Fig 1: Schwann cells lacking YAP/TAZ transdifferentiate into repair Schwann cells.Longitudinal sections of transected sciatic nerves of WT and Yap/Taz iDKO immunostained by various markers of growth-promoting repair SCs at five dpi. SCs are marked by Sox10 (red). (A) Representative sections showing upregulation of c-Jun in iDKO SCs, as in WT SCs. (B) Upregulation of active phospho-S63 c-Jun in iDKO SCs, as in WT. (C) Upregulation of p75 in iDKO SCs, as in WT SCs. (D) Upregulation of Oct-6 in iDKO SCs, as in WT SCs. (E) Quantification of c-Jun+ SCs in WT and iDKO. n = 3 mice per genotype. ns, not significant, p=0.1266, Mann-Whitney. (F) Quantification of pc-Jun+ SCs in WT and iDKO. n = 3 mice per genotype. *p=0.0495, Mann-Whitney. (G) Quantification of p75+ SCs in WT and iDKO. n = 3 mice per genotype. ns, not significant, p=0.5127, Mann-Whitney. (H) Quantification of Oct-6+ SCs in WT and iDKO. n = 3 mice per genotype. ns, not significant, p=0.8273, Mann-Whitney. Scale bars = 30 μm (A–D).Figure 4—source data 1.Source files for c-Jun+ SC data.This zip archive contains the IHC for one WT and one iDKO used for quantitative analysis shown in Figure 4E. Leica SP8 confocal lif images were processed using Imaris software and saved as tiffs.Figure 4—source data 2.Source files for graphs quantifying c-Jun+ SCs, pc-Jun+ SCs, p75+ SCs, and Oct6+ SCs.This zip archive contains the raw data for WT and iDKO used for the quantitative analysis shown in Figure 4E, F, G and H. The data are contained in both a text document and an Excel file, both labeled as Mann Whitney data. These files also contain data for Figure 3—figure supplement 1, Figures 5, 6, 7, 8 and Figure 8—figure supplement 1E.Figure 4—source data 3.Western blotting analysis of repair Schwann cell markers.Uncropped Western blots of images used to make Figure 4—figure supplement 1. Individually processed samples from 2 WT (#656, #625) and 2 iDKO mice (#378, #379) are shown and used for quantification. The following figure supplements are available for Figure 4.
Fig 2: Redifferentiation of Schwann cells lacking YAP/TAZ.Longitudinal sections of crushed nerves of WT and Yap/Taz iDKO at 12 dpi, immunostained by various markers of SC dedifferentiation (c-Jun and Oct-6), proliferation (Ki67) and redifferentiation (Krox20). SCs are marked by Sox10. (A) Representative sections showing c-Jun+ SCs markedly reduced in iDKO, as in WT. (B) Representative sections showing rarely observed Ki67+ proliferating SCs in iDKO, as in WT. (C) Representative sections showing Oct-6+ SCs reduced in iDKO, as in WT. (D) Representative sections showing failed upregulation of Krox20 in iDKO SCs. (E) Quantitative comparison of c-Jun+ SCs at 5 and 12 dpi, showing similar downregulation of c-Jun in WT and iDKO SC. n = 3 mice per genotype, 2-way ANOVA, ns = not significant. WT five dpi vs WT 12 dpi, **p=0.0069; WT five dpi vs iDKO five dpi, p=0.4260; WT 12 dpi vs iDKO 12 dpi, p=0.9574; iDKO five dpi vs iDKO 12 dpi, **p=0.0018. (F) Quantitative comparison of Ki67+ SCs, showing similar reduction in proliferating SCs in WT and iDKO nerves between 5 dpi and 12 dpi. n = 3 mice per genotype, 2-way ANOVA, ns = not significant. WT five dpi vs WT 12 dpi, ****p<0.0001; WT five dpi vs iDKO five dpi, p>0.9999; WT 12 dpi vs iDKO 12 dpi, p=0.6775; iDKO five dpi vs iDKO 12 dpi, ****p<0.0001. (G) Quantitative comparison of Oct-6+ SCs, showing significant downregulation of Oct-6 in WT and iDKO SCs between 5 dpi and 12 dpi. n = 3 mice per genotype, ns = not significant, 2-way ANOVA. WT five dpi vs WT 12 dpi, ***p=0.0005; WT five dpi vs iDKO five dpi, p=0.9817; WT 12 dpi vs iDKO 12 dpi, *p=0.0221; iDKO five dpi vs iDKO 12 dpi, *p=0.0299. (H) Quantitative comparison of Krox20+ SCs, showing upregulation of Krox20 in WT SCs, but not in iDKO SCs between 5 dpi and 12 dpi. n = 3 mice per genotype, 2-way ANOVA, ns = not significant. WT five dpi vs WT 12 dpi, ****p<0.0001; WT five dpi vs iDKO five dpi, p>0.9999; WT 12 dpi vs iDKO 12 dpi, ****p<0.0001; iDKO five dpi vs iDKO 12 dpi, p>0.9999. Scale bar = 10 μm (A–D).Figure 9—source data 1.Source files for Krox20+ SC data.This zip archive contains the IHC for one WT and one iDKO used for quantitative analysis shown in Figure 9E. Leica SP8 confocal lif images were processed using Imaris software and saved as tiffs.Figure 9—source data 2.Source files for graphs quantifying c-Jun+ SCs, Ki67+ SCs, Oct6+ SCs and Krox20+ SCs.This zip archive contains the raw data for WT and iDKO used for the quantitative analysis shown in Figure 9E, F, G and H. The data are contained in GraphPad Prism files, as indicated.
Fig 3: Progressive loss of phrenic but not other spinal motor neurons in Tshz1MNΔ mice. Phrenic motor neurons were identified in the cervical spinal cord using Pou3f1 and Isl1/2 (E12.5) or ChAT (P0.5) antibodies. (A) Total number of phrenic motor neurons at E12.5 in Tshz1MNΔ and control embryos. (B) Total number of phrenic motor neurons at P0.5 in Tshz1MNΔ and control embryos. (C) Motor neurons of the lateral and medial motor columns (LMC and MMC) in the lumbar spinal cord were identified using an anti-ChAT antibody and quantified in control and Tshz1MNΔ mice at P0.5. At least three animals per condition were analyzed. Each dot represents one animal, bars represent the mean±s.d. Unpaired t-test: n.s., non-significant; ***P<0.001.
Fig 4: Tshz1 is expressed in specific motor neuron populations during development. Tshz1 expression was assessed by analysis of GFP expression in Tshz1GFP/+ heterozygous mice. (A) Coronal sections of E12.5 and P0.5 hindbrain showed that Tshz1-driven GFP expression (green) was detected persistently in developing and mature Isl1/2+ hypoglossal motor neurons (red). GFP was not expressed in Olig2+ progenitors (blue). At P0.5, the midline and the limits of the hypoglossal nuclei are indicated by dashed lines. (B) Quantification of Isl1/2+ hypoglossal motor neurons that co-express GFP at the indicated stages. (C) 3D reconstruction of the hypoglossal motor nucleus (nXII) derived from serial hindbrain sections (P0.5) of a Tshz1GFP/+ animal; the dorsal motor nucleus of the vagus (nX) serves as a landmark. Neuron distributions in each slice analyzed are shown; note that slices are angled for better visualization of the neuronal organization. Blue dots: nX neurons (ChAT+); red/green dots: green and red dots display hypoglossal motor neurons (ChAT+) that do and do not express GFP, respectively. (D) Restricted Tshz1 expression in hypoglossal motor neuronal subpopulations at different levels of the rostro-caudal axis. (E) In the cervical spinal cord, GFP (green) was detected in Pou3f1+ phrenic motor neurons (red) at E12.5 and P0.5. Isl1/2 (blue, upper panels) or ChAT (blue, lower panels) were used as general motor neuron markers. (F) Quantification of the percentage of phrenic motor neurons expressing GFP at the indicated stages. (G) GFP expression (green) in postmitotic neurons of the LMC (Isl1/2+ red); Olig2+ progenitors are shown in blue. (H) Quantification of the percentage of motor neurons in the LMC expressing GFP at the indicated stages. (I) Scheme summarizing Tshz1 expression in different motor neuronal populations. Tshz1 is expressed persistently in the hypoglossal nucleus (nXII) and PMC (solid green) but is only transiently expressed in LMCs (dashed green). Scale bars: 50 µm. Data are represented as mean±s.d.; n=3.
Fig 5: YAP/TAZ are required for Krox20 upregulation in differentiating Schwann cells.(A) Identification of Oct6+ SC nuclei at P4 and P60 in WT and Yap/Taz cDKO, as determined by co-staining for Oct6 (red), Sox10 (SC nuclei; green) and DAPI (all cell nuclei; blue). (B) Identification of Krox20+ SC nuclei at P4 and P60 in WT and Yap/Taz cDKO, as determined by co-staining for Krox20 (red), Sox10 (SC nuclei, green) and DAPI (all cell nuclei). (C) Quantification of Oct6 expression in SC nuclei. n = 4 mice per genotype (WT and cDKO P4) or n = 3 mice per genotype (WT and cDKO P0, (P18 and P60). **p<0.01 (P0), ***p<0.001, ****p<0.0001, 2-way ANOVA with Sidak’s multiple comparison test. (D) Quantification of Krox20 expression in SC nuclei. n = 3 mice per genotype (WT P0, WT P4, mutant P18) or n = 2 mice per genotype (mutant P0, mutant P4, WT P18, WT and mutant P60). ****p<0.0001, n.s. = non-significant, 2-way ANOVA, with Sidak’s multiple comparison test. (E) Western blotting of P4 and P60 WT and Yap/Taz cDKO sciatic nerve lysates, using the indicated antibodies and anti-βactin as a loading control. n = 3 experiments. (F) Quantitative RT-PCR using Krox20 and β-actin-specific primers and total RNA isolated from WT and Yap/Taz cDKO P60 sciatic nerves. Expression of Krox20 is normalized to that of β-actin as an internal control, and WT expression is arbitrarily given the value 1. n = 3 mice per genotype. **p<0.01, unpaired Student’s t-test.DOI: http://dx.doi.org/10.7554/eLife.20982.014
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