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.

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.

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.

Coordination of tissue homeostasis and growth by the Scribble-α-Catenin-Septate junction complex.

Maintaining apicobasal polarity (ABP) is crucial for epithelial integrity and homeostasis during tissue development. Although intracellular mechanisms underlying ABP establishment have been well studied, it remains to be addressed how the ABP coordinates tissue growth and homeostasis. By studying Scribble, a key ABP determinant, we address molecular mechanisms underlying ABP-mediated growth control in the Drosophila wing imaginal disc. Our data reveal that genetic and physical interactions between Scribble, Septate junction complex and α-Catenin appear to be key for sustaining ABP-mediated growth control. Cells with conditional scribble knockdown instigate the loss of α-Catenin, ultimately leading to the formation of neoplasia accompanying with activation of Yorkie. In contrast, cells expressing wild type scribble progressively restore ABP in scribble hypomorphic mutant cells in a non-autonomous manner. Our findings provide unique insights into cellular communication among optimal and sub-optimal cells to regulate epithelial homeostasis and growth.

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.

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.

The Eleventh ENBDC Workshop: Advances in Technology Help to Unveil Mechanisms of Mammary Gland Development and Cancerogenesis.

The eleventh annual workshop of the European Network for Breast Development and Cancer, Methods in mammary gland biology and breast cancer, took place on the 16th to 18th of May 2019 in Weggis, Switzerland. The main topics of the meeting were high resolution genomics and proteomics for the study of mammary gland development and cancer, breast cancer signaling, tumor microenvironment, preclinical models of breast cancer, and tissue morphogenesis. Exciting novel findings in, or highly relevant to, mammary gland biology and breast cancer field were presented, with insights into the methods used to obtain them. Among others, the discussed methods included single-cell RNA sequencing, genetic barcoding, lineage tracing, spatial transcriptomics, optogenetics, genetic mouse models and organoids.

Inductive signals in branching morphogenesis - lessons from mammary and salivary glands.

Branching morphogenesis is a fundamental developmental program that generates large epithelial surfaces in a limited three-dimensional space. It is regulated by inductive tissue interactions whose effects are mediated by soluble signaling molecules, and cell-cell and cell-extracellular matrix interactions. Here, we will review recent studies on inductive signaling interactions governing branching morphogenesis in light of phenotypes of mouse mutants and ex vivo organ culture studies with emphasis on developing mammary and salivary glands. We will highlight advances in understanding how cell fate decisions are intimately linked with branching morphogenesis. We will also discuss novel insights into the molecular control of cellular mechanisms driving the formation of these arborized ductal structures and reflect upon how distinct spatial patterns are generated.

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.

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 ß-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 nM), BPA significantly increased ductal growth, as previously observed in vivo, while 1 µM 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.

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 κB 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.

Foxi3 Deficiency Compromises Hair Follicle Stem Cell Specification and Activation.

The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ (HG). Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here, we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary HG marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary HG activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. Stem Cells 2016;34:1896-1908.

Ectodysplasin/NF-κB Promotes Mammary Cell Fate via Wnt/ß-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-κB 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-κB 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.

Ectodysplasin research--where to next?

Ectodysplasin (Eda) is the most studied tumor necrosis ligand in the field of developmental biology. In all vertebrates studied so far, inactivating germline mutations in Eda lead to the genetic disease called hypohidrotic ectodermal dysplasia (HED). In humans, HED is a life-threatening condition in particular in infants due to absent or severely reduced sweating leading to hyperthermia. HED is also characterized by sparse hair, and oligo- or anodontia. Research of the Eda pathway has not only increased our knowledge on ectodermal appendage development and etiology of developmental disorders, but also on evolution of several vertebrate species including humankind. Studies on mouse and dog models of HED has led to one of the most stunning breakthroughs in applied developmental biology research by showing that a short-term treatment of neonates with a synthetic ligand corrects many of the HED-associated traits. Eighteen years after the identification of EDA as the causative gene in HED, a phase II trial aiming at permanent correction of the disease is now ongoing. This review summarizes the latest discoveries in the Eda field and points to areas that need further investigation such as the possible involvement of Eda in cell migration, stem cell maintenance, or cancer.

Protocol: ex vivo culture of mouse embryonic mammary buds.

The explant culture techniques of embryonic tissues allow continuous monitoring of organ growth and morphogenesis ex vivo. The effect of growth factors and other soluble molecules can be examined by applying them to the culture medium. Relatively few studies have reported application of tissue culture techniques to analysis of embryonic mammary glands. Here we describe a protocol for murine mammary rudiments that permits ex vivo development up to branching stage.

Ectodysplasin/NF-κB signaling in embryonic mammary gland development.

The ectodysplasin (Eda) signaling pathway consists of a TNF-like ligand Eda, its receptor Edar, and an adaptor protein Edaradd and its activation leads to NF-κB mediated transcription. In humans, mutations in the EDA pathway genes cause hypohidrotic ectodermal dysplasia, a disorder characterized by defective formation of hair follicles, teeth, and several exocrine glands including the breast. Embryonic mammary gland development proceeds via placode, bud, bulb and sprout stages before the onset of branching morphogenesis. Studies on mouse models have linked Eda with two aspects of embryonic mammary gland morphogenesis: placode induction and ductal growth and branching. Here we summarize the current knowledge on the role of Eda/NF-κB in mammary gland development.

Fgf20 governs formation of primary and secondary dermal condensations in developing hair follicles.

In hair follicle development, a placode-derived signal is believed to induce formation of the dermal condensation, an essential component of ectodermal organs. However, the identity of this signal is unknown. Furthermore, although induction and patterning of hair follicles are intimately linked, it is not known whether the mesenchymal condensation is necessary for inducing the initial epithelial pattern. Here, we show that fibroblast growth factor 20 (Fgf20) is expressed in hair placodes and is induced by and functions downstream from epithelial ectodysplasin (Eda)/Edar and Wnt/ß-Catenin signaling to initiate formation of the underlying dermal condensation. Fgf20 governs formation of primary and secondary dermal condensations in developing hair follicles and subsequent formation of guard, awl, and auchene hairs. Although primary dermal condensations are absent in Fgf20 mutant mice, a regular array of hair placodes is formed, demonstrating that the epithelial patterning process is independent of known histological and molecular markers of underlying mesenchymal patterns during the initial stages of hair follicle development.

Expression of Foxi3 is regulated by ectodysplasin in skin appendage placodes.

BACKGROUND: Foxi3 is a member of the large forkhead box family of transcriptional regulators, which have a wide range of biological activities including manifold developmental processes. Heterozygous mutation in Foxi3 was identified in several hairless dog breeds characterized by sparse fur coat and missing teeth. A related phenotype called hypohidrotic ectodermal dysplasia (HED) is caused by mutations in the ectodysplasin (Eda) pathway genes. RESULTS: Expression of Foxi3 was strictly confined to the epithelium in developing ectodermal appendages in mouse embryos, but no expression was detected in the epidermis. Foxi3 was expressed in teeth and hair follicles throughout embryogenesis, but in mammary glands only during the earliest stages of development. Foxi3 expression was decreased and increased in Eda loss- and gain-of-function embryos, respectively, and was highly induced by Eda protein in embryonic skin explants. Also activin A treatment up-regulated Foxi3 mRNA levels in vitro. CONCLUSIONS: Eda and activin A were identified as upstream regulators of Foxi3. Foxi3 is a likely transcriptional target of Eda in ectodermal appendage placodes suggesting that HED phenotype may in part be produced by compromised Foxi3 activity. In addition to hair and teeth, Foxi3 may have a role in nail, eye, and mammary, sweat, and salivary gland development.