Developmental stratification of the mammary epithelium occurs through symmetry-breaking vertical divisions of apically positioned luminal cells

Abstract

Mammary ducts are elongated during development by stratified epithelial structures, known as terminal end buds (TEBs). TEBs exhibit reduced apicobasal polarity and extensive proliferation. A major unanswered question concerns the mechanism by which the simple ductal epithelium stratifies during TEB formation. We sought to elucidate this mechanism using real-time imaging of growth factorinduced stratification in 3D cultures of mouse primary epithelial organoids. We hypothesized that stratification could result from vertical divisions in either the apically positioned luminal epithelial cells or the basally positioned myoepithelial cells. Stratification initiated exclusively from vertical apical cell divisions, both in 3D culture and in vivo. During vertical apical divisions, only the mother cell retained tight junctions and segregated apical membranes. Vertical daughter cells initiated an unpolarized cell population located between the luminal and myoepithelial cells, similar to the unpolarized body cells in the TEB. As stratification and loss of apicobasal polarity are early hallmarks of cancer, we next determined the cellular mechanism of oncogenic stratification. Expression of activated ERBB2 induced neoplastic stratification through analogous vertical divisions of apically positioned luminal epithelial cells. However, ERBB2-induced stratification was accompanied by tissue overgrowth and acute loss of both tight junctions and apical polarity. Expression of phosphomimetic MEK (MEK1DD), a major ERBB2 effector, also induced stratification through vertical apical cell divisions. However, MEK1DD-expressing organoids exhibited normal levels of growth and retained apicobasal polarity. We conclude that both normal and neoplastic stratification are accomplished through receptor tyrosine kinase signaling dependent vertical cell divisions within the luminal epithelial cell layer. KEY WORDS: Apicobasal polarity, Breast cancer, Epithelial development, Mammary gland, Mouse INTRODUCTIONSimple epithelial cells are characterized by extensive intercellular junctions and strong polarity along the apicobasal axis (Bryant and Mostov, 2008;Zegers et al., 2003). This polarity is manifested both in specialized morphological features, such as apically localized microvilli, and by segregated plasma membrane domains with distinct apical and basolateral polarity proteins (Nelson, 2003;Yeaman et al., RESEARCH ARTICLE 1999). Yet these highly polarized cells emerge during development from less polarized, multilayered embryonic tissues. Early embryonic morphogenesis in the mammary and salivary glands is accomplished by a tissue with lower levels of polarity and adhesion than the mature epithelium in the adult organ (Andrew and Ewald, 2010;Hsu and Yamada, 2010). A fundamental and unresolved question in biology is how epithelial tissues transition between these different states of organization and polarity (Nelson, 2009). These transitions are also of biomedical interest, as epithelial tumors lose apicobasal polarity and simple organi...

Mammary gland development: cell fate specification, stem cells and the microenvironment

“…Similar properties, mediated by the integrin α6β4 complex, are exhibited by mammary luminal cells when cultured in laminin-rich, three-dimensional (3D) ECM gels (Weaver et al, 2002). In vivo, the two layers of the duct express different cell-cell contact mediators (Chanson et al, 2011;Huebner et al, 2014;Mroue et al, 2014) and cell-ECM connections (Brizzi et al, 2012); as a result, the layered structure of the duct selforganizes when luminal and myoepithelial cells are mixed in 3D cultures (Chanson et al, 2011;Gudjonsson et al, 2002;Huebner et al, 2014;Runswick et al, 2001). Furthermore, these connections are necessary for normal cell function: mammary cells in the lactating gland require E-cadherin to survive (Boussadia et al, 2002).…”

3D Culture Assays of Murine Mammary Branching Morphogenesis and Epithelial Invasion

“…Building on our previously published methods [17, 22, 23, 31, 33–35], this protocol seeks to provide a comprehensive guide to utilizing the mammary organoid assay (Fig. 1a).…”

Cellular foundations of mammary tubulogenesis

“…These simple tubes are then essentially quiescent until the rise in steroid hormones that triggers the onset of puberty 12,13 . Before puberty the mammary ducts are all simple (Figure 2A,A’) and they then stratify to produce stratified terminal end buds 14,15,16 (TEBs) (Figure 2B,B’). Terminal end buds have multiple layers of epithelial cells, referred to as body cells, surrounded by a basally positioned cap cell layer 15,16 .…”