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.

Spatially coordinated cell cycle activity and motility govern bifurcation of mammary branches.

Branching morphogenesis is an evolutionary solution to maximize epithelial function in a compact organ. It involves successive rounds of branch elongation and branch point formation to generate a tubular network. In all organs, branch points can form by tip splitting, but it is unclear how tip cells coordinate elongation and branching. Here, we addressed these questions in the embryonic mammary gland. Live imaging revealed that tips advance by directional cell migration and elongation relies upon differential cell motility that feeds a retrograde flow of lagging cells into the trailing duct, supported by tip proliferation. Tip bifurcation involved localized repression of cell cycle and cell motility at the branch point. Cells in the nascent daughter tips remained proliferative but changed their direction to elongate new branches. We also report the fundamental importance of epithelial cell contractility for mammary branching morphogenesis. The co-localization of cell motility, non-muscle myosin II, and ERK activities at the tip front suggests coordination/cooperation between these functions.

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.

PPARß/δ ligands regulate the expression of immune response mediators in the porcine endometrium - An in vitro study.

The peroxisome proliferator-activated receptor (PPAR) ß/δ belongs to a group of nuclear receptors that act as transcription factors. PPAR ß/δ plays a significant role in the regulation of female reproductive processes. It has been demonstrated that PPARß/δ is expressed in mouse, rat and porcine endometrium during the estrous cycle and pregnancy. The current study aimed to investigate the effect of selected PPARß/δ ligands on the expression of nuclear factor kappa (NF-κB) and selected cytokines - interleukin (IL)-1ß, IL-6, IL-8, IL-4, IL-10, leukemia inhibitory factor (LIF), in the porcine endometrium on days 10-12 and 14-16 of the estrous cycle (mid- and late-luteal phases corresponding to the full activity and luteolysis of the corpus luteum, respectively) and pregnancy (maternal recognition of pregnancy and beginning of implantation, respectively). Endometrial slices were incubated in vitro in the presence of PPARß/δ agonist L-165,041 (1 or 10 µM) or antagonist GW9662 (10 µM). The expression of mRNA and protein of the immune response mediator in the tissues was determined by real-time PCR and Western Blot. In general, the PPARß/δ agonist inhibited endometrial NF-κB mRNA expression during all analyzed reproductive stages, but it did not change protein expression. In turn, the PPARß/δ antagonist increased NF-κB protein levels on days 10-12 of the estrous cycle or pregnancy. The presence of the PPARß/δ agonist stimulated mRNA expression of LIF, IL-1ß and IL-8 and decreased the expression of IL-6. The presence of PPARß/δ ligands had a varied effect on protein expression in different stages on the analyzed period. The obtained results indicate that PPARß/δ regulates the expression of endometrial NF-κB and selected cytokines in pigs. The effects of PPARß/δ ligands on immune response mediators varied subject to the reproductive status of females and could be associated with differences in endometrial receptivity.