Fig. 3: Gli genes regulate BMP signaling via activin receptor type 1-like expression.

a The experimental scheme of the unbiased high-throughput genomic screening (see Methods). From CNE14:egfp; gli3^+/+ and CNE14:egfp; gli3^14ins/14ins embryos, EGFP-positive cells were separately isolated by FACS. The sorted cells were subjected to RNA-seq and ATAC-seq. Three biological replicates were conducted for RNA-sequencing and two were for ATAC-seq. b The heat map of top 2000 differentially expressed genes in CNE14:egfp; gli3^+/+ and CNE14:egfp; gli3^14ins/14ins embryos. Color code: red indicates high and blue indicates low enrichment. c The volcano plot of the RNA-seq result. The genes with p < 0.5 and >two-fold expression change are coded by red. The genes on the left side (Log2 fold change <0) are down-regulated genes in gli3^14ins/14ins embryos while the genes on the right side (Log2 fold change > 0) are up-regulated genes in the mutant embryos. Acvr11 is one of the prominent Gli3 candidate genes. The statistical analysis was conducted by EdgeR program. d The genome browser visualization of zebrafish RNA-seq (wildtype and gli3^14ins/14ins samples), ATAC-seq, and HiC results, and skate RNA-seq and ATAC-seq results at the acvr1l locus. In zebrafish, the transcript level is lower at the exons of acvr1l in gli3^14ins/14ins embryos than gli3^+/+ embryos. Zebrafish ATAC-seq and HiC showed that the promoter region of acvr1l is ACR and in a TAD. Zebrafish HiC result was produced from the previously published data⁸¹. Skate RNA-seq and ATAC-seq data were generated from the previous paper¹¹⁸. e, f The expression pattern of acvr1l in gli3^+/+ and gli3^14ins/14ins embryos. Expression in the pectoral fin is decreased in gli3^14ins/14ins embryos compared to gli3^+/+ embryos. n = 24. The scale is the same in (e, f). g–i Immunofluorescence of phosphorylated-Smad 1/5/8. The strong signal is observed in cells surrounding the cleithrum of wildtype embryos (g, arrows), which decreases to gli3^14ins/14ins (h) and to gli2b^17ins/17ins; gli3^14ins/14ins embryos (i). n = 6 for wildtype, 9 for gli3^14ins/14ins, and 5 for gli2b^17ins/17ins; gli3^14ins/14ins embryos. j a bar graph of phosphorylated Smad1/5/8 staining level in gli3^+/+, gli3^14ins/14ins and gli2b^17ins/17ins embryos. The fluorescence intensity in the cleithrum was quantified and standardized by the area size in the cleithrum (“Method”). All raw data points were plotted. Raw data are in Supplementary Table 9. The data were analyzed and compared among samples by two-tailed student’s t-test. * indicates p < 0.05. p-value between gli3^+/+ and gli3^14ins/14ins is 0.049 and between gli3^+/+ (WT) and gli3^14ins/14ins is 0.025. While gli3^14ins/14ins and gli2b^17ins/17ins; gli3^14ins/14ins embryos exhibit statistically significant reduction of the PSmad1/5/8 signal from wildtype embryos, gli3^14ins/14ins and gli2b^17ins/17ins; gli3^14ins/14ins embryos do not show statistically significant change (p = 0.28). The unit of vertical axis is arbitrary unit. n = 6, 9, and 5 biologically independent samples for wildtype embryos, gli3^14ins/14ins and gli2b^17ins/17ins; gli3^14ins/14ins embryos, respectively. Error bars show standard deviations. k-l) LDN193189-treated pectoral fins stained by HCR with sp7 and col2a1a probes and DAPI staining. Sp7 expression was diminished by 10 μM of the inhibitor treatment. Arrows indicate sp7 expression, and arrowheads show its weak or no expression. sc scapula. n = 23 for 0 and 10 μM. The scale bar in (e, g) are 50 μm. g–i, k, l are the same scale.