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.

Lamellipodia-like protrusions and focal adhesions contribute to collective cell migration in zebrafish.

Collective cell migration is a process where cohorts of cells exhibit coordinated migratory behavior. During individual and collective cellular migration, cells must extend protrusions to interact with the extracellular environment, sense chemotactic cues, and act as points of attachment. The mechanisms and regulators of protrusive behavior have been widely studied in individually migrating cells; however, how this behavior is regulated throughout collectives is not well understood. To address this, we used the zebrafish posterior lateral line primordium (pLLP) as a model. The pLLP is a cluster of ~150 â€‹cells that migrates along the zebrafish trunk, depositing groups of cells that will become sensory organs. To define protrusive behavior, we performed mosaic analysis to sparsely label pLLP cells with a transgene marking filamentous actin. This approach revealed an abundance of brush-like protrusions throughout the pLLP that orient in the direction of migration. Formation of these protrusions depends on the Arp2/3 complex, a regulator of dendritic actin. This argues that these brush-like protrusions are an in vivo example of lamellipodia. Mosaic analysis demonstrated that these lamellipodia-like protrusions are located in a close proximity to the overlying skin. Immunostaining revealed an abundance of focal adhesion complexes surrounding the pLLP. Disruption of these complexes specifically in pLLP cells led to impaired pLLP migration. Finally, we show that Erk signaling, a known regulator of focal adhesions, is required for proper formation of lamellipodia-like protrusions and pLLP migration. Altogether, our results suggest a model where the coordinated dynamics of lamellipodia-like protrusions, making contact with either the overlying skin or the extracellular matrix through focal adhesions, promotes migration of pLLP cells.

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.

Nothing to Be Sniffed At: Anosmin1 Tunes Fgf Diffusivity.

The extracellular matrix plays both positive and negative roles in growth factor diffusion, a process critical for organ formation. In this issue of Developmental Cell, Wang et al. (2018) identify the extracellular matrix protein Anosmin1 as a key regulator of Fgf diffusion during sensory organ formation in zebrafish.