RhoA GEF Mcf2lb regulates rosette integrity during collective cell migration.

Multicellular rosettes are transient epithelial structures that serve as important cellular intermediates in the formation of diverse organs. Using the zebrafish posterior lateral line primordium (pLLP) as a model system, we investigated the role of the RhoA GEF Mcf2lb in rosette morphogenesis. The pLLP is a group of ∼150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA-sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This resulted in an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are properly polarized. In contrast, RhoA activity, as well as signaling components downstream of RhoA, Rock2a and non-muscle Myosin II, were diminished apically. Thus, Mcf2lb-dependent RhoA activation maintains the integrity of epithelial rosettes.

RhoA GEF Mcf2lb regulates rosette integrity during collective cell migration.

During development, multicellular rosettes serve as important cellular intermediates in the formation of diverse organ systems. Multicellular rosettes are transient epithelial structures that are defined by the apical constriction of cells towards the rosette center. Due to the important role these structures play during development, understanding the molecular mechanisms by which rosettes are formed and maintained is of high interest. Utilizing the zebrafish posterior lateral line primordium (pLLP) as a model system, we identify the RhoA GEF Mcf2lb as a regulator of rosette integrity. The pLLP is a group of ~150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Given the known role of RhoA in rosette formation, we asked whether Mcf2lb plays a role in regulating apical constriction of cells within rosettes. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This in turn resulted in a unique posterior Lateral Line phenotype: an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are normally polarized. In contrast, signaling components that mediate apical constriction downstream of RhoA, Rock-2a and non-muscle Myosin II were diminished apically. Altogether our results suggest a model whereby Mcf2lb activates RhoA, which in turn activates downstream signaling machinery to induce and maintain apical constriction in cells incorporated into rosettes.

Using Zebrafish to Study Collective Cell Migration in Development and Disease.

Cellular migration is necessary for proper embryonic development as well as maintenance of adult health. Cells can migrate individually or in groups in a process known as collective cell migration. Collectively migrating cohorts maintain cell-cell contacts, group polarization, and exhibit coordinated behavior. This mode of migration is important during numerous developmental processes including tracheal branching, blood vessel sprouting, neural crest cell migration and others. In the adult, collective cell migration is important for proper wound healing and is often misappropriated during cancer cell invasion. A variety of genetic model systems are used to examine and define the cellular and molecular mechanisms behind collective cell migration including border cell migration and tracheal branching in Drosophila melanogaster, neural crest cell migration in chick and Xenopus embryos, and posterior lateral line primordium (pLLP) migration in zebrafish. The pLLP is a group of about 100 cells that begins migrating around 22 hours post-fertilization along the lateral aspect of the trunk of the developing embryo. During migration, clusters of cells are deposited from the trailing end of the pLLP; these ultimately differentiate into mechanosensory organs of the lateral line system. As zebrafish embryos are transparent during early development and the pLLP migrates close to the surface of the skin, this system can be easily visualized and manipulated in vivo. These advantages together with the amenity to advance genetic methods make the zebrafish pLLP one of the premier model systems for studying collective cell migration. This review will describe the cellular behaviors and signaling mechanisms of the pLLP and compare the pLLP to collective cell migration in other popular model systems. In addition, we will examine how this type of migration is hijacked by collectively invading cancer cells.