Wnt target gene Ascl4 is dispensable for skin appendage development.

The development of skin appendages, including hair follicles, teeth and mammary glands is initiated through the formation of the placode, a local thickening of the epithelium. The Wnt/ß-catenin signaling cascade is an evolutionary conserved pathway with an essential role in placode morphogenesis, but its downstream targets and their exact functions remain ill defined. In this study, we identify Achaete-scute complex-like 4 (Ascl4) as a novel target of the Wnt/ß-catenin pathway and demonstrate its expression pattern in the signaling centers of developing hair follicles and teeth. Ascl transcription factors belong to the superfamily of basic helix-loop-helix transcriptional regulators involved in cell fate determination in many tissues. However, their specific role in the developing skin remains largely unknown. We report that Ascl4 null mice have no overt phenotype. Absence of Ascl4 did not impair hair follicle morphogenesis or hair shaft formation suggesting that it is non-essential for hair follicle development. No tooth or mammary gland abnormalities were detected either. We suggest that other transcription factors may functionally compensate for the absence of Ascl4, but further research is warranted to assess this possibility.

Mesenchyme instructs growth while epithelium directs branching in the mouse mammary gland.

The mammary gland is a unique organ that undergoes dynamic alterations throughout a female's reproductive life, making it an ideal model for developmental, stem cell and cancer biology research. Mammary gland development begins in utero and proceeds via a quiescent bud stage before the initial outgrowth and subsequent branching morphogenesis. How mammary epithelial cells transit from quiescence to an actively proliferating and branching tissue during embryogenesis and, importantly, how the branch pattern is determined remain largely unknown. Here, we provide evidence indicating that epithelial cell proliferation and onset of branching are independent processes, yet partially coordinated by the Eda signaling pathway. Through heterotypic and heterochronic epithelial-mesenchymal recombination experiments between mouse mammary and salivary gland tissues and ex vivo live imaging, we demonstrate that unlike previously concluded, the mode of branching is an intrinsic property of the mammary epithelium whereas the pace of growth and the density of ductal tree are determined by the mesenchyme. Transcriptomic profiling and ex vivo and in vivo functional studies in mice disclose that mesenchymal Wnt/ß-catenin signaling, and in particular IGF-1 downstream of it critically regulate mammary gland growth. These results underscore the general need to carefully deconstruct the different developmental processes producing branched organs.

Stabilization of Epithelial ß-Catenin Compromises Mammary Cell Fate Acquisition and Branching Morphogenesis.

The Wnt/ß-catenin pathway plays a critical role in cell fate specification, morphogenesis, and stem cell activation across diverse tissues, including the skin. In mammals, the embryonic surface epithelium gives rise to the epidermis as well as the associated appendages including hair follicles and mammary glands, both of which depend on epithelial Wnt/ß-catenin activity for initiation of their development. Later on, Wnts are thought to enhance mammary gland growth and branching, whereas in hair follicles, they are essential for hair shaft formation. In this study, we report a strong downregulation of epithelial Wnt/ß-catenin activity as the mammary bud progresses to branching. We show that forced activation of epithelial ß-catenin severely compromises embryonic mammary gland branching. However, the phenotype of conditional Lef1-deficient embryos implies that a low level of Wnt/ß-catenin activity is necessary for mammary cell survival. Transcriptomic profiling suggests that sustained high ß-catenin activity leads to maintenance of mammary bud gene signature at the expense of outgrowth/branching gene signature. In addition, it leads to upregulation of epidermal differentiation genes. Strikingly, we find a partial switch to hair follicle fate early on upon stabilization of ß-catenin, suggesting that the level of epithelial Wnt/ß-catenin signaling activity may contribute to the choice between skin appendage identities.

Protocol for Studying Embryonic Mammary Gland Branching Morphogenesis Ex Vivo.

Mammary gland development starts during embryogenesis, and the process continues after birth. During development, the mammary gland undergoes massive morphological and physiological alterations including growth, invasion, and branching morphogenesis providing an ideal model for stem cell and cancer biology studies. Great efforts have been made in understanding mammary gland development during puberty and adulthood; however, the process during embryogenesis is still elusive. One reason is that the tools to study tissue dynamics during development are limited, which is partially due to the lack of an ex vivo culture method. Here we describe an updated organ culture protocol of the murine embryonic mammary gland. This powerful tool allows monitoring of growth and branching morphogenesis of mammary gland ex vivo by live imaging. In addition, we introduce a novel method for culturing intact, stroma-free mammary rudiments from late gestation mouse embryos in 3D in Matrigel. This approach can be used to identify the direct stromal cues for branching morphogenesis.