DEV033795 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression of Shh and Ptch1 in the developing hChP at various stages. (A-F) Shh transcript is robustly expressed by hChPe cells at E14 (A-C) and E16 (D-F). The high magnification views show a small subset of hChPe cells with low level or no Shh expression, as indicated by red arrows in B and E. The putative Shh⁻ but Gli1⁺ hChPe progenitor domain can be identified, as highlighted by black arrows and dotted lines in C and F. (G-I) Ptch1 transcript expression, another readout for Shh pathway activity, can be detected in the putative hChPe progenitor domain at E12 (G), E14 (H) and E16 (I). Black dotted lines are continuous with the rest of the hChPe. Scale bars: 20 µm.

• Supplemental Figure S2 -

  Fig. S2. Expression of Ptch2, sprouty 1 and sprouty 2 in hChPs of E14.5 control and Wnt1-Cre; Shh^fl/− embryos. (A-D) Ptch2 transcript is not expressed in either the hChP or hChPe progenitor domain of control (A) and Wnt1-Cre; Shh^fl/− embryos (C), although robust signal can be seen in the developing hair follicles from the same sections (B,D). (E-L) Expression of sprouty 1 and sprouty 2, target genes of the Fgf signaling pathway, cannot be detected in either E14.5 control (E,F,I,J) or Wnt1-Cre; Shh^fl/− (G,H,K,L) hChPs, whereas midbrain and/or cerebellar ventricular zones display strong expression. Scale bars: 20 µm. M, midbrain; CB, cerebellum.

• Supplemental Figure S3 -

  Fig. S3. Developing peripheral nerve and intercostal muscle in control and Wnt1-Cre; Shh^fl/− embryos display apoptotic activity. (A-D′) Peripheral nervous tissue at E12.5 (A-B′) and intercostal muscle between the developing ribs at E14.5 (C-D′) in control and Wnt1-Cre; Shh^fl/− embryos display apoptotic activity, as shown by apparent cleaved-caspase 3 expression. Blue-boxed regions in A-D indicate the hChPs shown in Fig. 3. Black-boxed regions in A-D are magnified and shown in A′-D′ as positive internal controls. Scale bars: 20 µm.

• Supplemental Figure S4 -

  Fig. S4. Hypomorphic Gdf7^Cre/+; Shh^fl/− mutants exhibit reduced proliferation in the hChPe progenitor domain. (A,B) Shh in situ staining analysis of E14.5 controls (A) and Gdf7^Cre/+; Shh^fl/- mutants (B) shows that knock-in Gdf7^Cre fails to completely abrogate Shh expression in the hChPe. (C,D) The Gdf7^Cre/+; Shh^fl/− mutant hChPe, like the Wnt1-Cre; Shh^fl/− mutant, undergoes normal differentiation as indicated by robust Aqp1 expression. (E-K) Consistent with the incomplete deletion of Shh in the Gdf7^Cre/+; Shh^fl/− mutant, its hChPe progenitor domain displays moderate, but statistically significant, reduction in proliferation at E14.5. Scale bars: 20 µm.

• Supplemental Figure S5 -

  Fig. S5. Gli1-Cre^ERT2; R-eYFP marking the EGL. (A-C) As shown in Fig. 5, temporal fate-mapping of Gli1⁺ Shh-responding cells at postnatal day 0, with tamoxifen injections at E15 and E17, demonstrate that at later developmental stages, Gli1⁺ hChPe progenitor cells still contribute to hChPe. Here, we present a positive internal control showing the cerebellar external granule layer (EGL) of the same section as in Fig. 5, marked by extensive YFP fluorescence (A). Scale bars: 20 µm.