Fig. 4: Most genes are not de-repressed in Gli3 mutants prior to HH induction.
From: GLI transcriptional repression is inert prior to Hedgehog pathway activation
a Schematic for testing whether loss of Gli3 can prematurely activate target genes through GLI3 de-repression. b, c Volcano plot of differentially expressed genes (DEGs) detected in RNA-seq between WT and Gli3−/− limbs E9.25 (21-23 S) (b) and E10.5 (32–35 S) anterior limb buds (c). d Fluorescent in situ hybridization showing maximum intensity projections for Hand2, Foxf1 and Ptch1 in E9.25 WT and Gli3−/− limb buds indicating the absence of detectable Foxf1 and Ptch1 in pre-HH limb buds. Dashed white lines outline the limb bud region. e In situ hybridization for the GLI3 target Hand2, in WT and Gli3−/− limbs pre- (21, 23 S) and immediately post-HH induction (24–27 S; n = 3). Note that Hand2 is expressed almost uniformly across the limb bud in 21 S embryos in both WT and Gli3−/− limbs. At 23 S, slight reduction in anterior expression of Hand2 is observed in both WT and Gli3−/− limbs (white arrowhead). The red brackets denote anterior limb. Images acquired on a Zeiss LSM 710 Confocal visualized using maximal intensity projections with pseudo-coloring for merged images. f Schematic identifying H3K27me3 enriched regions in E9.25, E10.5 anterior (GLI3-repressed), and E10.5 posterior (HH signaling, loss of GLI-R) limb buds using CUT&Tag. g–j Orange shading in tracks indicates HH-responsive GBRs defined in Supplementary Fig. 1e. g Example of a HH-responsive GBR at a HH target gene promoter with H3K27me3 enrichment specifically at E9.25 but not E10.5. h Representative distal limb-specific HH-responsive GBR downstream of Ptch1 with no H3K27me3 enrichment in E9.25 or E10.5 limb buds. i Examples of HH target genes associated with polycomb repression that could potentially be incompetent for GLl3 repression. j Examples of genes that are bound by GLl3 at E9.25, contain H3K27ac enrichment and lack H3K27me3 enrichment, suggesting they are competent for GLl3 repression but are not de-repressed in the absence of Gli3. For f–j see Supplementary Data 1. Scale bars for tracks indicate 1 kb.
