Experiment
The goal of this study was to focus on a specific aspect of wound healing, the early stages of angiogenesis, to reduce the complexity of the experimental wounding model. MRL/MpJ (MRL) inbred mice are well known for their unique abilities to achieve a scarless healing in juvenile and adult mice. MRL mice are known to remove and reestablish basement membrane in the resolution phase of healing, whereas other strains do not.
To localize the healing differences to specific QTL a (C57BL/6J x MRL/MpJ)F1 intercross was used to generate 178 F2 animals that were analyzed for vessel growth with an ex vivo subcutaneous assay. Genome wide expression analyses of skin biopsies were subjected to a culture model. After 7 days of culture explants were fixed, rinsed, and then visualized with a light microscope. A maximum local contrast projection was generated from each image stack by ImagePro 2.6. Nuclei were counted using a size exclusion paradigm designed to count objects of similar size.
Each animal from the F2 cohort was genotyped with a genome wide panel of 172 informative markers. The resulting data was used to perform linkage analysis in R/qtl using a normalized growth response for individuals as the phenotypic trait measured. Genome wide significance levels were determined by permutations of the entire dataset using 2000 replicates. A p-value of <0.05 was considered significant, while p<0.063 was considered suggestive.
Two significant QTL were detected on Chromosome 3 and 14 respectively. A suggestive QTL mapped to Chromosome 4.
QTL Scvg2, subcutaneous vessel growth QTL 2, mapped to Chr 3 with a 95% confidence interval between 7,384,755 and 26,195,496 bp GRCm38, LOD=4.09, accounting for 12% of total phenotypic variance. The growth effect of Svcg2 was transgressive, with the allele from the lower growth B6 parental strain providing a positive influence on vessel growth in the F2 progeny.
QTL Scvg3, subcutaneous vessel growth QTL 3, mapped to Chr 14, with a 95% confidence interval between 105,307,202 and 117,038,577 bp GRCm38, LOD=3.90, accounting for 11.5% of trait variance. The MRL allele conveyed positive growth at this locus.
The effects of both QTL were additive in nature, with two copies providing roughly twice the influence on growth.
Potentially relevant genes that were differentially expressed within the 95% confidence interval of Scvg2 were Car2, Cpa3, and Rprl2; within in the Scvg3 interval were Spry3, Slirk6, and Clybl. While expression differences could potentially account for divergent vessel growth, no clear connection between these genes and the observed phenotypes were obvious in this study.
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