We next compared the gene expression of HBCs activated via PMA in vitro and HBCs activating natively under injury conditions in vivo. Given the potential wide-ranging effects of PMA on cultured cells, we assessed transcriptomic similarity using specific established marker genes and by analyzing the expression of entire gene sets. For our in vivo reference, we re-analyzed a single-cell dataset of resting and activated HBCs from the injured, regenerating wild-type mouse OE harvested at multiple time points after MTZ injury (Gadye et al., 2017).
We clustered Krt5(+) HBCs from the Gadye dataset into three groups based on their expression of three marker genes: dormancy regulator Tp63, proliferation marker Mki67, and activation marker Hopx. Hopx was additionally chosen in part because of its known role in cellular differentiation in other tissues (Hng et al., 2020; Palpant et al., 2017). These markers identify three developmentally sequential HBC subpopulations: Tp63(+) resting HBCs, Mki67(+) transitioning/ cycling HBCs, and Hopx(+) fully activated HBCs (Figure 2A). A set of ten candidate genes that were highlighted by Gadye as key during the regeneration process in vivo were mapped onto the re-analyzed, native HBC UMAP (Uniform Manifold Approximation and Projection) to highlight the gene list’s correlation with HBC status in vivo, confirming the clustering into three subpopulations (Figure 2B). In keeping with the in vivo analysis, PMA-activated HBCs demonstrated, by immunostaining, an inverse correlation between increasing HOPX protein and decreasing TP63 protein at 12 HPT (Figure 2C).
This result is corroborated by prior work demonstrating enrichment of HOPX in human keratinocytes following PMA treatment (Yang et al., 2010). In contrast with the results in vivo, HOPX labeled a very high percentage of the PMA-activated HBCs. Nonetheless, PMA treatment does induce a specific state highly analogous to in vivo injury-activated HBCs. Using the module detection function of Seurat (Tirosh et al., 2016), we plotted the collective expression of the top 100 DEGs induced by PMA treatment, including both 6 HPT and 12 HPT sets given the high degree of concordance between the sets (Figure S2).
Expression of this signature was highly elevated in the same in vivo-activated HBC cluster (Figure 2D), reaffirming transcriptomic similarity between PMA-activated HBCs in vitro and injury-activated HBCs in vivo. Importantly, while the PMA-induced gene set was enriched for signaling, development, and differentiation GO terms (Figure 2E), only one gene overlapped with the previously published wound-response gene set (Sprr1a), thus indicating broader transcriptomic similarity between the two activated HBC populations.
We examined the overrepresented ontology categories across five distinct HBC populations to compare changes between in vitro and in vivo activation more comprehensively: PMA-treated HBCs in vitro (6 HPT and 12 HPT) and in vivo post-injury HBCs (resting, HBC*1, and HBC*2, the latter two representing the two stages of activated HBCs) (Gadye et al., 2017).
All five HBC populations show upregulation of various epithelial remodeling categories. In addition, all five HBC populations show downregulation of developmental, microtubule polymerization, and neuron projection categories. PMA-treated HBCs, irrespective of length of treatment, could be defined by overlapping upregulated and downregulated ontology categories.
