Slit2 and netrin 1 act synergistically as adhesive cues to generate tubular bi-layers during ductal morphogenesis

Abstract

Development of many organs, including the mammary gland, involves ductal morphogenesis. Mammary ducts are bi-layered tubular structures comprising an outer layer of cap/myoepithelial cells (MECs) and an inner layer of luminal epithelial cells (LECs). Slit2 is expressed by cells in both layers, with secreted SLIT2 broadly distributed throughout the epithelial compartment. By contrast, Robo1 is expressed specifically by cap/MECs. Loss-of-function mutations in Slit2 and Robo1 yield similar phenotypes, characterized by disorganized end buds (EBs) reminiscent of those present in Ntn1 -/-glands, suggesting that SLIT2 and NTN1 function in concert during mammary development. Analysis of Slit2 -/-;Ntn1 -/-glands demonstrates an enhanced phenotype that extends through the ducts and is characterized by separated cell layers and occluded lumens. Aggregation assays show that Slit2-/-cells, in contrast to wild-type cells, do not form bi-layered organoids, a defect rescued by addition of SLIT2. NTN1 has no effect alone, but synergistically enhances this rescue. Thus, our data establish a novel role for SLIT2 as an adhesive cue, acting in parallel with NTN1 to generate cell boundaries along ducts during bi-layered tube formation.

Vascular Robo4 restricts proangiogenic VEGF signaling in breast

“…We previously demonstrated the expression of SLITs in mammary gland epithelium (24). Here, we identify a second source of SLIT, mural cells associated with blood vessels.…”

“…The study conformed to guidelines set by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Santa Cruz. Slit2, Slit3, Robo1, and Robo4 null mice were generated as described (13,24). Transplant techniques, antibodies, immunohistochemistry, migration assays, RT-PCR, immunoprecipitation, immunoblotting, image processing, statistical analyses and determination of blood vessel density, branchpoints and tortuosity are described in detail in SI Materials and Methods.…”

“…Alternatively, there may be an epithelial effect because these analyses were performed on intact, rather than transplanted, glands. ROBO1 is expressed in the epithelium (24), as well as the endothelium ( Fig. 2B and Fig S2 C and D), raising the possibility that loss of Robo1 in the epithelium contributes to the observed increase in blood vessel density in the Robo1 −/− ;Robo4 −/− mice.…”

“…To determine SLIT function during mammary gland development, we initially focused on epithelial SLITs as a target-derived source because previous studies have reported directional migration of endothelial cells in response to exogenous or tumor-supplied SLIT protein (2,4,7,8). Only Slit2 and Slit3 are expressed by mammary epithelia (24); therefore, to evaluate the consequences of losing epithelial SLIT expression, we generated Slit2 −/− ;Slit3 −/− chimeric mammary outgrowths by transplantation because the Slit2 −/− mutation is perinatal lethal (Fig. 1A) (25).…”

Intraislet SLIT–ROBO signaling is required for beta-cell survival and potentiates insulin secretion

“…Mammalian homologs of Slit and Robo with functions outside of axon guidance have since been identified (18, 19). Slit ligands have been implicated in liver, kidney, lung, and mammary development by modulating cell adhesion, migration, differentiation, and death (18,20,21). It was not known whether Slit-Robo signaling functions in beta cells.…”