Neutral lineage tracing of proliferative embryonic and adult mammary stem/progenitor cells

Abstract

Mammary gland development occurs over multiple phases, beginning in the mammalian embryo and continuing throughout reproductive life. The remarkable morphogenetic capacity of the mammary gland at each stage of development is attributed to the activities of distinct populations of mammary stem cells (MaSCs) and progenitor cells. However, the relationship between embryonic and adult MaSCs, and their fate during different waves of mammary gland morphogenesis, remains unclear. By employing a neutral, low-density genetic labelling strategy, we characterised the contribution of proliferative stem/progenitor cells to embryonic, pubertal and reproductive mammary gland development. Our findings further support a model of lineage restriction of MaSCs in the postnatal mammary gland, and highlight extensive redundancy and heterogeneity within the adult stem/progenitor cell pool. Furthermore, our data suggest extensive multiplicity in their foetal precursors that give rise to the primordial mammary epithelium before birth. In addition, using a single-cell labelling approach, we revealed the extraordinary capacity of a single embryonic MaSC to contribute to postnatal ductal development. Together, these findings provide tantalising new insights into the disparate and stage-specific contribution of distinct stem/progenitor cells to mammary gland development.

Statistical theory of branching morphogenesis

“…First, detailed analysis of mammary gland epithelium in whole‐mount stained for the basal or luminal cell markers, Keratin 14 and Keratin 8, respectively, allowed clones to be resolved by size and cell composition. From clonal maps showing both the representation of confetti labelled cells and their cell identity, it was apparent that, in line with similar clonal tracing studies (Davis et al., ; Lloyd‐Lewis, Davis, Harris, Hitchcock, & Watson, ; Van Keymeulen et al., ; Wuidart et al., ), by this stage of development, mammary stem cells have already become “compartmentalized”, lineage‐restricted to the basal or luminal compartments. However, based on these observations alone, further sublineage restriction within these compartments could not be ruled out.…”

Mammary mechanobiology: mechanically-activated ion channels in lactation and involution

“…To induce recombination in WAPCre mice, animals were taken through one full pregnancy-lactation-involution cycle. Pups (average 8; range [6][7][8][9][10] were weaned on postnatal day 21. Mothers were mated one week later in their second (experimental) mating.…”

“…The ductal epithelium contains both luminal and basal cells, which are produced and maintained postnatally by lineage-restricted precursors (2)(3)(4)(5)(6)(7). During gestation, a coordinated program of epithelial proliferation, side-branching, differentiation and tissue remodeling takes place (1,8), resulting in the generation of thousands of alveolar structures that adorn the central ductal epithelium in lobular clusters (9).…”

Mammary stem cells, where art thou?

“…Remarkably, using this method conflicting and sometime opposite data have been generated. Specifically, various research groups thus far have shown that there are no adult stem cells in the mammary gland because both basal and luminal cells are unipotent (Blanpain & Fuchs, 2014;Davis et al, 2016;Joshi & Khokha, 2012;Lloyd-Lewis, Davis, Harris, Hitchcock, & Watson, 2018;Morozov et al, 2016;Scheele et al, 2017;Smith & Medina, 2018;Sreekumar et al, 2017;van Amerongen, Bowman, & Nusse, 2012;Van Keymeulen et al, 2011;Van Keymeulen et al, 2017;Wang, Christin, Oktay, & Guo, 2017;Wuidart et al, 2016;Wuidart et al, 2018). However, other groups have shown that bipotent MaSCs exist in the adult mammary gland (Fu et al, 2017;Fu et al, 2018;Rios et al, 2014;Song et al, 2019;Wang et al, 2015).…”

“…Indeed, the Blainpain group has recently found that promoter infidelity is the cause where both luminal and basal lineages were labeled unexpectedly early during the "pulse phase" in various lineage-tracing studies, leading to the erroneous conclusion that bipotent MaSCs exist in the adult gland (Wuidart et al, 2016). Furthermore, tracing activator, which may inhibit mammary gland physiology such as Tamoxifen even at a low dose; clonal tracking strategies, whose resolution may be limited due to the small number of fluorescent protein trackers used, and clonal density, which often does not label 100% of the cells in the intended cell population and thus miss the rare but important stem cell population, could all explain these conflicting data (Davis et al, 2016;Lloyd-Lewis et al, 2018;Wuidart et al, 2016). Interestingly, these potential caveats do not appear to be at play in a recent report showing the presence of bipotent MaSCs in the luminal epithelium (Song et al, 2019).…”