Contractile fibroblasts form a transient niche of mammary epithelium undergoing branching morphogenesis 

Mammary gland is a highly dynamic branched organ that undergoes major development and remodeling postnatally. It consists of branched mammary epithelium and complex stroma (fat pad), which comprises multiple cell types, including fibroblasts. The complex branched structure of mammary epithelium develops from the most part during puberty, upon activation of dormant epithelium to highly proliferative bulb-shaped structures, terminal end buds (TEBs), that drive mammary epithelial branching morphogenesis. TEBs are regulated systemically and by local epithelial-stromal interactions, though the latter have remained poorly defined. 
Using single-cell RNA sequencing, spatial mapping and lineage tracing, we discovered that TEBs are surrounded by a transient niche-forming population of specialized contractile (αSMA+) fibroblasts that exclusively localize around the TEBs and are recruited from Fgf10+ preadipocytes in the surrounding fat pad. Despite being proliferative, the αSMA+ peri-TEB fibroblasts do not expand but give rise to peri-ductal and fat pad fibroblasts and adipocytes. Transcriptomic analysis revealed elevated WNT signaling in the peri-TEB fibroblasts, and in vivo chemical inhibition of WNT signaling led to depletion of peri-TEB fibroblasts, disrupted TEB morphology, and impaired epithelial outgrowth. Further studies using fibroblast-specific manipulation of WNT signaling are ongoing to test WNT signaling as a key regulator of the peri-TEB fibroblast population and, consequently, of mammary epithelial morphogenesis.
Together, our study contributes to understanding functional relevance, developmental relationships and regulation of fibroblast subtypes in mammary gland stroma and the role of contractile fibroblasts in the niche of actively growing epithelium undergoing branching morphogenesis.

Acknowledgement:

This project was supported by ERC-CZ LL2323 FIBROFORCE from the Ministry of Education, Youth and Sports (MEYS CR). We acknowledge Imaging Methods Core Facility at BIOCEV, supported by the MEYS CR (LM2023050 Czech-BioImaging) for their support and assistance with confocal microscopy and FACS in this work, and services of the Czech Centre for Phenogenomics at the Institute of Molecular Genetics supported by the Czech Academy of Sciences RVO 68378050 and by the MEYS CR (LM2023036 Czech Centre for Phenogenomics).