Essential roles of G{alpha}12/13 signaling in distinct cell behaviors driving zebrafish convergence and extension gastrulation movements

Galpha(12/13) have been implicated in numerous cellular processes, however, their roles in vertebrate gastrulation are largely unknown. Here, we show that during zebrafish gastrulation, suppression of both Galpha(12) and Galpha(13) signaling by overexpressing dominant negative proteins and application of antisense morpholino-modified oligonucleotide translation interference disrupted convergence and extension without changing embryonic patterning. Analyses of mesodermal cell behaviors revealed that Galpha(12/13) are required for cell elongation and efficient dorsalward migration during convergence independent of noncanonical Wnt signaling. Furthermore, Galpha(12/13) function cell-autonomously to mediate mediolateral cell elongation underlying intercalation during notochord extension, likely acting in parallel to noncanonical Wnt signaling. These findings provide the first evidence that Galpha(12) and Galpha(13) have overlapping and essential roles in distinct cell behaviors that drive vertebrate gastrulation.     

A beta1,4-galactosyltransferase is required for convergent extension movements in zebrafish

Our understanding of how complex carbohydrates function during embryonic development is still very limited, primarily due to the large number of glycosyltransferases now known to be involved in their synthesis. To overcome these limitations, we have taken advantage of the zebrafish system to analyze the function of complex carbohydrates during development by down-regulating the expression of specific glycosyltransferases. Herein, we report the identification of the zebrafish ortholog of mammalian beta1,4-galactosyltransferase I, beta4GalT1, and its requirement for proper convergent extension movements during gastrulation. beta4GalT1 is expressed in the oocyte and throughout the embryo during the first 24 h of development. Knockdown of zebrafish beta4GalT1 by two independent morpholino oligonucleotides results in embryos with a truncated anterior-posterior axis, as well as elongated somites and moderate defects in the patterning of the head mesenchyme. Co-injection of zebrafish beta4GalT1 mRNA returns galactosyltransferase activity to control levels and rescues the defects produced by morpholino oligonucleotides. In situ hybridizations of various molecular markers reveal that the axial mesoderm of epiboly stage embryos is abnormally widened in beta4GalT1 morphants, indicative of abnormal convergent extension. Consistent with this, the rate of anterior-posterior axis elongation is reduced relative to control-injected embryos, similar to that seen in known convergent extension mutants. Among the many potential substrates for beta4GalT1 is laminin, a principle component of the extracellular matrix that supports cell movements such as those that occur during convergent extension. Previous in vitro studies have shown that the galactosylation status of laminin directly influences its ability to support cell spreading and migration. In this regard, laminin isolated from beta4GalT1 morphant embryos is poorly galactosylated, which may contribute to defective cell migration during convergent extension movements. This work demonstrates that zebrafish can be used to identify critical developmental roles for specific glycosyltransferases that would not be obvious otherwise, such as an absolute requirement for beta4GalT1 during convergent extension movements.     

Cadherin-mediated adhesion regulates posterior body formation

Background  

The anterior-posterior axis of the vertebrate embryo undergoes a dramatic elongation during early development. Convergence and extension of the mesoderm, occurring during gastrulation, initiates the narrowing and lengthening of the embryo. However the lengthening of the axis continues during post-gastrula stages in the tailbud region, and is thought to involve convergent extension movements as well as other cell behaviors specific to posterior regions.     

Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements

Embryonic morphogenesis is driven by a suite of cell behaviours, including coordinated shape changes, cellular rearrangements and individual cell migrations, whose molecular determinants are largely unknown. In the zebrafish, Dani rerio, trilobite mutant embryos have defects in gastrulation movements and posterior migration of hindbrain neurons. Here, we have used positional cloning to demonstrate that trilobite mutations disrupt the transmembrane protein Strabismus (Stbm)/Van Gogh (Vang), previously associated with planar cell polarity (PCP) in Drosophila melanogaster, and PCP and canonical Wnt/beta-catenin signalling in vertebrates. Our genetic and molecular analyses argue that during gastrulation, trilobite interacts with the PCP pathway without affecting canonical Wnt signalling. Furthermore, trilobite may regulate neuronal migration independently of PCP molecules. We show that trilobite mediates polarization of distinct movement behaviours. During gastrulation convergence and extension movements, trilobite regulates mediolateral cell polarity underlying effective intercalation and directed dorsal migration at increasing velocities. In the hindbrain, trilobite controls effective migration of branchiomotor neurons towards posterior rhombomeres. Mosaic analyses show trilobite functions cell-autonomously and non-autonomously in gastrulae and the hindbrain. We propose Trilobite/Stbm mediates cellular interactions that confer directionality on distinct movements during vertebrate embryogenesis.     

Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants.

We have analyzed cell behavior in the organizer region of the Xenopus laevis gastrula by making high resolution time-lapse recordings of cultured explants. The dorsal marginal zone, comprising among other tissues prospective notochord and somitic mesoderm, was cut from early gastrulae and cultured in a way that permits high resolution microscopy of the deep mesodermal cells, whose organized intercalation produces the dramatic movements of convergent extension. At first, the explants extend without much convergence. This initial expansion results from rapid radial intercalation, or exchange of cells between layers. During the second half of gastrulation, the explants begin to converge strongly toward the midline while continuing to extend vigorously. This second phase of extension is driven by mediolateral cell intercalation, the rearrangement of cells within each layer to lengthen and narrow the array. Toward the end of gastrulation, fissures separate the central notochord from the somitic mesoderm on each side, and cells in both tissues elongate mediolaterally as they intercalate. A detailed analysis of the spatial and temporal pattern of these behaviors shows that both radial and mediolateral intercalation begin first in anterior tissue, demonstrating that the anterior-posterior timing gradient so evident in the mesoderm of the neurula is already forming in the gastrula. Finally, time-lapse recordings of intact embryos reveal that radial intercalation takes places primarily before involution, while mediolateral intercalation begins as the mesoderm goes around the lip. We discuss the significance of these findings to our understanding of both the mechanics of gastrulation and the patterning of the dorsal axis.     

Integrin alpha5beta1 and fibronectin regulate polarized cell protrusions required for Xenopus convergence and extension

BACKGROUND: Integrin recognition of fibronectin is required for normal gastrulation including the mediolateral cell intercalation behaviors that drive convergent extension and the elongation of the frog dorsal axis; however, the cellular and molecular mechanisms involved are unclear. RESULTS: We report that depletion of fibronectin with antisense morpholinos blocks both convergent extension and mediolateral protrusive behaviors in explant preparations. Both chronic depletion of fibronectin and acute disruptions of integrin alpha5beta1 binding to fibronectin increases the frequency and randomizes the orientation of polarized cellular protrusions, suggesting that integrin-fibronectin interactions normally repress frequent random protrusions in favor of fewer mediolaterally oriented ones. In the absence of integrin alpha5beta1 binding to fibronectin, convergence movements still occur but result in convergent thickening instead of convergent extension. CONCLUSIONS: These findings support a role for integrin signaling in regulating the protrusive activity that drives axial extension. We hypothesize that the planar spatial arrangement of the fibrillar fibronectin matrix, which delineates tissue compartments within the embryo, is critical for promoting productive oriented protrusions in intercalating cells.     

Cell rearrangement and segmentation in Xenopus: direct observation of cultured explants

We make use of a novel system of explant culture and high resolution video-film recording to analyse for the first time the cell behaviour underlying convergent extension and segmentation in the somitic mesoderm of Xenopus. We find that a sequence of activities sweeps through the somitic mesoderm from anterior to posterior during gastrulation and neurulation, beginning with radial cell intercalation or thinning, continuing with mediolateral intercalation and cell elongation, and culminating in segmentation and somite rotation. Radial intercalation at the posterior tip lengthens the tissue, while mediolateral intercalation farther anterior converges it toward the midline. This extension of the somitic mesoderm helps to elongate the dorsal side of intact neurulae. By separating tissues, we demonstrate that cell rearrangement is independent of the notochord, but radial intercalation - and thus the bulk of extension - requires the presence of an epithelium, either endodermal or ectodermal. Segmentation, on the other hand, can proceed in somitic mesoderm isolated at the end of gastrulation. Finally, we discuss the relationship between cell rearrangement and segmentation.     

Essential role for Csk upstream of Fyn and Yes in zebrafish gastrulation

Morphogenetic cell movements during gastrulation shape the vertebrate embryo bodyplan. Non-canonical Wnt signaling has been established to regulate convergence and extension cell movements that mediate anterior-posterior axis elongation. In recent years, many other factors have been implicated in the process by modulation of non-canonical Wnt signaling or by different, unknown mechanisms. We have found that the Src family kinases, Fyn and Yes, are required for normal convergence and extension cell movements in zebrafish embryonic development and they signal in parallel to non-canonical Wnts, eventually converging on a common downstream factor, RhoA. Here, we report that Csk, a negative regulator of Src family kinases has a role in gastrulation cell movements as well. Csk knock down induced a phenotype that was similar to the defects observed after knock down of Fyn and Yes, in that gastrulation cell movements were impaired, without affecting cell fate. The Csk knock down phenotype was rescued by simultaneous partial knock down of Fyn and Yes. We conclude that Csk acts upstream of Fyn and Yes to control vertebrate gastrulation cell movements.     

Patterns of cell motility in the organizer and dorsal mesoderm of Xenopus laevis.

In a companion paper (Shih, J. and Keller, R. (1992) Development 116, 901-914), we described a sequence of cell behaviors, called mediolateral intercalation behavior (MIB), that produces mediolateral cell intercalation, the process that drives convergence and extension of the axial and paraxial mesoderm of Xenopus. In this paper, we describe the pattern of expression of MIB in the mesoderm during gastrulation, using video image processing and recording of cell behavior in 'shaved', open-faced explants of the marginal zone. At midgastrula stage (10.5), MIB begins at two dorsolateral sites in the prospective anterior mesoderm and progresses medially along two arcs that lengthen toward and meet at the midline to form a single arc of cells expressing MIB, called the vegetal alignment zone (VgAZ). The notochordal-somitic mesodermal boundary forms within the VgAZ at stage 11, and then progresses animally and laterally, along the prospective anterior-posterior axis, eventually bounding a trapezoidal area the shape of the fate-mapped notochord. Meanwhile, from its origin in the VgAZ, MIB spreads in the prospective posterior direction along the lateral boundaries of both the notochordal and somitic mesoderm. From there it spreads medially in both tissues. Subsequently, vacuolation of notochord cells, and segmentation and expression of a somite-specific marker repeat the progression of mediolateral intercalation behavior. Thus cells in the posterior, medial regions of the notochordal and the somitic territories are the last to express mediolateral intercalation behavior and subsequent tissue differentiations. In explants that do not converge, these cells neither express mediolateral intercalation behavior nor differentiate. These facts suggest that progressions of MIB in the anterior-posterior and lateral-medial directions may be organized by signals emanating from the lateral somitic and notochordal boundaries. These signals may have limited range and may be dependent on convergence, driven by mediolateral cell intercalation, to bring cells within their range. In the embryo, the posterior progression of MIB results in arcs of convergence, anchored in the vegetal endoderm at each end, acting on the inside of the blastoporal lip to produce involution of the IMZ.     

Zebrafish Rho kinase 2 acts downstream of Wnt11 to mediate cell polarity and effective convergence and extension movements

BACKGROUND: During vertebrate gastrulation convergence and extension (CE), movements narrow and lengthen embryonic tissues. In Xenopus and zebrafish, a noncanonical Wnt signaling pathway constitutes the vertebrate counterpart to the Drosophila planar cell polarity pathway and regulates mediolateral cell polarization underlying CE. Despite the identification of several signaling molecules required for normal CE, the downstream transducers regulating individual cell behaviors driving CE are only beginning to be elucidated. Moreover, how defective mediolateral cell polarity impacts CE is not understood.RESULTS: Here, we show that overexpression of zebrafish dominant-negative Rho kinase 2 (dnRok2) disrupts CE without altering cell fates, phenocopying noncanonical Wnt signaling mutants. Moreover, Rho kinase 2 (Rok2) overexpression partially suppresses the slb/wnt11 gastrulation phenotype, and ectopic expression of noncanonical Wnts modulates Rok2 intracellular distribution. In addition, time-lapse analyses associate defective dorsal convergence movements with impaired cell elongation, mediolateral orientation, and consequently failure to migrate along straight paths. Transplantation experiments reveal that dnRok2 cells in wild-type hosts neither elongate nor orient their axes. In contrast, wild-type cells are able to elongate their cell bodies in dnRok2 hosts, even though they fail to orient their axes.CONCLUSIONS: During zebrafish gastrulation, Rok2 acts downstream of noncanonical Wnt11 signaling to mediate mediolateral cell elongation required for dorsal cell movement along straight paths. Furthermore, elongation and orientation of the cell body are independent properties that require both cell-autonomous and nonautonomous Rok2 function.     

Signaling by FGF4 and FGF8 is required for axial elongation of the mouse embryo

Fibroblast growth factor (FGF) signaling has been shown to play critical roles in vertebrate segmentation and elongation of the embryonic axis. Neither the exact roles of FGF signaling, nor the identity of the FGF ligands involved in these processes, has been conclusively determined. Fgf8 is required for cell migration away from the primitive streak when gastrulation initiates, but previous studies have shown that drastically reducing the level of FGF8 later in gastrulation has no apparent effect on somitogenesis or elongation of the embryo. In this study, we demonstrate that loss of both Fgf8 and Fgf4 expression during late gastrulation resulted in a dramatic skeletal phenotype. Thoracic vertebrae and ribs had abnormal morphology, lumbar and sacral vertebrae were malformed or completely absent, and no tail vertebrae were present. The expression of Wnt3a in the tail and the amount of nascent mesoderm expressing Brachyury were both severely reduced. Expression of genes in the NOTCH signaling pathway involved in segmentation was significantly affected, and somite formation ceased after the production of about 15-20 somites. Defects seen in the mutants appear to result from a failure to produce sufficient paraxial mesoderm, rather than a failure of mesoderm precursors to migrate away from the primitive streak. Although the epiblast prematurely decreases in size, we did not detect evidence of a change in the proliferation rate of cells in the tail region or excessive apoptosis of epiblast or mesoderm cells. We propose that FGF4 and FGF8 are required to maintain a population of progenitor cells in the epiblast that generates mesoderm and contributes to the stem cell population that is incorporated in the tailbud and required for axial elongation of the mouse embryo after gastrulation.     

Local shifts in position and polarized motility drive cell rearrangement during sea urchin gastrulation

This study examines the mechanisms of epithelial cell rearrangement during archenteron elongation in the sea urchin embryo using scanning electron microscopy, differential interference contrast videomicroscopy, cell marking, and fluorescently labeled chimaeric clones. Archenteron elongation involves two major processes: local shifts in position of cells in the archenteron wall and polarized motility of the cells as they rearrange. Fluorescently labeled chimaeric clones introduced into the archenteron of Lytechinus pictus are initially 4-5 cells wide; by the end of gastrulation the clones elongate and narrow, so that they are one cell wide in the narrowest region of the archenteron. The extent of clonal mixing indicates that cells in the archenteron change their relative positions by only 1-2 cell diameters during cell rearrangement. Cells at the blastopore rearrange concomitantly with cells in the archenteron, resulting in a 35% decrease in blastopore diameter. Endoderm cells undergo polarized, stage-specific changes in shape and motility as they rearrange; (1) they flatten markedly along their apical-basal axis throughout archenteron elongation; (2) just prior to the onset of cell rearrangement, basal surfaces of all cells in the archenteron extend long, polarized lamellipodial protrusions along the axis of extension of the archenteron; (3) as cell rearrangement begins, basal surfaces round up and the cells become isodiametric; (4) by the 3/4 gastrula stage the cells become stretched along the animal-vegetal axis, apparently due to filopodial traction, and finally (5) they continue to rearrange, returning to a less elongated shape by the end of gastrulation. Direct observation of gastrulation in the cidaroid Eucidaris tribuloides indicates that in this species cell rearrangement is accomplished by progressive circumferential intercalation of cells without upwardly directed filopodia. This intercalation is accompanied by explosive, apparently stochastic, cortical blebbing activity at the boundaries between cells, suggesting that in addition to whatever cell rearrangement may be generated by filopodial tension, such activity is an important component of the active rearrangement process.     

Regulation of convergence and extension movements during vertebrate gastrulation by the Wnt/PCP pathway

Vertebrate gastrulation entails massive cell movements that establish and shape the germ layers. During gastrulation, the individual cell behaviors are strictly coordinated in time and space by various signaling pathways. These pathways instruct the cells about proliferation, shape, fate and migration into proper location. Convergence and extension (C&E) movements during vertebrate gastrulation play a major role in the shaping of the embryonic body. In vertebrates, the Wnt/Planar Cell Polarity (Wnt/PCP) pathway is a key regulator of C&E movements, essential for several polarized cell behaviors, including directed cell migration, and mediolateral and radial cell intercalation. However, the molecular mechanisms underlying the acquisition of Planar Cell Polarity by highly dynamic mesenchymal cells engaged in C&E are still not well understood. Here we review new evidence implicating the Wnt/PCP pathway in specific cell behaviors required for C&E during zebrafish gastrulation, in comparison to other vertebrates. We also discuss findings on the molecular regulation and the interaction of the Wnt/PCP pathway with other signaling pathways during gastrulation movements.     

Gastrulation in zebrafish — all just about adhesion?

During vertebrate gastrulation, the evolutionarily conserved morphogenetic movements of epiboly, internalization, convergence and extension cooperate to generate germ layers and to sculpt the body plan. In zebrafish, these movements are driven by a variety of cell behaviors, including slow and fast directed migration, radial and mediolateral intercalation, and cell shape changes. Whereas some signaling pathways are required for a subset of these behaviors, other molecules, such as E-cadherin or Galpha12 and Galpha13 proteins, appear to have a widespread role in different gastrulation cell behaviors.     

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.     

Eph regulates dorsoventral asymmetry of the notochord plate and convergent extension-mediated notochord formation

Background

The notochord is a signaling center required for the patterning of the vertebrate embryic midline, however, the molecular and cellular mechanisms involved in the formation of this essential embryonic tissue remain unclear. The urochordate Ciona intestinalis develops a simple notochord from 40 specific postmitotic mesodermal cells. The precursors intercalate mediolaterally and establish a single array of disk-shaped notochord cells along the midline. However, the role that notochord precursor polarization, particularly along the dorsoventral axis, plays in this morphogenetic process remains poorly understood.

Methodology/Principal Findings

Here we show that the notochord preferentially accumulates an apical cell polarity marker, aPKC, ventrally and a basement membrane marker, laminin, dorsally. This asymmetric accumulation of apicobasal cell polarity markers along the embryonic dorsoventral axis was sustained in notochord precursors during convergence and extension. Further, of several members of the Eph gene family implicated in cellular and tissue morphogenesis, only Ci-Eph4 was predominantly expressed in the notochord throughout cell intercalation. Introduction of a dominant-negative Ci-Eph4 to notochord precursors diminished asymmetric accumulation of apicobasal cell polarity markers, leading to defective intercalation. In contrast, misexpression of a dominant-negative mutant of a planar cell polarity gene Dishevelled preserved asymmetric accumulation of aPKC and laminin in notochord precursors, although their intercalation was incomplete.

Conclusions/Significance

Our data support a model in which in ascidian embryos Eph-dependent dorsoventral polarity of notochord precursors plays a crucial role in mediolateral cell intercalation and is required for proper notochord morphogenesis.     

The synergistic function of long and short forms of β4GalT1 in p53-mediated drug resistance in bladder cancer cells

β1,4-galactosyltransferase-1 (β4GalT1) is a type II membrane protein that catalyzes the transfer of galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc) and forms a LacNAc structure. β4GalT1 has a long form (termed β4GalT1-L) and a short form (termed β4GalT1-S) in mammalian cells. Although β4GalT1 has been proven to play an important role in many biological and pathological processes, such as differentiation, immune responses and cancer development, the different functions of the two β4GalT1 forms remain ambiguous. In this study, we demonstrated that total β4GalT1 was upregulated in bladder cancer. Overexpression of β4GalT1-S, but not β4GalT1-L, increased drug resistance in bladder epithelial cells by upregulating p53 expression. Glycoproteomic analysis revealed that the substrate specificities of the two β4GalT1 forms were different. Among the LacNAcylated proteins, the E3 ligase MDM2 could be preferentially modified by β4GalT1-L compared to β4GalT1-S, and this modification could increase the binding of MDM2 and p53 and further facilitate the degradation of p53. Our data proved that the two forms of β4GalT1 could synergistically regulate p53-mediated cell survival under chemotherapy treatment. These results provide insights into the role of β4GalT1-L and β4GalT1-S and suggest their differentially important implications in the development of bladder cancer.     

Coordination of cell polarity during Xenopus gastrulation

Cell polarity is an essential feature of animal cells contributing to morphogenesis. During Xenopus gastrulation, it is known that chordamesoderm cells are polarized and intercalate each other allowing anterior-posterior elongation of the embryo proper by convergent extension (CE). Although it is well known that the cellular protrusions at both ends of polarized cells exert tractive force for intercalation and that PCP pathway is known to be essential for the cell polarity, little is known about what triggers the cell polarization and what the polarization causes to control intracellular events enabling the intercalation that leads to the CE. In our research, we used EB3 (end-binding 3), a member of +TIPs that bind to the plus end of microtubule (MT), to visualize the intracellular polarity of chordamesoderm cells during CE to investigate the trigger of the establishment of cell polarity. We found that EB3 movement is polarized in chordamesoderm cells and that the notochord-somite tissue boundary plays an essential role in generating the cell polarity. This polarity was generated before the change of cell morphology and the polarized movement of EB3 in chordamesoderm cells was also observed near the boundary between the chordamesoderm tissue and na?ve ectoderm tissue or lateral mesoderm tissues induced by a low concentration of nodal mRNA. These suggest that definitive tissue separation established by the distinct levels of nodal signaling is essential for the chordamesodermal cells to acquire mediolateral cell polarity.     

Cell motility driving mediolateral intercalation in explants of Xenopus laevis.

In Xenopus, convergence and extension are produced by active intercalation of the deep mesodermal cells between one another along the mediolateral axis (mediolateral cell intercalation), to form a narrower, longer array. The cell motility driving this intercalation is poorly understood. A companion paper shows that the endodermal epithelium organizes the outermost mesodermal cells immediately beneath it to undergo convergence and extension, and other evidence suggests that these deep cells are the most active participants in mediolateral intercalation (Shih, J. and Keller, R. (1992) Development 116, 887-899). In this paper, we shave off the deeper layers of mesodermal cells, which allows us to observe the protrusive activity of the mesodermal cells next to the organizing epithelium with high resolution video microscopy. These mesodermal cells divide in the early gastrula and show rapid, randomly directed protrusive activity. At the early midgastrula stage, they begin to express a characteristic sequence of behaviors, called mediolateral intercalation behavior (MIB): (1) large, stable, filiform and lamelliform protrusions form in the lateral and medial directions, thus making the cells bipolar; (2) these protrusions are applied directly to adjacent cell surfaces and exert traction on them, without contact inhibition; (3) as a result, the cells elongate and align parallel to the mediolateral axis and perpendicular to the axis of extension; (4) the elongate, aligned cells intercalate between one another along the mediolateral axis, thus producing a longer, narrower array. Explants of essentially a single layer of deep mesodermal cells, made at stage 10.5, converge and extend by mediolateral intercalation. Thus by stage 10.5 (early midgastrula), expression of MIB among deep mesodermal cells is physiologically and mechanically independent of the organizing influence of the endodermal epithelium, described previously (Shih, J. and Keller, R. (1992) Development 116 887-899), and is the fundamental cell motility underlying mediolateral intercalation and convergence and extension of the body axis.     

Fyn/Yes and non-canonical Wnt signalling converge on RhoA in vertebrate gastrulation cell movements

Convergent extension (CE) cell movements during gastrulation mediate extension of the anterior-posterior body axis of vertebrate embryos. Non-canonical Wnt5 and Wnt11 signalling is essential for normal CE movements in vertebrate gastrulation. Here, we show that morpholino (MO)-mediated double knock-down of the Fyn and Yes tyrosine kinases in zebrafish embryos impaired normal CE cell movements, resembling the silberblick and pipetail mutants, caused by mutations in wnt11 and wnt5, respectively. Co-injection of Fyn/Yes- and Wnt11- or Wnt5-MO was synergistic, but wnt11 or wnt5 RNA did not rescue the Fyn/Yes knockdown or vice versa. Remarkably, active RhoA rescued the Fyn/Yes knockdown as well as the Wnt11 knockdown, indicating that Fyn/Yes and Wnt11 signalling converged on RhoA. Our results show that Fyn and Yes act together with non-canonical Wnt signalling via RhoA in CE cell movements during gastrulation.