Figure 3. Shh is required for LMC formation in developing mouse spinal cord.

(A) IHC analyses of E12.5 Shh-cKO (Shh^f/f;Olig2-Cre) mutant embryos (n = 4) (lower panel) and control littermates (n = 4) (upper panel). The cervical level of ventral spinal cord is shown. LMCm (Isl1⁺/FoxP1⁺) neurons and LMCl (Hb9⁺/FoxP1⁺ or Lhx1⁺/FoxP1⁺) neurons (yellow bracket) in Shh conditional knock-out (Shh-cKO) were significantly reduced. On the other hand, the number of MMC (Hb9⁺/Lhx3⁺) and HMC (Hb9⁺/Isl1⁺) neurons did not change (white bracket). (B) IHC analyses of Olig2, Sox2, and Ki67 in E12.5 Shh-cKO mutant embryo and control littermates (cervical level). No significant difference in the expression of Sox2, Olig2 and Ki67 within the spinal cord. Scale bars: 100 μm. (C) Quantification of the number of LMCm (Isl1⁺/FoxP1⁺), LMCl (Hb9⁺/FoxP1⁺ or Lhx1⁺/FoxP1⁺), MMC (Hb9⁺/Lhx3⁺) and HMC (Hb9⁺/Isl1⁺) neurons, Olig2⁺, Sox2⁺, Ki67⁺ cells and total MNs at cervical level in E12.5 mouse embryonic spinal cord. Data are mean ± s.d. ***p<0.0001, ****p<0.00001; ns, non-significant (Student’s t-test). n = 5 ~ 28 independent images per each sample.

Figure 3—figure supplement 1. Reduced expression of ARHGAP3 does not affect progenitor cell proliferation in Shh-cKO mouse spinal cord.

(A) IHC analysis for ARHGAP36 in E12.5 Shh-cKO (n = 4) and littermate control (n = 4) embryos showing the reduced expression of ARHGAP36 in ventro-lateral region of the spinal cord at cervical level. (B) IHC analysis for BrdU incorporation in E11.5 Shh-cKO (n = 4) and littermate control (n = 5) embryos showing no defect in proliferation of progenitor cells within the spinal cord. Scale bars: 100 μm. Data are mean ± s.d. ****p<0.00001; ns, non-significant (Student's t-test). n = 14 ~ 32 independent images per each sample.