UC Santa Cruz

ABSTRACT

The SLIT2/ROBO1 signaling axis regulates ductal development and stem cell self-renewal by controlling Inscuteable through the SNAIL transcription factor

by

MIMMI SEWERIN BALLARD

The breast, or mammary gland in mice, is an epithelial bilayered ductal system that produces milk during pregnancy. It is composed of two main cell types, basal and luminal epithelial cells, and develops postnatally, primarily during puberty. In the mouse, this occurs between 3-6 weeks of age and is driven by stem cells in the end buds, located at the tips of the primary and secondary ductal branches. SLIT2, signaling through ROBO1 expressed on the surface of basal cells during development, regulates many aspects of mammary gland morphogenesis, including basal cell proliferation and branching, as well tumor cell transformation. Here, I first summarize how SLIT2/ROBO1 signaling drives, and in some cases, counteracts tumor progression, and then investigate if this signaling axis plays a role in the self-renewal of end bud stem cells. We show that mammary stem cells self-renew via asymmetric cell divisions (ACDs) and that SLIT2/ROBO1 prevents precocious stem cell expansion, thus maintaining the stem cell population, by controlling the expression of the ACD regulator Inscuteable. Immunostaining, immunoblotting and quantitative real-time PCR (RT-qPCR) experiments demonstrate increased expression of Inscuteable in Robo1-/- animals, while SLIT2 treatment of transformed basal cells leads to decreased Inscuteable levels. Furthermore, we demonstrate that this regulatory relationship is mediated by the SNAIL (SNAI1) transcription factor, which is present at higher levels in the Robo1-/- tissue. Through luciferase promoter assays, we find that SNAI1 enhances Inscuteable expression by binding to a non-canonical DNA binding site (TCACA) in the Inscuteable promoter. To investigate the consequences of elevated Inscuteable levels during ductal development, we characterized mammary gland morphology in an Inscuteable over-expressing mouse model (mInsc KI/KI) and found enhanced ductal outgrowth, without increased cell proliferation, in the transgenic, compared to wild type, mice. Using in vivo immunostaining and an in vitro live-cell labeling assay, we found an increased frequency of symmetric cell divisions, and limiting dilution assays revealed an expanded stem cell population in the mInsc KI/KI compared to wild type mouse. The consequence of this precocious stem cell expansion in puberty is decreased alveogenesis during pregnancy. Taken together, these results suggest that SLIT2/ROBO1 regulates the self-renewal of mammary stem cells during mammary gland development, and that this in turn influences ductal outgrowth, stem cell numbers and ultimately alveogenesis.