Harnessing Novel Gene Expression Analyses To Identify Drivers Of Ear Regeneration In MRL Mice
Heather E. desJardins-Park, AB1,2, Katya L. Mack, PhD3, Michael F. Davitt, MD1, Michelle Griffin, MD1, Hunter B. Fraser, PhD3, Michael T. Longaker, MD, MBA, FACS1,2.
1Stanford School of Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford, CA, USA, 2Stanford School of Medicine, Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA, USA, 3Stanford University, Department of Biology, Stanford, CA, USA.
PURPOSE: The “super-healing” MRL/MpJ mouse is uniquely able to regenerate ear punch wounds, while dorsal wounds heal in a typical scarring fashion. Identification of the genes responsible for ear regeneration in these mice could provide insights toward novel anti-scarring therapies. However, previous methods for identifying the genetic basis of species-/strain-specific traits are imprecise and extremely resource-intensive. Recent studies have revealed that, by outbreeding a strain with a trait of interest (here, regenerative ear healing), assessing the hybrid offspring for spatial/tissue-specific differences in relative gene expression from the two parent alleles (“differential allele-specific expression”) can reveal genes underlying that phenotype. We hypothesized that differential allele-specific expression could represent a novel approach to identify genes driving regenerative ear healing in MRL mice.
METHODS: MRL/MpJ (MRL) and CAST/EiJ (CAST) mouse genome sequences were obtained. Variant calling was performed to identify sites where the sequences differed. MRL and CAST mice were cross-bred, F1 offspring underwent dorsal excisional and ear punch wounding (standard protocols), and wounds were harvested on POD7. Following incubation in ammonium thiocyanate, epidermis and underlying cartilage (for ear wounds) were removed under a surgical microscope. Dorsal and ear wound dermis were digested, and inflammatory/immune cells (CD45+; chosen because prior studies have implicated altered wound inflammatory response in MRL regeneration) were isolated by FACS and subjected to bulk RNA-sequencing. Reads were mapped to the MRL or CAST genome based on strain-specific variants, and genes with significantly differing expression (Benjamini-Hochberg method, FDR=5%) from the MRL vs. CAST allele and from dorsal vs. ear wounds were identified.
RESULTS: Ear wounds in MRLxCAST hybrid mice exhibited grossly apparent regeneration over time (Fig. 1A), consistent with prior reports. Roughly equal proportions of RNA-seq reads mapped to MRL vs. CAST alleles, indicating minimal mapping bias. Principal component analysis demonstrated that reads clustered by parent allele (MRL vs. CAST) and wound site (ear vs. dorsum) (Fig. 1B), with good reproducibility between biological replicates (n=4). Importantly, the number of genes with significantly different expression from MRL vs. CAST allele was substantially (>10-fold) greater in the ear than the dorsum (2531 vs. 159 unique genes; Fig. 1C). The majority of allele-specific expression in the dorsum was also observed in the ear (Fig. 1C, overlapping region; 878 shared genes vs. 159 unique to dorsum).
CONCLUSIONS: In MRL hybrid mice, inflammatory cells exhibit markedly greater allele-specific gene expression in ear wounds (regenerative) than dorsal wounds (scarring). This finding would be consistent with MRL-specific gene cis-regulatory activity leading to the improved healing phenotype seen in the ear. Our results suggest that probing differential allele-specific gene expression may represent a powerful computational approach for identifying the genetic basis of complex traits in mammals. We will examine inflammatory and other cell types (e.g., fibroblasts) in order to identify specific genes with differential allele-specific expression in regenerative versus scarring wounds, with the ultimate goal of identifying novel targets for preventing scarring.
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