Supplemental Material: Allele-specific expression reveals genetic drivers of tissue regeneration in mice

In adult mammals, skin wounds typically heal by scarring rather than through regeneration. In contrast, “super-healer” MRL mice have the unusual ability to regenerate ear punch wounds, yet the molecular basis for this regeneration remains elusive. Here, in hybrid crosses between MRL and non-regenerating mice, we use allele-specific gene expression to identify cis-regulatory variation associated with ear regeneration. Analyzing three major wound cell populations, we identified extensive strain- and tissue- specific cis-regulatory divergence associated with differences in healing outcomes. Genes with cis-regulatory differences specifically in fibroblasts were associated with wound healing phenotypes and pathways, and were enriched near genetic markers associated with ear-healing in a genetic cross. We further demonstrated that one of these genes, CFH, could be applied ectopically to accelerate wound repair and induce regeneration in typically fibrotic wounds. Through single-cell RNA-seq, we observed that CFH treatment strongly dramatically reduced immune cell recruitment to wounds, suggesting a potential mechanism for CFH’s effect. In support of this, CFH strongly down-regulated the neutrophil chemoattractant CXCL2, and chemical inhibition of CXCL2’s receptor in dorsal wounds mimicked the regenerative phenotype of MRL ear wounds. Overall, our results provide insight into the molecular drivers of regeneration in MRL mice with potential clinical implications.

在成年哺乳动物中，皮肤伤口通常通过瘢痕愈合而非再生。与此形成鲜明对比的是，“超级愈合者”MRL小鼠具有再生耳穿孔伤口的异常能力，然而这种再生的分子基础仍难以捉摸。在本研究中，我们通过MRL与不具再生能力小鼠的杂交，利用等位基因特异性基因表达来识别与耳再生相关的顺式调控变异。分析三大伤口细胞群体，我们发现了与愈合结果差异相关的广泛菌株和组织特异性顺式调控分歧。在成纤维细胞中具有顺式调控差异的基因与伤口愈合表型和通路相关，并且这些基因在遗传交叉中靠近与耳愈合相关的遗传标记。我们进一步证明，其中一种基因CFH可以通过外源应用加速伤口修复并诱导通常纤维化的伤口再生。通过单细胞RNA测序，我们发现CFH处理显著降低了免疫细胞对伤口的募集，这表明CFH效应的可能机制。为此，CFH强烈下调了中性粒细胞趋化因子CXCL2，而在背部伤口中CXCL2受体的化学抑制模拟了MRL耳伤口的再生表型。总体而言，我们的研究结果揭示了MRL小鼠再生分子驱动的机制，并具有潜在的临床意义。