Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland

Branching morphogenesis of the murine mammary gland starts during late embryogenesis. It is regulated by the signals emanating both from the epithelium and the mesenchyme, yet the molecular mechanisms regulating this process remain poorly understood. We have previously developed a unique whole organ culture technique for embryonic mammary glands, which provides a powerful tool to monitor and manipulate branching morphogenesis ex vivo. Nowadays, RNA sequencing and other transcriptional profiling techniques provide robust methods to identify components of gene regulatory networks driving branching morphogenesis. However, validation of the candidate genes still mainly depends on the use of the transgenic mouse models, especially in mammary gland studies. By comparing different serotypes of recombinant adeno-associated virus (rAAVs), we found out that rAAVs provide sufficient efficiency for gene transfer with different tissue preferences depending on the serotypes of the virus. AAV-2 and AAV-8 preferentially target epithelial and mesenchymal compartments, respectively, while AAV-9 infects both tissues. Here, we describe a protocol for AAV-mediated gene transfer in ex vivo cultured murine embryonic mammary gland facilitating gene function studies on mammary gland branching morphogenesis.Peer reviewe

New insights into fetal mammary gland morphogenesis : differential effects of natural and environmental estrogens

An increased breast cancer risk during adulthood has been linked to estrogen exposure during fetal life. However, the impossibility of removing estrogens from the feto-maternal unit has hindered the testing of estrogen's direct effect on mammary gland organogenesis. To overcome this limitation, we developed an ex vivo culture method of the mammary gland where the direct action of estrogens can be tested during embryonic days (E) 14 to 19. Mouse mammary buds dissected at E14 and cultured for 5 days showed that estrogens directly altered fetal mammary gland development. Exposure to 0.1 pM, 10 pM, and 1 nM 17 beta-estradiol (E2) resulted in monotonic inhibition of mammary buds ductal growth. In contrast, Bisphenol-A (BPA) elicited a non-monotonic response. At environmentally relevant doses (1 mu M), BPA significantly increased ductal growth, as previously observed in vivo, while 1 mu M BPA significantly inhibited ductal growth. Ductal branching followed the same pattern. This effect of BPA was blocked by Fulvestrant, a full estrogen antagonist, while the effect of estradiol was not. This method may be used to study the hormonal regulation of mammary gland development, and to test newly synthesized chemicals that are released into the environment without proper assessment of their hormonal action on critical targets like the mammary gland.Peer reviewe

Ectodysplasin/NF-kappa B Promotes Mammary Cell Fate via Wnt/beta-catenin Pathway

Mammary gland development commences during embryogenesis with the establishment of a species typical number of mammary primordia on each flank of the embryo. It is thought that mammary cell fate can only be induced along the mammary line, a narrow region of the ventro-lateral skin running from the axilla to the groin. Ectodysplasin (Eda) is a tumor necrosis factor family ligand that regulates morphogenesis of several ectodermal appendages. We have previously shown that transgenic overexpression of Eda (K14-Eda mice) induces formation of supernumerary mammary placodes along the mammary line. Here, we investigate in more detail the role of Eda and its downstream mediator transcription factor NF-kappa B in mammary cell fate specification. We report that K14-Eda mice harbor accessory mammary glands also in the neck region indicating wider epidermal cell plasticity that previously appreciated. We show that even though NF-kappa B is not required for formation of endogenous mammary placodes, it is indispensable for the ability of Eda to induce supernumerary placodes. A genome-wide profiling of Eda-induced genes in mammary buds identified several Wnt pathway components as potential transcriptional targets of Eda. Using an ex vivo culture system, we show that suppression of canonical Wnt signalling leads to a dose-dependent inhibition of supernumerary placodes in K14-Eda tissue explants.Peer reviewe

Ectodysplasin target gene Fgf20 regulates mammary bud growth and ductal invasion and branching during puberty

Mammary gland development begins with the appearance of epithelial placodes that invaginate, sprout, and branch to form small arborized trees by birth. The second phase of ductal growth and branching is driven by the highly invasive structures called terminal end buds (TEBs) that form at ductal tips at the onset of puberty. Ectodysplasin (Eda), a tumor necrosis factor-like ligand, is essential for the development of skin appendages including the breast. In mice, Eda regulates mammary placode formation and branching morphogenesis, but the underlying molecular mechanisms are poorly understood. Fibroblast growth factor (Fgf) receptors have a recognized role in mammary ductal development and stem cell maintenance, but the ligands involved are ill-defined. Here we report that Fgf20 is expressed in embryonic mammary glands and is regulated by the Eda pathway. Fgf20 deficiency does not impede mammary gland induction, but compromises mammary bud growth, as well as TEB formation, ductal outgrowth and branching during puberty. We further show that loss of Fgf20 delays formation of Eda-induced supernumerary mammary buds and normalizes the embryonic and postnatal hyperbranching phenotype of Eda overexpressing mice. These findings identify a hitherto unknown function for Fgf20 in mammary budding and branching morphogenesis.Peer reviewe

Publisher Correction: FGF signalling controls the specification of hair placode-derived SOX9 positive progenitors to Merkel cells.

The originally published version of this Article contained an error in Figure 2. In panel e, the blue bar was incorrectly labelled 'KRT8(+)/TOMATO(-)'. Furthermore, during the process of preparing a correction, the publication date of the Article was inadvertently changed to June 20th 2018. Both of these errors have been corrected in the PDF and HTML versions of the Article

Imagine beyond: recent breakthroughs and next challenges in mammary gland biology and breast cancer research

On 8 December 2022 the organizing committee of the European Network for Breast Development and Cancer labs (ENBDC) held its fifth annual Think Tank meeting in Amsterdam, the Netherlands. Here, we embraced the opportunity to look back to identify the most prominent breakthroughs of the past ten years and to reflect on the main challenges that lie ahead for our field in the years to come. The outcomes of these discussions are presented in this position paper, in the hope that it will serve as a summary of the current state of affairs in mammary gland biology and breast cancer research for early career researchers and other newcomers in the field, and as inspiration for scientists and clinicians to move the field forward

FGF signalling controls the specification of hair placode-derived SOX9 positive progenitors to Merkel cells

Merkel cells are innervated mechanosensory cells responsible for light-touch sensations. In murine dorsal skin, Merkel cells are located in touch domes and found in the epidermis around primary hairs. While it has been shown that Merkel cells are skin epithelial cells, the progenitor cell population that gives rise to these cells is unknown. Here, we show that during embryogenesis, SOX9-positive (+) cells inside hair follicles, which were previously known to give rise to hair follicle stem cells (HFSCs) and cells of the hair follicle lineage, can also give rise to Merkel Cells. Interestingly, while SOX9 is critical for HFSC specification, it is dispensable for Merkel cell formation. Conversely, FGFR2 is required for Merkel cell formation but is dispensable for HFSCs. Together, our studies uncover SOX9(+) cells as precursors of Merkel cells and show the requirement for FGFR2-mediated epithelial signalling in Merkel cell specification.Peer reviewe

Early epithelial signaling center governs tooth budding morphogenesis

During organogenesis, cell fate specification and patterning are regulated by signaling centers, specialized clusters of morphogen-expressing cells. In many organs, initiation of development is marked by bud formation, but the cellular mechanisms involved are ill defined. Here, we use the mouse incisor tooth as a model to study budding morphogenesis. We show that a group of nonproliferative epithelial cells emerges in the early tooth primordium and identify these cells as a signaling center. Confocal live imaging of tissue explants revealed that although these cells reorganize dynamically, they do not reenter the cell cycle or contribute to the growing tooth bud. Instead, budding is driven by proliferation of the neighboring cells. We demonstrate that the activity of the ectodysplasin/Edar/nuclear factor kappa B pathway is restricted to the signaling center, and its inactivation leads to fewer quiescent cells and a smaller bud. These data functionally link the signaling center size to organ size and imply that the early signaling center is a prerequisite for budding morphogenesis.Peer reviewe

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.This study shows that mammary gland branching morphogenesis is orchestrated by spatiotemporally coordinated changes in tip cell motility and proliferation to alternate between branch elongation and branch point formation.Peer reviewe