Figure 5. Gli1⁺ PMCs have a unique transcriptomic signature driven by hedgehog signaling.

(A) After 4 daily doses of tamoxifen and an additional 10 day washout, isolated cells underwent flow sorting or BDL for 5 days and then flow sorting. Of the live nonparenchymal population, stellate cells made up 6.8% of cells, whereas Gli1:tdTomato⁺ cells made up a smaller fraction at 0.5%. (B) Principle component analysis. (C) Volcano plot, showing differential gene expression between flow sorted stellate cells and Gli1+ PMCs. 2523 genes were differentially expressed using an FDR of 0.05, a Benjamini-Hochberg adjusted p-value < 0.01, with a 1-fold log₂ change. (D) 336 genes in stellate cells and 592 genes in Gli1⁺ continued to maintain high differential expression after injury. qPCR analysis of fibrogenic genes shows that Gli1⁺PMCs markedly induce fibrogenic genes after BDL compared to stellate cells (p < 0.01 for uninjured Gli1 compared to uninjured stellate cells and injured Gli1 compared to injured stellate cells for all 5 genes). (E, F) Heatmaps of identified genes that maintain differential expression in stellate cells (E) and Gli1⁺ PMCs (F) from uninjured (U1–2) and injured (B1–2) samples. (G) qPCR analysis of fibrogenic genes shows that Gli1⁺ PMCs markedly induce fibrogenic genes after BDL compared to stellate cells (p < 0.01 for uninjured Gli1 compared to uninjured stellate cells and injured Gli1 compared to injured stellate cells for all 5 genes). (H) Boxplots of Log2 fold change of gene expression values between Gli1⁺ PMCs compared to stellate cells shows that genes in the Hedgehog pathway (BSID 137950) are significantly upregulated (bars represent 1.5 times the width of a quartile in either direction. Two-sided unpaired Wilcoxon ranked sum, p=0.0384). (I) The expression differential for known hedgehog target genes within the hedgehog signaling gene set between Gli1⁺ PMCs compared to stellate cells. **P<0.05. Scale bars, 50 pm.