Structured Review

Abcam mouse anti isl1
Figure 6. Structure of cardiomyocyte colonies grown in the primary culture of rat neonatal myocardial cells. ( A–C ) Different stages of development of the colonies stemming from <t>Isl1</t> + CSCs. ( A ) Cell division, DIV 2. Isl1 + (FITC, green), GATA-4 (phycoerythrin, red). ( B ) Colony consisting of approximately 8 cells, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( C ) Large Isl1 + colony, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( D ) The optical sections of colonies formed by Isl1 + , c-kit + , and Sca1 + CSCs on the 11th DIV. Isl1 + CSCs (Alexa 405, blue), Z = 12. c-kit + CSCs (FITC, green), Z = 12. Sca1 + CSCs (Alexa 405, blue), Z = 11. Actin was stained using rhodamine-phalloidin (red). ( E ) Differentiation of c-kit + CSCs inside the colony on the 13th DIV. Overlaid optical section of transmitted light and fluorescent images in 2 emitting wavelengths: 488 nm (FITC) and 543 nm (Alexa) in the bottom (Z = 5), in the middle (Z = 10), and the top (Z = 20) parts of the colony. c-kit + expression was revealed by FITC-conjugated antibodies (green), and α-sarcomeric actinin was revealed by Alexa-conjugated antibodies (red). Confocal microscope, Leica TCS SP5 (Germany), objective ×63, oil.
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Images

1) Product Images from "Characterization of contracting cardiomyocyte colonies in the primary culture of neonatal rat myocardial cells"

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

Journal: Cell Cycle

doi: 10.4161/cc.27768

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

Techniques Used: Staining, Expressing, Microscopy

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

Techniques Used: Microscopy

2) 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

3) Product Images from "α2-Chimaerin Is Required for Eph Receptor-Class-Specific Spinal Motor Axon Guidance and Coordinate Activation of Antagonistic Muscles"

Article Title: α2-Chimaerin Is Required for Eph Receptor-Class-Specific Spinal Motor Axon Guidance and Coordinate Activation of Antagonistic Muscles

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4151-14.2015

Expression of α2-chimaerin in LMC motor neurons. A – F , Detection of mRNA in consecutive sections of the spinal cord. All mouse sections ( A – C ) are from e11.5 lumbar spinal cord; all chick sections ( D – F ) are from HH st. 25/26 lumbar spinal cord. A , B , Detection of Lim1 ( A ) and Isl1 ( B ) mRNA in lateral and medial LMC neurons, respectively, in the mouse spinal cord. Dark pixels denote high signal. C , Detection of α2-chimaerin mRNA in both medial and lateral mouse LMC neurons. D , E , Detection of Lim1 ( D ) and Isl1 ( E ) mRNA in lateral and medial chick LMC neurons, respectively. F , Detection of α-chimaerin mRNA in both medial and lateral chick LMC neurons. M, Medial; L, lateral. Scale bars in A – F , 50 μm. G , α2-chimaerin interacts with EphA4 and EphB1 in mouse spinal cord. Lysates from wild-type P3 mouse spinal cord were immunoprecipitated with anti-α2-chimaerin antibody or anti-HA antibody (negative control). Bound and total proteins were analyzed by immunoblotting with antibodies against EphA4, EphB1, α2-chimaerin. Input represents a 1% aliquot of the total load.
Figure Legend Snippet: Expression of α2-chimaerin in LMC motor neurons. A – F , Detection of mRNA in consecutive sections of the spinal cord. All mouse sections ( A – C ) are from e11.5 lumbar spinal cord; all chick sections ( D – F ) are from HH st. 25/26 lumbar spinal cord. A , B , Detection of Lim1 ( A ) and Isl1 ( B ) mRNA in lateral and medial LMC neurons, respectively, in the mouse spinal cord. Dark pixels denote high signal. C , Detection of α2-chimaerin mRNA in both medial and lateral mouse LMC neurons. D , E , Detection of Lim1 ( D ) and Isl1 ( E ) mRNA in lateral and medial chick LMC neurons, respectively. F , Detection of α-chimaerin mRNA in both medial and lateral chick LMC neurons. M, Medial; L, lateral. Scale bars in A – F , 50 μm. G , α2-chimaerin interacts with EphA4 and EphB1 in mouse spinal cord. Lysates from wild-type P3 mouse spinal cord were immunoprecipitated with anti-α2-chimaerin antibody or anti-HA antibody (negative control). Bound and total proteins were analyzed by immunoblotting with antibodies against EphA4, EphB1, α2-chimaerin. Input represents a 1% aliquot of the total load.

Techniques Used: Expressing, Immunoprecipitation, Negative Control

Cell-autonomous function of α2-chimaerin in LMC axon guidance in the limb. A – F , Detection of Foxp1, Isl1, GFP, and α2-chimaerin mRNA in LMC neurons of chick HH st. 28/29 embryos electroporated with GFP ( A – C ) and [α 2-chi ] siRNA + GFP ( D – F ). G , Quantification of effects of GFP and [α 2-chi ] siRNA + GFP ). H , Number of LMC motor neurons expressed as the average number of total (Foxp1 + ) LMC neurons per section (#Foxp1 + /section). I , J , Number of total or electroporated medial (Foxp1 + Isl1 + ) and lateral (Foxp1 + Isl1 − ) LMC motor neurons in lumbar spinal cord expressed as the percentage of total motor neurons [Foxp1 + MNs (%)] ( I ) or electroporated motor neurons [GFP + MNs (%)] ( J ). K – L , GFP and neurofilament detection in the limb nerves in the crural plexus of embryos electroporated with GFP ( K ), [α 2-chi ] siRNA + GFP ( L ), α 2-chi::GFP ( M ), α 2-chi N94H ::GFP ( N ), or α 2-chi ΔYRV ::GFP ( O ). Quantification of GFP signals in both groups expressed as, respectively, percentage in dorsal and ventral limb nerves [GFP Fluo (%)]. Number of embryos: n = 5 for all groups. All images are at chick HH st. 28/29 lumbar levels. Error bars indicate SD. *** p
Figure Legend Snippet: Cell-autonomous function of α2-chimaerin in LMC axon guidance in the limb. A – F , Detection of Foxp1, Isl1, GFP, and α2-chimaerin mRNA in LMC neurons of chick HH st. 28/29 embryos electroporated with GFP ( A – C ) and [α 2-chi ] siRNA + GFP ( D – F ). G , Quantification of effects of GFP and [α 2-chi ] siRNA + GFP ). H , Number of LMC motor neurons expressed as the average number of total (Foxp1 + ) LMC neurons per section (#Foxp1 + /section). I , J , Number of total or electroporated medial (Foxp1 + Isl1 + ) and lateral (Foxp1 + Isl1 − ) LMC motor neurons in lumbar spinal cord expressed as the percentage of total motor neurons [Foxp1 + MNs (%)] ( I ) or electroporated motor neurons [GFP + MNs (%)] ( J ). K – L , GFP and neurofilament detection in the limb nerves in the crural plexus of embryos electroporated with GFP ( K ), [α 2-chi ] siRNA + GFP ( L ), α 2-chi::GFP ( M ), α 2-chi N94H ::GFP ( N ), or α 2-chi ΔYRV ::GFP ( O ). Quantification of GFP signals in both groups expressed as, respectively, percentage in dorsal and ventral limb nerves [GFP Fluo (%)]. Number of embryos: n = 5 for all groups. All images are at chick HH st. 28/29 lumbar levels. Error bars indicate SD. *** p

Techniques Used:

α2-chimaerin is required for the selection of LMC axon trajectory. A – H , Detection of Isl1 (green), and Foxp1 (red) protein and α2-chimaerin mRNA in the LMC region at the lumbar level of control α 2-chi gt /+ ( A – D ) or α 2-chi gt/gt ( E – F ) e12.5 mouse embryos. I , Number of LMC motor neurons expressed as the average number of total (Foxp1 + ) LMC neurons per section (# FoxP1 + /section). J , Number of total medial (FoxP1 + Isl1 + ) and lateral (FoxP1 + Isl1 − ) LMC motor neurons in lumbar spinal cord expressed as the percentage of total motor neurons [FoxP1 + MNs (%)]. K – R , Detection of Isl1 (green) and EphA4 (red) protein, Isl1 and EphB1 mRNA in the spinal cord of mouse e12.5 α 2-chi gt /+ ( K – L ) or α 2-chi gt/gt ( O – R ) embryos. The expression and localization of EphA4, EphB1 , and Isl1 in LMC are not obviously changed compared with control littermates. Number of embryos quantified: n = 7 for all experimental groups. Error bars indicate SD; n.s., not significant. Scale bars: A – H , 20 μm; K – R , 15 μm. S , T , LacZ and neurofilament detection in the hindlimb nerves of e12.5 α 2-chi gt /+ ; Lim1 tlz /+ ( S ) or α 2-chi gt/gt ; Lim1 tlz /+ ( T ) mice. Quantification of LacZ signals in both groups expressed as, respectively, percentage in dorsal and ventral limb nerves [lacZ Fluo Int (%)]. Number of embryos: n = 6 ( S ), 7 ( T ). d, Dorsal; v, ventral; NF, neurofilament; α2-chi, α2-chimaerin. Error bars indicate SD; *** p
Figure Legend Snippet: α2-chimaerin is required for the selection of LMC axon trajectory. A – H , Detection of Isl1 (green), and Foxp1 (red) protein and α2-chimaerin mRNA in the LMC region at the lumbar level of control α 2-chi gt /+ ( A – D ) or α 2-chi gt/gt ( E – F ) e12.5 mouse embryos. I , Number of LMC motor neurons expressed as the average number of total (Foxp1 + ) LMC neurons per section (# FoxP1 + /section). J , Number of total medial (FoxP1 + Isl1 + ) and lateral (FoxP1 + Isl1 − ) LMC motor neurons in lumbar spinal cord expressed as the percentage of total motor neurons [FoxP1 + MNs (%)]. K – R , Detection of Isl1 (green) and EphA4 (red) protein, Isl1 and EphB1 mRNA in the spinal cord of mouse e12.5 α 2-chi gt /+ ( K – L ) or α 2-chi gt/gt ( O – R ) embryos. The expression and localization of EphA4, EphB1 , and Isl1 in LMC are not obviously changed compared with control littermates. Number of embryos quantified: n = 7 for all experimental groups. Error bars indicate SD; n.s., not significant. Scale bars: A – H , 20 μm; K – R , 15 μm. S , T , LacZ and neurofilament detection in the hindlimb nerves of e12.5 α 2-chi gt /+ ; Lim1 tlz /+ ( S ) or α 2-chi gt/gt ; Lim1 tlz /+ ( T ) mice. Quantification of LacZ signals in both groups expressed as, respectively, percentage in dorsal and ventral limb nerves [lacZ Fluo Int (%)]. Number of embryos: n = 6 ( S ), 7 ( T ). d, Dorsal; v, ventral; NF, neurofilament; α2-chi, α2-chimaerin. Error bars indicate SD; *** p

Techniques Used: Selection, Expressing, Mouse Assay

α2-chimaerin function is required for EphA forward signaling in cultured LMC neurons. A – C , Detection of medial (GFP + ) LMC neurites of e [ Isl1 ] ::GFP ( A ), [α 2-chi ] siRNA + e [ Isl1 ] ::GFP ( B ), or α 2-chi + e [ Isl1 ] ::GFP ( C ) on ephrinB2-Fc/Fc stripes and superimposed images of five representative explants from each experimental group highlighting the distribution of LMC neurites. Quantification of medial (GFP + ) LMC neurites on first (red) and second (pale) stripes expressed as a percentage of total GFP signals. Minimal number of neurites and explants: 79 and 11 for all groups. D – G , Detection of lateral (EphA4 + ) LMC neurites of GFP ( D ), [α 2-chi ] siRNA + GFP ( E ), α 2-chi::GFP ( F ), or α 2-chi Δ YRV ::GFP ( G ) on ephrinA5-Fc/Fc stripes and superimposed images of five representative explants from each experimental group highlighting the distribution of LMC neurites. Quantification of electroporated lateral (EphA4 + ) LMC neurites on first (blue) and second (pale) stripes expressed as a percentage of total EphA4 signals. Minimal number of neurites and explants: 72 and 10 for all groups. eB2, ephrin-B2; eA5, ephrin-A5. Error bars indicate SD. *** p
Figure Legend Snippet: α2-chimaerin function is required for EphA forward signaling in cultured LMC neurons. A – C , Detection of medial (GFP + ) LMC neurites of e [ Isl1 ] ::GFP ( A ), [α 2-chi ] siRNA + e [ Isl1 ] ::GFP ( B ), or α 2-chi + e [ Isl1 ] ::GFP ( C ) on ephrinB2-Fc/Fc stripes and superimposed images of five representative explants from each experimental group highlighting the distribution of LMC neurites. Quantification of medial (GFP + ) LMC neurites on first (red) and second (pale) stripes expressed as a percentage of total GFP signals. Minimal number of neurites and explants: 79 and 11 for all groups. D – G , Detection of lateral (EphA4 + ) LMC neurites of GFP ( D ), [α 2-chi ] siRNA + GFP ( E ), α 2-chi::GFP ( F ), or α 2-chi Δ YRV ::GFP ( G ) on ephrinA5-Fc/Fc stripes and superimposed images of five representative explants from each experimental group highlighting the distribution of LMC neurites. Quantification of electroporated lateral (EphA4 + ) LMC neurites on first (blue) and second (pale) stripes expressed as a percentage of total EphA4 signals. Minimal number of neurites and explants: 72 and 10 for all groups. eB2, ephrin-B2; eA5, ephrin-A5. Error bars indicate SD. *** p

Techniques Used: Cell Culture

4) Product Images from "Src Family Kinases Are Required for Limb Trajectory Selection by Spinal Motor Axons"

Article Title: Src Family Kinases Are Required for Limb Trajectory Selection by Spinal Motor Axons

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0265-09.2009

Expression of Src , Fyn , and Csk in chick and mouse LMC motor neurons. A–J , Detection of mRNA in consecutive sections of the spinal cord. All chick sections ( A–E ) are HH st. 25/26 lumbar spinal cord; all mouse sections ( F–J ) are e11.5 lumbar spinal cord. A , B , Detection of Lim1 and Isl1 mRNA in the chick spinal cord. Lim1 ( A ) and Isl1 ( B ) expression highlights lateral and medial LMC motor neurons, respectively. C , D , Detection of Src ( C ) and Fyn ( D ) mRNA, in both medial and lateral chick LMC neurons. E , Detection of Csk mRNA in LMC in both medial and lateral chick LMC neurons. F , G , Detection of Lim1 ( F ) and Isl1 ( G ) mRNA in mouse LMC motor neurons. H–J , Detection of Src ( H ), Fyn ( I ), and Csk ( J ) mRNA in medial and lateral mouse LMC neurons. Scale bar, 25 μm ( A–J ).
Figure Legend Snippet: Expression of Src , Fyn , and Csk in chick and mouse LMC motor neurons. A–J , Detection of mRNA in consecutive sections of the spinal cord. All chick sections ( A–E ) are HH st. 25/26 lumbar spinal cord; all mouse sections ( F–J ) are e11.5 lumbar spinal cord. A , B , Detection of Lim1 and Isl1 mRNA in the chick spinal cord. Lim1 ( A ) and Isl1 ( B ) expression highlights lateral and medial LMC motor neurons, respectively. C , D , Detection of Src ( C ) and Fyn ( D ) mRNA, in both medial and lateral chick LMC neurons. E , Detection of Csk mRNA in LMC in both medial and lateral chick LMC neurons. F , G , Detection of Lim1 ( F ) and Isl1 ( G ) mRNA in mouse LMC motor neurons. H–J , Detection of Src ( H ), Fyn ( I ), and Csk ( J ) mRNA in medial and lateral mouse LMC neurons. Scale bar, 25 μm ( A–J ).

Techniques Used: Expressing

SFK function is required for the fidelity of LMC motor axon limb trajectories. A , I , Diagram depicting retrograde labeling of LMC neurons by HRP injections into dorsal ( A ) or ventral ( I ) hindlimb shank muscles of chick HH st. 28/29 embryos overexpressing Csk::GFP or GFP . B–G , Detection of HRP (red), Isl1 (blue), and GFP (green) in the LMC region of GFP ( B–D ) or Csk::GFP ( E–G ) electroporated embryos injected with HRP into dorsal shank muscles. Examples of electroporated medial LMC motor neurons labeled with HRP are indicated by arrows ( F , G ) and arrowheads ( F , G ). Examples indicated by arrowheads are shown at a higher magnification (insets of F , G ). H , Proportions (%) of electroporated medial LMC motor neurons labeled with HRP in embryos injected with HRP into dorsal shank muscles. In dorsally filled GFP expressing embryos, 8 ± 2% of HRP + GFP + LMC neurons were Isl1 + . In dorsally filled Csk::GFP expressing embryos, 33 ± 2% of HRP + GFP + LMC neurons were Isl1 + . Number of embryos analyzed: n = 4 for both GFP and Csk::GFP expressing embryos. J–O , Detection of HRP (red), Lim1 (blue), and GFP (green) in the LMC region of GFP ( J–L ) or Csk::GFP ( M–O ) electroporated embryos injected with HRP into ventral shank muscles. Examples of electroporated lateral LMC motor neuron labeled with HRP are indicated by arrows ( N , O ) and arrowheads ( N , O ). Examples indicated by arrowheads are shown at a higher magnification (insets of N , O ). P , Proportions (%) of electroporated lateral LMC motor neurons labeled with HRP in embryos injected with HRP into ventral shank muscles. In ventrally filled GFP expressing embryos, 6 ± 5% of HRP + GFP + LMC neurons were Lim1 + . In ventrally filled Csk::GFP expressing embryos, 17 ± 3% of HRP + GFP + LMC neurons were Lim1 + . Number of embryos quantified: n = 4 for GFP and Csk::GFP expressing embryos. Proportions of HRP + GFP + medial LMC neurons in dorsally filled Csk::GFP expressing embryos and those in dorsally filled GFP expressing embryos are significantly different ( p
Figure Legend Snippet: SFK function is required for the fidelity of LMC motor axon limb trajectories. A , I , Diagram depicting retrograde labeling of LMC neurons by HRP injections into dorsal ( A ) or ventral ( I ) hindlimb shank muscles of chick HH st. 28/29 embryos overexpressing Csk::GFP or GFP . B–G , Detection of HRP (red), Isl1 (blue), and GFP (green) in the LMC region of GFP ( B–D ) or Csk::GFP ( E–G ) electroporated embryos injected with HRP into dorsal shank muscles. Examples of electroporated medial LMC motor neurons labeled with HRP are indicated by arrows ( F , G ) and arrowheads ( F , G ). Examples indicated by arrowheads are shown at a higher magnification (insets of F , G ). H , Proportions (%) of electroporated medial LMC motor neurons labeled with HRP in embryos injected with HRP into dorsal shank muscles. In dorsally filled GFP expressing embryos, 8 ± 2% of HRP + GFP + LMC neurons were Isl1 + . In dorsally filled Csk::GFP expressing embryos, 33 ± 2% of HRP + GFP + LMC neurons were Isl1 + . Number of embryos analyzed: n = 4 for both GFP and Csk::GFP expressing embryos. J–O , Detection of HRP (red), Lim1 (blue), and GFP (green) in the LMC region of GFP ( J–L ) or Csk::GFP ( M–O ) electroporated embryos injected with HRP into ventral shank muscles. Examples of electroporated lateral LMC motor neuron labeled with HRP are indicated by arrows ( N , O ) and arrowheads ( N , O ). Examples indicated by arrowheads are shown at a higher magnification (insets of N , O ). P , Proportions (%) of electroporated lateral LMC motor neurons labeled with HRP in embryos injected with HRP into ventral shank muscles. In ventrally filled GFP expressing embryos, 6 ± 5% of HRP + GFP + LMC neurons were Lim1 + . In ventrally filled Csk::GFP expressing embryos, 17 ± 3% of HRP + GFP + LMC neurons were Lim1 + . Number of embryos quantified: n = 4 for GFP and Csk::GFP expressing embryos. Proportions of HRP + GFP + medial LMC neurons in dorsally filled Csk::GFP expressing embryos and those in dorsally filled GFP expressing embryos are significantly different ( p

Techniques Used: Labeling, Injection, Expressing

Src is required for the fidelity of LMC motor axon trajectory selection. A , G , Diagram depicting retrograde labeling of LMC neurons by HRP injections into dorsal ( A ) or ventral ( G ) limb muscles of mouse e12.5 embryos. B–E , Detection of HRP (red), Isl1 (blue), and FoxP1 (green), which marks all LMC motor neurons, in the LMC region of Src +/+ ( B , C ) or Src −/− ( D , E ) embryos injected with HRP into dorsal forelimb muscles. Examples of medial LMC motor neurons labeled with HRP are indicated by arrows and arrowheads ( E ). Examples indicated by arrowheads are shown at a higher magnification (inset of E ). F , Proportions (%) of medial LMC motor neurons labeled with HRP in embryos injected with HRP into dorsal forelimb muscles. In dorsally filled Src +/+ embryos, 5 ± 3% of HRP + LMC neurons were Isl1 + . In dorsally filled Src −/− embryos, 36 ± 14% of HRP + LMC neurons were Isl1 + . Number of embryos quantified: n = 5. H–K , Detection of HRP (red), Lim1 (blue), and FoxP1 (green) in the LMC region of Src +/+ ( H , I ) or Src −/− ( J , K ) embryos injected with HRP into ventral forelimb muscles. Examples of lateral LMC motor neurons labeled are indicated by arrows and arrowheads ( K ). Examples indicated by arrowheads are shown at a higher magnification (inset of K ). L , Proportions (%) of lateral LMC motor neurons filled with HRP in embryos injected with HRP into ventral forelimb muscles. In ventrally filled Src +/+ embryos, 4 ± 3% of HRP + LMC neurons were Lim1 + . In ventrally filled Src −/− embryos, 15 ± 11% of HRP + LMC neurons were Lim1 + . Number of embryos quantified: n = 4. Proportions of HRP + medial LMC neurons in dorsally filled Src −/− embryos and those in dorsally filled Src +/+ embryos are significantly different ( p
Figure Legend Snippet: Src is required for the fidelity of LMC motor axon trajectory selection. A , G , Diagram depicting retrograde labeling of LMC neurons by HRP injections into dorsal ( A ) or ventral ( G ) limb muscles of mouse e12.5 embryos. B–E , Detection of HRP (red), Isl1 (blue), and FoxP1 (green), which marks all LMC motor neurons, in the LMC region of Src +/+ ( B , C ) or Src −/− ( D , E ) embryos injected with HRP into dorsal forelimb muscles. Examples of medial LMC motor neurons labeled with HRP are indicated by arrows and arrowheads ( E ). Examples indicated by arrowheads are shown at a higher magnification (inset of E ). F , Proportions (%) of medial LMC motor neurons labeled with HRP in embryos injected with HRP into dorsal forelimb muscles. In dorsally filled Src +/+ embryos, 5 ± 3% of HRP + LMC neurons were Isl1 + . In dorsally filled Src −/− embryos, 36 ± 14% of HRP + LMC neurons were Isl1 + . Number of embryos quantified: n = 5. H–K , Detection of HRP (red), Lim1 (blue), and FoxP1 (green) in the LMC region of Src +/+ ( H , I ) or Src −/− ( J , K ) embryos injected with HRP into ventral forelimb muscles. Examples of lateral LMC motor neurons labeled are indicated by arrows and arrowheads ( K ). Examples indicated by arrowheads are shown at a higher magnification (inset of K ). L , Proportions (%) of lateral LMC motor neurons filled with HRP in embryos injected with HRP into ventral forelimb muscles. In ventrally filled Src +/+ embryos, 4 ± 3% of HRP + LMC neurons were Lim1 + . In ventrally filled Src −/− embryos, 15 ± 11% of HRP + LMC neurons were Lim1 + . Number of embryos quantified: n = 4. Proportions of HRP + medial LMC neurons in dorsally filled Src −/− embryos and those in dorsally filled Src +/+ embryos are significantly different ( p

Techniques Used: Selection, Labeling, Injection

SFK inhibition by Csk overexpression apparently randomizes medial LMC motor axon projections. A , D , Neurofilament detection in the limb nerves of chick HH st. 28/29 embryos electroporated with the medial LMC marker, e[Isl1]::GFP ( A ), or Csk and e[Isl1]::GFP ( D ) expression plasmids. B , E , GFP detection in the limb nerves of chick HH st. 28/29 embryos electroporated with e[Isl1]::GFP ( B ) or Csk and e[Isl1]::GFP ( E ) expression plasmids. C , F , Quantification of GFP + axons within dorsal and ventral limb nerves expressed as the percentage of total GFP + axons [GFP Fluo (%)]. In e[Isl1]::GFP electroporated embryos, 10 ± 8% of GFP + axons were detected in the dorsal limb nerves, and 90 ± 8% of GFP + axons were detected in the ventral limb nerves (v/d ratio of 9.0; C). In Csk and e[Isl1]::GFP coelectroporated embryos, 44 ± 15% of GFP + axons were detected in the dorsal limb nerves and 56 ± 15% of GFP + axons were detected in the ventral limb nerves, resulting (v/d ratio of 1.3; F). Number of embryos quantified: ( C ) n = 6, ( F ) n = 6. Proportions of GFP + axons in Csk and e[Isl1]::GFP expressing embryos and those in e[Isl1]::GFP expressing embryos are significantly different ( p
Figure Legend Snippet: SFK inhibition by Csk overexpression apparently randomizes medial LMC motor axon projections. A , D , Neurofilament detection in the limb nerves of chick HH st. 28/29 embryos electroporated with the medial LMC marker, e[Isl1]::GFP ( A ), or Csk and e[Isl1]::GFP ( D ) expression plasmids. B , E , GFP detection in the limb nerves of chick HH st. 28/29 embryos electroporated with e[Isl1]::GFP ( B ) or Csk and e[Isl1]::GFP ( E ) expression plasmids. C , F , Quantification of GFP + axons within dorsal and ventral limb nerves expressed as the percentage of total GFP + axons [GFP Fluo (%)]. In e[Isl1]::GFP electroporated embryos, 10 ± 8% of GFP + axons were detected in the dorsal limb nerves, and 90 ± 8% of GFP + axons were detected in the ventral limb nerves (v/d ratio of 9.0; C). In Csk and e[Isl1]::GFP coelectroporated embryos, 44 ± 15% of GFP + axons were detected in the dorsal limb nerves and 56 ± 15% of GFP + axons were detected in the ventral limb nerves, resulting (v/d ratio of 1.3; F). Number of embryos quantified: ( C ) n = 6, ( F ) n = 6. Proportions of GFP + axons in Csk and e[Isl1]::GFP expressing embryos and those in e[Isl1]::GFP expressing embryos are significantly different ( p

Techniques Used: Inhibition, Over Expression, Marker, Expressing

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Immunohistochemistry:

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    Abcam mouse anti isl1
    Figure 6. Structure of cardiomyocyte colonies grown in the primary culture of rat neonatal myocardial cells. ( A–C ) Different stages of development of the colonies stemming from <t>Isl1</t> + CSCs. ( A ) Cell division, DIV 2. Isl1 + (FITC, green), GATA-4 (phycoerythrin, red). ( B ) Colony consisting of approximately 8 cells, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( C ) Large Isl1 + colony, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( D ) The optical sections of colonies formed by Isl1 + , c-kit + , and Sca1 + CSCs on the 11th DIV. Isl1 + CSCs (Alexa 405, blue), Z = 12. c-kit + CSCs (FITC, green), Z = 12. Sca1 + CSCs (Alexa 405, blue), Z = 11. Actin was stained using rhodamine-phalloidin (red). ( E ) Differentiation of c-kit + CSCs inside the colony on the 13th DIV. Overlaid optical section of transmitted light and fluorescent images in 2 emitting wavelengths: 488 nm (FITC) and 543 nm (Alexa) in the bottom (Z = 5), in the middle (Z = 10), and the top (Z = 20) parts of the colony. c-kit + expression was revealed by FITC-conjugated antibodies (green), and α-sarcomeric actinin was revealed by Alexa-conjugated antibodies (red). Confocal microscope, Leica TCS SP5 (Germany), objective ×63, oil.
    Mouse Anti Isl1, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    isl1  (Abcam)
    94
    Abcam isl1
    Loss of EED does not affect the hair follicle microenvironment, but leads to upregulation of Merkel cell differentiation genes. (A-E) Shh and Wnt pathways do not appear to be majorly altered in PRC-null developing skin. (A) RT-qPCR analysis of Shh pathway genes shows no significant difference in their expression in P0 EED cKO (K14-Cre; EED flox/flox ) compared to control epidermis, while Shh expression is slightly reduced in EED-null compared to control epidermis (Gli1, p = 0.2000; Gli2, p = 0.1143; Gli3, p = 0.3429; Ptch1, p = 0.1143; Shh, p = 0.0286). RT-qPCR analysis of Wnt pathway genes shows no significant difference in expression of most genes in P0 EED cKO compared to control epidermis (Wnt3, p = 0.4857; Wnt4, p = 0.1143; Wnt7a, p = 0.3429; Wnt7b, p = 0.4857; Wnt10a, p = 0.2000; Wnt10b, p = 0.0286; Tcf3, p = 0.6857; Tcf4, p = 0.4857; Dkkl1, p = 0.6857; Axin2, p = 0.2000; Sp5, p = 0.0286). (B,C) Immunohistochemistry staining for β-catenin does not show major differences in expression or nuclear staining in first wave (B) or second wave (C) hair follicles in EED cKO skin compared to control at E16. Note that, as has been previously described, the stratum corneum is prematurely acquired in the PRC2-null E16 embryo [ 51 ]. (D,E) In situ hybridization for Gli1 RNA (D) and Shh RNA (E) shows no major changes in expression in Ezh1/2 2KO (K14-Cre; Ezh1 del/del ;Ezh2 flox/flox ) skin compared to control at E16. (F) FACS scheme for Merkel cell (MC) sorting. After gating on singlets and live cells, EpCAM-APC staining was used to gate on all epidermal cells and Atoh1-GFP labels Merkel cells. EpCAM-APC(+) Atoh1-GFP(-) cells were sorted as epidermal controls. (G) ChIP-qPCR showing significantly lower levels of H3K27me3 at Merkel genes, <t>Isl1</t> , Sox2 , and Atoh1 , in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells. (Neuro, p = 0.0411; Olig1, p = 0.0200; Isl1, p = 0.0022; Sox2, p = 0.0194; Atoh1, p = 0.0050). (H) RT-qPCR showing specific expression of MC signature genes Isl1 , Sox2 , and Atoh1 in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells ( Isl1 , p = 0.0004; Sox2 , p = 0.0004; Atoh1 , p = 0.0004) (I) IF staining showing that Krt8(+) (K8) MCs have the H3K27me3 mark in P0 control Krt14(+) (K14) epidermis. Krt14(+) cells serve as a positive control for H3K27me3 staining. Quantification of H3K27me3 staining intensity (below) (Kruskal-Wallis test p
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    Figure 6. Structure of cardiomyocyte colonies grown in the primary culture of rat neonatal myocardial cells. ( A–C ) Different stages of development of the colonies stemming from Isl1 + CSCs. ( A ) Cell division, DIV 2. Isl1 + (FITC, green), GATA-4 (phycoerythrin, red). ( B ) Colony consisting of approximately 8 cells, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( C ) Large Isl1 + colony, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( D ) The optical sections of colonies formed by Isl1 + , c-kit + , and Sca1 + CSCs on the 11th DIV. Isl1 + CSCs (Alexa 405, blue), Z = 12. c-kit + CSCs (FITC, green), Z = 12. Sca1 + CSCs (Alexa 405, blue), Z = 11. Actin was stained using rhodamine-phalloidin (red). ( E ) Differentiation of c-kit + CSCs inside the colony on the 13th DIV. Overlaid optical section of transmitted light and fluorescent images in 2 emitting wavelengths: 488 nm (FITC) and 543 nm (Alexa) in the bottom (Z = 5), in the middle (Z = 10), and the top (Z = 20) parts of the colony. c-kit + expression was revealed by FITC-conjugated antibodies (green), and α-sarcomeric actinin was revealed by Alexa-conjugated antibodies (red). Confocal microscope, Leica TCS SP5 (Germany), objective ×63, oil.

    Journal: Cell Cycle

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

    doi: 10.4161/cc.27768

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

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

    Techniques: Staining, Expressing, Microscopy

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

    Journal: Cell Cycle

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

    doi: 10.4161/cc.27768

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

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

    Techniques: Microscopy

    BMP signaling enhances Isl1-CPC cardiac differentiation. Freshly sorted EB day 5.5 GFP + Isl1-CPC were treated with control (A, D) or 25 ng/ml Bmp4 (B, E) for 6 days, followed by immunostaining with antibodies against cardiomyocyte-specific protein cTnT (red in A and B) or labelled with cTnT and Alexa fluor 647 for FACS analysis (D, E). Nuclei: blue (Hoechst). (C) Quantification of A and B (over 2000 cells counted from 3 independent experiments). (F) Quantification of D and E (20000 cells FACS analyzed for each of 3 independent experiments). Scale bar: 100 µm; *statistical significance p

    Journal: PLoS ONE

    Article Title: Functional Cardiomyocytes Derived from Isl1 Cardiac Progenitors via Bmp4 Stimulation

    doi: 10.1371/journal.pone.0110752

    Figure Lengend Snippet: BMP signaling enhances Isl1-CPC cardiac differentiation. Freshly sorted EB day 5.5 GFP + Isl1-CPC were treated with control (A, D) or 25 ng/ml Bmp4 (B, E) for 6 days, followed by immunostaining with antibodies against cardiomyocyte-specific protein cTnT (red in A and B) or labelled with cTnT and Alexa fluor 647 for FACS analysis (D, E). Nuclei: blue (Hoechst). (C) Quantification of A and B (over 2000 cells counted from 3 independent experiments). (F) Quantification of D and E (20000 cells FACS analyzed for each of 3 independent experiments). Scale bar: 100 µm; *statistical significance p

    Article Snippet: Immunofluorescence stainings were performed with mouse anti-Isl1 antibody (Hybridoma Bank) and rabbit anti-alpha smooth muscle actin (SMA from Abcam), cardiac troponin T (cTnT from Abcam) or CD31 (Abcam).

    Techniques: Immunostaining, FACS

    Isl1-CPC derived cultures have cardiomyocyte-like electrophysiological properties. Action potentials of differentiated Isl1-CPCs from control (A) or Bmp4-treated cultures (B) were recorded after 6 days in culture under whole-cell current clamp mode. (A, B) Representative action potentials demonstrate nodal-like, atrial-like and ventricular-like cardiomyocytes in the absence and presence of Bmp4. (C) Action potential amplitude (APA), action potential duration at the 90% of repolarization (APD 90 ) and maximum diastolic potential (MDP) for ventricular-like cardiomyocytes were quantified and depicted as bar graph for both control and Bmp4-treated cultures. A total of 29 cardiomyocytes derived from Isl1-CPCs was analyzed from 4 independent experiments. There were no significant differences for APA, MDP and APD 90 between control and Bmp4 treated samples. The details for AP measurement setting are under Materials and Methods .

    Journal: PLoS ONE

    Article Title: Functional Cardiomyocytes Derived from Isl1 Cardiac Progenitors via Bmp4 Stimulation

    doi: 10.1371/journal.pone.0110752

    Figure Lengend Snippet: Isl1-CPC derived cultures have cardiomyocyte-like electrophysiological properties. Action potentials of differentiated Isl1-CPCs from control (A) or Bmp4-treated cultures (B) were recorded after 6 days in culture under whole-cell current clamp mode. (A, B) Representative action potentials demonstrate nodal-like, atrial-like and ventricular-like cardiomyocytes in the absence and presence of Bmp4. (C) Action potential amplitude (APA), action potential duration at the 90% of repolarization (APD 90 ) and maximum diastolic potential (MDP) for ventricular-like cardiomyocytes were quantified and depicted as bar graph for both control and Bmp4-treated cultures. A total of 29 cardiomyocytes derived from Isl1-CPCs was analyzed from 4 independent experiments. There were no significant differences for APA, MDP and APD 90 between control and Bmp4 treated samples. The details for AP measurement setting are under Materials and Methods .

    Article Snippet: Immunofluorescence stainings were performed with mouse anti-Isl1 antibody (Hybridoma Bank) and rabbit anti-alpha smooth muscle actin (SMA from Abcam), cardiac troponin T (cTnT from Abcam) or CD31 (Abcam).

    Techniques: Derivative Assay

    Bmp4 signaling enhances transcription factor Tbx5 and Tbx20 mRNA expression. FACS-sorted GFP+ Isl1-CPCs were cultured in the presence of Bmp4 or vehicle control. mRNA was collected following 2, 4 and 6 days of Bmp4 treatment. qRT-PCR analysis of duplicates from three independent experiments for tbx5 (A) and tbx20 (B) expression is normalized by housekeeping gene, Gapdh. *p

    Journal: PLoS ONE

    Article Title: Functional Cardiomyocytes Derived from Isl1 Cardiac Progenitors via Bmp4 Stimulation

    doi: 10.1371/journal.pone.0110752

    Figure Lengend Snippet: Bmp4 signaling enhances transcription factor Tbx5 and Tbx20 mRNA expression. FACS-sorted GFP+ Isl1-CPCs were cultured in the presence of Bmp4 or vehicle control. mRNA was collected following 2, 4 and 6 days of Bmp4 treatment. qRT-PCR analysis of duplicates from three independent experiments for tbx5 (A) and tbx20 (B) expression is normalized by housekeeping gene, Gapdh. *p

    Article Snippet: Immunofluorescence stainings were performed with mouse anti-Isl1 antibody (Hybridoma Bank) and rabbit anti-alpha smooth muscle actin (SMA from Abcam), cardiac troponin T (cTnT from Abcam) or CD31 (Abcam).

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

    Calcium transients of cardiomyocytes derived from Isl1-CPC after cell loading with Ca 2+ indicator fura-2. (A) Representative recordings for increase in [Ca 2+ ]i transient amplitude and frequency was measured following loading of control and Bmp4-treated cardiomyocyte cultures with Ca 2+ indicator fura-2. Cytosolic Ca 2+ of spontaneous beating cardiomyocyte was measured by ratio of fluorescence intensity at 340 nm and 380 nm (F340/F380). The measurements were recorded 6 days of incubation after Isl1-CPC isolation. (B) The quantitative representation of beating frequency (BF) of calcium transients per minute is shown by bar graphs (n = 3). Experiments were repeated 3 times and 10–15 cells were measured in each experiment. (C) Change of [Ca 2+ ] was calculated from Ca 2+ transient recordings for cardiomyocyte cultures differentiated in the absence or presence of Bmp4. *p

    Journal: PLoS ONE

    Article Title: Functional Cardiomyocytes Derived from Isl1 Cardiac Progenitors via Bmp4 Stimulation

    doi: 10.1371/journal.pone.0110752

    Figure Lengend Snippet: Calcium transients of cardiomyocytes derived from Isl1-CPC after cell loading with Ca 2+ indicator fura-2. (A) Representative recordings for increase in [Ca 2+ ]i transient amplitude and frequency was measured following loading of control and Bmp4-treated cardiomyocyte cultures with Ca 2+ indicator fura-2. Cytosolic Ca 2+ of spontaneous beating cardiomyocyte was measured by ratio of fluorescence intensity at 340 nm and 380 nm (F340/F380). The measurements were recorded 6 days of incubation after Isl1-CPC isolation. (B) The quantitative representation of beating frequency (BF) of calcium transients per minute is shown by bar graphs (n = 3). Experiments were repeated 3 times and 10–15 cells were measured in each experiment. (C) Change of [Ca 2+ ] was calculated from Ca 2+ transient recordings for cardiomyocyte cultures differentiated in the absence or presence of Bmp4. *p

    Article Snippet: Immunofluorescence stainings were performed with mouse anti-Isl1 antibody (Hybridoma Bank) and rabbit anti-alpha smooth muscle actin (SMA from Abcam), cardiac troponin T (cTnT from Abcam) or CD31 (Abcam).

    Techniques: Derivative Assay, Fluorescence, Incubation, Isolation

    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

    Loss of EED does not affect the hair follicle microenvironment, but leads to upregulation of Merkel cell differentiation genes. (A-E) Shh and Wnt pathways do not appear to be majorly altered in PRC-null developing skin. (A) RT-qPCR analysis of Shh pathway genes shows no significant difference in their expression in P0 EED cKO (K14-Cre; EED flox/flox ) compared to control epidermis, while Shh expression is slightly reduced in EED-null compared to control epidermis (Gli1, p = 0.2000; Gli2, p = 0.1143; Gli3, p = 0.3429; Ptch1, p = 0.1143; Shh, p = 0.0286). RT-qPCR analysis of Wnt pathway genes shows no significant difference in expression of most genes in P0 EED cKO compared to control epidermis (Wnt3, p = 0.4857; Wnt4, p = 0.1143; Wnt7a, p = 0.3429; Wnt7b, p = 0.4857; Wnt10a, p = 0.2000; Wnt10b, p = 0.0286; Tcf3, p = 0.6857; Tcf4, p = 0.4857; Dkkl1, p = 0.6857; Axin2, p = 0.2000; Sp5, p = 0.0286). (B,C) Immunohistochemistry staining for β-catenin does not show major differences in expression or nuclear staining in first wave (B) or second wave (C) hair follicles in EED cKO skin compared to control at E16. Note that, as has been previously described, the stratum corneum is prematurely acquired in the PRC2-null E16 embryo [ 51 ]. (D,E) In situ hybridization for Gli1 RNA (D) and Shh RNA (E) shows no major changes in expression in Ezh1/2 2KO (K14-Cre; Ezh1 del/del ;Ezh2 flox/flox ) skin compared to control at E16. (F) FACS scheme for Merkel cell (MC) sorting. After gating on singlets and live cells, EpCAM-APC staining was used to gate on all epidermal cells and Atoh1-GFP labels Merkel cells. EpCAM-APC(+) Atoh1-GFP(-) cells were sorted as epidermal controls. (G) ChIP-qPCR showing significantly lower levels of H3K27me3 at Merkel genes, Isl1 , Sox2 , and Atoh1 , in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells. (Neuro, p = 0.0411; Olig1, p = 0.0200; Isl1, p = 0.0022; Sox2, p = 0.0194; Atoh1, p = 0.0050). (H) RT-qPCR showing specific expression of MC signature genes Isl1 , Sox2 , and Atoh1 in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells ( Isl1 , p = 0.0004; Sox2 , p = 0.0004; Atoh1 , p = 0.0004) (I) IF staining showing that Krt8(+) (K8) MCs have the H3K27me3 mark in P0 control Krt14(+) (K14) epidermis. Krt14(+) cells serve as a positive control for H3K27me3 staining. Quantification of H3K27me3 staining intensity (below) (Kruskal-Wallis test p

    Journal: PLoS Genetics

    Article Title: Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin DevelopmentA Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel cell Development

    doi: 10.1371/journal.pgen.1006151

    Figure Lengend Snippet: Loss of EED does not affect the hair follicle microenvironment, but leads to upregulation of Merkel cell differentiation genes. (A-E) Shh and Wnt pathways do not appear to be majorly altered in PRC-null developing skin. (A) RT-qPCR analysis of Shh pathway genes shows no significant difference in their expression in P0 EED cKO (K14-Cre; EED flox/flox ) compared to control epidermis, while Shh expression is slightly reduced in EED-null compared to control epidermis (Gli1, p = 0.2000; Gli2, p = 0.1143; Gli3, p = 0.3429; Ptch1, p = 0.1143; Shh, p = 0.0286). RT-qPCR analysis of Wnt pathway genes shows no significant difference in expression of most genes in P0 EED cKO compared to control epidermis (Wnt3, p = 0.4857; Wnt4, p = 0.1143; Wnt7a, p = 0.3429; Wnt7b, p = 0.4857; Wnt10a, p = 0.2000; Wnt10b, p = 0.0286; Tcf3, p = 0.6857; Tcf4, p = 0.4857; Dkkl1, p = 0.6857; Axin2, p = 0.2000; Sp5, p = 0.0286). (B,C) Immunohistochemistry staining for β-catenin does not show major differences in expression or nuclear staining in first wave (B) or second wave (C) hair follicles in EED cKO skin compared to control at E16. Note that, as has been previously described, the stratum corneum is prematurely acquired in the PRC2-null E16 embryo [ 51 ]. (D,E) In situ hybridization for Gli1 RNA (D) and Shh RNA (E) shows no major changes in expression in Ezh1/2 2KO (K14-Cre; Ezh1 del/del ;Ezh2 flox/flox ) skin compared to control at E16. (F) FACS scheme for Merkel cell (MC) sorting. After gating on singlets and live cells, EpCAM-APC staining was used to gate on all epidermal cells and Atoh1-GFP labels Merkel cells. EpCAM-APC(+) Atoh1-GFP(-) cells were sorted as epidermal controls. (G) ChIP-qPCR showing significantly lower levels of H3K27me3 at Merkel genes, Isl1 , Sox2 , and Atoh1 , in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells. (Neuro, p = 0.0411; Olig1, p = 0.0200; Isl1, p = 0.0022; Sox2, p = 0.0194; Atoh1, p = 0.0050). (H) RT-qPCR showing specific expression of MC signature genes Isl1 , Sox2 , and Atoh1 in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells ( Isl1 , p = 0.0004; Sox2 , p = 0.0004; Atoh1 , p = 0.0004) (I) IF staining showing that Krt8(+) (K8) MCs have the H3K27me3 mark in P0 control Krt14(+) (K14) epidermis. Krt14(+) cells serve as a positive control for H3K27me3 staining. Quantification of H3K27me3 staining intensity (below) (Kruskal-Wallis test p

    Article Snippet: Antibodies Antibodies were used as follows: Krt14 (generous gift of Julie Segre, National Human Genome Research Institute, MD, USA, 1:20,000); Krt8 (Developmental Studies Hybridoma Bank, TROMA-1, 1:500); Krt18 (abcam, ab668, 1:100); Krt20 (Dako, M7019, 1:70); Sox2 (Stemgent, 09–0024, 1:150); Isl1 (abcam, ab109517, 1:250); GFP (abcam, ab13970, 1/1000); Krt5 (generous gift of Elaine Fuchs, The Rockefeller University, NY, USA, 1:500); Phospho-histone H3 (Upstate, 06–570, 1/1000); BrdU (abcam, ab1893, 1:250); Integrin α8 (Santa Cruz, sc-30982, 1:100); Activated Caspase 3 (R & D, AF835, 1:250); β-Catenin (BD Biosciences, 610153, 1:100); H3K27me3 (Millipore, 07–449, 1:300).

    Techniques: Cell Differentiation, Quantitative RT-PCR, Expressing, Immunohistochemistry, Staining, In Situ Hybridization, FACS, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control

    Shh signaling activity is required for Merkel cell formation. (A-C) IF stainings for Merkel cell (MC) markers Krt20 (K20) (A,C), Isl1 (B), Krt8 (K8) (B), and Sox2 (C) show a complete absence of Merkel cells in E18 Shh KO (Shh EGFPcre/EGFPcre ) mice compared to control (ctrl). Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Shh KO E18 skin (right panel of B) (Krt8 p = 0.0005 and Krt20 p = 0.0001). (D) TUNEL staining shows no increase in apoptosis in the skin of E18 Shh KO mice. (E) IF stainings for Merkel cell markers Krt18 (K18) and Sox2 show a complete absence of Merkel cells in E16 Shh KO mice when compared to control. Sox2 also labels the dermal condensate (dc). (F) TUNEL staining shows no increase in apoptosis in the Krt14(+) basal layer of E16 Shh KO mice when compared to control. Note that cells undergoing cornification in the suprabasal layers are TUNEL(+), as previously reported [ 33 ]. (G,I,J) IF stainings for Merkel cell (MC) markers Krt20 (G,I), Krt8 (J), Isl1 (J), and Sox2 (I) show a complete absence of Merkel cells in P0 Shh epidermis-conditional knockout (Shh cKO) (K14-Cre; Shh flox/flox ) mice compared to control. Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Shh cKO P0 skin (right panel of J) (both p

    Journal: PLoS Genetics

    Article Title: Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin DevelopmentA Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel cell Development

    doi: 10.1371/journal.pgen.1006151

    Figure Lengend Snippet: Shh signaling activity is required for Merkel cell formation. (A-C) IF stainings for Merkel cell (MC) markers Krt20 (K20) (A,C), Isl1 (B), Krt8 (K8) (B), and Sox2 (C) show a complete absence of Merkel cells in E18 Shh KO (Shh EGFPcre/EGFPcre ) mice compared to control (ctrl). Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Shh KO E18 skin (right panel of B) (Krt8 p = 0.0005 and Krt20 p = 0.0001). (D) TUNEL staining shows no increase in apoptosis in the skin of E18 Shh KO mice. (E) IF stainings for Merkel cell markers Krt18 (K18) and Sox2 show a complete absence of Merkel cells in E16 Shh KO mice when compared to control. Sox2 also labels the dermal condensate (dc). (F) TUNEL staining shows no increase in apoptosis in the Krt14(+) basal layer of E16 Shh KO mice when compared to control. Note that cells undergoing cornification in the suprabasal layers are TUNEL(+), as previously reported [ 33 ]. (G,I,J) IF stainings for Merkel cell (MC) markers Krt20 (G,I), Krt8 (J), Isl1 (J), and Sox2 (I) show a complete absence of Merkel cells in P0 Shh epidermis-conditional knockout (Shh cKO) (K14-Cre; Shh flox/flox ) mice compared to control. Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Shh cKO P0 skin (right panel of J) (both p

    Article Snippet: Antibodies Antibodies were used as follows: Krt14 (generous gift of Julie Segre, National Human Genome Research Institute, MD, USA, 1:20,000); Krt8 (Developmental Studies Hybridoma Bank, TROMA-1, 1:500); Krt18 (abcam, ab668, 1:100); Krt20 (Dako, M7019, 1:70); Sox2 (Stemgent, 09–0024, 1:150); Isl1 (abcam, ab109517, 1:250); GFP (abcam, ab13970, 1/1000); Krt5 (generous gift of Elaine Fuchs, The Rockefeller University, NY, USA, 1:500); Phospho-histone H3 (Upstate, 06–570, 1/1000); BrdU (abcam, ab1893, 1:250); Integrin α8 (Santa Cruz, sc-30982, 1:100); Activated Caspase 3 (R & D, AF835, 1:250); β-Catenin (BD Biosciences, 610153, 1:100); H3K27me3 (Millipore, 07–449, 1:300).

    Techniques: Activity Assay, Mouse Assay, TUNEL Assay, Staining, Knock-Out

    Shh signaling activity in the epidermis is required for Merkel cell formation. (A-C) IF stainings for Merkel cell markers Krt8 (K8) (A,C), Krt20 (K20) (B), Sox2 (B), and Isl1 (C) show a highly significant reduction in the number of Merkel cells in P0 Smoothened epidermis-conditional knockout (Smo cKO) (K14-Cre; Smo flox/flox ) mice when compared to control (ctrl). Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Smo cKO P0 skin (right panel of B) (both p

    Journal: PLoS Genetics

    Article Title: Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin DevelopmentA Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel cell Development

    doi: 10.1371/journal.pgen.1006151

    Figure Lengend Snippet: Shh signaling activity in the epidermis is required for Merkel cell formation. (A-C) IF stainings for Merkel cell markers Krt8 (K8) (A,C), Krt20 (K20) (B), Sox2 (B), and Isl1 (C) show a highly significant reduction in the number of Merkel cells in P0 Smoothened epidermis-conditional knockout (Smo cKO) (K14-Cre; Smo flox/flox ) mice when compared to control (ctrl). Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Smo cKO P0 skin (right panel of B) (both p

    Article Snippet: Antibodies Antibodies were used as follows: Krt14 (generous gift of Julie Segre, National Human Genome Research Institute, MD, USA, 1:20,000); Krt8 (Developmental Studies Hybridoma Bank, TROMA-1, 1:500); Krt18 (abcam, ab668, 1:100); Krt20 (Dako, M7019, 1:70); Sox2 (Stemgent, 09–0024, 1:150); Isl1 (abcam, ab109517, 1:250); GFP (abcam, ab13970, 1/1000); Krt5 (generous gift of Elaine Fuchs, The Rockefeller University, NY, USA, 1:500); Phospho-histone H3 (Upstate, 06–570, 1/1000); BrdU (abcam, ab1893, 1:250); Integrin α8 (Santa Cruz, sc-30982, 1:100); Activated Caspase 3 (R & D, AF835, 1:250); β-Catenin (BD Biosciences, 610153, 1:100); H3K27me3 (Millipore, 07–449, 1:300).

    Techniques: Activity Assay, Knock-Out, Mouse Assay