DEV00851 Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. (A) Expression of Pax1 and Pax9 was assessed by whole-mount in situ hybridization in wild-type and Gli2^+/–Gli3^–/– E9.5 embryos. Pax1 and Pax9 are expressed normally in Gli2^+/–Gli3^–/– mutants. (B) Expression of Sox9 was assessed by whole-mount in situ hybridization in wild-type, Gli2^–/–, Gli3^–/– and Gli2^–/–Gli3^+/– embryos at E9.5. Transverse sections through the anterior trunk reveal loss of Sox9 to be most dramatic in the dorsal sclerotome domain of Gli3^–/– mutants, while both dorsal and ventral domains are strongly affected in Gli2^–/– and Gli2^–/–Gli3^+/– mutants (Loss of expression indicated by brackets). (C) Transverse sections through the trunk of Gli2^+/–Gli3^–/– (top) and Gli2^–/–Gli3^+/– (bottom) embryos at E10.5 were stained to reveal tissue morphology. Dermomyotome (dm), myotome (my) and sclerotome (sc) appear normal. (D) Psm tissue isolated from wild-type or Gli2^+/–Gli3^–/– embryos at E9.5 was cultured in the presence (+) or absence (–) of Shh-N for 24 hours (n=3). Induction of target genes was assessed by RT-PCR. Gli2^+/–Gli3^–/– psm responds to Shh-N by inducing target genes. Note a 2.1-fold increase in Ptc1, and a 1.4-fold increase in Pax9 expression in Gli2^+/–Gli3^–/– psm in the absence of Shh-N.

• Supplemental Figure 2 -

  Fig. S2. Gli-GFP fusion proteins are functional. (A) The diagram depicts adenoviral vectors harboring Gli1, Gli2, Gli3 or a deleted form of Gli2 (DNG2), fused to EGFP at the C terminus, driven by a CMV promoter for overexpression in somitic tissues. (B) Western blot analysis confirms that Gli-GFP fusion proteins of the expected sizes are generated. (C) In the presence of 100 mM forskolin, Gli3-GFP is processed into a truncated form marked by an arrowhead, as described (Wang et al., 2000). (D) Smo and Smo* are expressed and glycosylated normally (arrowheads). (E) A CH310T1/2 cell line carrying a stably integrated 8XGli-Luciferase reporter (Sasaki et al., 1997) was generated. Cells were plated in a six-well dish at 80% confluency, infected with the indicated virus and a virus carrying lacZ (to normalize protein sample recovery) for 48 hours at 0.5´10⁸ pfu and assayed using the Luciferase Assay System (Promega). Luciferase activity is presented as fold induction normalized to luciferase activity in cells infected with control EGFP adenovirus. Infection of ~30% of cells was observed for each experiment. Experiments were performed in triplicate. Gli1, DNG2 and Smo* act as strong activators, while Gli2 and wild-type Smo act as moderate activators. Gli3 acts as a repressor.

  Reference

  Wang, B., Fallon, J. F. and Beachy, P. A. (2000). Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100, 423-434.