SDF-1 alpha regulates mesendodermal cell migration during frog gastrulation

During frog gastrulation, mesendodermal cells become apposed to the blastocoel roof (BCR) by endoderm rotation, and migrate towards the animal pole. The leading edge of the mesendodermal cells (LEM) contributes to the directional migration of involuting marginal zone (IMZ) cells, but the molecular mechanism of this process is not well understood. Here we show that CXCR4/SDF-1 signaling mediates the directional movement of the LEM in Xenopus embryos. Expression of xCXCR4 was detected in the IMZ, and was complemented by xSDF-1alpha expression in the inner surface of the BCR. Over-expression of xCXCR4 and xSDF-1alpha caused gastrulation defects. An xCXCR4 N-terminus deletion construct and xSDF-1alpha-MO also inhibited gastrulation. Furthermore, explants of LEM migrate towards the dorsal BCR in the presence of xSDF-1alpha, and altered xCXCR4 expression in the LEM inhibited LEM migration. These results suggest that CXCR4/SDF-1 signaling is necessary for the migrations of massive numbers of cells during gastrulation.     

Cell movements during epiboly and gastrulation in zebrafish

Beginning during the late blastula stage in zebrafish, cells located beneath a surface epithelial layer of the blastoderm undergo rearrangements that accompany major changes in shape of the embryo. We describe three distinctive kinds of cell rearrangements. (1) Radial cell intercalations during epiboly mix cells located deeply in the blastoderm among more superficial ones. These rearrangements thoroughly stir the positions of deep cells, as the blastoderm thins and spreads across the yolk cell. (2) Involution at or near the blastoderm margin occurs during gastrulation. This movement folds the blastoderm into two cellular layers, the epiblast and hypoblast, within a ring (the germ ring) around its entire circumference. Involuting cells move anteriorwards in the hypoblast relative to cells that remain in the epiblast; the movement shears the positions of cells that were neighbors before gastrulation. Involuting cells eventually form endoderm and mesoderm, in an anterior-posterior sequence according to the time of involution. The epiblast is equivalent to embryonic ectoderm. (3) Mediolateral cell intercalations in both the epiblast and hypoblast mediate convergence and extension movements towards the dorsal side of the gastrula. By this rearrangement, cells that were initially neighboring one another become dispersed along the anterior-posterior axis of the embryo. Epiboly, involution and convergent extension in zebrafish involve the same kinds of cellular rearrangements as in amphibians, and they occur during comparable stages of embryogenesis.     

MicroRNA-206 regulates cell movements during zebrafish gastrulation by targeting prickle1a and regulating c-Jun N-terminal kinase 2 phosphorylation

During vertebrate gastrulation, both concurrent inductive events and cell movements are required for axis formation. Convergence and extension (CE) movements contribute to narrowing and lengthening the forming embryonic axis. MicroRNAs (miRNAs) play a critical role in regulating fundamental cellular functions and developmental processes, but their functions in CE movements are not well known. Zebrafish mir206 is maternally expressed and present throughout blastulation and gastrulation periods. Either gain or loss of function of mir206 leads to severe defects of convergent extension movements both cell autonomously and non-cell autonomously. Mosaic lineage tracing studies reveal that the formation of membrane protrusions and actin filaments is disturbed in mir206-overexpressing embryos or mir206 morphants. Mechanistically, mir206 targets prickle1a (pk1a) mRNA and as a result regulates c-Jun N-terminal protein kinase 2 (JNK2) phosphorylation. pk1a overexpression or knockdown can rescue convergent extension defects induced by mir206 overexpression or knockdown, respectively. Therefore, mir206 is an essential, novel regulator for normal convergent and extension movements by regulating mitogen-activated protein kinase (MAPK) JNK signaling.     

Membrane-type 1 matrix metalloproteinase regulates cell migration during zebrafish gastrulation: evidence for an interaction with non-canonical Wnt signaling

Key to invasiveness is the ability of tumor cells to modify the extracellular matrix, become motile, and engage in directed migration towards the vasculature. One significant protein associated with metastatic progression is membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP14). How MMP14 activity is coordinated with other signaling pathways to regulate cell migration in vivo is largely unknown. Here we have used zebrafish embryogenesis as a model to understand the potential relationship between MMP14-dependent pericellular proteolysis, cell polarity, and motility. Knockdown of zebrafish Mmp14 function disrupted gastrulation convergence and extension cell movements and craniofacial morphogenesis. Using time-lapse imaging and morphometric analyses, we show that Mmp14 is required for proper cell polarity underlying the directed migration of mesodermal cells during gastrulation. We have identified a genetic interaction between mmp14 and non-canonical Wnt signaling, a pathway that also regulates cell polarity in embryonic tissues and is increasingly being linked with tumor cell migration. Finally, we demonstrate that Van Gogh-like 2, a key regulator of the non-canonical Wnt pathway, co-localizes with MMP14 and becomes redistributed towards the leading edge of polarized human cancer cells. Together, our results support the notion that pathways regulating pericellular proteolysis and cell polarity converge to promote efficient cell migration.     

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.     

E-cadherin is required for gastrulation cell movements in zebrafish

E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish.     

Ptenb mediates gastrulation cell movements via Cdc42/AKT1 in zebrafish

Phosphatidylinositol 3-kinase (PI3 kinase) mediates gastrulation cell migration in zebrafish via its regulation of PIP(2)/PIP(3) balance. Although PI3 kinase counter enzyme PTEN has also been reported to be essential for gastrulation, its role in zebrafish gastrulation has been controversial due to the lack of gastrulation defects in pten-null mutants. To clarify this issue, we knocked down a pten isoform, ptenb by using anti-sense morpholino oligos (MOs) in zebrafish embryos and found that ptenb MOs inhibit convergent extension by affecting cell motility and protrusion during gastrulation. The ptenb MO-induced convergence defect could be rescued by a PI3-kinase inhibitor, LY294002 and by overexpressing dominant negative Cdc42. Overexpression of human constitutively active akt1 showed similar convergent extension defects in zebrafish embryos. We also observed a clear enhancement of actin polymerization in ptenb morphants under cofocal microscopy and in actin polymerization assay. These results suggest that Ptenb by antagonizing PI3 kinase and its downstream Akt1 and Cdc42 to regulate actin polymerization that is critical for proper cell motility and migration control during gastrulation in zebrafish.     

Bmp activity gradient regulates convergent extension during zebrafish gastrulation

During vertebrate gastrulation, a ventral to dorsal gradient of bone morphogenetic protein (Bmp) activity establishes cell fates. Concomitantly, convergent extension movements narrow germ layers mediolaterally while lengthening them anteroposteriorly. Here, by measuring movements of cell populations in vivo, we reveal the presence of three domains of convergent extension movements in zebrafish gastrula. Ventrally, convergence and extension movements are absent. Lateral cell populations converge and extend at increasing speed until they reach the dorsal domain where convergence speed slows but extension remains strong. Using dorsalized and ventralized mutants, we demonstrate that these domains are specified by the Bmp activity gradient. In vivo cell morphology and behavior analyses indicated that low levels of Bmp activity might promote extension with little convergence by allowing mediolateral cell elongation and dorsally biased intercalation. Further, single cell movement analyses revealed that the high ventral levels of Bmp activity promote epibolic migration of cells into the tailbud, increasing tail formation at the expense of head and trunk. We show that high Bmp activity limits convergence and extension by negatively regulating expression of the wnt11 (silberblick) and wnt5a (pipetail) genes, which are required for convergent extension but not cell fate specification. Therefore, during vertebrate gastrulation, a single gradient of Bmp activity, which specifies cell fates, also regulates the morphogenetic process of convergent extension.     

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cortactin, a filamentous actin (F-actin) associated protein and a prominent substrate of protein tyrosine kinase Src[1,2], is composed of several functional do-mains, including an amino terminal domain (NTA) that is rich in acidic residues, six and one half 37-amino-acid tandem repeating segments, an al-pha-helical motif, a less conserved region but rich in tyrosine, proline, serine and threonine residues, and a Src homology 3 (SH3) domain at the distal carboxyl terminus. In mammalian cells …     

The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension.

Mutations in the zebrafish knypek locus impair gastrulation movements of convergent extension that narrow embryonic body and elongate it from head to tail. We demonstrate that knypek regulates cellular movements but not cell fate specification. Convergent extension movement defects in knypek are associated with abnormal cell polarity, as mutant cells fail to elongate and align medio-laterally. Positional cloning reveals that knypek encodes a member of the glypican family of heparan sulfate proteoglycans. Double mutant and overexpression analyses show that Knypek potentiates Wnt11 signaling, mediating convergent extension. These studies provide experimental and genetic evidence that glypican Knypek acts during vertebrate gastrulation as a positive modulator of noncanonical Wnt signaling to establish polarized cell behaviors underlying convergent extension movements.     

Loss of cofilin 1 disturbs actin dynamics, adhesion between enveloping and deep cell layers and cell movements during gastrulation in zebrafish

During gastrulation, cohesive migration drives associated cell layers to the completion of epiboly in zebrafish. The association of different layers relies on E-cadherin based cellular junctions, whose stability can be affected by actin turnover. Here, we examined the effect of malfunctioning actin turnover on the epibolic movement by knocking down an actin depolymerizing factor, cofilin 1, using antisense morpholino oligos (MO). Knockdown of cfl1 interfered with epibolic movement of deep cell layer (DEL) but not in the enveloping layer (EVL) and the defect could be specifically rescued by overexpression of cfl1. It appeared that the uncoordinated movements of DEL and EVL were regulated by the differential expression of cfl1 in the DEL, but not EVL as shown by in situ hybridization. The dissociation of DEL and EVL was further evident by the loss of adhesion between layers by using transmission electronic and confocal microscopy analyses. cfl1 morphants also exhibited abnormal convergent extension, cellular migration and actin filaments, but not involution of hypoblast. The cfl1 MO-induced cell migration defect was found to be cell-autonomous in cell transplantation assays. These results suggest that proper actin turnover mediated by Cfl1 is essential for adhesion between DEL and EVL and cell movements during gastrulation in zebrafish.     

Mesodermal cell migration during Xenopus gastrulation

The adhesive glycoprotein fibronectin (FN), which is a component of the network of extracellular matrix fibrils on the inner surface of the blastocoel roof (BCR), has been proposed to play a major role in directing mesodermal cell migration during amphibian gastrulation. In the first part of this paper, the adhesion of Xenopus mesodermal cells to FN in vitro is examined. Cells from several mesoderm regions, which differ in developmental fate and morphogenetic activity, are able to bind specifically to the RGD cell-binding site of FN. Dorsal mesodermal cell adhesion to FN varies along the anterior-posterior (a-p) axis: adhesion is strongest in the anterior head mesoderm, and gradually decreases posteriorly. This a-p gradient of mesodermal adhesiveness to FN does not change during mesodermal involution, and is reflected in the morphology of mesodermal explants on FN. An a-p strip of mesoderm develops a spreading, leading anterior margin and a compact, retracting posterior end, thus moving slowly and directionally over the FN substrate at about 0.8 micron/min. Although dissociated cells from all levels of the dorsal mesodermal axis adhere to FN, only the anterior, leading prospective head mesoderm cells migrate as single cells on a FN substrate in vitro. Locomotion by means of lamelliform protrusions occurs at an average rate of about 1.5 micron/min. Cells of the more posterior axial mesoderm merely shift position at random without substantial net translocation, and preinvolution mesoderm cells are completely stationary. On the BCR, the in vivo substrate for mesodermal cell migration, dissociated prospective head mesoderm cells spread and migrate as on FN in vitro, at 2.2 microns/min. In the presence of an RGD peptide which inhibits cell-FN interaction, cells remain globular and do not spread. They are still motile, but change direction frequently, which leads to less efficient net translocation. Apparently, interaction with the RGD cell-binding site of FN and concomitant spreading of head mesoderm cells is required for the stabilization of cell locomotion. In contrast to the directional migration of the mesoderm cell population toward the animal pole in the embryo, the pathways of dissociated cells on the BCR are randomly oriented. Coherent explants of migratory mesoderm do not move at all on the BCR, although they translocate on FN in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

The ERRalpha orphan nuclear receptor controls morphogenetic movements during zebrafish gastrulation

Gastrulation is a process involving cellular commitment and movements whereby the three fundamental germ layers are established in vertebrates embryos. Estrogen Receptor-Related (ERR) alpha is a nuclear receptor displaying high sequence identity to the Estrogen Receptors (ERs). However, ERRalpha is unable to bind and to be regulated by estrogens or any natural ligand to date. Whereas recent studies have suggested roles for ERRalpha in bone and adipose tissue metabolism in the mouse, little is known about its roles during embryonic development. In zebrafish embryos, ERRalpha is expressed from the beginning of gastrulation at the margin of the blastoderm that represents the presumptive mesendoderm. Using loss of function (morpholinos or a dominant-negative version of the protein) and gain of function (mRNA injection) strategies, we show here that ERRalpha is involved in epiboly and convergent-extension (CE) processes in the zebrafish. Altogether, these results propose ERRalpha as a new regulator of morphogenetic movement during gastrulation, independently of cell fate determination.     

Chemokine signaling regulates sensory cell migration in zebrafish

Chemokines play an important role in the migration of a variety of cells during development. Recent investigations have begun to elucidate the importance of chemokine signaling within the developing nervous system. To better appreciate the neural function of chemokines in vivo, the role of signaling by SDF-1 through its CXCR4 receptor was analyzed in zebrafish. The SDF-1-CXCR4 expression pattern suggested that SDF-1-CXCR4 signaling was important for guiding migration by sensory cells known as the migrating primordium of the posterior lateral line. Ubiquitous induction of the ligand in transgenic embryos, antisense knockdown of the ligand or receptor, and a genetic receptor mutation all disrupted migration by the primordium. Furthermore, in embryos in which endogenous SDF-1 was knocked down, the primordium migrated towards exogenous sources of SDF-1. These data demonstrate that SDF-1 signaling mediated via CXCR4 functions as a chemoattractant for the migrating primordium and that chemokine signaling is both necessary and sufficient for directing primordium migration.     

Conserved patterns of cell movements during vertebrate gastrulation

Vertebrate embryogenesis entails an exquisitely coordinated combination of cell proliferation, fate specification and movement. After induction of the germ layers, the blastula is transformed by gastrulation movements into a multilayered embryo with head, trunk and tail rudiments. Gastrulation is heralded by formation of a blastopore, an opening in the blastula. The axial side of the blastopore is marked by the organizer, a signaling center that patterns the germ layers and regulates gastrulation movements. During internalization, endoderm and mesoderm cells move via the blastopore beneath the ectoderm. Epiboly movements expand and thin the nascent germ layers. Convergence movements narrow the germ layers from lateral to medial while extension movements elongate them from head to tail. Despite different morphology, parallels emerge with respect to the cellular and genetic mechanisms of gastrulation in different vertebrate groups. Patterns of gastrulation cell movements relative to the blastopore and the organizer are similar from fish to mammals, and conserved molecular pathways mediate gastrulation movements.