Mammary epithelial tubes elongate through MAPK-dependent coordination of cell migration

Abstract

Mammary branching morphogenesis is regulated by receptor tyrosine kinases (RTKs). We sought to determine how these RTK signals alter proliferation and migration to accomplish tube elongation in mouse. Both behaviors occur but it has been difficult to determine their relative contribution to elongation in vivo, as mammary adipocytes scatter light and limit the depth of optical imaging. Accordingly, we utilized 3D culture to study elongation in an experimentally accessible setting. We first used antibodies to localize RTK signals and discovered that phosphorylated ERK1/2 ( pERK) was spatially enriched in cells near the front of elongating ducts, whereas phosphorylated AKT was ubiquitous. We next observed a gradient of cell migration speeds from rear to front of elongating ducts, with the front characterized by both high pERK and the fastest cells. Furthermore, cells within elongating ducts oriented both their protrusions and their migration in the direction of tube elongation. By contrast, cells within the organoid body were isotropically protrusive. We next tested the requirement for proliferation and migration. Early inhibition of proliferation blocked the creation of migratory cells, whereas late inhibition of proliferation did not block continued duct elongation. By contrast, pharmacological inhibition of either MEK or Rac1 signaling acutely blocked both cell migration and duct elongation. Finally, conditional induction of MEK activity was sufficient to induce collective cell migration and ductal elongation. Our data suggest a model for ductal elongation in which RTK-dependent proliferation creates motile cells with high pERK, the collective migration of which acutely requires both MEK and Rac1 signaling.

Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma

“…The ERK1/2-dependent re-awakening of collective motion and proliferation of terminal differentiated mammary glands has been associated with the initiation of a more complex program of branched morphogenesis that begins with the formation of multicellular buds 63, 64 . The latter process is thought to require in addition to specific growth factors, also the interaction of epithelial acini with the microenvironment and ECM components 65-67 .…”

Precise targeting of myotubes to muscle attachment sites patterns the musculoskeletal system

“…For example, FGF2 promotes mammary epithelial tube elongation in organoid cultures through Rac1-and MAPK-dependent mechanisms. In this system, Rac1 inhibition caused epithelial branches to collapse after FGF2-induced elongation, but Rac1 activation alone was insufficient to induce branch elongation (Huebner et al, 2016). Unlike myotubes, mammary epithelial tubes are comprised of dozens of cells and pERK was enriched in cells at the branch tips, which suggests cells in the mammary epithelia have a differential response to exogenous FGF2.…”

“…Htl suppresses Rho/Rac activity during myotube guidance FGF receptors activate multiple intracellular pathways. The vertebrate MAPK, AKT, and Rho/Rac signaling pathways are activated in response to FGF signals and all three pathways influence cell migration, cell elongation, and organ morphogenesis (Benazeraf et al, 2010;Fera et al, 2004;Harding and Nechiporuk, 2012;Huebner et al, 2016;Jeong et al, 2016;Sato et al, 2011). In Drosophila, the intracellular pathways downstream of FGF receptors have been remarkably understudied compared to most other signal transducing receptors (Muha and Muller, 2013).…”

Cell-based Simulations of Biased Epithelial Lung Growth

“…Alternatively, Fgf signaling could mediate elongating outgrowth by generating a pulling force at the tip. In fact, Fgf2 signaling has been shown to enhance motility and persistence of epithelial cells at the tip of epithelial tubes in the mammary gland [16]. A pulling force would act on the cells behind the tip via the cell-cell adhesion bonds, and could thus generate a bias in cell shape along the entire tube.…”

“…As an alternative to cell migration, biased outgrowth in the mammary gland has been linked to a mechanical constraint posed by the myoepithelium [18]. Such a mechanical constraint in circumferential direction would result in growth-dependent uniform expansion in longitudinal direction, and would thus explain the measured linear gradient in cell speeds that is observed along the elongating mammary gland tube [16]. The lung mesenchyme could, in principle, play a similar role in the embryonic lung though smooth muscles mature only at later stages and are not as tightly associated as in a myoepithelium [19].…”