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.

RETRACTED: Btbd7 is essential for region-specific epithelial cell dynamics and branching morphogenesis in vivo

“…Increased Ret signaling at the tips of kidney tubules promotes cell migration (Riccio et al, 2016), and FGF receptor activation at the tips of mammary terminal end buds signals to stimulate tip cell migration (Huebner et al, 2016). Based on the results presented here, we now propose that Btbd7 is a crucial regulator of differential cell migration in the outer bud cells of developing salivary glands.…”

“…In contrast, mammalian epithelial branching organs undergo collective migration in the absence of these invasive leading-edge membrane protrusions (Ewald et al, 2008;Schnatwinkel and Niswander, 2013). Live imaging of the salivary gland (Hsu et al, 2013;Larsen et al, 2006), lung (Schnatwinkel and Niswander, 2013), kidney (Riccio et al, 2016), mammary gland (Huebner et al, 2016) and pancreas (Shih et al, 2016) have all revealed that the peripheral bud tip cells in these organs are more dynamic compared with inner bud and ductal cells.…”

“…Live imaging of organ cultures has revealed that individual epithelial cells of major branching organs are transiently motile during early development (Huebner et al, 2016;Riccio et al, 2016;Schnatwinkel and Niswander, 2013). Cells at the peripheral tips of branching end buds are the most dynamic, with faster migration rates than inner bud cells in the developing salivary gland (Hsu et al, 2013;Larsen et al, 2006); in the pancreas, the outer bud cells migrate with more persistence than their inner bud counterparts (Shih et al, 2016).…”

Andrew Ewald: Understanding cellular cooperation

“…In the first, we seek to understand how mammary epithelial tubes elongate, bifurcate, and polarize during normal development. We have defined the basic cell movements during each process and identified key regulators of each step (1). We are now collaborating with engineers to integrate our experimental understanding into predictive models.…”

“…The second area focuses on using 3D culture and animal models to understand how the tumor microenvironment cooperates with genetic changes to affect cell behavior within intact tissues. We have demonstrated that the extracellular matrix is a potent regulator of cancer invasion (2), that activation of individual signaling nodes can drive complex tissue level behaviors (1), and that mammary cells can be induced to disseminate without losing their epithelial character (3). We are now extending these efforts by working with systems biologists to understand how information flows from DNA to protein to signaling as cells reprogram from normal to malignant phenotypes, and with oncologists to identify targets for anti-metastatic therapy.…”