INVESTIGATING THE SPATIOTEMPORAL WINDOW OF LINEAGE SPECIFICATION DURING EARLY MAMMARY GLAND DEVELOPMENT.

Abstract

The mammary gland is derived from an epidermal placode that receives reciprocal induction from the underlying mesenchyme. The mammary mesenchyme is crucial for the specification of the mammary gland. Studies have reported that co-culture of non-mammary epithelial cells (for instance, salivary gland1) with mammary mesenchyme/fat pad cells specify the epithelial cells into mammary epithelial cells. Even though mammary mesenchyme signaling is required for proper mammary specification and early development up to E14.5, subsequent growth, and maintenance beyond E14.5 occurs independently of mesenchymal signaling. In vitro culture of mammary epithelium alone after E14.5 can give rise to all mammary cell types. Sexual dimorphism of the mouse mammary gland also begins around E14.5. The generation of an autonomous, transient mammary stem cell niche and new response to androgen signaling at E14.5 indicates a major developmental stage in the fetal mouse mammary gland. Therefore, we aim to understand the gene networks expressed within fetal mouse mammary epithelial cells as they transition through this period. We also aim to identify different cell types within mammary epithelium and mammary mesenchyme. The mammary epithelial cells are not committed to either luminal or basal lineages until birth2, although mammary epithelial cells of three different hybrid states of specification (luminal-like, basal-like, and hybrid cells) have been reported to present at E15.5 2,3. To obtain a more detailed gene expression map of early mammary lineage regulators, we performed multiplexed RNA-FISH on mouse mammary tissues from E13.5 to E15.5 (n=3 for each stage). We observed asymmetric expression patterns of Notch1 (luminal specifier), Lef1 (mammary mesenchymal marker), and Pthlh (basal specifier) between the peripheral and center cells of the mammary rudiments at E14.5 and E15.5, which suggests that the mammary epithelium is primed for lineage specification by E14.5. We observed the low expression of Snail2 from E13.5-E15.5 where it was expressed both in the mammary epithelium and the mammary mesenchyme. We also observed the expression of Sox10 as early as E13.5 which has been reported to not be expressed until E18.5. Our RNA-FISH data showed that Sox9 and Gata3 are exclusively expressed in the mammary rudiments, while Lmo4 and Sox10 were found to be expressed at similar levels in the mammary rudiments and the surrounding mesenchyme. The mammary epithelial specifier Sox11 was observed to be upregulated at E15.5 as compared to E14.5 and E13.5. Qualitative analyses for Notch1 and Pthlh suggest there are varied hybrid states of specification in the mouse mammary rudiments by E13.5. ScRNA-seq is an unbiased approach that can be used to identify cells at different hybrid states of specification. The collection of sufficient cells via the dissociation of tissues is a prerequisite of scRNA-seq. We optimized the previously published protocol for single-cell dissociation2,3 to collect cells from the mammary epithelium and mammary mesenchyme from both male and female embryos at E14.5. Our results and method optimizations serve as pioneering steps towards performing quantitative, multiplexed gene expression analyses by coupling spatial techniques with single-cell-RNA-seq data. Together, our findings contribute towards the construction of an embryonic mouse mammary gene expression atlas as well as provide insight into fetal mammary reprogramming and dysfunction.