Collective Cell Behavior and Morphogenesis in Development

Minisymposium 29: Collective Cell Behavior and Morphogenesis in

Development

181

Regulation of collective cell movements and morphogenetic diversity in vivo.

D. Montell1, D. Cai1, L. He2, X. Wang1, J. Sawyer1, W. Yoon1, A. Cho1, J-H. Kim1, J. Jensen1;

1_{Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, MD,} 2_Johns

Hopkins School of Medicine, Baltimore, MD

Morphogenetic cell movements are very diverse and many cells travel in groups. We study the similarities and differences between the mechanisms of collective cell migrations and those of

single cells. We would also like to understand how cells self-organize into tissues. We use ovarian development in the fruit fly as our major experimental model. Border cell migration serves as a genetically tractable, in vivo model for collective cell migration. We have identified signals that regulate when, where and which cells migrate and invade. Recent studies suggest that E-cadherin plays diverse and central roles both in organizing the cluster as well as in guidance and movement of the cells, in contrast to the general notion that E-cadherin inhibits cell motility. We have also discovered a novel role for Src in cell motility in vivo. On the other hand, using a photo-activatable form of the small GTPase Rac, we have shown that local activation of Rac in one cell can guide the whole cluster in a new direction. This is very similar to the function of Rac in single cells. In addition to promoting protrusion locally, focal Rac activation inhibits protrusion of other cells in the cluster. We are exploring the mechanism by which activation or inactivation of Rac in one cell affects the behavior of other cells in the cluster. Taken together our results support the hypothesis that morphogenetic diversity arises from combinatorial use of modular mechanical properties including cell-cell and cell-matrix adhesion, myosin-mediated contractility, and Rac-mediated protrusion.

182

Actin turnover balances forces between cells during epithelial invagination.

M. Tworoger1, A. Martin1; 1Biology, Massachusetts Institute of Technology, Cambridge, MA Embryonic development requires that coordinated cell shape changes collectively deform tissues to generate organs with diverse forms and functions. Cell shape changes result from forces generated by actin networks that are coupled to adhesive complexes. During Drosophila gastrulation, pulsed contraction of an apical actin and myosin II network coupled to adherens junctions drives apical constriction of mesoderm cells, which is important for epithelial invagination. While the role of actin turnover is well established for individual cell migration, the importance of actin turnover for the coordinated movement of an epithelial sheet is not known. To examine the importance of actin turnover, we titrated the rate of actin polymerization by injecting different concentrations of cytochalasin D, which blocks barbed end growth, and imaged cell and cytoskeletal dynamics in live embryos. We observed a transition from a general disruption of contractility in all cells with high doses of cytochalasin D to a mesoderm specific disruption in cell-cell connections at low doses. At low cytochalasin D neighboring actomyosin networks continually lost and reformed connections, resulting in an unbalanced ‘tug-of-war’ between cells of the mesoderm. A similar phenotype was observed in mutants for the formin, dia, suggesting that formin mediated actin polymerization is required to maintain cell-cell connections. Live imaging of F-actin in wild-type embryos demonstrates that pulsed contractions generate transient holes in the actin meshwork across the apical surface. We propose that rapid actin turnover is required to fill holes generated by network contraction in order to balance forces across adhesive contacts.

183

Angular morphomechanics in the establishment of multicellular architecture. K. Tanner1, M. Bissell1; 1Life Sciences, LBNL, Berkeley, CA

We report a novel type of human cell motility in which cells undergo coherent angular motion to assemble into polarized multicellular spherical structures (acini) when placed in a 3D basement membrane surrogate gel. We visualize the complete evolution from the single cell to an acinus. We address a fundamental question: what are the physical laws that govern the geometry of acinar structures? We link the functional relevance of this distinct coherent angular motion (CAMo) to the ability to attain the resultant architecture, and determine the relevance of adhesion and tissue polarity to the outcome. CAMo is conserved from primary human cells to

established breast cell lines where the final realized geometry is spherical. Cancer cells do not display CAMo motility but random motility. Upon ‘phenotypic reversion’ of malignant cells, both CAMo motility and correct architecture are restored.

184

Self-organizing and stochastic behaviors during the regeneration of a large population of hair stem cells.

C. Chuong1, M. Plikus2, C. Chen3, D. de la Cruz2, R. Widelitz2; 1Pathology, University of Southern California, Los Angeles, CA, 2University of Southern California, Los Angeles, CA,

3

Yang Ming University, China

The hair follicle undergoes cyclic degeneration and regeneration cycle throughout life. The length of growing (anagen) and resting phase (telogen) can determine the hair length. Long resting phase mean shorter or no hair, i.e., alopecia. We have developed ways to visualize hair stem cell activation over entire skin in living mice. With this, we found cyclic BMP signaling from subcutaneous adipose layer regulates stem cell activation during hair regeneration (Plikus et al. 2008, Nature. 451:340-344. Cyclic dermal BMP signaling regulates stem cell activation during hair regeneration). More molecular analyses showed wnt serves as activators and hair growth patterns are governed by simple rules based on a pair of activator/inhibitor signaling. Regeneration in a population of hair follicles spreads like chain reaction, forming diverse wave patterns (Plikus et al. 2011, Science 332:586-589. Self organizing and stochastic behaviors during the regeneration of hair stem cells). Mathematical modeling reveals unexpected self- organizing and stochastic nature of this novel stem cell behavior, which emerge only at higher organ population level, allowing hair regeneration to become a very adaptable trait. These variations are seen among different animal species with different needs for hairs: robust spreading in rabbits, gradual wave spreading in mice, and random growth with loss of coupling among follicles in human skin. The hair wave can also vary under different physiological conditions of a same individual, such as in puberty, pregnancy and aging. We show that change in macro-environment is one major factor that contributes to alopecia in aging mice. We have developed ways to modify macro-environment for hair follicles, and hence can stimulate many more resting phase hair follicles to re-enter growing phase.

185

Par-1 Controls Non-Muscle Myosin II Activity and Dynamics to Regulate Collective Cell Migration.

P. Majumder1, G. Aranjuez1,2, J. McDonald1,2; 1Molecular Genetics, Cleveland Clinic Lerner Research Institute, Cleveland, OH, 2Genetics, Case Western Reserve University School of Medicine, Cleveland, OH

Many cells migrate in collective groups during tissue morphogenesis, wound healing and tumor invasion and metastasis. In single migrating cells, localized actomyosin contraction couples with actin polymerization and cell-matrix adhesion to regulate cell protrusions and retract trailing cell edges. In contrast, we have only a limited understanding of mechanisms that coordinate actomyosin dynamics in collective cell migration. We study the migration of Drosophila border cells, which move as a cohesive group of 6-10 epithelial-derived cells during late oogenesis. We previously observed that mutants of the cell polarity protein Par-1 (MARK), a serine-threonine kinase, result in defective border cell delamination and migration. We now show that these defects are caused by perturbations in cytoskeletal dynamics due to disruption of a previously unknown signaling pathway between Par-1 and non-muscle myosin-II (Myo-II). Using live time- lapse imaging, we show that Myo-II is required for two critical features of border cell migration: initial detachment of the border cells from the surrounding epithelium, and extension of cell

protrusions of normal length and direction. We identified a robust genetic interaction between the myosin regulatory light chain (MRLC) homolog spaghetti squash (sqh) and par-1 and, remarkably, found that active Sqh/MRLC strongly suppresses par-1-dependent mutant phenotypes. We show that Par-1 regulates dynamic subcellular localization of Sqh/MRLC in live border cells, and, furthermore, that Par-1 regulates the activity of Myo-II. We have discovered that Par-1 phosphorylates and inactivates myosin phosphatase, thus promoting phosphorylation of Sqh/MRLC and increasing Myo-II activity. Finally, while myosin phosphatase is distributed uniformly in the border cell cluster, we find that Par-1 localizes to the cluster rear along with active Myo-II; in the absence of Par-1, spatially distinct active Myo-II is lost. Our study reveals that Par-1 kinase is a principal regulator of polarized Myo-II activity within the border cell cluster through localized inhibition of myosin phosphatase. Polarity proteins such as Par-1, which intrinsically localize, can thus directly modulate the spatiotemporal actomyosin dynamics required for proper collective cell migration.

186

Spatiotemporal regulation of somitogenesis by the oscillator networks of the segmentation clock.

R. Kageyama1; 1Institute for Virus Research, Kyoto Univ, Kyoto, Japan

A bilateral pair of somites forms periodically by segmentation of the anterior ends of the presomitic mesoderm (PSM). This periodic event in time and space is regulated by a biological clock called the segmentation clock, which involves cyclic gene expression. In mice, Hes7 expression oscillates by negative feedback, and mathematical models have been used to generate and test hypotheses to aide elucidation of the role of negative feedback in regulating oscillatory expression. Hes7 induces coupled oscillations of Notch and Fgf signaling in the posterior PSM, while Notch and Fgf signaling cooperatively regulate Hes7 oscillation, indicating that Hes7 and Notch and Fgf signaling form the oscillator networks. Oscillations in Notch signaling generate traveling waves, thereby periodically segregating a group of synchronized cells. Notch signaling activates, but Fgf signaling represses, expression of the master regulator for somitogenesis Mesp2, and coupled oscillations in Notch and Fgf signaling dissociate in the anterior PSM, which allows Notch signaling-induced synchronized cells to express Mesp2 after these cells are freed from Fgf signaling. These results indicate that Notch signaling periodically defines the prospective somite region (spatial periodicity), while Fgf signaling regulates the pace of segmentation (temporal periodicity).