Breaking Barriers: Uncovering Gene Signatures Associated with Basement Membrane Crossing in Dermal Melanoblast Populations

Abstract

The basement membrane (BM) is a specialized extracellular matrix that underlies diverse tissues and shapes distinct biological environments. Though the BM acts as a selective barrier to cells and molecules, certain cells— such as immature melanocytes during embryogenesis and epithelial tumor cells during metastasis— transit across the BM to migrate through tissue compartments. Currently, the time- and context-dependent transcriptional regulation driving melanoblast (Mb) BM crossing is poorly understood. Moreover, potential differences in the crossing patterns of embryonic Mb populations due to oncogenic transformation have not yet been defined. Thus, an enriched understanding of the gene expression patterns underlying compartmentally distinct Mb identities, and how these characteristic patterns change upon introduction of an oncogenic mutation, is important from both a developmental and disease perspective. First, I aim to spatially validate transcriptional signatures linked to BM transit. Preliminary scRNA-seq findings support the existence of putative dermal and epidermal Mb subpopulations, each displaying distinct transcriptomes that match the cells’ activity in these respective compartments; scRNA-seq provides no spatial information, so it is necessary to spatially validate these BM crossing signatures, which are hypothesized to be exclusively expressed in dermal populations due to the unidirectional nature of Mb BM crossing. Utilizing HCR RNA-FISH technology on E13-E15 mouse embryonic backskins to perform spatial validation of putative dermal Mb gene candidates, this study documents the validation of selected mRNA probes targeting Dct and Cdh1— respective dermal and epidermal Mb signatures— that successfully function as positive control gene markers. Furthermore, the first experimental mRNA probe that was tested— targeting putative dermal Mb signature Sema3C— produced fluorescent signal in E13.5 backskins, however no Sema3C expression was detected in dermal Mbs, indicating the need for further optimization of the HCR technique before successfully validating putative dermal Mb expression profiles. Secondly, I characterize the patterns by which oncogenically transformed V600EBRaf-expressing Mbs cross the BM, by comparing the relative Mb numbers, compartmental distributions, and cell-division frequencies of wild-type (WT) and mutant models across E14.5 and E15.5 timepoints. I show that at both E14.5 and E15.5, V600EBRaf Mb counts were significantly elevated in the dermis compared to those of WT Mbs, with no significant differences in both the counts of epidermal Mb, and in the amount of Mb proliferation across mutant and WT groups. Results suggest that V600EBRaf selectively alters the dermal Mb population size. Future work, including additional biological replicates and earlier timepoints (E13.5), is needed to confirm these results and conclusively establish the mutation’s impact on Mb spatial distribution and BM crossing. Together with the spatial validation of dermal Mb gene signatures using HCR RNA-FISH, these findings pave the way for continued exploration of transcriptional regulation of Mb behavior during BM crossing and how oncogenic mutations may disrupt this process.