Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland

Abstract

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

Statistical theory of branching morphogenesis

“…From this behavior, it therefore follows that sublineage‐restricted mammary stem cells must constitute populations that, in the medium term, function as equipotent pools (Scheele et al., ). However, given that monoclonal conversion occurs very slowly throughout development, this finding does not rule out the potential for further sub‐compartmentalization of the basal and luminal stem cell populations (Lilja et al., ; Wuidart et al., ), nor heterogeneity in their short‐term proliferative potential. Finally, the ratio of basal and luminal stem cells was found to be proportional to the fraction of basal and luminal cells in ducts, indicating that their respective clonal outputs are also similar.…”

“…From this behavior, it therefore follows that sublineage-restricted mammary stem cells must constitute populations that, in the medium term, function as equipotent pools. However, given that monoclonal conversion occurs very slowly throughout development, this finding does not rule out the potential for further sub-compartmentalization of the basal and luminal stem cell populations(Lilja et al, 2018;Wuidart et al, 2018), nor heterogeneity in their short-term proliferative potential. Finally, the ratio of basal and luminal stem cells was found to be proportional to the fraction of basal and luminal cells in ducts, indicating that their respective clonal outputs are also similar.However, measurement of the average size of sub-clones in ducts as a function of the position of similarly-labelled cells in proximate active terminal end-buds shows that only clones with cells at the border of the end-bud contribute to the immediate expansion of the adjacent duct, while clones with marked cells only at the tip make little or no contribution on average.…”

“…However, based on these observations alone, further sublineage restriction within these compartments could not be ruled out. Indeed, recent clonal tracing studies targeting embryonic development show that the earliest phase of mammary growth (E12‐E18) is characterized by progressive sublineage restriction, with initially bipotent cells becoming gradually restricted to the basal (Wuidart et al., ) and luminal sublineages (Lilja et al., ), with luminal cells then becoming sublineage‐restricted into ER+ and ER‐ compartments (Rodilla et al., ). Whether there is further sub‐compartmentalization remains an open question, calling for lineage tracing studies based on targeted assays.…”

“…First, detailed analysis of mammary gland epithelium in wholemount 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, 2016; Lloyd-Lewis, Davis, Harris, Hitchcock, & Watson, 2018; Van Keymeulen et al, 2011; Wuidart et al, 2016), 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.Indeed, recent clonal tracing studies targeting embryonic development show that the earliest phase of mammary growth (E12-E18) is characterized by progressive sublineage restriction, with initially bipotent cells becoming gradually restricted to the basal(Wuidart et al, 2018) and luminal sublineages(Lilja et al, 2018), with luminal cells then becoming sublineage-restricted into ER+ and ER-compartments(Rodilla et al, 2015). Whether there is further sub-compartmentalization remains an open question, calling for lineage tracing studies based on targeted assays.…”

Mammary stem cells, where art thou?

“…Despite being a well-accepted concept and a long-time interest, research on embryonic mammary gland stem cells has not gained much tractions until recently (Giraddi et al, 2018;Lilja et al, 2018;Trejo, Luna, Dravis, Spike, & Wahl, 2017;Wuidart et al, 2018). This is in contrast to the research on adult stem cell biology, which has blossomed over the past decade due in a large part to a strong interest in its supposed therapeutic applications and in regenerative medicine.…”