP plus pCAGGS::Phox2b-IRES-EGFP were analyzed by anti-GFP and anti-Islet1,2 immunohistochemistry as indicated. Co-expression of Sfrp1 did not prevent Phox2b from inducing Islet1,2 (bracket). ( f-h') Chicken neural tubes electroporated with pCAGGS::Phox2b-IRES-EGFP (f, f'), pCAGGS::Sfrp1-IRES-EGFP (g, g') or with pCAGGS::Sfrp1-IRES-EGFP plus pCAGGS::Phox2b-IRES-EGFP (h, h') were analyzed by anti-axonin-1 and anti-GFP immunohistochemistry. In (f, g, h), the anti-axonin-1 staining is shown alone, and in (f', g', h') it is merged with the anti-GFP immunofluorescence. The fascicle formed by the commissural fibers en route to the floor plate is marked by an asterisk; it is absent after Phox2b transfection and partially restored by co-expressing Sfrp1 .

    Article Snippet: The following antibodies were used: rabbit anti-axonin-1 [ ], mouse monoclonal anti-BrdU (1/100; Sigma), mouse monoclonal (Roche, Basel, Switzerland) and rabbit (Chemicon, Temecula, CA, USA) anti-GFP (1/400), mouse anti-Islet1,2 (1/100) [ ], rabbit anti-Islet1 (1/500; Abcam, Paris, France), mouse anti-Lhx1,5 (Developmental Studies Hybridoma Bank), mouse anti-NeuN (1/500), rabbit anti-Sox2 (1/1,000; Abcam), rabbit anti-Sox13 [ ] and adequate fluorescent secondary antibodies.

    Techniques: Immunohistochemistry, Expressing, Plasmid Preparation, Transfection, Staining, Immunofluorescence

    MED17.11 express the early neuronal markers FOX3 (NeuN), Isl1 and Tuj1, and can be transfected with GFP. Immunolabelling of MED17.11 cells cultured in permissive conditions for large T antigen expression. GFP transgene expression in MED17.11. Scale bar is 100 μm.

    Journal: PLoS ONE

    Article Title: Mouse DRG Cell Line with Properties of Nociceptors

    doi: 10.1371/journal.pone.0128670

    Figure Lengend Snippet: MED17.11 express the early neuronal markers FOX3 (NeuN), Isl1 and Tuj1, and can be transfected with GFP. Immunolabelling of MED17.11 cells cultured in permissive conditions for large T antigen expression. GFP transgene expression in MED17.11. Scale bar is 100 μm.

    Article Snippet: The polyclonal antibodies used were Isl1 (1:250, Abcam), FOX3 (NeuN, 1:250, Millipore), SOX10 (1:250, Abcam), Advillin (1:500, Abcam), TrkA, TrkB and TrkC (1:500, Alomone), Nav1.3 (1:500, Alomone), and SV40 (1:250, Santa Cruz).

    Techniques: Transfection, Cell Culture, Expressing

    ISL1 directly regulates a number of genes required for normal pacemaker function in mice and human.

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: ISL1 directly regulates a number of genes required for normal pacemaker function in mice and human.

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Mouse Assay

    Bradycardia and reduced TBX3 and HCN4 expression following ablation of Isl1 during later SAN morphogenesis. ( A ) Ablation of Isl1 at E11.5 led to significantly slower heart rate at E12.5 and E14.5 ( n = 15 per group, P

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: Bradycardia and reduced TBX3 and HCN4 expression following ablation of Isl1 during later SAN morphogenesis. ( A ) Ablation of Isl1 at E11.5 led to significantly slower heart rate at E12.5 and E14.5 ( n = 15 per group, P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Expressing

    Bradycardia and loss of SAN cells in Isl1 compound mutants. ISL1-nLacZ was expressed in SV myocardium, including the SAN region (red arrow), and mesocardium at E9.5 ( A and C ) and E11.5 ( G and I ). Expression of ISL1 and HCN4 in the SV region of Isl1 compound mutant embryos was significantly reduced ( E and F ). Expression of ISL1 and the number of ISL1-expressing cells in the SV, SAN (red arrow), and DM was markedly reduced in Isl1 compound mutant embryos at E9.5 ( B and D ) and E11.5 ( H and J ). BrdU staining revealed significantly reduced proliferation of SV myocardium in Isl1 compound mutants at E9.5 ( K – M ). TUNEL labeling showed significantly increased cell death in the SV of Isl1 compound mutant embryos at E10.5 ( N – P ) ( n = 4 per group. Scale bars as shown). Echocardiography revealed a significant reduction in the heart rate of Isl1 compound mutant embryos at E9.5 and E11.5 ( Q ). n = 15 per group; * P

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: Bradycardia and loss of SAN cells in Isl1 compound mutants. ISL1-nLacZ was expressed in SV myocardium, including the SAN region (red arrow), and mesocardium at E9.5 ( A and C ) and E11.5 ( G and I ). Expression of ISL1 and HCN4 in the SV region of Isl1 compound mutant embryos was significantly reduced ( E and F ). Expression of ISL1 and the number of ISL1-expressing cells in the SV, SAN (red arrow), and DM was markedly reduced in Isl1 compound mutant embryos at E9.5 ( B and D ) and E11.5 ( H and J ). BrdU staining revealed significantly reduced proliferation of SV myocardium in Isl1 compound mutants at E9.5 ( K – M ). TUNEL labeling showed significantly increased cell death in the SV of Isl1 compound mutant embryos at E10.5 ( N – P ) ( n = 4 per group. Scale bars as shown). Echocardiography revealed a significant reduction in the heart rate of Isl1 compound mutant embryos at E9.5 and E11.5 ( Q ). n = 15 per group; * P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Expressing, Mutagenesis, BrdU Staining, TUNEL Assay, Labeling

    Reduced expression of Hcn4 , Tbx3 , and Shox2 in the SAN region of Isl1 compound mutant embryos. At E9.5, Hcn4 and Shox2 were expressed in the SV, and SAN region (red arrow; A , C , E , and G ). Tbx3 was expressed in the SV and surrounding mesenchyme (red arrow; I and K ). In Isl1 compound mutant embryos, expression of Hcn4, Shox2, and Tbx3 in the SV and SAN region was markedly reduced ( B , D , F , H , J , and L ). Cx40 and Nkx2-5 were expressed in working myocardium but not in the SAN region ( M , O , Q , and S In Isl1 compound mutant embryos, expression of Cx40 and Nkx2-5 was markedly reduced in atrial myocardium, but no expansion or ectopic expression of Cx40 or Nkx2-5 was observed in the SAN region ( N , P , R , and T ). n = 4 per group, Scale bars as shown.

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: Reduced expression of Hcn4 , Tbx3 , and Shox2 in the SAN region of Isl1 compound mutant embryos. At E9.5, Hcn4 and Shox2 were expressed in the SV, and SAN region (red arrow; A , C , E , and G ). Tbx3 was expressed in the SV and surrounding mesenchyme (red arrow; I and K ). In Isl1 compound mutant embryos, expression of Hcn4, Shox2, and Tbx3 in the SV and SAN region was markedly reduced ( B , D , F , H , J , and L ). Cx40 and Nkx2-5 were expressed in working myocardium but not in the SAN region ( M , O , Q , and S In Isl1 compound mutant embryos, expression of Cx40 and Nkx2-5 was markedly reduced in atrial myocardium, but no expansion or ectopic expression of Cx40 or Nkx2-5 was observed in the SAN region ( N , P , R , and T ). n = 4 per group, Scale bars as shown.

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Expressing, Mutagenesis

    Bradycardia and loss of SAN cells following ablation of Isl1 in SAN during early developmental stages using Hcn4-CreERT2 . Isl1 mutant ( Hcn4-CreERT2 Isl1 fl/fl ) and control ( Hcn4-CreERT2 Isl1 fl/+ or +/+ ) embryos were given tamoxifen at E9.5. Embryos were analyzed 36 and 48 hours after induction. ( A ) Echocardiography revealed that the heart rate of Isl1 mutants was significantly reduced at E11 and was further reduced at E11.5 ( n = 20 per group). ( B – D ) Whole-mount X-gal staining and quantitative analysis revealed a significantly reduced number of X-gal + and Tomato + cells in the SAN (red arrow) of Isl1 mutants relative to control littermates at E11.5 ( n = 4. Scale bars as shown). ( D – H ) Immunostaining demonstrated significantly reduced expression of HCN4 and TBX3 in the SAN of Isl1 mutants compared with controls marked by Tomato + at E11.5. However, a slight but not significant reduction in the number of Hcn4 lineage–labeled cells in Isl1 mutant SAN region was observed when analyzed at E11 ( D , I , and J ). ( K – M ) TUNEL revealed increased cell death in Isl1 mutant SAN marked by Tomato + . ( N – P ) BrdU revealed decreased proliferation in Isl1 mutant SAN marked by Tomato. n = 4; * P

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: Bradycardia and loss of SAN cells following ablation of Isl1 in SAN during early developmental stages using Hcn4-CreERT2 . Isl1 mutant ( Hcn4-CreERT2 Isl1 fl/fl ) and control ( Hcn4-CreERT2 Isl1 fl/+ or +/+ ) embryos were given tamoxifen at E9.5. Embryos were analyzed 36 and 48 hours after induction. ( A ) Echocardiography revealed that the heart rate of Isl1 mutants was significantly reduced at E11 and was further reduced at E11.5 ( n = 20 per group). ( B – D ) Whole-mount X-gal staining and quantitative analysis revealed a significantly reduced number of X-gal + and Tomato + cells in the SAN (red arrow) of Isl1 mutants relative to control littermates at E11.5 ( n = 4. Scale bars as shown). ( D – H ) Immunostaining demonstrated significantly reduced expression of HCN4 and TBX3 in the SAN of Isl1 mutants compared with controls marked by Tomato + at E11.5. However, a slight but not significant reduction in the number of Hcn4 lineage–labeled cells in Isl1 mutant SAN region was observed when analyzed at E11 ( D , I , and J ). ( K – M ) TUNEL revealed increased cell death in Isl1 mutant SAN marked by Tomato + . ( N – P ) BrdU revealed decreased proliferation in Isl1 mutant SAN marked by Tomato. n = 4; * P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Mutagenesis, Staining, Immunostaining, Expressing, Labeling, TUNEL Assay

    RNA-seq analyses reveal dysregulation of a number of genes important for SAN function in Hcn4-CreERT2 Isl1 fl/fl mutants. ( A ) Scatterplot illustrating relative gene expression of polyA-selected RNA transcripts from RNA-seq comparison of control and Hcn4-CreERT2 Isl1 fl/fl mutant SAN cells. Genes upregulated or downregulated 1.5-fold in Isl1 mutant SAN cells are shown in red and green, respectively. Values are presented as log2 of tag counts normalized to 10 7 uniquely mapped tags. ( B ) RNA-seq comparison of control and Hcn4-CreERT2 Isl1 fl/fl mutant SAN transcriptomes revealed a total of 12,441 genes expressed (RPKM ≥ 1) in SAN cells, of which 1,035 upregulated and 3,690 downregulated in Isl1 mutant SAN cells ( |fold-change mutant vs. ctrl| ≥ 1.5). ( C ) GO functional clustering of genes down- and upregulated in Isl1 mutant, highlighting cellular processes most significantly affected in mutant SAN (top 10 not redundant categories are shown). ( D ) qPCR validation analysis. mRNA expression of ion channels and associated genes, and genes involved in transcription regulation, cell cycle, and signaling pathways are shown. ( E ) qRT-PCR validation analysis. mRNA expression of atrial myocardial specific genes. Results are shown as fold-change Isl1 mutant vs. ctrl. n = 4 per group, P

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: RNA-seq analyses reveal dysregulation of a number of genes important for SAN function in Hcn4-CreERT2 Isl1 fl/fl mutants. ( A ) Scatterplot illustrating relative gene expression of polyA-selected RNA transcripts from RNA-seq comparison of control and Hcn4-CreERT2 Isl1 fl/fl mutant SAN cells. Genes upregulated or downregulated 1.5-fold in Isl1 mutant SAN cells are shown in red and green, respectively. Values are presented as log2 of tag counts normalized to 10 7 uniquely mapped tags. ( B ) RNA-seq comparison of control and Hcn4-CreERT2 Isl1 fl/fl mutant SAN transcriptomes revealed a total of 12,441 genes expressed (RPKM ≥ 1) in SAN cells, of which 1,035 upregulated and 3,690 downregulated in Isl1 mutant SAN cells ( |fold-change mutant vs. ctrl| ≥ 1.5). ( C ) GO functional clustering of genes down- and upregulated in Isl1 mutant, highlighting cellular processes most significantly affected in mutant SAN (top 10 not redundant categories are shown). ( D ) qPCR validation analysis. mRNA expression of ion channels and associated genes, and genes involved in transcription regulation, cell cycle, and signaling pathways are shown. ( E ) qRT-PCR validation analysis. mRNA expression of atrial myocardial specific genes. Results are shown as fold-change Isl1 mutant vs. ctrl. n = 4 per group, P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: RNA Sequencing Assay, Expressing, Mutagenesis, Functional Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Expression of ISL1 in pacemaker cells of the SAN during development and after birth. ISL1 was coexpressed with HCN4 in myocardium of the SV at E9.5 ( A ), and in the majority of SAN cells from E10.5–P7 ( B – G ). ISL1 expression did not overlap with Cx40, which is expressed in atrial myocardium ( E and G ). The boxed area in H delineates regions depicted in F and G . The fraction of HCN4 cells that expressed Isl1 remained constant at early stages from E11.5–E14.5, but decreased at E18 ( I ). After birth, the fraction of HCN4 cells that expressed Isl1 decreased significantly ( I ). n = 4, P

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: Expression of ISL1 in pacemaker cells of the SAN during development and after birth. ISL1 was coexpressed with HCN4 in myocardium of the SV at E9.5 ( A ), and in the majority of SAN cells from E10.5–P7 ( B – G ). ISL1 expression did not overlap with Cx40, which is expressed in atrial myocardium ( E and G ). The boxed area in H delineates regions depicted in F and G . The fraction of HCN4 cells that expressed Isl1 remained constant at early stages from E11.5–E14.5, but decreased at E18 ( I ). After birth, the fraction of HCN4 cells that expressed Isl1 decreased significantly ( I ). n = 4, P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Expressing

    PSCs to cardiogenic mesoderm expressing KDR and ISL1 a Schematic of differentiating hPSCs to CPCs, endocardial progenitor cells and VEPs. b The qPCR analysis of day 3 hPSC-derived CPCs for the indicated markers. c IF staining of day 3 CPCs showing that the majority of cells were ISL1 positive. Scale bar: 50 μm. d Quantification analysis of panel (c) by ImageJ. Bar graph represents percentage of ISL1 positive cells ± S.D of three independent experiments. e WB analysis of day 3 CPCs using the indicated antibodies. f Flow cytometry analysis showing percentage of KDR and ISL1 positive cells. g The representative morphology of cells daily during hPSC differentiation to CPCs. All experiments were repeated 3 times. Significant levels are: * p

    Journal: bioRxiv

    Article Title: Generation of cardiac valve endocardial like cells from human pluripotent stem cells

    doi: 10.1101/2020.04.20.050161

    Figure Lengend Snippet: PSCs to cardiogenic mesoderm expressing KDR and ISL1 a Schematic of differentiating hPSCs to CPCs, endocardial progenitor cells and VEPs. b The qPCR analysis of day 3 hPSC-derived CPCs for the indicated markers. c IF staining of day 3 CPCs showing that the majority of cells were ISL1 positive. Scale bar: 50 μm. d Quantification analysis of panel (c) by ImageJ. Bar graph represents percentage of ISL1 positive cells ± S.D of three independent experiments. e WB analysis of day 3 CPCs using the indicated antibodies. f Flow cytometry analysis showing percentage of KDR and ISL1 positive cells. g The representative morphology of cells daily during hPSC differentiation to CPCs. All experiments were repeated 3 times. Significant levels are: * p

    Article Snippet: The blots were incubated over night at 4℃ with primary antibodies against Anti-CD31 (Abcam, ab28364, 1:500), Anti VE-cadherin (Abcam, ab33168, 1:1000), Anti-NFATc1 (Abcam, ab2796, 1:2000), Anti-ISLET1 (Abcam, ab20670, 1:1000), FSP1 (Abcam, ab124805, 1:1000), anti-TBX2 (Proteintech, 22346-1-AP, 1: 200), Anti-BMP4 (Abcam, ab39973, 1:1000) and Anti-GAPDH (Santa Cruz, sc-25778, 1:1000), followed by incubation with a HRP-conjugated goat anti-rabbit IgG (GtxRb-003-DHRPX, ImmunoReagents, 1:5000), a HRP-linked anti-mouse IgG (7076S, Cell Signaling Technology, 1:5000) for 1 hour at room temperature.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Staining, Western Blot, Flow Cytometry

    Effect of RCASBP(B)-RFP-GluA2 RNAi infection on chicken embryos. A–B ) Expression of red fluorescence protein (RFP) transgene in the lumbar spinal cord of E6 ( A ) and E11 ( B ) chicken embryos following retroviral infection with an RCASBP(B)-RFP-GluA2 RNAi construct. Embryos infected with the RCASBP(B)-RFP-GluA2 RNAi construct show strong fluorescent labeling throughout the whole spinal cord cross section. cc = central canal, nc = notochord. C ) Averaged number of labeled neurons for the RCASBP(B) viral protein p27 gag as a percent of the total number of neuron labeled with the motoneuron marker Islet1/2 in chicken embryos infected with an RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP- GluA2 RNAi construct. Lumbar ventral neurons were isolated from E6, E8 or E11 chicken embryos and immunolabeled with p27 gag and Islet1/2 in order to assess the extent of viral infection of spinal motoneurons. Notice that ≥60% of infected cells also tested positive for the motoneuron marker Islet1/2 at all ages tested. D ) Representative example of Western Blot data collected from the E6 and E11 ventral spinal cords from control chicken embryos (non-infected) or embryos infected with an RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi constructs. The anti-GluA2 antibody detected a band with a relative molecular weight of ∼102 kD. To normalize for changes in protein loading in each well, membranes were reprobed for β-actin (∼42 kD). Infection of chicken embryos with an RCASBP(B)-RFP- GluA2 RNAi construct causes a significant reduction in GluA2 expression at E11 as determined by immunoblot analysis. E ) Expression of GluA2 protein as a function of β-actin in chicken ventral spinal cords. The age-dependent increase in GluA2 protein expression between E6 and E11 chicken spinal cords was reversed by infection of chicken embryos with an RCASBP(B)-RFP- GluA2 RNAi construct. In these experiments, RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi viral particles were injected into the developing neural tube at E2 (approximately 36 hr after incubation). Embryos were allowed to develop up to E11 before tissue isolation and processing.

    Journal: PLoS ONE

    Article Title: Downregulation of GluA2 AMPA Receptor Subunits Reduces the Dendritic Arborization of Developing Spinal Motoneurons

    doi: 10.1371/journal.pone.0049879

    Figure Lengend Snippet: Effect of RCASBP(B)-RFP-GluA2 RNAi infection on chicken embryos. A–B ) Expression of red fluorescence protein (RFP) transgene in the lumbar spinal cord of E6 ( A ) and E11 ( B ) chicken embryos following retroviral infection with an RCASBP(B)-RFP-GluA2 RNAi construct. Embryos infected with the RCASBP(B)-RFP-GluA2 RNAi construct show strong fluorescent labeling throughout the whole spinal cord cross section. cc = central canal, nc = notochord. C ) Averaged number of labeled neurons for the RCASBP(B) viral protein p27 gag as a percent of the total number of neuron labeled with the motoneuron marker Islet1/2 in chicken embryos infected with an RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP- GluA2 RNAi construct. Lumbar ventral neurons were isolated from E6, E8 or E11 chicken embryos and immunolabeled with p27 gag and Islet1/2 in order to assess the extent of viral infection of spinal motoneurons. Notice that ≥60% of infected cells also tested positive for the motoneuron marker Islet1/2 at all ages tested. D ) Representative example of Western Blot data collected from the E6 and E11 ventral spinal cords from control chicken embryos (non-infected) or embryos infected with an RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi constructs. The anti-GluA2 antibody detected a band with a relative molecular weight of ∼102 kD. To normalize for changes in protein loading in each well, membranes were reprobed for β-actin (∼42 kD). Infection of chicken embryos with an RCASBP(B)-RFP- GluA2 RNAi construct causes a significant reduction in GluA2 expression at E11 as determined by immunoblot analysis. E ) Expression of GluA2 protein as a function of β-actin in chicken ventral spinal cords. The age-dependent increase in GluA2 protein expression between E6 and E11 chicken spinal cords was reversed by infection of chicken embryos with an RCASBP(B)-RFP- GluA2 RNAi construct. In these experiments, RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi viral particles were injected into the developing neural tube at E2 (approximately 36 hr after incubation). Embryos were allowed to develop up to E11 before tissue isolation and processing.

    Article Snippet: Sections were incubated overnight at 4°C with an anti-Islet1/2 (1∶100 mouse hybridoma supernatant, clone 39.405, Developmental Studies Hybridoma Bank, University of Iowa) diluted in blocking solution.

    Techniques: Infection, Expressing, Fluorescence, Construct, Labeling, Marker, Isolation, Immunolabeling, Western Blot, Molecular Weight, Injection, Incubation

    Effect of GluA2 RNAi on motoneurons survival. A ) Islet1/2 -staining in the lumbar spinal cord of E11 chicken embryos. Only Islet1/2 -positive neurons in the motoneurons pool were counted (circled area). Islet1/2 -positive interneurons located in dorsal and medial portions of the spinal cord were not included in our measurements. B ) Total number of Islet1/2 -positive neurons in the lumbar spinal cord of E11 chicken embryos in control (non-infected) and in RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi–infected embryos. Infection of chicken embryos with the RCASBP(B)-RFP-GluA2 RNAi has no overall effect on motoneuron survival when compared with control or RCASBP(B)-RFP-scrambled RNAi-infected embryos.

    Journal: PLoS ONE

    Article Title: Downregulation of GluA2 AMPA Receptor Subunits Reduces the Dendritic Arborization of Developing Spinal Motoneurons

    doi: 10.1371/journal.pone.0049879

    Figure Lengend Snippet: Effect of GluA2 RNAi on motoneurons survival. A ) Islet1/2 -staining in the lumbar spinal cord of E11 chicken embryos. Only Islet1/2 -positive neurons in the motoneurons pool were counted (circled area). Islet1/2 -positive interneurons located in dorsal and medial portions of the spinal cord were not included in our measurements. B ) Total number of Islet1/2 -positive neurons in the lumbar spinal cord of E11 chicken embryos in control (non-infected) and in RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi–infected embryos. Infection of chicken embryos with the RCASBP(B)-RFP-GluA2 RNAi has no overall effect on motoneuron survival when compared with control or RCASBP(B)-RFP-scrambled RNAi-infected embryos.

    Article Snippet: Sections were incubated overnight at 4°C with an anti-Islet1/2 (1∶100 mouse hybridoma supernatant, clone 39.405, Developmental Studies Hybridoma Bank, University of Iowa) diluted in blocking solution.

    Techniques: Staining, Infection

    Evf-2  and Dlx-2 form a complex in vivo. ( a ) Nuclear extracts made from C17 neural cells transfected with Flag-tagged Emx-1, Flag-tagged Dlx-2, or pcDNA control were analyzed for the presence of  Evf-2 –Dlx-2 complexes by immunoprecipitation with anti-Flag antibody, followed by RT–PCR against  Evf-2 -specific primers and S17 control primers. Western analysis shows that both Flag-Emx-1 and Flag-Dlx-2 are present in nuclear extracts transfected with constructs expressing these proteins. ( b ) Nuclear extracts made from rat E11.5 BAs were analyzed for the presence of  Evf-2 /Dlx-2 complexes by immunoprecipitation with anti- dll,  anti-Islet 1/2, or anti-IgG antibody, followed by RT–PCR against  Evf-2 -specific primers or GAPDH. ( c ) Single-cell suspensions made from mouse E12.5 dorsal and ventral telencephalon were dissected as shown in the schematic, centrifuged onto slides, and processed for fluorescent in situ/immunolocalization using  Evf-2  antisense RNA probe and anti- dll  antibody. DAPI staining (blue) reveals nuclei.  Evf-2  RNA (Alexa fluor 568) is in red, Dlx (Alexa fluor 488) is in green, and regions of overlap are in yellow. ( d ) A model proposing that a complex of  Evf-2  and Dlx-2 affects ei activity, ultimately affecting transcription of the Dlx-5 and Dlx-6 genes.

    Journal: Genes & Development

    Article Title: The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator

    doi: 10.1101/gad.1416106

    Figure Lengend Snippet: Evf-2 and Dlx-2 form a complex in vivo. ( a ) Nuclear extracts made from C17 neural cells transfected with Flag-tagged Emx-1, Flag-tagged Dlx-2, or pcDNA control were analyzed for the presence of Evf-2 –Dlx-2 complexes by immunoprecipitation with anti-Flag antibody, followed by RT–PCR against Evf-2 -specific primers and S17 control primers. Western analysis shows that both Flag-Emx-1 and Flag-Dlx-2 are present in nuclear extracts transfected with constructs expressing these proteins. ( b ) Nuclear extracts made from rat E11.5 BAs were analyzed for the presence of Evf-2 /Dlx-2 complexes by immunoprecipitation with anti- dll, anti-Islet 1/2, or anti-IgG antibody, followed by RT–PCR against Evf-2 -specific primers or GAPDH. ( c ) Single-cell suspensions made from mouse E12.5 dorsal and ventral telencephalon were dissected as shown in the schematic, centrifuged onto slides, and processed for fluorescent in situ/immunolocalization using Evf-2 antisense RNA probe and anti- dll antibody. DAPI staining (blue) reveals nuclei. Evf-2 RNA (Alexa fluor 568) is in red, Dlx (Alexa fluor 488) is in green, and regions of overlap are in yellow. ( d ) A model proposing that a complex of Evf-2 and Dlx-2 affects ei activity, ultimately affecting transcription of the Dlx-5 and Dlx-6 genes.

    Article Snippet: The C17 nuclear extracts were then incubated with mouse anti-Flag (Sigma) prebound to protein G-agarose, whereas the BA nuclear extracts were incubated with anti- dll (affinity-purified as described by ), anti-Islet 1/2 (40.2D6, Developmental Hybridoma Studies Bank), or anti-rabbit IgG prebound to protein G-agarose overnight at 4°C.

    Techniques: In Vivo, Transfection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Construct, Expressing, In Situ, Staining, Activity Assay

    Loss of SMN reactivates the cell cycle. a Ki67 and ISL1 immunostaining analysis of wild-type (BJ iPS and 18a), SMA Type I (1-38 G), and SMA Type II (1-51 N) motor neuron cultures at day 28. The percentages of ISL1 + Ki67 + cells amongst all ISL1 + motor neurons are shown. b Knockdown of SMN in wild-type cell line (BJ-iPS) increased the percentage of ISL1 + motor neurons co-expressing Ki67. c Co-staining of ISL1 (red) and Ki67 (green) showing increased Ki67 + cells upon SMN knockdown in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Ki67 and cCASP3 immunostaining analysis of wild-type motor neurons demonstrated higher cCASP3 expression in Ki67 + motor neurons than Ki67 − motor neurons. *** p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: Loss of SMN reactivates the cell cycle. a Ki67 and ISL1 immunostaining analysis of wild-type (BJ iPS and 18a), SMA Type I (1-38 G), and SMA Type II (1-51 N) motor neuron cultures at day 28. The percentages of ISL1 + Ki67 + cells amongst all ISL1 + motor neurons are shown. b Knockdown of SMN in wild-type cell line (BJ-iPS) increased the percentage of ISL1 + motor neurons co-expressing Ki67. c Co-staining of ISL1 (red) and Ki67 (green) showing increased Ki67 + cells upon SMN knockdown in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Ki67 and cCASP3 immunostaining analysis of wild-type motor neurons demonstrated higher cCASP3 expression in Ki67 + motor neurons than Ki67 − motor neurons. *** p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Immunostaining, Expressing, Staining

    Inhibition of CDKs prolongs SMA motor neuron survival. a Graphical representation of ISL1 + SMA type I motor neurons (1-38 G) treated with various CDKs inhibitors treatment. The blue dotted line indicates percentage of motor neurons relative to DMSO-treated motor neurons. b Quantification of ISL1 + SMA type II motor neurons (1-51 N) treated with various CDKs inhibitors treatment. The blue dotted line indicates percentage of motor neurons relative to DMSO-treated motor neurons. c ISL1 immunostaining analysis of various CDKs knockdown in SMA type II motor neurons. The blue dotted line indicates percentage of motor neurons survival relative to non-targeting siRNA treated motor neurons. d Representative images of SMA type II motor neurons treated with various CDKs siRNA and stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 50 μm. e Western blot of SMA type II motor neurons treated with various CDKs inhibitors, indicating that SMN levels remained the same. f Quantification of SMN levels of SMA type II motor neurons treated with various CDKs inhibitors relative to α-tubulin expression. The values were not significant. * p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: Inhibition of CDKs prolongs SMA motor neuron survival. a Graphical representation of ISL1 + SMA type I motor neurons (1-38 G) treated with various CDKs inhibitors treatment. The blue dotted line indicates percentage of motor neurons relative to DMSO-treated motor neurons. b Quantification of ISL1 + SMA type II motor neurons (1-51 N) treated with various CDKs inhibitors treatment. The blue dotted line indicates percentage of motor neurons relative to DMSO-treated motor neurons. c ISL1 immunostaining analysis of various CDKs knockdown in SMA type II motor neurons. The blue dotted line indicates percentage of motor neurons survival relative to non-targeting siRNA treated motor neurons. d Representative images of SMA type II motor neurons treated with various CDKs siRNA and stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 50 μm. e Western blot of SMA type II motor neurons treated with various CDKs inhibitors, indicating that SMN levels remained the same. f Quantification of SMN levels of SMA type II motor neurons treated with various CDKs inhibitors relative to α-tubulin expression. The values were not significant. * p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Inhibition, Immunostaining, Staining, Western Blot, Expressing

    CDK inhibitor reversed motor neuron death in SMA spinal organoids. a Co-staining of ISL1 (red) and SMI-32 (green) in SMA type I spinal organoids treated with DMSO and PD0332991. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. b SMA type I and c SMA type II spinal organoids shows increased MN survival. * p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: CDK inhibitor reversed motor neuron death in SMA spinal organoids. a Co-staining of ISL1 (red) and SMI-32 (green) in SMA type I spinal organoids treated with DMSO and PD0332991. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. b SMA type I and c SMA type II spinal organoids shows increased MN survival. * p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Staining

    Generation of three-dimensional spinal organoids from human iPSCs. a Schematic illustration of spinal organoids differentiation from iPSC. b Co-staining of SOX1 (red) and Nestin (green) illustrating successful generation of neural progenitors in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 50 μm. c Representative images BJ-iPS spinal organoids at respective time points stained with SOX1 (red) and TUJ1 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Quantification of SOX1 + levels percentage of BJ-iPS spinal organoids at respective time points relative to total cell number. e Representative images of BJ-iPS spinal organoids demonstrating SOX1 + (green) and ISL1 + (red) in an apical-to-basal patterning. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. f Representative images of BJ-iPS spinal organoids at respective time points stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. g Graph shows percentage of ISL1 + at day 21, 28, and 35 in BJ-iPS spinal organoids relative to total cell number. * p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: Generation of three-dimensional spinal organoids from human iPSCs. a Schematic illustration of spinal organoids differentiation from iPSC. b Co-staining of SOX1 (red) and Nestin (green) illustrating successful generation of neural progenitors in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 50 μm. c Representative images BJ-iPS spinal organoids at respective time points stained with SOX1 (red) and TUJ1 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Quantification of SOX1 + levels percentage of BJ-iPS spinal organoids at respective time points relative to total cell number. e Representative images of BJ-iPS spinal organoids demonstrating SOX1 + (green) and ISL1 + (red) in an apical-to-basal patterning. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. f Representative images of BJ-iPS spinal organoids at respective time points stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. g Graph shows percentage of ISL1 + at day 21, 28, and 35 in BJ-iPS spinal organoids relative to total cell number. * p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Staining

    Spinal organoids consists of various spinal cord cell types. Representative images illustrating the presence of a HOXB4 + and b HOXC8 + cells in spinal organoids. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. c Co-staining of FOXP1 (green) and ISL1 (red) demonstrates presence of limb-innervating neurons in spinal organoids. Scale bars, 100 μm. d Representative images of spinal organoids at day 42 stained with ISL1 (red) and ChAT (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. Spinal organoids are stained with e CHX10 + cells (RED) and f CALB + cells (green). Scale bars, 100 μm. g Co-staining of S100β and TUJ1 shows presence of astrocytes in spinal organoids. Scale bars, 100 μm. h Quantitative-PCR analysis demonstrates a lack of dorsal cell types in the spinal organoids generated

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: Spinal organoids consists of various spinal cord cell types. Representative images illustrating the presence of a HOXB4 + and b HOXC8 + cells in spinal organoids. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. c Co-staining of FOXP1 (green) and ISL1 (red) demonstrates presence of limb-innervating neurons in spinal organoids. Scale bars, 100 μm. d Representative images of spinal organoids at day 42 stained with ISL1 (red) and ChAT (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. Spinal organoids are stained with e CHX10 + cells (RED) and f CALB + cells (green). Scale bars, 100 μm. g Co-staining of S100β and TUJ1 shows presence of astrocytes in spinal organoids. Scale bars, 100 μm. h Quantitative-PCR analysis demonstrates a lack of dorsal cell types in the spinal organoids generated

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Staining, Real-time Polymerase Chain Reaction, Generated

    Cell cycle genes are upregulated in SMA motor neurons. a Motor neurons were purified based on HB9 immunoreactivity. mRNA expression levels of CDKs and cyclins measured by RNA-seq and qPCR respectively. Graph shows fold change comparing SMA HB9 + motor neurons to wild-type HB9 + motor neurons. The blue dotted line indicates relative expression of wild-type HB9 + motor neurons. b qPCR analysis of ISL1 + motor neurons derived from day 28 organoids. Graph shows mRNA fold change, relative to BJ ISL1 + motor neurons. c Knockdown of SMN in wild-type motor neuron cultures revealed upregulation of cell cycle genes. * p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: Cell cycle genes are upregulated in SMA motor neurons. a Motor neurons were purified based on HB9 immunoreactivity. mRNA expression levels of CDKs and cyclins measured by RNA-seq and qPCR respectively. Graph shows fold change comparing SMA HB9 + motor neurons to wild-type HB9 + motor neurons. The blue dotted line indicates relative expression of wild-type HB9 + motor neurons. b qPCR analysis of ISL1 + motor neurons derived from day 28 organoids. Graph shows mRNA fold change, relative to BJ ISL1 + motor neurons. c Knockdown of SMN in wild-type motor neuron cultures revealed upregulation of cell cycle genes. * p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Purification, Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Derivative Assay

    SMA organoids shows reduced motor neuron survival. a Co-staining of SOX1 (red) and TUJ1 (green) in SMA Type I (1-38 G) and SMA Type II (1-51 N) spinal organoids at respective time points. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. b Quantification of SOX1 + levels percentage of SMA Type I and Type II spinal organoids at respective time points relative to total cell number. The values were not significant. c Representative images of SMA Type I and Type II spinal organoids at respective time points stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Graph shows percentage of ISL1 + at day 21, 28, and 35 in SMA Type I and Type II spinal organoids relative to total cell number. ** p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: SMA organoids shows reduced motor neuron survival. a Co-staining of SOX1 (red) and TUJ1 (green) in SMA Type I (1-38 G) and SMA Type II (1-51 N) spinal organoids at respective time points. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. b Quantification of SOX1 + levels percentage of SMA Type I and Type II spinal organoids at respective time points relative to total cell number. The values were not significant. c Representative images of SMA Type I and Type II spinal organoids at respective time points stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Graph shows percentage of ISL1 + at day 21, 28, and 35 in SMA Type I and Type II spinal organoids relative to total cell number. ** p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Staining

    TDP-43 localization in patient iPSC-derived motor neurons (A) iPSC-MN stained for TDP-43 shows TDP-43 is ubiquitously expressed and nuclear in all cells including in ISLET1-positive neurons from control (left panel) and ALS (right panel) patients. ISLET1-positive cells from sporadic ALS iPSC-MN have nuclear staining as well as nuclear aggregates that stain with higher intensity for TDP-43 (arrowheads, right panel) but aggregates are not present in healthy controls (left panel). Fibroblasts from healthy control and sALS patients do not show nuclear aggregates. Scale bar: 30 µm. (B) Sporadic ALS patient-derived iPSC-MN cells stained with nuclear envelop marker LAMIN-A (green) and TDP-43 (red) shows TDP-43 aggregates are inside the nuclear envelope; scale bar: 20µm. (C) Quantification of healthy control IPRN.0013 and sALS IPRN.0048 clone 1 iPSC-MN cultures shows TDP-43 aggregation is present in higher fraction of ISLET1 or HB9-positive cells compared to negative cells. (30.7% of ISLET1/HB9-positive motor neurons as compared to 16.2% of ISLET1/HB9-negative cells in sALS iPSC-MN cultures. Bars are standard deviation 9% and 8% respectively; P

    Journal: Molecular and cellular neurosciences

    Article Title: A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells

    doi: 10.1016/j.mcn.2013.07.007

    Figure Lengend Snippet: TDP-43 localization in patient iPSC-derived motor neurons (A) iPSC-MN stained for TDP-43 shows TDP-43 is ubiquitously expressed and nuclear in all cells including in ISLET1-positive neurons from control (left panel) and ALS (right panel) patients. ISLET1-positive cells from sporadic ALS iPSC-MN have nuclear staining as well as nuclear aggregates that stain with higher intensity for TDP-43 (arrowheads, right panel) but aggregates are not present in healthy controls (left panel). Fibroblasts from healthy control and sALS patients do not show nuclear aggregates. Scale bar: 30 µm. (B) Sporadic ALS patient-derived iPSC-MN cells stained with nuclear envelop marker LAMIN-A (green) and TDP-43 (red) shows TDP-43 aggregates are inside the nuclear envelope; scale bar: 20µm. (C) Quantification of healthy control IPRN.0013 and sALS IPRN.0048 clone 1 iPSC-MN cultures shows TDP-43 aggregation is present in higher fraction of ISLET1 or HB9-positive cells compared to negative cells. (30.7% of ISLET1/HB9-positive motor neurons as compared to 16.2% of ISLET1/HB9-negative cells in sALS iPSC-MN cultures. Bars are standard deviation 9% and 8% respectively; P

    Article Snippet: The following antibodies were used ISLET1 at 1:1000 (Abcam, ab86501), phospho-S409/S410-TDP-43 (Sigma), LAMIN-A at 1:200 (Cell Signaling, 4777), MNR2/HB9 at 1:100 (Developmental Studies Hybridoma Bank, 81.5C10), SMI31 at 1:1000 (Covance, SMI-31R) and/or TARDBP at 1:500 (ProteinTech, 10782-2-AP), CTIP2 at 1:500 (Abcam).

    Techniques: Derivative Assay, Staining, Marker, Standard Deviation

    Patient-specific iPSC and iPSC-derived motor neurons Representative images of: (A) iPSC colonies from one control and one ALS patient in phase-contrast. (B) iPSC colonies stained with antibodies for pluripotency markers NANOG and TRA-1-60. (C) iPSC-MN cultures stained with nuclear marker DAPI (blue) and antibodies to motor neuron markers ISLET1 (red) and HB9 (green). (D) iPSC-MN cultures stained with antibody to axonal marker Neurofilament (SMI31). iPSC and iPSC-MN shown are from healthy control IPRN.0013 and fALS patient IPRN.0028. (Scale bars a-b: 200 µm, c-d: 75 µm). (E) (i-ii), Representative trace showing electrical activity of iPS-MN from a healthy individual (Scale bars: 100 mV; 100 ms. 8 neurons recorded from fired robust and repetitive action potentials (bursts) to depolarizing current steps. (Iii) Example trace showing action potential elicited by the rebound depolarization following 300 pA hyperpolarizing step for 100 ms (Scales bars: 100 mV; 100 ms) and example trace of spontaneous bursts of action potentials and spontaneous post-synaptic potentials from iPS derived motor neurons (50 mV; 3 Sec). (Insets) Examples of EPSP (scales: 3 mV; 250 ms), and IPSP (scales: 30 mV, 100 ms). (F) (i) Sporadic ALS iPSC-MN responses −10, 0, and 10 pA current steps for 300 msec (scale: 25 mV). (ii) APs elicited by rebound depolarization following hyperpolarizing current step (300 pA; 300 msec). (iii) Spontaneous activity of sALS iPSC-MN (scale bars; 50 mV; 500 msec) (insets example of sEPSP and sIPSP (scale bars: 5 mV; 200 msec))( n = 7).

    Journal: Molecular and cellular neurosciences

    Article Title: A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells

    doi: 10.1016/j.mcn.2013.07.007

    Figure Lengend Snippet: Patient-specific iPSC and iPSC-derived motor neurons Representative images of: (A) iPSC colonies from one control and one ALS patient in phase-contrast. (B) iPSC colonies stained with antibodies for pluripotency markers NANOG and TRA-1-60. (C) iPSC-MN cultures stained with nuclear marker DAPI (blue) and antibodies to motor neuron markers ISLET1 (red) and HB9 (green). (D) iPSC-MN cultures stained with antibody to axonal marker Neurofilament (SMI31). iPSC and iPSC-MN shown are from healthy control IPRN.0013 and fALS patient IPRN.0028. (Scale bars a-b: 200 µm, c-d: 75 µm). (E) (i-ii), Representative trace showing electrical activity of iPS-MN from a healthy individual (Scale bars: 100 mV; 100 ms. 8 neurons recorded from fired robust and repetitive action potentials (bursts) to depolarizing current steps. (Iii) Example trace showing action potential elicited by the rebound depolarization following 300 pA hyperpolarizing step for 100 ms (Scales bars: 100 mV; 100 ms) and example trace of spontaneous bursts of action potentials and spontaneous post-synaptic potentials from iPS derived motor neurons (50 mV; 3 Sec). (Insets) Examples of EPSP (scales: 3 mV; 250 ms), and IPSP (scales: 30 mV, 100 ms). (F) (i) Sporadic ALS iPSC-MN responses −10, 0, and 10 pA current steps for 300 msec (scale: 25 mV). (ii) APs elicited by rebound depolarization following hyperpolarizing current step (300 pA; 300 msec). (iii) Spontaneous activity of sALS iPSC-MN (scale bars; 50 mV; 500 msec) (insets example of sEPSP and sIPSP (scale bars: 5 mV; 200 msec))( n = 7).

    Article Snippet: The following antibodies were used ISLET1 at 1:1000 (Abcam, ab86501), phospho-S409/S410-TDP-43 (Sigma), LAMIN-A at 1:200 (Cell Signaling, 4777), MNR2/HB9 at 1:100 (Developmental Studies Hybridoma Bank, 81.5C10), SMI31 at 1:1000 (Covance, SMI-31R) and/or TARDBP at 1:500 (ProteinTech, 10782-2-AP), CTIP2 at 1:500 (Abcam).

    Techniques: Derivative Assay, Staining, Marker, Activity Assay, Mass Spectrometry, Size-exclusion Chromatography

    Figure 6. Structure of cardiomyocyte colonies grown in the primary culture of rat neonatal myocardial cells. ( A–C ) Different stages of development of the colonies stemming from Isl1 + CSCs. ( A ) Cell division, DIV 2. Isl1 + (FITC, green), GATA-4 (phycoerythrin, red). ( B ) Colony consisting of approximately 8 cells, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( C ) Large Isl1 + colony, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( D ) The optical sections of colonies formed by Isl1 + , c-kit + , and Sca1 + CSCs on the 11th DIV. Isl1 + CSCs (Alexa 405, blue), Z = 12. c-kit + CSCs (FITC, green), Z = 12. Sca1 + CSCs (Alexa 405, blue), Z = 11. Actin was stained using rhodamine-phalloidin (red). ( E ) Differentiation of c-kit + CSCs inside the colony on the 13th DIV. Overlaid optical section of transmitted light and fluorescent images in 2 emitting wavelengths: 488 nm (FITC) and 543 nm (Alexa) in the bottom (Z = 5), in the middle (Z = 10), and the top (Z = 20) parts of the colony. c-kit + expression was revealed by FITC-conjugated antibodies (green), and α-sarcomeric actinin was revealed by Alexa-conjugated antibodies (red). Confocal microscope, Leica TCS SP5 (Germany), objective ×63, oil.

    Journal: Cell Cycle

    Article Title: Characterization of contracting cardiomyocyte colonies in the primary culture of neonatal rat myocardial cells

    doi: 10.4161/cc.27768

    Figure Lengend Snippet: Figure 6. Structure of cardiomyocyte colonies grown in the primary culture of rat neonatal myocardial cells. ( A–C ) Different stages of development of the colonies stemming from Isl1 + CSCs. ( A ) Cell division, DIV 2. Isl1 + (FITC, green), GATA-4 (phycoerythrin, red). ( B ) Colony consisting of approximately 8 cells, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( C ) Large Isl1 + colony, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( D ) The optical sections of colonies formed by Isl1 + , c-kit + , and Sca1 + CSCs on the 11th DIV. Isl1 + CSCs (Alexa 405, blue), Z = 12. c-kit + CSCs (FITC, green), Z = 12. Sca1 + CSCs (Alexa 405, blue), Z = 11. Actin was stained using rhodamine-phalloidin (red). ( E ) Differentiation of c-kit + CSCs inside the colony on the 13th DIV. Overlaid optical section of transmitted light and fluorescent images in 2 emitting wavelengths: 488 nm (FITC) and 543 nm (Alexa) in the bottom (Z = 5), in the middle (Z = 10), and the top (Z = 20) parts of the colony. c-kit + expression was revealed by FITC-conjugated antibodies (green), and α-sarcomeric actinin was revealed by Alexa-conjugated antibodies (red). Confocal microscope, Leica TCS SP5 (Germany), objective ×63, oil.

    Article Snippet: In the second series, the primary mouse anti-Isl1 (Abcam) and anti-Sca1 (Abcam) monoclonal antibodies were preliminary conjugated with Alexa 405 according to Zenon technology (Invitrogen) and then used for immunostaining at a 1:100 dilution.

    Techniques: Staining, Expressing, Microscopy

    Figure 7. Differentiation of Isl1 + and c-kit + CSCs inside the colonies on the 13th DIV. The optical sections of colonies on 2 levels: ( A ) Isl1 + middle (Z = 14) and ( B ) bottom (Z = 0). ( C ) c-kit + top (Z = 10) and ( D ) bottom (Z = 0). Confocal microscope, LEICA TCS SL, objective ×63, oil.

    Journal: Cell Cycle

    Article Title: Characterization of contracting cardiomyocyte colonies in the primary culture of neonatal rat myocardial cells

    doi: 10.4161/cc.27768

    Figure Lengend Snippet: Figure 7. Differentiation of Isl1 + and c-kit + CSCs inside the colonies on the 13th DIV. The optical sections of colonies on 2 levels: ( A ) Isl1 + middle (Z = 14) and ( B ) bottom (Z = 0). ( C ) c-kit + top (Z = 10) and ( D ) bottom (Z = 0). Confocal microscope, LEICA TCS SL, objective ×63, oil.

    Article Snippet: In the second series, the primary mouse anti-Isl1 (Abcam) and anti-Sca1 (Abcam) monoclonal antibodies were preliminary conjugated with Alexa 405 according to Zenon technology (Invitrogen) and then used for immunostaining at a 1:100 dilution.

    Techniques: Microscopy