Dkk1 Controls Cell-Cell Interaction through Regulation of Non-nuclear ß-Catenin Pools.

Dickkopf-1 (Dkk1) is a secreted Wnt antagonist with a well-established role in head induction during development. Numerous studies have emerged implicating Dkk1 in various malignancies and neurodegenerative diseases through an unknown mechanism. Using zebrafish gastrulation as a model for collective cell migration, we unveil such a mechanism, identifying a role for Dkk1 in control of cell connectivity and polarity in vivo, independent of its known function. We find that Dkk1 localizes to adhesion complexes at the plasma membrane and regions of concentrated actomyosin, suggesting a direct involvement in regulation of local cell adhesion. Our results show that Dkk1 represses cell polarization and integrity of cell-cell adhesion, independently of its impact on ß-catenin protein degradation. Concurrently, Dkk1 prevents nuclear localization of ß-catenin by restricting its distribution to a discrete submembrane pool. We propose that redistribution of cytosolic ß-catenin by Dkk1 concomitantly drives repression of cell adhesion and inhibits ß-catenin-dependent transcriptional output.

Pineal progenitors originate from a non-neural territory limited by FGF signalling.

The embryonic development of the pineal organ, a neuroendocrine gland on top of the diencephalon, remains enigmatic. Classic fate-mapping studies suggested that pineal progenitors originate from the lateral border of the anterior neural plate. We show here, using gene expression and fate mapping/lineage tracing in zebrafish, that pineal progenitors originate, at least in part, from the non-neural ectoderm. Gene expression in chick indicates that this non-neural origin of pineal progenitors is conserved in amniotes. Genetic repression of placodal, but not neural crest, cell fate results in pineal hypoplasia in zebrafish, while mis-expression of transcription factors known to specify placodal identity during gastrulation promotes the formation of ectopic pineal progenitors. We also demonstrate that fibroblast growth factors (FGFs) position the pineal progenitor domain within the non-neural border by repressing pineal fate and that the Otx transcription factors promote pinealogenesis by inhibiting this FGF activity. The non-neural origin of the pineal organ reveals an underlying similarity in the formation of the pineal and pituitary glands, and suggests that all CNS neuroendocrine organs may require a non-neural contribution to form neurosecretory cells.

The Birth of the Eye Vesicle: When Fate Decision Equals Morphogenesis.

As the embryonic ectoderm is induced to form the neural plate, cells inside this epithelium acquire restricted identities that will dictate their behavior and progressive differentiation. The first behavior adopted by most neural plate cells is called neurulation, a morphogenetic movement shaping the neuroepithelium into a tube. One cell population is not adopting this movement: the eye field. Giving eye identity to a defined population inside the neural plate is therefore a key neural fate decision. While all other neural population undergo neurulation similarly, converging toward the midline, the eye field moves outwards, away from the rest of the forming neural tube, to form vesicles. Thus, while delay in acquisition of most other fates would not have significant morphogenetic consequences, defect in the establishment of the eye field would dramatically impact the formation of the eye. Yet, very little is understood of the molecular and cellular mechanisms driving them. Here, we summarize what is known across vertebrate species and propose a model highlighting what is required to form the essential vesicles that initiate the vertebrate eyes.

Analysis of In Vivo Cell Migration in Mosaic Zebrafish Embryos.

Being optically clear, the zebrafish embryo is a nice model system to analyze cell migration in vivo. This chapter describes a combination of injection and cell transplant procedures that allows creation of mosaic embryos, containing a few cells labeled differently from their neighbors. Rapid 5D confocal imaging of these embryos permits to simultaneously track and quantify the movement of large cell groups, as well as analyze the cellular or subcellular dynamics of transplanted cells during their migration. In addition, expression of a candidate gene can be modified in transplanted cells. Comparing behavior of these cells to control or neighboring cells allows determination of the role of the candidate gene in cell migration. We describe the procedure, focusing on one specific cell population during gastrulation, but it can easily be adapted to other cell populations and other migration events during early embryogenesis.

Endodermal germ-layer formation through active actin-driven migration triggered by N-cadherin.

Germ-layer formation during gastrulation is both a fundamental step of development and a paradigm for tissue formation and remodeling. However, the cellular and molecular basis of germ-layer segregation is poorly understood, mostly because of the lack of direct in vivo observations. We used mosaic zebrafish embryos to investigate the formation of the endoderm. High-resolution live imaging and functional analyses revealed that endodermal cells reach their characteristic innermost position through an active, oriented, and actin-based migration dependent on Rac1, which contrasts with the previously proposed differential adhesion cell sorting. Rather than being attracted to their destination, the yolk syncytial layer, cells appear to migrate away from their neighbors. This migration depends on N-cadherin that, when imposed in ectodermal cells, is sufficient to trigger their internalization without affecting their fate. Overall, these results lead to a model of germ-layer formation in which, upon N-cadherin expression, endodermal cells actively migrate away from their epiblastic neighbors to reach their internal position, revealing cell-contact avoidance as an unexplored mechanism driving germ-layer formation.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Inhibitory signalling to the Arp2/3 complex steers cell migration.

Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.