A. L. Moore1,2, C. D. Marshall2, U. Litzenburger2, E. A. Brett2, L. A. Barnes2, R. C. Ransom2, M. S. Hu2,3, G. Walmsley2, T. Leavitt2,4, H. Y. Chang2, H. P. Lorenz2, M. T. Longaker2 1Brigham And Women’s Hospital,Department Of Surgery,Boston, MA, USA 2Stanford University School Of Medicine,Department Of Surgery,Palo Alto, CA, USA 3University Of Hawaii , John A. Burns School Of Medicine,Department Of Surgery,Honolulu, HI, USA 4Boston University School Of Medicine,Boston, MA, USA
Introduction:
Scarring of the skin poses a major burden to the US healthcare system. While scarring is the expected outcome of tissue damage, it may lead to several negative outcomes including painful contractures, keloids, and hypertrophic scars. Skin wounds in early gestational age heal without a scar, but factors allowing for this scarless phenotype are still unknown. We have shown that in murine dorsal skin, Engrailed positive fibroblasts (EPFs) appear in the dermis at the same time that the transition from a scarless to a scarring phenotype occurs. Additionally, EPFs are responsible for the formation of dermal scar in adult mice. We hypothesize that EPFs are responsible for the transition from scarless to scar-forming phenotype in the late gestational fetus, and that the cells are functionally distinct.
Methods:
Dorsal dermal fibroblasts from En1Cre; R26mTmG mice were isolated at gestational ages E10, E16, E18, P1 and P30. EPFs and ENFs from these time points were sorted using fluorescence-activated cell sorting (FACS) and were analyzed using assay for transposase-accessible chromatin using sequencing (ATAC-seq). This assay compared the accessibility of segments of genomic DNA.
Results:
Preliminary ATAC-seq data comparing EPFs to ENFs reveals a total of 3008 genes more accessible in EPFs than in ENFs. For example, vimentin and α -SMA are both expressed in EPFs and ENFs, however the accessibility is significantly higher in EPFs. Genetic segments more accessible in EPFs are correlated with fibrosis, mesenchymal tumors, systemic scleroderma, pulmonary fibrosis, and endometriosis.
Additionally, male and female EPFs have several gender specific differences. Males tend to have more open domains in cell and germline differentiation, whereas female mice have a predominance of genes implicated in leukocyte development, as well as diseases associated with a dysfunctional immune system.
Conclusion:
ATAC-seq analysis of EPFs and ENFs confirms that they are functionally distinct cells, consistent with our prior published data. Additionally, male and female EPFs and ENFs possess different genetic signatures in areas associated with gender specific disorders. This may indicate that fibroblasts function in gender biased ways. Alternatively, the gender differences may demonstrate the plasticity and responsiveness of fibroblasts to external signals such as androgens.
Based on these data, we will perform reciprocal transplantation experiments in which EPFs and ENFs isolated from the dorsal skin of postnatal mice are transplanted into fetal skin and vice versa. These experiments will determine whether EPFs intrinsically carry the ability to form scar, or whether scar-forming behavior depends on local factors.
