rabbit anti isl1  (Abcam)

 
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    Anti Islet 1 antibody Neural Stem Cell Marker
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    ab20670
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    Structured Review

    Abcam rabbit anti isl1
    <t>ISL1</t> directly regulates a number of genes required for normal pacemaker function in mice and human.

    https://www.bioz.com/result/rabbit anti isl1/product/Abcam
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rabbit anti isl1 - by Bioz Stars, 2020-07
    94/100 stars

    Images

    1) Product Images from "Transcription factor ISL1 is essential for pacemaker development and function"

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

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI68257

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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: Expressing

    2) Product Images from "Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification"

    Article Title: Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.078501

    Re-evaluated model for the control of MN differentiation. ( A ) Isl1 ensures the survival of newly born MNs, whereas Hb9 and Isl cooperate to consolidate MN identity and simultaneously repress the V2a differentiation program. ( B ) At thoracic levels of the
    Figure Legend Snippet: Re-evaluated model for the control of MN differentiation. ( A ) Isl1 ensures the survival of newly born MNs, whereas Hb9 and Isl cooperate to consolidate MN identity and simultaneously repress the V2a differentiation program. ( B ) At thoracic levels of the

    Techniques Used:

    OC factors directly stimulate the expression of Isl1 . ( A-C ) Overexpression of Hnf6 or Oc2 in chick embryonic spinal cord at HH14 results in ectopic Isl1 expression 6 hours after electroporation. ( D ) Binding of endogenous OC factors to potential binding
    Figure Legend Snippet: OC factors directly stimulate the expression of Isl1 . ( A-C ) Overexpression of Hnf6 or Oc2 in chick embryonic spinal cord at HH14 results in ectopic Isl1 expression 6 hours after electroporation. ( D ) Binding of endogenous OC factors to potential binding

    Techniques Used: Expressing, Over Expression, Electroporation, Binding Assay

    OC factors are required to maintain Isl1 expression during MN subtype diversification. ( A-H ) Triple label immunofluorescence analysis of Olig2, Hb9 and Isl1 on a single spinal cord section of control mouse embryos or embryos double mutant for Hnf6 and
    Figure Legend Snippet: OC factors are required to maintain Isl1 expression during MN subtype diversification. ( A-H ) Triple label immunofluorescence analysis of Olig2, Hb9 and Isl1 on a single spinal cord section of control mouse embryos or embryos double mutant for Hnf6 and

    Techniques Used: Expressing, Immunofluorescence, Mutagenesis

    3) Product Images from "Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria"

    Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

    Journal: eLife

    doi: 10.7554/eLife.32282

    Validation of the CRISPR/Cas9 Alsin -/- cells in iPSC, NPC and iPSC-sMN. ( A ) Electrophoresis result of the PCR reaction using primers flanking exon three and within exon 3. Homozygous deletion was confirmed by the absence of a 2.8 kb. ( B ) Protein lysates from WT KOLF and Alsin -/- iPSC were loaded onto SDS-PAGE and immunoblotted with Alsin antibody. A band detected at ~184 kDa, but absent in Alsin -/- , corresponds to full-length Alsin. ( C ) WT KOLF, Alsin -/- iPSC, and neuroprogenitor cells (NPC) were immunostained with pluripotency markers Oct4 and Lin28, and neuroprogenitor markers Sox2 and Pax6, respectively. ( D–E ) WT KOLF and Alsin -/- iPSC-sMN were immunostained with motor neuron markers such as ChAT, HB9, and ISL1, nuclear dye DAPI, and cytoskeletal marker MAP2. ( F ) WT KOLF and Alsin -/- iPSC-sMN were immunostained with antibodies against Rab5 and Alsin, along with DAPI (nuclear) and phalloidin (actin) stains. Scale bars, 10 μm.
    Figure Legend Snippet: Validation of the CRISPR/Cas9 Alsin -/- cells in iPSC, NPC and iPSC-sMN. ( A ) Electrophoresis result of the PCR reaction using primers flanking exon three and within exon 3. Homozygous deletion was confirmed by the absence of a 2.8 kb. ( B ) Protein lysates from WT KOLF and Alsin -/- iPSC were loaded onto SDS-PAGE and immunoblotted with Alsin antibody. A band detected at ~184 kDa, but absent in Alsin -/- , corresponds to full-length Alsin. ( C ) WT KOLF, Alsin -/- iPSC, and neuroprogenitor cells (NPC) were immunostained with pluripotency markers Oct4 and Lin28, and neuroprogenitor markers Sox2 and Pax6, respectively. ( D–E ) WT KOLF and Alsin -/- iPSC-sMN were immunostained with motor neuron markers such as ChAT, HB9, and ISL1, nuclear dye DAPI, and cytoskeletal marker MAP2. ( F ) WT KOLF and Alsin -/- iPSC-sMN were immunostained with antibodies against Rab5 and Alsin, along with DAPI (nuclear) and phalloidin (actin) stains. Scale bars, 10 μm.

    Techniques Used: CRISPR, Electrophoresis, Polymerase Chain Reaction, SDS Page, Marker

    4) Product Images from "Celsr3 and Fzd3 Organize a Pioneer Neuron Scaffold to Steer Growing Thalamocortical Axons"

    Article Title: Celsr3 and Fzd3 Organize a Pioneer Neuron Scaffold to Steer Growing Thalamocortical Axons

    Journal: Cerebral Cortex (New York, NY)

    doi: 10.1093/cercor/bhw132

    Isl1-positive cells in the prethalamic region are positive for Pax6 and negative for Reln, Calretinin, and Calbindin. ( A – D ) E13.5 sections double-stained by Pax6 and Isl1. In the prethalamic region, the distribution area of Pax6-positive cells was wider than that of Isl1-positive cells ( B and C ) and almost all Isl1-positive cells were Pax6-positive ( D ). ( E – H ) Double staining for Isl1 and Reln. Isl1-positive cells were consistently negative for Reln, indicating that they do not belong to the reticular thalamus. ( I – P ) Double staining for Isl1 and Calretinin (CR) or Calbindin (CB) showed no colocalization, suggesting that Isl1-cells are not part of the thalamic eminences. Ctx, neocortex; Th, thalamus; IC, internal capsule; pTh, prethalamus. Scale bars: 400 μm ( A , E , I , and M ); 100 μm ( B – D , F – H , J – L , and N – P ).
    Figure Legend Snippet: Isl1-positive cells in the prethalamic region are positive for Pax6 and negative for Reln, Calretinin, and Calbindin. ( A – D ) E13.5 sections double-stained by Pax6 and Isl1. In the prethalamic region, the distribution area of Pax6-positive cells was wider than that of Isl1-positive cells ( B and C ) and almost all Isl1-positive cells were Pax6-positive ( D ). ( E – H ) Double staining for Isl1 and Reln. Isl1-positive cells were consistently negative for Reln, indicating that they do not belong to the reticular thalamus. ( I – P ) Double staining for Isl1 and Calretinin (CR) or Calbindin (CB) showed no colocalization, suggesting that Isl1-cells are not part of the thalamic eminences. Ctx, neocortex; Th, thalamus; IC, internal capsule; pTh, prethalamus. Scale bars: 400 μm ( A , E , I , and M ); 100 μm ( B – D , F – H , J – L , and N – P ).

    Techniques Used: Staining, Double Staining

    Defective forebrain axonal projections in Isl1-Cre;Celsr3 f/− mice. ( A and B ) E18.5 sections stained by NF antibodies. Unlike in control mice ( A ), in Isl1-Cre;Celsr3 f/− mutant mice ( B ), fibers looped aberrantly in the middle area of the ventral telencephalon (arrow) and were misrouted at the level of the DTJ (arrowhead). ( C – F ) DiI tracing at P0. Upon DiI injection in thalamus (Th), unlike in control brain ( C ), in mutants, only a minority of thalamic axons followed a normal path ( D , arrow) and many were misrouted ventrally ( D , arrowhead). Upon DiI injection in cortex (Ctx), cortical axons traversed the internal capsule (IC) in the control ( E ) and retrogradely labeled neurons were seen in thalamus. In contrast, in Isl1-Cre;Celsr3 f/− mice, cortical axons stalled and formed whorls ( F , arrowhead), and rare thalamic neurons were labeled ( F ′). ( G and H ) Unlike control axons ( G ), mutant corticospinal axons ( H ), traced using Thy1-YFP at P20, formed whorls in the IC (arrowheads), and some projected aberrantly to thalamus (arrow). ( I – L ) Cortical barrels in P7 tangential and coronal sections stained with anti-VGLUT2. In the control, barrels were well organized ( I and K ). In mutants with partial phenotype ( J and L ), barrels were blurry although a few were visible and their anatomical localization was preserved. Scale bars: 500 μm ( A – F and I – J ); 200 μm ( G – H and K – L ).
    Figure Legend Snippet: Defective forebrain axonal projections in Isl1-Cre;Celsr3 f/− mice. ( A and B ) E18.5 sections stained by NF antibodies. Unlike in control mice ( A ), in Isl1-Cre;Celsr3 f/− mutant mice ( B ), fibers looped aberrantly in the middle area of the ventral telencephalon (arrow) and were misrouted at the level of the DTJ (arrowhead). ( C – F ) DiI tracing at P0. Upon DiI injection in thalamus (Th), unlike in control brain ( C ), in mutants, only a minority of thalamic axons followed a normal path ( D , arrow) and many were misrouted ventrally ( D , arrowhead). Upon DiI injection in cortex (Ctx), cortical axons traversed the internal capsule (IC) in the control ( E ) and retrogradely labeled neurons were seen in thalamus. In contrast, in Isl1-Cre;Celsr3 f/− mice, cortical axons stalled and formed whorls ( F , arrowhead), and rare thalamic neurons were labeled ( F ′). ( G and H ) Unlike control axons ( G ), mutant corticospinal axons ( H ), traced using Thy1-YFP at P20, formed whorls in the IC (arrowheads), and some projected aberrantly to thalamus (arrow). ( I – L ) Cortical barrels in P7 tangential and coronal sections stained with anti-VGLUT2. In the control, barrels were well organized ( I and K ). In mutants with partial phenotype ( J and L ), barrels were blurry although a few were visible and their anatomical localization was preserved. Scale bars: 500 μm ( A – F and I – J ); 200 μm ( G – H and K – L ).

    Techniques Used: Mouse Assay, Staining, Mutagenesis, Injection, Labeling

    Interactions between growing axons and Isl1-positive cells. ( A ) Confocal stack showing DiI-labeled cortical axons (red) and guidepost cells labeled using Isl1-Cre;Rosa26 YFP mice (green) in E13.5 ventral telencephalon. Axons ran parallel to one another, in close juxtaposition with YFP-positive cells. ( B and C ) Axons (NF-IHC, red) and Isl1-positive cells (green) came in close contact ( B , arrowheads), which was confirmed by IHC-EM ( C ), where the NF-labeled axon was seen in direct contact (red arrowheads) with the Isl1-positive cell (immunopositive nucleus). Blue in B is DAPI; IHC, immunohistochemistry. Scale bars: 50 μm ( A ); 20 μm ( B ); 1 μm ( C ).
    Figure Legend Snippet: Interactions between growing axons and Isl1-positive cells. ( A ) Confocal stack showing DiI-labeled cortical axons (red) and guidepost cells labeled using Isl1-Cre;Rosa26 YFP mice (green) in E13.5 ventral telencephalon. Axons ran parallel to one another, in close juxtaposition with YFP-positive cells. ( B and C ) Axons (NF-IHC, red) and Isl1-positive cells (green) came in close contact ( B , arrowheads), which was confirmed by IHC-EM ( C ), where the NF-labeled axon was seen in direct contact (red arrowheads) with the Isl1-positive cell (immunopositive nucleus). Blue in B is DAPI; IHC, immunohistochemistry. Scale bars: 50 μm ( A ); 20 μm ( B ); 1 μm ( C ).

    Techniques Used: Labeling, Mouse Assay, Immunohistochemistry

    Summary of defective axonal bundles in Isl1-Cre;Celsr3 f/− mice. ( A ) In control embryos at E12.5, prior to growth of thalamic and cortical axons, Isl1-positive cells (dark mauve) form an early scaffold that extends from prosomere 3 (p3), across the DTJ, in ventral telencephalon, and across the PSPB to subcortex (light gray area). Reciprocal projections across the diencephalon–telencephalon junction (DTJ) form a “bridge” which is absent in Celsr3 and Fzd3 mutant embryos. Dark gray: ventricular zones. ( B – D ) At later stages, for example, E14.5, thalamocortical (green) and corticothalamic (red) axons extend in the corridor in vTel (light gray) and reach their respective targets in control mice ( B ). In Isl1-Cre;Celsr3 f/− mice ( C ), the early scaffold is disorganized in regions of Isl1 expression (light mauve cells). Thalamocortical axons (green) are misrouted ventrally, and corticothalamic axons (red) stall and form a whorl in the corridor, more medially than in Dlx5/6-Cre;Celsr3 f/− mutants ( D ). Ctx, cortex; pTh, prethalamus; Th, thalamus; DTJ, diencephalon–telencephalon junction; PSPB, pallium–subpallium boundary.
    Figure Legend Snippet: Summary of defective axonal bundles in Isl1-Cre;Celsr3 f/− mice. ( A ) In control embryos at E12.5, prior to growth of thalamic and cortical axons, Isl1-positive cells (dark mauve) form an early scaffold that extends from prosomere 3 (p3), across the DTJ, in ventral telencephalon, and across the PSPB to subcortex (light gray area). Reciprocal projections across the diencephalon–telencephalon junction (DTJ) form a “bridge” which is absent in Celsr3 and Fzd3 mutant embryos. Dark gray: ventricular zones. ( B – D ) At later stages, for example, E14.5, thalamocortical (green) and corticothalamic (red) axons extend in the corridor in vTel (light gray) and reach their respective targets in control mice ( B ). In Isl1-Cre;Celsr3 f/− mice ( C ), the early scaffold is disorganized in regions of Isl1 expression (light mauve cells). Thalamocortical axons (green) are misrouted ventrally, and corticothalamic axons (red) stall and form a whorl in the corridor, more medially than in Dlx5/6-Cre;Celsr3 f/− mutants ( D ). Ctx, cortex; pTh, prethalamus; Th, thalamus; DTJ, diencephalon–telencephalon junction; PSPB, pallium–subpallium boundary.

    Techniques Used: Mouse Assay, Mutagenesis, Expressing

    The early scaffold “bridge” between the ventral telencephalon and the prethalamus. ( A ) Schema illustrating the preparation of oblique slices. ( B – E ) Merged stacks of sections generated by 2-photon microscopy, using E12.5 Isl1-Cre;Rosa26 Tomato mice to label Isl1-positive cells and fibers in control ( B ), Isl1-Cre;Celsr3 f/− ( C and D ) and Isl1-Cre;Fzd3 f/− embryos ( E ) at E12.5. In all control embryos, a connecting “bridge” ( B , arrows) was evident across the DTJ (interrupted line), between ventral telencephalon (vTel) and prethalamus (pTh). The bridge was completely absent in 14 Isl1-Cre;Celsr3 f/− samples ( C ), and partial in 5 other Isl1-Cre;Celsr3 f/− samples ( D , arrowheads) The bridge was fully absent in all Isl1-Cre;Fzd3 f/− samples ( E ). ( F ) Summary histogram. ( G and H ) Axon bundles from Isl1-positive cells crossing the DTJ were visualized in sections of E15.5 ( G ) and P0 Isl1-Cre;Rosa26 Tomato embryos. Ctx, cortex; Th, thalamus. Scale bars: 50 μm ( B – E ); 200 μm ( G and H ).
    Figure Legend Snippet: The early scaffold “bridge” between the ventral telencephalon and the prethalamus. ( A ) Schema illustrating the preparation of oblique slices. ( B – E ) Merged stacks of sections generated by 2-photon microscopy, using E12.5 Isl1-Cre;Rosa26 Tomato mice to label Isl1-positive cells and fibers in control ( B ), Isl1-Cre;Celsr3 f/− ( C and D ) and Isl1-Cre;Fzd3 f/− embryos ( E ) at E12.5. In all control embryos, a connecting “bridge” ( B , arrows) was evident across the DTJ (interrupted line), between ventral telencephalon (vTel) and prethalamus (pTh). The bridge was completely absent in 14 Isl1-Cre;Celsr3 f/− samples ( C ), and partial in 5 other Isl1-Cre;Celsr3 f/− samples ( D , arrowheads) The bridge was fully absent in all Isl1-Cre;Fzd3 f/− samples ( E ). ( F ) Summary histogram. ( G and H ) Axon bundles from Isl1-positive cells crossing the DTJ were visualized in sections of E15.5 ( G ) and P0 Isl1-Cre;Rosa26 Tomato embryos. Ctx, cortex; Th, thalamus. Scale bars: 50 μm ( B – E ); 200 μm ( G and H ).

    Techniques Used: Generated, Microscopy, Mouse Assay

    Isl1-positive cells reciprocally migrate across the DTJ from the prethalamus and ventral telencephalon. ( A and B ) The early scaffold bridge is composed of Isl1-positive fibers and migrating cells disclosed using anti-neurofilament (NF) IHC and Isl1-Cre;Rosa26 Tomato tracing. Oblique section at E12.5, with Isl1-positive cells revealed using the Tomato transgene, and axons stained with anti-neurofilaments (green) ( A ).The selected area from the scafold bridge in A contained both yellow fibers and red cells ( B ). ( C – F ) In E12.5 oblique sections from Isl1-Cre;Rosa26 Tomato embryos, CMFDA (green) was placed into the prethalamus ( C ) or the ventral telencephalon ( E ), and slices were cultured for 2 days (2DIV). Isl1-positive cells were red due to Tomato fluorescence. Double labeled cells could be seen in both ventral telencephalon (arrows in D ) and prethalamus (arrows in F ). ( D and F ) are selected areas from ( C and E ), respectively. pTh, prethalamus; Ctx, cortex; vTel, ventral telencephalon; Th, thalamus; LV, lateral ventricle. Scale bars: 200 μm ( A , C , and E ); 20 μm ( B ); 50 μm ( D , F ).
    Figure Legend Snippet: Isl1-positive cells reciprocally migrate across the DTJ from the prethalamus and ventral telencephalon. ( A and B ) The early scaffold bridge is composed of Isl1-positive fibers and migrating cells disclosed using anti-neurofilament (NF) IHC and Isl1-Cre;Rosa26 Tomato tracing. Oblique section at E12.5, with Isl1-positive cells revealed using the Tomato transgene, and axons stained with anti-neurofilaments (green) ( A ).The selected area from the scafold bridge in A contained both yellow fibers and red cells ( B ). ( C – F ) In E12.5 oblique sections from Isl1-Cre;Rosa26 Tomato embryos, CMFDA (green) was placed into the prethalamus ( C ) or the ventral telencephalon ( E ), and slices were cultured for 2 days (2DIV). Isl1-positive cells were red due to Tomato fluorescence. Double labeled cells could be seen in both ventral telencephalon (arrows in D ) and prethalamus (arrows in F ). ( D and F ) are selected areas from ( C and E ), respectively. pTh, prethalamus; Ctx, cortex; vTel, ventral telencephalon; Th, thalamus; LV, lateral ventricle. Scale bars: 200 μm ( A , C , and E ); 20 μm ( B ); 50 μm ( D , F ).

    Techniques Used: Immunohistochemistry, Staining, Cell Culture, Fluorescence, Labeling

    The bridge is required for thalamocortical axons to cross the DTJ from E13.5. Early thalamocortical axons were labeled at E13.5, following NeuroVue implantation in the dorsal thalamus, and Isl1-positive cells were visualized by the Isl1-Cre;Rosa26 Tomato transgene, in oblique slices. ( A and B ) In control embryos, thalamocortical axons ( A , green arrowheads) followed the bridge ( A , red arrowheads) to cross the DTJ ( A ) and then ran in the ventral telencephalon uniformly ( B , arrows). ( C – F ) In 2 mutants, a few thalamocortical fibers ( C , green arrowhead) could cross the DTJ along a partial bridge ( C , red arrowhead) and then ran randomly in the ventral telencephalon ( D , arrowheads). No thalamocortical axons crossed the DTJ in 8 other mutant embryos with the absence of the bridge ( E ). ( F ) Summary histogram. ( B and D ) are higher magnification of selected areas in ( A and C ) respectively. Th, dorsal thalamus; LV, lateral ventricle; pTh, prethalamus; vTel, ventral telencephalon. Scale bars: 200 μm ( A , C , E ); 50 μm ( B , D ).
    Figure Legend Snippet: The bridge is required for thalamocortical axons to cross the DTJ from E13.5. Early thalamocortical axons were labeled at E13.5, following NeuroVue implantation in the dorsal thalamus, and Isl1-positive cells were visualized by the Isl1-Cre;Rosa26 Tomato transgene, in oblique slices. ( A and B ) In control embryos, thalamocortical axons ( A , green arrowheads) followed the bridge ( A , red arrowheads) to cross the DTJ ( A ) and then ran in the ventral telencephalon uniformly ( B , arrows). ( C – F ) In 2 mutants, a few thalamocortical fibers ( C , green arrowhead) could cross the DTJ along a partial bridge ( C , red arrowhead) and then ran randomly in the ventral telencephalon ( D , arrowheads). No thalamocortical axons crossed the DTJ in 8 other mutant embryos with the absence of the bridge ( E ). ( F ) Summary histogram. ( B and D ) are higher magnification of selected areas in ( A and C ) respectively. Th, dorsal thalamus; LV, lateral ventricle; pTh, prethalamus; vTel, ventral telencephalon. Scale bars: 200 μm ( A , C , E ); 50 μm ( B , D ).

    Techniques Used: Labeling, Mutagenesis

    Development of Isl1-positive cells in forebrain. Isl1 expression was studied using anti-Isl1 immunohistochemistry ( A , B , C , E, and G ) and by mapping tomato fluorescent protein expression using Isl1-Cre;Rosa26 Tomato mice ( B , D , F , and H ). Inset in A1 ( A 1′) is a magnification of the boxed area. ( A and B ) Isl1-positive cells appeared at E9.5–E10.5, close to midline. ( C and D ) At E11.5, cells expressing Isl1 and tomato protein appeared in ventral telencephalon (vTel) and hypothalamic anlage (arrows). ( E – H ) At E12.5 ( E and F ), the number of Isl1-positive cells increased in ventral telencephalon and hypothalamic anlage, and another population appears in the prethalamic region (pTh, arrowheads), a pattern that became better defined at E13.5 ( G and H ). I: schematic summary of development of Isl1-positive cells. *, trigeminal ganglion. Th, thalamic anlage; Ctx, cortex; IHC, immunohistochemistry. Scale bars: 400 μm.
    Figure Legend Snippet: Development of Isl1-positive cells in forebrain. Isl1 expression was studied using anti-Isl1 immunohistochemistry ( A , B , C , E, and G ) and by mapping tomato fluorescent protein expression using Isl1-Cre;Rosa26 Tomato mice ( B , D , F , and H ). Inset in A1 ( A 1′) is a magnification of the boxed area. ( A and B ) Isl1-positive cells appeared at E9.5–E10.5, close to midline. ( C and D ) At E11.5, cells expressing Isl1 and tomato protein appeared in ventral telencephalon (vTel) and hypothalamic anlage (arrows). ( E – H ) At E12.5 ( E and F ), the number of Isl1-positive cells increased in ventral telencephalon and hypothalamic anlage, and another population appears in the prethalamic region (pTh, arrowheads), a pattern that became better defined at E13.5 ( G and H ). I: schematic summary of development of Isl1-positive cells. *, trigeminal ganglion. Th, thalamic anlage; Ctx, cortex; IHC, immunohistochemistry. Scale bars: 400 μm.

    Techniques Used: Expressing, Immunohistochemistry, Mouse Assay

    Early reciprocal pioneer projections between ventral telencephalon and prethalamus. NeuroVue was inserted in E12.5 vibratome slices. Isl1-positive cells were detected using the Isl1-Cre;Rosa26 Tomato transgene (red), and DAPI (blue) was used as a general nuclear stain. ( A – D ) Upon NeuroVue implantation in prethalamus ( A ), many Isl1-positive cells were back labeled in the ventral telencephalon in control ( B and B ′, arrows), a few in one mutant ( C and C ′, arrowhead) and none in 4 other mutants ( D ). ( E ) Summary of results; P = 0.0022 (Mann–Whitney U test). ( F – I ) NeuroVue implantation in ventral telencephalon ( F ) resulted in retrograde labeling of many Isl1-positive cells in the prethalamus in control ( G and G ′, arrows), a few in one mutant ( H and H ′, arrowhead) and none in 4 other mutant embryos ( I ). ( J ) Summary of results; P = 0.0007 (Mann–Whitney U test). ( B ′, C ′, G ′, and H ′) are higher magnification of boxed areas in ( B , C , G , and H ), respectively. pTh: prethalamus; Ctx, cortex; vTel, ventral telencephalon; Th, thalamus. Scale bars: 400 μm ( B – D and G – I ); 50 μm ( B ′, C ′, G ′, and H ′).
    Figure Legend Snippet: Early reciprocal pioneer projections between ventral telencephalon and prethalamus. NeuroVue was inserted in E12.5 vibratome slices. Isl1-positive cells were detected using the Isl1-Cre;Rosa26 Tomato transgene (red), and DAPI (blue) was used as a general nuclear stain. ( A – D ) Upon NeuroVue implantation in prethalamus ( A ), many Isl1-positive cells were back labeled in the ventral telencephalon in control ( B and B ′, arrows), a few in one mutant ( C and C ′, arrowhead) and none in 4 other mutants ( D ). ( E ) Summary of results; P = 0.0022 (Mann–Whitney U test). ( F – I ) NeuroVue implantation in ventral telencephalon ( F ) resulted in retrograde labeling of many Isl1-positive cells in the prethalamus in control ( G and G ′, arrows), a few in one mutant ( H and H ′, arrowhead) and none in 4 other mutant embryos ( I ). ( J ) Summary of results; P = 0.0007 (Mann–Whitney U test). ( B ′, C ′, G ′, and H ′) are higher magnification of boxed areas in ( B , C , G , and H ), respectively. pTh: prethalamus; Ctx, cortex; vTel, ventral telencephalon; Th, thalamus. Scale bars: 400 μm ( B – D and G – I ); 50 μm ( B ′, C ′, G ′, and H ′).

    Techniques Used: Staining, Labeling, Mutagenesis, MANN-WHITNEY

    5) Product Images from "Cardiac cell proliferation assessed by EdU, a novel analysis of cardiac regeneration"

    Article Title: Cardiac cell proliferation assessed by EdU, a novel analysis of cardiac regeneration

    Journal: Cytotechnology

    doi: 10.1007/s10616-014-9827-8

    EdU-labeled cardiac resident stem cells and neotatal cardiomyocytes in vitro. EdU sensitively labels resident stem cells with IsL1 ( a ), Tbx18 ( b ), and WT1 ( c ) (×200) (Scale bars, 25 µm), and noetatal cardiomyocytes in vitro (×400)
    Figure Legend Snippet: EdU-labeled cardiac resident stem cells and neotatal cardiomyocytes in vitro. EdU sensitively labels resident stem cells with IsL1 ( a ), Tbx18 ( b ), and WT1 ( c ) (×200) (Scale bars, 25 µm), and noetatal cardiomyocytes in vitro (×400)

    Techniques Used: Labeling, In Vitro

    Related Articles

    Immunohistochemistry:

    Article Title: Intravitreal homocysteine-thiolactone injection leads to the degeneration of multiple retinal cells, including photoreceptors
    Article Snippet: .. Immunohistochemical analysis and quantification of labeled cells For immunohistochemical analysis, the retinal tissue sections were boiled in a citrate buffer (pH 6.0) for 20 min for antigen retrieval and were then incubated, respectively, with one of the following antibodies: rabbit anti-homocysteine (1/50, cat. ab15154; Abcam); mouse anti-rhodopsin (1/400, cat. Ab81702; Abcam ) ; mouse anti-calbindin (1/50, cat. sc-74462; Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-Pax-6 antibody (1/50, cat. sc-32766; Santa Cruz Biotechnology); rabbit anti-Islet-1 (1/150, cat. Ab20670; Abcam); sheep anti-Chx-10 (1/150, cat. ab16141; Abcam); or rabbit anti-GFAP (1/250, cat. 2301–1; Epitomics, Burlingame, CA). .. The preparations were then incubated with a horseradish peroxidase-conjugated secondary antibody (1/200), either antimouse, antirabbit, or antisheep IgG (Jackson ImmunoResearch Laboratories, Inc.).

    Immunocytochemistry:

    Article Title: Stimulation of endogenous cardioblasts by exogenous cell therapy after myocardial infarction
    Article Snippet: .. Fluorescent immunocytochemistry was performed with antibodies against GFP (ab13970, Abcam), α-sarcomeric actin (A7811, Sigma), α-myosin heavy chain (ab15, Abcam), NKX2-5 (ab35842, Abcam), GATA4 (ab84593, Abcam), MEF2C (ab64644, Abcam), TBX5 (ab101227, Abcam), Isl1 (ab20670, Abcam), Ki67 (RM-9106, Thermo Scientific), H3P (ab5176, Abcam), lacZ (ab9361, Abcam), Sca-1 (ab25195, Abcam), Nestin (ab6142, Abcam), c-kit (ab5506, Abcam), SSEA1 (ab16285, Abcam), CD34 (ab8158, Abcam), PDGFRα (ab61219, Abcam), CD31 (ab28364, Abcam), CD 45 (ab10558, Abcam), CXCR4 (ab2074, Abcam), VEGFR1 (ab32152, Abcam), VEGFR2 (ab2349, Abcam), and VEGFR3 (ab27278, Abcam). ..

    Incubation:

    Article Title: Intravitreal homocysteine-thiolactone injection leads to the degeneration of multiple retinal cells, including photoreceptors
    Article Snippet: .. Immunohistochemical analysis and quantification of labeled cells For immunohistochemical analysis, the retinal tissue sections were boiled in a citrate buffer (pH 6.0) for 20 min for antigen retrieval and were then incubated, respectively, with one of the following antibodies: rabbit anti-homocysteine (1/50, cat. ab15154; Abcam); mouse anti-rhodopsin (1/400, cat. Ab81702; Abcam ) ; mouse anti-calbindin (1/50, cat. sc-74462; Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-Pax-6 antibody (1/50, cat. sc-32766; Santa Cruz Biotechnology); rabbit anti-Islet-1 (1/150, cat. Ab20670; Abcam); sheep anti-Chx-10 (1/150, cat. ab16141; Abcam); or rabbit anti-GFAP (1/250, cat. 2301–1; Epitomics, Burlingame, CA). .. The preparations were then incubated with a horseradish peroxidase-conjugated secondary antibody (1/200), either antimouse, antirabbit, or antisheep IgG (Jackson ImmunoResearch Laboratories, Inc.).

    Labeling:

    Article Title: Intravitreal homocysteine-thiolactone injection leads to the degeneration of multiple retinal cells, including photoreceptors
    Article Snippet: .. Immunohistochemical analysis and quantification of labeled cells For immunohistochemical analysis, the retinal tissue sections were boiled in a citrate buffer (pH 6.0) for 20 min for antigen retrieval and were then incubated, respectively, with one of the following antibodies: rabbit anti-homocysteine (1/50, cat. ab15154; Abcam); mouse anti-rhodopsin (1/400, cat. Ab81702; Abcam ) ; mouse anti-calbindin (1/50, cat. sc-74462; Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-Pax-6 antibody (1/50, cat. sc-32766; Santa Cruz Biotechnology); rabbit anti-Islet-1 (1/150, cat. Ab20670; Abcam); sheep anti-Chx-10 (1/150, cat. ab16141; Abcam); or rabbit anti-GFAP (1/250, cat. 2301–1; Epitomics, Burlingame, CA). .. The preparations were then incubated with a horseradish peroxidase-conjugated secondary antibody (1/200), either antimouse, antirabbit, or antisheep IgG (Jackson ImmunoResearch Laboratories, Inc.).

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    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: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti isl1/product/Abcam
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    rabbit anti isl1 - by Bioz Stars, 2020-07
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    Abcam rabbit isl1
    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
    Rabbit Isl1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit isl1/product/Abcam
    Average 94 stars, based on 3 article reviews
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    rabbit isl1 - by Bioz Stars, 2020-07
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    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

    The cytotoxic effects of homocysteine-thiolactone (Hcy-T) on specific retinal cells as reflected by alterations of A - D : rhodopsin positive photoreceptors, E - H : calbindin positive horizontal cells, I - L : Chx- 10 positive bipolar cells, M - P : Islet-1 positive bipolar cells, Q - T : Pax-6 positive amacrine cells, and U - X : glial fibrillary acidic protein (GFAP) positive Müller cells at day 15 and day 90 following the intravitreal injections. Abbreviations: GCLrepresents ganglion cell layer; INL represents inner nuclear layer; IPL represents inner plexiform layer; ONL represents outer nuclear layer; OPL represents outer plexiform layer; OS/IS represents outer and inner segments of photoreceptors. Scale bar: 45 μm.

    Journal: Molecular Vision

    Article Title: Intravitreal homocysteine-thiolactone injection leads to the degeneration of multiple retinal cells, including photoreceptors

    doi:

    Figure Lengend Snippet: The cytotoxic effects of homocysteine-thiolactone (Hcy-T) on specific retinal cells as reflected by alterations of A - D : rhodopsin positive photoreceptors, E - H : calbindin positive horizontal cells, I - L : Chx- 10 positive bipolar cells, M - P : Islet-1 positive bipolar cells, Q - T : Pax-6 positive amacrine cells, and U - X : glial fibrillary acidic protein (GFAP) positive Müller cells at day 15 and day 90 following the intravitreal injections. Abbreviations: GCLrepresents ganglion cell layer; INL represents inner nuclear layer; IPL represents inner plexiform layer; ONL represents outer nuclear layer; OPL represents outer plexiform layer; OS/IS represents outer and inner segments of photoreceptors. Scale bar: 45 μm.

    Article Snippet: Immunohistochemical analysis and quantification of labeled cells For immunohistochemical analysis, the retinal tissue sections were boiled in a citrate buffer (pH 6.0) for 20 min for antigen retrieval and were then incubated, respectively, with one of the following antibodies: rabbit anti-homocysteine (1/50, cat. ab15154; Abcam); mouse anti-rhodopsin (1/400, cat. Ab81702; Abcam ) ; mouse anti-calbindin (1/50, cat. sc-74462; Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-Pax-6 antibody (1/50, cat. sc-32766; Santa Cruz Biotechnology); rabbit anti-Islet-1 (1/150, cat. Ab20670; Abcam); sheep anti-Chx-10 (1/150, cat. ab16141; Abcam); or rabbit anti-GFAP (1/250, cat. 2301–1; Epitomics, Burlingame, CA).

    Techniques:

    Quantification of specific retinal cells per 100 μm length according to their specific markers. The markers were used to detect at day 90 following the intravitreal injections. A : Rhodopsin positive cells per 100 μm length in outer nuclear layer (ONL). B : Calbindin positive cells per 100 μm length in the retina. C : Chx-10 positive cells per 100 μm length in inner nuclear layer (INL). D : Islet-1 positive cells per 100 μm length in INL. E : Pax-6 positive cells per 100 μm length in INL. F : Glial fibrillary acidic protein (GFAP) positive cells per 100 μm length in the retina. The sample size for all markers was 5 in all groups. The error bars indicate standard error of the means (SEMs).

    Journal: Molecular Vision

    Article Title: Intravitreal homocysteine-thiolactone injection leads to the degeneration of multiple retinal cells, including photoreceptors

    doi:

    Figure Lengend Snippet: Quantification of specific retinal cells per 100 μm length according to their specific markers. The markers were used to detect at day 90 following the intravitreal injections. A : Rhodopsin positive cells per 100 μm length in outer nuclear layer (ONL). B : Calbindin positive cells per 100 μm length in the retina. C : Chx-10 positive cells per 100 μm length in inner nuclear layer (INL). D : Islet-1 positive cells per 100 μm length in INL. E : Pax-6 positive cells per 100 μm length in INL. F : Glial fibrillary acidic protein (GFAP) positive cells per 100 μm length in the retina. The sample size for all markers was 5 in all groups. The error bars indicate standard error of the means (SEMs).

    Article Snippet: Immunohistochemical analysis and quantification of labeled cells For immunohistochemical analysis, the retinal tissue sections were boiled in a citrate buffer (pH 6.0) for 20 min for antigen retrieval and were then incubated, respectively, with one of the following antibodies: rabbit anti-homocysteine (1/50, cat. ab15154; Abcam); mouse anti-rhodopsin (1/400, cat. Ab81702; Abcam ) ; mouse anti-calbindin (1/50, cat. sc-74462; Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-Pax-6 antibody (1/50, cat. sc-32766; Santa Cruz Biotechnology); rabbit anti-Islet-1 (1/150, cat. Ab20670; Abcam); sheep anti-Chx-10 (1/150, cat. ab16141; Abcam); or rabbit anti-GFAP (1/250, cat. 2301–1; Epitomics, Burlingame, CA).

    Techniques:

    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

    The ER chaperone DNAJC10 ) (A) Schematic showing the 5 isoforms of DNAJC10 , and primers used in this study to distinguish between DNAJC10 pseudogenes (blue) and total DNAJC10 transcripts that detects all 5 isoforms (red). (B–C) qPCR measuring total DNAJC10 transcript levels and DNAJC10 pseudogene levels in HB9 + purified MNs and ISL1 + purified MNs respectively indicating that SMA MNs express more pseudogene transcripts even though total DNAJC10 levels were unchanged. Notably, Type I MNs, which has least SMN, express the most DNAJC10 pseudogene transcripts. (D) Western blot showing that SMA MN cultures express less DNAJC10 protein compared to wild-type MNs. In addition, when SMN is depleted in wild-type MNs (BJ si-SMN), DNAJC10 protein levels also decreased. (E) Overexpression of SMN in Type I SMA MNs reduces DNAJC10 pseudogene expression without changing total DNAJC10 levels. (F) Depletion of DNAJC10 by siRNAs in wild-type MNs led to induction of chronic ER stress markers ATF4 and CHOP. (G) Knockdown of DNAJC10 in two wild-type lines increased percentage of ISL1 + MNs co-expressing ATF4. (H) Depletion of DNAJC10 reduced number of ISL1 + MNs.

    Journal: Cell stem cell

    Article Title: Genome-Wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy

    doi: 10.1016/j.stem.2015.08.003

    Figure Lengend Snippet: The ER chaperone DNAJC10 ) (A) Schematic showing the 5 isoforms of DNAJC10 , and primers used in this study to distinguish between DNAJC10 pseudogenes (blue) and total DNAJC10 transcripts that detects all 5 isoforms (red). (B–C) qPCR measuring total DNAJC10 transcript levels and DNAJC10 pseudogene levels in HB9 + purified MNs and ISL1 + purified MNs respectively indicating that SMA MNs express more pseudogene transcripts even though total DNAJC10 levels were unchanged. Notably, Type I MNs, which has least SMN, express the most DNAJC10 pseudogene transcripts. (D) Western blot showing that SMA MN cultures express less DNAJC10 protein compared to wild-type MNs. In addition, when SMN is depleted in wild-type MNs (BJ si-SMN), DNAJC10 protein levels also decreased. (E) Overexpression of SMN in Type I SMA MNs reduces DNAJC10 pseudogene expression without changing total DNAJC10 levels. (F) Depletion of DNAJC10 by siRNAs in wild-type MNs led to induction of chronic ER stress markers ATF4 and CHOP. (G) Knockdown of DNAJC10 in two wild-type lines increased percentage of ISL1 + MNs co-expressing ATF4. (H) Depletion of DNAJC10 reduced number of ISL1 + MNs.

    Article Snippet: Primary antibodies used in this study (and their respective dilutions) are as follow: rabbit ISL1 (Abcam ab109517; 1:1000), mouse TUJ1 (Covance MMS-435P; 1:1000), mouse HB9 (DSHB 81.5C10; 1:200), mouse ISL1 (DSHB 39.4D5), mouse SMI-32 (Calbiochem NE-1023; 1:1000), goat ChAT (Millipore AB114P; 1:100), rabbit ATF6 (Abcam ab37149; 1:500), rabbit ATF4 (Cell Signaling #11815; 1:100), rabbit cleaved Caspase-3 (Cell Signaling #9661) and mouse monoclonal SMN (BD Pharmingen; 1:250).

    Techniques: Real-time Polymerase Chain Reaction, Purification, Western Blot, Over Expression, Expressing

    ) (A) Quantitative PCR of FACS-purified HB9 + MNs derived from wild-type and SMA cultures at day 31. SMA MNs from 1-38G and 1-51N show higher expression of ER stress markers. Only 1-38G MNs show increased expression of markers characteristic of chronic ER stress: PERK , CHOP and CASP3 . Gene expression is normalized to GAPDH . (B) Co-staining of MN marker ISL1 (green) and ER stress marker ATF6 (red) in wild-type and SMA MN cultures. Scale bar indicates 25 μm. Nuclear ATF6 intensities of ISL1 + and ISL1 − cells are also measured. (C) Co-staining of motor neuron marker ISL1 (green) and ER stress marker ATF4 (red) in wild-type and SMA MN cultures. Scale bar indicates 100 μm. The graph depicts percentage of ATF4 + cells in whole cultures, as well as percentage of ISL1 + MNs co-expressing ATF4. (D–E) Quantification of ISL1 + MNs co-expressing ATF4 and cCASP3 respectively at days 23, 28 and 31, showing increasing ER stress over time, co-incident with increased apoptosis in SMA MNs. (F) Immunostaining analysis indicating that majority of MNs undergoing ER stress (ISL1 + ATF4 + MNs) also co-express ChAT. (G–H) qPCR and western blot analyses respectively show that knockdown of SMN in wild-type BJ-riPS MNs increased UPR target gene expression in a dose-dependent manner. (I) Co-staining of ISL1 (green) and ATF6 (red) in wild-type MNs transfected with non-targeting siRNA (si-NT) and SMN siRNA (si-SMN) at the indicated dose. The scale bar indicates 50 μm. Increase in nuclear ATF6 intensity upon SMN knockdown is graphically represented. (J) Co-staining of ISL1 (green) and ATF4 (red) in SMN knockdown BJ-riPS cultures. Scale bar indicates 100 μm. The graph shows increase in percentage of total cells and ISL1 + MNs co-expressing ATF4 in SMN knockdown conditions.

    Journal: Cell stem cell

    Article Title: Genome-Wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy

    doi: 10.1016/j.stem.2015.08.003

    Figure Lengend Snippet: ) (A) Quantitative PCR of FACS-purified HB9 + MNs derived from wild-type and SMA cultures at day 31. SMA MNs from 1-38G and 1-51N show higher expression of ER stress markers. Only 1-38G MNs show increased expression of markers characteristic of chronic ER stress: PERK , CHOP and CASP3 . Gene expression is normalized to GAPDH . (B) Co-staining of MN marker ISL1 (green) and ER stress marker ATF6 (red) in wild-type and SMA MN cultures. Scale bar indicates 25 μm. Nuclear ATF6 intensities of ISL1 + and ISL1 − cells are also measured. (C) Co-staining of motor neuron marker ISL1 (green) and ER stress marker ATF4 (red) in wild-type and SMA MN cultures. Scale bar indicates 100 μm. The graph depicts percentage of ATF4 + cells in whole cultures, as well as percentage of ISL1 + MNs co-expressing ATF4. (D–E) Quantification of ISL1 + MNs co-expressing ATF4 and cCASP3 respectively at days 23, 28 and 31, showing increasing ER stress over time, co-incident with increased apoptosis in SMA MNs. (F) Immunostaining analysis indicating that majority of MNs undergoing ER stress (ISL1 + ATF4 + MNs) also co-express ChAT. (G–H) qPCR and western blot analyses respectively show that knockdown of SMN in wild-type BJ-riPS MNs increased UPR target gene expression in a dose-dependent manner. (I) Co-staining of ISL1 (green) and ATF6 (red) in wild-type MNs transfected with non-targeting siRNA (si-NT) and SMN siRNA (si-SMN) at the indicated dose. The scale bar indicates 50 μm. Increase in nuclear ATF6 intensity upon SMN knockdown is graphically represented. (J) Co-staining of ISL1 (green) and ATF4 (red) in SMN knockdown BJ-riPS cultures. Scale bar indicates 100 μm. The graph shows increase in percentage of total cells and ISL1 + MNs co-expressing ATF4 in SMN knockdown conditions.

    Article Snippet: Primary antibodies used in this study (and their respective dilutions) are as follow: rabbit ISL1 (Abcam ab109517; 1:1000), mouse TUJ1 (Covance MMS-435P; 1:1000), mouse HB9 (DSHB 81.5C10; 1:200), mouse ISL1 (DSHB 39.4D5), mouse SMI-32 (Calbiochem NE-1023; 1:1000), goat ChAT (Millipore AB114P; 1:100), rabbit ATF6 (Abcam ab37149; 1:500), rabbit ATF4 (Cell Signaling #11815; 1:100), rabbit cleaved Caspase-3 (Cell Signaling #9661) and mouse monoclonal SMN (BD Pharmingen; 1:250).

    Techniques: Real-time Polymerase Chain Reaction, FACS, Purification, Derivative Assay, Expressing, Staining, Marker, Immunostaining, Western Blot, Transfection

    ) (A) Experimental outline of lentiviral infection to achieve SMN overexpression in Types I and II SMA MN cultures. (B) Quantitative measurement of SMN intensity indicates that SMN expression is 2.6-fold higher upon doxycycline addition in Type I MNs and 3.2-fold higher in Type II MNs. (C) Immunostaining showing SMN overexpression (in green) after 3 days induction with 500 ng/ml doxycycline. Cellular nuclei are counterstained with DAPI. The scale bar indicates 50 μm. (D) Quantitative assessment of nuclear ATF6 intensity in various MN populations (HB9 + , ISL1 + and ChAT + ) with and without doxycycline induction SMN overexpression showing significant reduction of nuclear ATF6 levels in each of the MN populations, while the non-MNs (HB9 − , ISL1 − and ChAT − populations) remain unchanged. (E) Overexpression of SMN in Type I MN cultures reduces UPR target gene expression by qPCR. (F) Western blot showing reduced ER stress markers upon SMN overexpression in Type II MNs. (G) Co-staining of ISL1, SMN and ATF4 showing fewer ATF4 + cells upon SMN overexpression in Type II MN cultures. (H) Quantification of ATF4 + MNs in Types I and II SMA MN cultures upon SMN overexpression.

    Journal: Cell stem cell

    Article Title: Genome-Wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy

    doi: 10.1016/j.stem.2015.08.003

    Figure Lengend Snippet: ) (A) Experimental outline of lentiviral infection to achieve SMN overexpression in Types I and II SMA MN cultures. (B) Quantitative measurement of SMN intensity indicates that SMN expression is 2.6-fold higher upon doxycycline addition in Type I MNs and 3.2-fold higher in Type II MNs. (C) Immunostaining showing SMN overexpression (in green) after 3 days induction with 500 ng/ml doxycycline. Cellular nuclei are counterstained with DAPI. The scale bar indicates 50 μm. (D) Quantitative assessment of nuclear ATF6 intensity in various MN populations (HB9 + , ISL1 + and ChAT + ) with and without doxycycline induction SMN overexpression showing significant reduction of nuclear ATF6 levels in each of the MN populations, while the non-MNs (HB9 − , ISL1 − and ChAT − populations) remain unchanged. (E) Overexpression of SMN in Type I MN cultures reduces UPR target gene expression by qPCR. (F) Western blot showing reduced ER stress markers upon SMN overexpression in Type II MNs. (G) Co-staining of ISL1, SMN and ATF4 showing fewer ATF4 + cells upon SMN overexpression in Type II MN cultures. (H) Quantification of ATF4 + MNs in Types I and II SMA MN cultures upon SMN overexpression.

    Article Snippet: Primary antibodies used in this study (and their respective dilutions) are as follow: rabbit ISL1 (Abcam ab109517; 1:1000), mouse TUJ1 (Covance MMS-435P; 1:1000), mouse HB9 (DSHB 81.5C10; 1:200), mouse ISL1 (DSHB 39.4D5), mouse SMI-32 (Calbiochem NE-1023; 1:1000), goat ChAT (Millipore AB114P; 1:100), rabbit ATF6 (Abcam ab37149; 1:500), rabbit ATF4 (Cell Signaling #11815; 1:100), rabbit cleaved Caspase-3 (Cell Signaling #9661) and mouse monoclonal SMN (BD Pharmingen; 1:250).

    Techniques: Infection, Over Expression, Expressing, Immunostaining, Real-time Polymerase Chain Reaction, Western Blot, Staining

    ) (A) Experimental outline of ER stress inhibitor treatment on MN cultures. Cultures were treated for three days, starting from day 28, and analyzed at day 31. (B–C) Percentage of ISL1 + MN after ER stress inhibitor treatment in SMA Type I (1-38G), and Type II (1-51N) cultures respectively. Values are normalized to number of MNs plated at day 28. (D) Percentage of ISL1 + MNs after ER stress inhibitor treatment in wild-type (BJ) MN cultures. The values were not significant ( n.s ). (E) Western blot of Type I MN cultures treated with the various ER stress inhibitors indicating that SMN levels remained the same while CHOP protein is reduced. (F) Spliced XBP1 assay performed on SMA Type I MN cultures treated with DMSO, ER stress inhibitors and 1 μM tunicamycin (TM) as positive control. (G) Measurement of nuclear ATF6 intensity in SMA Type I MN cultures treated with the indicated compound. (H) Quantification of cleaved Caspase3 + MNs in SMA Type I MN cultures treated with the indicated compound. (I–J) ER stress inhibitor treatment increases soma size of Type I and Type II SMA MNs respectively. (K) Representative images of Type I SMA MN cultures treated with DMSO, SAL or GUA, stained with ISL1 and TUJ1. Magnified images of the white boxes are shown in the bottom panel. The white-dotted circumference represents the measured soma sizes.

    Journal: Cell stem cell

    Article Title: Genome-Wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy

    doi: 10.1016/j.stem.2015.08.003

    Figure Lengend Snippet: ) (A) Experimental outline of ER stress inhibitor treatment on MN cultures. Cultures were treated for three days, starting from day 28, and analyzed at day 31. (B–C) Percentage of ISL1 + MN after ER stress inhibitor treatment in SMA Type I (1-38G), and Type II (1-51N) cultures respectively. Values are normalized to number of MNs plated at day 28. (D) Percentage of ISL1 + MNs after ER stress inhibitor treatment in wild-type (BJ) MN cultures. The values were not significant ( n.s ). (E) Western blot of Type I MN cultures treated with the various ER stress inhibitors indicating that SMN levels remained the same while CHOP protein is reduced. (F) Spliced XBP1 assay performed on SMA Type I MN cultures treated with DMSO, ER stress inhibitors and 1 μM tunicamycin (TM) as positive control. (G) Measurement of nuclear ATF6 intensity in SMA Type I MN cultures treated with the indicated compound. (H) Quantification of cleaved Caspase3 + MNs in SMA Type I MN cultures treated with the indicated compound. (I–J) ER stress inhibitor treatment increases soma size of Type I and Type II SMA MNs respectively. (K) Representative images of Type I SMA MN cultures treated with DMSO, SAL or GUA, stained with ISL1 and TUJ1. Magnified images of the white boxes are shown in the bottom panel. The white-dotted circumference represents the measured soma sizes.

    Article Snippet: Primary antibodies used in this study (and their respective dilutions) are as follow: rabbit ISL1 (Abcam ab109517; 1:1000), mouse TUJ1 (Covance MMS-435P; 1:1000), mouse HB9 (DSHB 81.5C10; 1:200), mouse ISL1 (DSHB 39.4D5), mouse SMI-32 (Calbiochem NE-1023; 1:1000), goat ChAT (Millipore AB114P; 1:100), rabbit ATF6 (Abcam ab37149; 1:500), rabbit ATF4 (Cell Signaling #11815; 1:100), rabbit cleaved Caspase-3 (Cell Signaling #9661) and mouse monoclonal SMN (BD Pharmingen; 1:250).

    Techniques: Western Blot, Positive Control, Staining

    ) (A) Differentiation scheme of iPSCs to MNs in 31 days. (B) MN cultures at day 23, showing that both wild-type (BJ-riPS) and SMA Type I (1-38G) iPSCs differentiate into MNs expressing ISL1 and TUJ1. Many ISL1 + cells also co-express HB9, another MN transcription factor. The scale bar indicates 50 μm. (C) Percentage overlap between ISL1 and HB9 expression in MNs assessed by immunostaining. The percentages of ISL1 + /HB9 − , ISL1 + /HB9 + and ISL1 − /HB9 + MNs were similar between wild-type and SMA iPSC-derived cultures. (D) Immunostaining showing HB9 + and/or ISL1 + co-expressing ChAT at day 28. (E) Quantification of HB9 + MNs co-expressing ChAT at days 28 and 31. (F) Quantification of ISL1 + MNs co-expressing ChAT at days 28 and 31. (G–J) Quantification of respective ISL1 + , HB9 + , ISL1 + and/or HB9 + and ChAT + MN numbers from wild-type (BJ-riPS), SMA Type I (1-38G) and SMA Type II (1-51N) cultures at days 28 and 31. Numbers are shown as percentages normalized to respective MN count at day 28. (K) Representative images of BJ-riPS and 1-38G-derived MNs co-stained with ISL1 and TUJ1 at day 23 and day 31. The white-dotted circumference represents the measured soma sizes. (L) Measurements of ISL1 + MN soma sizes at day 23 and day 31. At day 23, soma sizes of wild-type and SMA MNs were similar – between 210 μm 2 and 230 μm 2 . By day 31, wild-type MNs remained in size while SMA MNs were significantly smaller. (M) Measurements of healthy (ISL1 + cCASP3 − ) MN soma sizes and apoptotic (ISL1 + cCASP3 + ) MN soma sizes at day 31 showing that apoptotic MNs were significantly smaller, regardless of genotype (about 165 μm 2 ), and that non-apoptotic SMA MNs remain smaller compared to wild-type counterparts.

    Journal: Cell stem cell

    Article Title: Genome-Wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy

    doi: 10.1016/j.stem.2015.08.003

    Figure Lengend Snippet: ) (A) Differentiation scheme of iPSCs to MNs in 31 days. (B) MN cultures at day 23, showing that both wild-type (BJ-riPS) and SMA Type I (1-38G) iPSCs differentiate into MNs expressing ISL1 and TUJ1. Many ISL1 + cells also co-express HB9, another MN transcription factor. The scale bar indicates 50 μm. (C) Percentage overlap between ISL1 and HB9 expression in MNs assessed by immunostaining. The percentages of ISL1 + /HB9 − , ISL1 + /HB9 + and ISL1 − /HB9 + MNs were similar between wild-type and SMA iPSC-derived cultures. (D) Immunostaining showing HB9 + and/or ISL1 + co-expressing ChAT at day 28. (E) Quantification of HB9 + MNs co-expressing ChAT at days 28 and 31. (F) Quantification of ISL1 + MNs co-expressing ChAT at days 28 and 31. (G–J) Quantification of respective ISL1 + , HB9 + , ISL1 + and/or HB9 + and ChAT + MN numbers from wild-type (BJ-riPS), SMA Type I (1-38G) and SMA Type II (1-51N) cultures at days 28 and 31. Numbers are shown as percentages normalized to respective MN count at day 28. (K) Representative images of BJ-riPS and 1-38G-derived MNs co-stained with ISL1 and TUJ1 at day 23 and day 31. The white-dotted circumference represents the measured soma sizes. (L) Measurements of ISL1 + MN soma sizes at day 23 and day 31. At day 23, soma sizes of wild-type and SMA MNs were similar – between 210 μm 2 and 230 μm 2 . By day 31, wild-type MNs remained in size while SMA MNs were significantly smaller. (M) Measurements of healthy (ISL1 + cCASP3 − ) MN soma sizes and apoptotic (ISL1 + cCASP3 + ) MN soma sizes at day 31 showing that apoptotic MNs were significantly smaller, regardless of genotype (about 165 μm 2 ), and that non-apoptotic SMA MNs remain smaller compared to wild-type counterparts.

    Article Snippet: Primary antibodies used in this study (and their respective dilutions) are as follow: rabbit ISL1 (Abcam ab109517; 1:1000), mouse TUJ1 (Covance MMS-435P; 1:1000), mouse HB9 (DSHB 81.5C10; 1:200), mouse ISL1 (DSHB 39.4D5), mouse SMI-32 (Calbiochem NE-1023; 1:1000), goat ChAT (Millipore AB114P; 1:100), rabbit ATF6 (Abcam ab37149; 1:500), rabbit ATF4 (Cell Signaling #11815; 1:100), rabbit cleaved Caspase-3 (Cell Signaling #9661) and mouse monoclonal SMN (BD Pharmingen; 1:250).

    Techniques: Expressing, Immunostaining, Derivative Assay, Staining