Exploring the principles of embryonic mammary gland branching morphogenesis

Abstract

Branching morphogenesis is a characteristic feature of many essential organs such as the lung, kidney, and most glands, and the net result of two tissue behaviors: branch point initiation and elongation. Each branched organ has a distinct architecture customized to its physiological function, but how patterning occurs in these ramified tubular structures is a fundamental problem of development. The conserved molecular pathways that control branching morphogenesis are well defined, but their contribution to growth versus branch patterning is often challenging to discern. Here we use quantitative 3D morphometrics, time-lapse imaging and manipulation of ex vivo cultured embryonic organs, and genetically modified mice to address these questions in the developing mammary gland. Our results show that the embryonic epithelial trees are highly complex in topology owing to the flexible use of two distinct modes of branch point initiation: lateral branching and tip bifurcation. This non-stereotypy was contrasted by the remarkably constant average branch frequency indicating a time-invariant, yet stochastic propensity to branch. The probability to branch was malleable, and could be increased or decreased by exogenous Fgf10 and Tgf-β1, respectively. Our in vivo and ex vivo time-lapse imaging also suggest involvement of convergent extension in mammary branch elongation, a conclusion supported by the phenotype analysis of mice deficient in the PCP component Vangl2.

Spatially coordinated cell cycle activity and motility govern mammary ductal growth and tip bifurcation

“…To discern cell behaviors during these processes, we took advantage of an ex vivo culture system that preserves the native tissue architecture with its epithelial-mesenchymal interactions (Kratochwil 1969, Voutilainen et al 2012). Branching morphogenesis in this setup is consistent with morphometric analysis of embryonic mammary glands and includes both terminal bifurcation and side-branching events (Lindström et al 2022). For a comprehensive view and mechanistic understanding of cell behaviors contributing to epithelial branching and elongation, we utilized a non-lineage specific approach to indiscriminately label and image the majority of embryonic mammary epithelial cells.…”

“…Changes in cell motility were spatiotemporally coordinated with changes in cell cycle activity, implying that they may be controlled by a localized inductive/repressive cue rather than by passive forces. The conditions that might trigger such a cue however are enigmatic, given the variation in branch point frequency in the mammary gland (Lindström et al 2022). Bifurcations are limited by their distance to the previous branch point, suggesting that a certain minimal tip size or branch length is required (Lindström et al 2022).…”

“…The conditions that might trigger such a cue however are enigmatic, given the variation in branch point frequency in the mammary gland (Lindström et al 2022). Bifurcations are limited by their distance to the previous branch point, suggesting that a certain minimal tip size or branch length is required (Lindström et al 2022). Several molecular pathways have been implicated in regulation of branching frequency in the mammary gland (Gjorevski & Nelson 2011, Myllymäki & Mikkola 2019).…”

“…Mammary gland is a unique arborized organ as branching morphogenesis takes place in three developmental stages – embryonic, pubertal, and reproductive. In mice, branching morphogenesis begins when a sprout rooted to the embryonic surface ectoderm is induced to branch around E16 and yields a rudimentary tree of 10-20 branches by birth (E19-20) (Macias & Hinck 2012, Lindström et al 2022). Lumen formation commences shortly before birth, and soon after, the final, bilayered structure of mammary ducts is established (Hogg et al, 1983).…”

On growth and form of the mammary gland: Mesenchyme instructs growth while epithelium directs branching

“…4c), as expected. Surprisingly, over 60% of mammary branching events were generated by lateral branching, similar to normal embryonic mammary gland branching 7 . We conclude that although salivary gland mesenchyme boosts growth of the mammary epithelium, the mode of branching is an intrinsic property of the mammary epithelium that is not altered by the growth-promoting salivary gland mesenchyme environment.…”