Background and Summary

Epithelial cells in a tissue live a crowded life connected to, and interacting with, other cell types, the extracellular matrix, and diverse signaling molecules. A fundamental question in biology is: how do the constituent cells of an epithelium collaborate to build and remodel its structure? This seemingly simple question also has great relevance to human disease. Cancer is a disease of disregulated proliferation, but also of disregulated tissue growth and invasion. Tumors could grow through novel cellular mechanisms, or they could grow through normal cellular mechanisms occurring in abnormal contexts or to abnormal extents. Until we understand the cellular mechanisms of normal epithelial morphogenesis, it is not possible to distinguish these possibilities.

Experimental Approach: We combine advanced time-lapse microscopy techniques and 3D organotypic cultures to study the cell behavioral basis of normal and neoplastic epithelial growth and invasion. Organotypic culture allows us to contrast different epithelia under identical conditions or to contrast similar epithelia under different microenvironmental conditions. We use a common set of imaging, genetic, and molecular interference tools to study epithelial morphogenesis, whether from normal or cancerous epithelium.

Summary of Recent Work: We seek to answer a simple question: how does an epithelium grow and invade? To answer this question, we observed the cell behaviors that drive mammary branching morphogenesis. We found that ductal elongation was accomplished by a multilayered epithelium, within which cells rearranged vigorously. Surprisingly, cells at the elongation front lacked forward oriented actin protrusions. We have shown that during morphogenesis, mammary epithelium transitions from a bilayered to a multilayered organization, with dramatic, reversible changes in epithelial polarity and cell motility. We are collaborating with Manfred Auer's electron microscopy group at the Lawrence Berkeley Lab to study the intercellular junctional basis of this transition. We have now dissected the process of branching morphogenesis into discrete, observable subprocesses and have identified molecular regulators of each subprocess. (For further information see Ewald et al, Developmental Cell, April 2008).

Project 1. Quantitative analysis of the cell behavioral basis of epithelial morphogenesis

The foundation for a cell biological understanding of epithelial morphogenesis is to resolve the tissue level process of ductal initiation, elongation and bifurcation into discrete changes in the properties and behaviors of individual cells. We are currently using 4D confocal microscopy to dissect the relative contributions of cell movement, cell proliferation, cell shape change and extracellular matrix dynamics to mammary branching morphogenesis.

Project 2. Molecular and cellular regulation of epithelial morphogenesis

Building on a our understanding of the cell behavioral basis of epithelial morphogenesis, we are taking a combined candidate and systematic approach to identify molecules regulators of these behaviors. We are currently using microarrays to identify potential regulatory molecules and we will use pharmacologic inhibitors, function blocking antibodies, and virally mediated gene inactivation to validate targets. We also want to understand the role of microenvironmental factors such as extracellular matrix (ECM) and stromal cell populations. Preliminary data suggest that the cellular mechanisms of invasion depend on the protein composition of the ECM. We predict that the genetic regulation of epithelial morphogenesis will intimately depend on the ECM and stromal cell context in the epithelial microenvironment.

Project 3. Contrasting epithelial morphogenesis in mammary development and breast cancer

The varied morphologic appearance of invasive tumors could reflect differences in the fundamental mechanisms of cellular invasion. Alternately, similar underlying invasion mechanisms might generate different outcomes, and different morphologic appearances, in response to a changing tumor microenvironment. It is difficult to distinguish these possibilities in fixed sections, as the tumor and stroma are both changing dramatically as a function of stage of progression. We are addressing this question by comparing the cellular mechanisms of growth and invasion of normal and neoplastic epithelia, cultured in identical solution, matrix, and stromal cellular conditions. We have developed protocols to culture epithelium from normal ducts, hyperplasias, adenomas, and advanced tumors, either alone or in co-culture with corresponding stromal cells. We are committed to identifying molecular strategies for limiting the growth and spread of epithelial tumors, with a specific focus on identifying extracellular inhibitors of breast tumor invasion and metastasis.

Publications:

Ewald, AJ, Brenot, A, Duong, M, Chan, BC, Werb, Z, "Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis", Developmental Cell, 2008, 14: 570-81.

Egeblad*, M, Ewald*, AJ, Asketraud, HA, Truitt, M, Welm, B, Bainbridge, E, Peeters, G, Krummel, M, Werb, Z, "Imaging stromal cell in intact tumor microenvironments", Disease Models and Mechanisms. 2008, 1: 155-67. * = Co-First Authors.

Lu, PF, Ewald, AJ, Werb, Z, Martin, G, "Genetic mosaic analysis reveals FGF receptor 2 is required in terminal end buds during mammary gland branching morphogenesis", Developmental Biology, 2008; 321:77-87.

Kouros-Mehr, H, Bechis, SK, Slorach, EM, Littlepage, LE, Egeblad, M, Ewald, AJ, Pai, SY, Ho, IC, Werb, Z, "Gata-3 links tumor differentiation and dissemination in a luminal breast cancer model", Cancer Cell, 2008; 13: 141-52.

Fata, J, Mora, H, Ewald, AJ, Zhang, H, Yao, E, Werb, Z, Bissell, M, "The MAPK ERK-1,2 pathway integrates distinct and antagonistic signals from TGFa and FGF7 in morphogenesis of mouse mammary epithelium", Developmental Biology, 2007; 306:193-207.

Page-McCaw*, A, Ewald*, AJ, Werb, Z, "Matrixmetalloproteinases and the regulation of tissue remodeling", Nature Reviews Molecular Cell Biology, 2007, Mar; 8(3): 221-33. * = Co-First Authors

Song S, Ewald, AJ, Stallcup B. Werb, Z. and G. Bergers, "PDGFRb+ perivascular progenitor cells in tumors regulate pericyte differentiation and vascular survival", Nature Cell Biology, 2005; 7 :870-9.

Chen, H, Detmer, SA, Ewald AJ, Griffin, EE, Fraser SE, and Chan, DC, "Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development," Journal of Cell Biology, 2003; 160: 189-200.

Ewald, AJ, Peyrot, S, Tyszka, JM, Fraser, SE, Wallingford, J, "Regional requirements for Dishevelled signaling during Xenopus gastrulation: Separable effects on blastopore closure, mesendoderm internalization, and archenteron formation," Development, 2004; 131: 6195-6209.

Wallingford, JB, Ewald, AJ, Harland, RM and Fraser, SE, "Calcium signaling during convergent extension in Xenopus," Current Biology, 2001; 11:652-661.