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.

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.

Cell influx and contractile actomyosin force drive mammary bud growth and invagination.

The mammary gland develops from the surface ectoderm during embryogenesis and proceeds through morphological phases defined as placode, hillock, bud, and bulb stages followed by branching morphogenesis. During this early morphogenesis, the mammary bud undergoes an invagination process where the thickened bud initially protrudes above the surface epithelium and then transforms to a bulb and sinks into the underlying mesenchyme. The signaling pathways regulating the early morphogenetic steps have been identified to some extent, but the underlying cellular mechanisms remain ill defined. Here, we use 3D and 4D confocal microscopy to show that the early growth of the mammary rudiment is accomplished by migration-driven cell influx, with minor contributions of cell hypertrophy and proliferation. We delineate a hitherto undescribed invagination mechanism driven by thin, elongated keratinocytes-ring cells-that form a contractile rim around the mammary bud and likely exert force via the actomyosin network. Furthermore, we show that conditional deletion of nonmuscle myosin IIA (NMIIA) impairs invagination, resulting in abnormal mammary bud shape.

Gene expression analysis of Drosophilaa Manf mutants reveals perturbations in membrane traffic and major metabolic changes.

BACKGROUND: MANF and CDNF are evolutionarily conserved neurotrophic factors that specifically support dopaminergic neurons. To date, the receptors and signalling pathways of this novel MANF/CDNF family have remained unknown. Independent studies have showed upregulation of MANF by unfolded protein response (UPR). To enlighten the role of MANF in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of Drosophila Manf. RESULTS: The most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. When evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic Manf mutants, the endoplasmic reticulum (ER) stress and membrane traffic alterations were evident. In Drosophila Manf mutants the expression of several genes involved in Parkinson's disease (PD) was altered as well. CONCLUSIONS: We conclude that besides a neurotrophic factor, Manf is an important cellular survival factor needed to overcome the UPR especially in tissues with high secretory function. In the absence of Manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. In neurodegenerative diseases, such as PD, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. The degeneration of dopaminergic neurons is the hallmark for PD and our work provides a clue on the mechanisms by which the novel neurotrophic factor MANF protects these neurons.