Gastrulation: making and shaping germ layers.

Gastrulation is a fundamental phase of animal embryogenesis during which germ layers are specified, rearranged, and shaped into a body plan with organ rudiments. Gastrulation involves four evolutionarily conserved morphogenetic movements, each of which results in a specific morphologic transformation. During emboly, mesodermal and endodermal cells become internalized beneath the ectoderm. Epibolic movements spread and thin germ layers. Convergence movements narrow germ layers dorsoventrally, while concurrent extension movements elongate them anteroposteriorly. Each gastrulation movement can be achieved by single or multiple motile cell behaviors, including cell shape changes, directed migration, planar and radial intercalations, and cell divisions. Recent studies delineate cyclical and ratchet-like behaviors of the actomyosin cytoskeleton as a common mechanism underlying various gastrulation cell behaviors. Gastrulation movements are guided by differential cell adhesion, chemotaxis, chemokinesis, and planar polarity. Coordination of gastrulation movements with embryonic polarity involves regulation by anteroposterior and dorsoventral patterning systems of planar polarity signaling, expression of chemokines, and cell adhesion molecules.

CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish.

Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.

Postembryonic screen for mutations affecting spine development in zebrafish.

The spine gives structural support for the adult body, protects the spinal cord, and provides muscle attachment for moving through the environment. The development and maturation of the spine and its physiology involve the integration of multiple musculoskeletal tissues including bone, cartilage, and fibrocartilaginous joints, as well as innervation and control by the nervous system. One of the most common disorders of the spine in human is adolescent idiopathic scoliosis (AIS), which is characterized by the onset of an abnormal lateral curvature of the spine of <10° around adolescence, in otherwise healthy children. The genetic basis of AIS is largely unknown. Systematic genome-wide mutagenesis screens for embryonic phenotypes in zebrafish have been instrumental in the understanding of early patterning of embryonic tissues necessary to build and pattern the embryonic spine. However, the mechanisms required for postembryonic maturation and homeostasis of the spine remain poorly understood. Here we report the results from a small-scale forward genetic screen for adult-viable recessive and dominant zebrafish mutations, leading to overt morphological abnormalities of the adult spine. Germline mutations induced with N-ethyl N-nitrosourea (ENU) were transmitted and screened for dominant phenotypes in 1229 F1 animals, and subsequently bred to homozygosity in F3 families; from these, 314 haploid genomes were screened for adult-viable recessive phenotypes affecting general body shape. We cumulatively found 40 adult-viable (3 dominant and 37 recessive) mutations each leading to a defect in the morphogenesis of the spine. The largest phenotypic group displayed larval onset axial curvatures, leading to whole-body scoliosis without vertebral dysplasia in adult fish. Pairwise complementation testing of 16 mutant lines within this phenotypic group revealed at least 9 independent mutant loci. Using massively-parallel whole genome or whole exome sequencing and meiotic mapping we defined the molecular identity of several loci for larval onset whole-body scoliosis in zebrafish. We identified a new mutation in the skolios/kinesin family member 6 (kif6) gene, causing neurodevelopmental and ependymal cilia defects in mouse and zebrafish. We also report multiple recessive alleles of the scospondin and a disintegrin and metalloproteinase with thrombospondin motifs 9 (adamts9) genes, which all display defects in spine morphogenesis. Our results provide evidence of monogenic traits that are essential for normal spine development in zebrafish, that may help to establish new candidate risk loci for spine disorders in humans.

Gon4l/Udu regulates cardiomyocyte proliferation and maintenance of ventricular chamber identity during zebrafish development.

Vertebrate heart development requires spatiotemporal regulation of gene expression to specify cardiomyocytes, increase the cardiomyocyte population through proliferation, and to establish and maintain atrial and ventricular cardiac chamber identities. The evolutionarily conserved chromatin factor Gon4-like (Gon4l), encoded by the zebrafish ugly duckling (udu) locus, has previously been implicated in cell proliferation, cell survival, and specification of mesoderm-derived tissues including blood and somites, but its role in heart formation has not been studied. Here we report two distinct roles of Gon4l/Udu in heart development: regulation of cell proliferation and maintenance of ventricular identity. We show that zygotic loss of udu expression causes a significant reduction in cardiomyocyte number at one day post fertilization that becomes exacerbated during later development. We present evidence that the cardiomyocyte deficiency in udu mutants results from reduced cell proliferation, unlike hematopoietic deficiencies attributed to TP53-dependent apoptosis. We also demonstrate that expression of the G1/S-phase cell cycle regulator, cyclin E2 (ccne2), is reduced in udu mutant hearts, and that the Gon4l protein associates with regulatory regions of the ccne2 gene during early embryogenesis. Furthermore, udu mutant hearts exhibit a decrease in the proportion of ventricular cardiomyocytes compared to atrial cardiomyocytes, concomitant with progressive reduction of nkx2.5 expression. We further demonstrate that udu and nkx2.5 interact to maintain the proportion of ventricular cardiomyocytes during development. However, we find that ectopic expression of nkx2.5 is not sufficient to restore ventricular chamber identity suggesting that Gon4l regulates cardiac chamber patterning via multiple pathways. Together, our findings define a novel role for zygotically-expressed Gon4l in coordinating cardiomyocyte proliferation and chamber identity maintenance during cardiac development.

Stat3/Cdc25a-dependent cell proliferation promotes embryonic axis extension during zebrafish gastrulation.

Cell proliferation has generally been considered dispensable for anteroposterior extension of embryonic axis during vertebrate gastrulation. Signal transducer and activator of transcription 3 (Stat3), a conserved controller of cell proliferation, survival and regeneration, is associated with human scoliosis, cancer and Hyper IgE Syndrome. Zebrafish Stat3 was proposed to govern convergence and extension gastrulation movements in part by promoting Wnt/Planar Cell Polarity (PCP) signaling, a conserved regulator of mediolaterally polarized cell behaviors. Here, using zebrafish stat3 null mutants and pharmacological tools, we demonstrate that cell proliferation contributes to anteroposterior embryonic axis extension. Zebrafish embryos lacking maternal and zygotic Stat3 expression exhibit normal convergence movements and planar cell polarity signaling, but transient axis elongation defect due to insufficient number of cells resulting largely from reduced cell proliferation and increased apoptosis. Pharmacologic inhibition of cell proliferation during gastrulation phenocopied axis elongation defects. Stat3 regulates cell proliferation and axis extension in part via upregulation of Cdc25a expression during oogenesis. Accordingly, restoring Cdc25a expression in stat3 mutants partially suppressed cell proliferation and gastrulation defects. During later development, stat3 mutant zebrafish exhibit stunted growth, scoliosis, excessive inflammation, and fail to thrive, affording a genetic tool to study Stat3 function in vertebrate development, regeneration, and disease.

Intracellular Golgi Complex organization reveals tissue specific polarity during zebrafish embryogenesis.

BACKGROUND: Cell polarity is essential for directed migration of mesenchymal cells and morphogenesis of epithelial tissues. Studies in cultured cells indicate that a condensed Golgi Complex (GC) is essential for directed protein trafficking to establish cell polarity underlying directed cell migration. Dynamic changes of the GC intracellular organization during early vertebrate development remain to be investigated. RESULTS: We used antibody labeling and fusion proteins in vivo to study the organization and intracellular placement of the GC during early zebrafish embryogenesis. We found that the GC was dispersed into several puncta containing cis- and trans-Golgi Complex proteins, presumably ministacks, until the end of the gastrula period. By early segmentation stages, the GC condensed in cells of the notochord, adaxial mesoderm, and neural plate, and its intracellular position became markedly polarized away from borders between these tissues. CONCLUSIONS: We find that GC is dispersed in early zebrafish cells, even when cells are engaged in massive gastrulation movements. The GC accumulates into patches in a stage and cell-type specific manner, and becomes polarized away from borders between the embryonic tissues. With respect to tissue borders, intracellular GC polarity in notochord is independent of mature apical/basal polarity, Wnt/PCP, or signals from adaxial mesoderm. Developmental Dynamics 245:678-691, 2016. © 2016 Wiley Periodicals, Inc.

Gpr125 modulates Dishevelled distribution and planar cell polarity signaling.

During vertebrate gastrulation, Wnt/planar cell polarity (PCP) signaling orchestrates polarized cell behaviors underlying convergence and extension (C&E) movements to narrow embryonic tissues mediolaterally and lengthen them anteroposteriorly. Here, we have identified Gpr125, an adhesion G protein-coupled receptor, as a novel modulator of the Wnt/PCP signaling system. Excess Gpr125 impaired C&E movements and the underlying cell and molecular polarities. Reduced Gpr125 function exacerbated the C&E and facial branchiomotor neuron (FBMN) migration defects of embryos with reduced Wnt/PCP signaling. At the molecular level, Gpr125 recruited Dishevelled to the cell membrane, a prerequisite for Wnt/PCP activation. Moreover, Gpr125 and Dvl mutually clustered one another to form discrete membrane subdomains, and the Gpr125 intracellular domain directly interacted with Dvl in pull-down assays. Intriguingly, Dvl and Gpr125 were able to recruit a subset of PCP components into membrane subdomains, suggesting that Gpr125 may modulate the composition of Wnt/PCP membrane complexes. Our study reveals a role for Gpr125 in PCP-mediated processes and provides mechanistic insight into Wnt/PCP signaling.

Chemokine GPCR signaling inhibits ß-catenin during zebrafish axis formation.

Embryonic axis formation in vertebrates is initiated by the establishment of the dorsal Nieuwkoop blastula organizer, marked by the nuclear accumulation of maternal ß-catenin, a transcriptional effector of canonical Wnt signaling. Known regulators of axis specification include the canonical Wnt pathway components that positively or negatively affect ß-catenin. An involvement of G-protein coupled receptors (GPCRs) was hypothesized from experiments implicating G proteins and intracellular calcium in axis formation, but such GPCRs have not been identified. Mobilization of intracellular Ca(2+) stores generates Ca(2+) transients in the superficial blastomeres of zebrafish blastulae when the nuclear accumulation of maternal ß-catenin marks the formation of the Nieuwkoop organizer. Moreover, intracellular Ca(2+) downstream of non-canonical Wnt ligands was proposed to inhibit ß-catenin and axis formation, but mechanisms remain unclear. Here we report a novel function of Ccr7 GPCR and its chemokine ligand Ccl19.1, previously implicated in chemotaxis and other responses of dendritic cells in mammals, as negative regulators of ß-catenin and axis formation in zebrafish. We show that interference with the maternally and ubiquitously expressed zebrafish Ccr7 or Ccl19.1 expands the blastula organizer and the dorsoanterior tissues at the expense of the ventroposterior ones. Conversely, Ccr7 or Ccl19.1 overexpression limits axis formation. Epistatic analyses demonstrate that Ccr7 acts downstream of Ccl19.1 ligand and upstream of ß-catenin transcriptional targets. Moreover, Ccl19/Ccr7 signaling reduces the level and nuclear accumulation of maternal ß-catenin and its axis-inducing activity and can also inhibit the Gsk3ß -insensitive form of ß-catenin. Mutational and pharmacologic experiments reveal that Ccr7 functions during axis formation as a GPCR to inhibit ß-catenin, likely by promoting Ca(2+) transients throughout the blastula. Our study delineates a novel negative, Gsk3ß-independent control mechanism of ß-catenin and implicates Ccr7 as a long-hypothesized GPCR regulating vertebrate axis formation.

Wnt/PCP signaling controls intracellular position of MTOCs during gastrulation convergence and extension movements.

During vertebrate gastrulation, convergence and extension cell movements are coordinated with the anteroposterior and mediolateral embryonic axes. Wnt planar cell polarity (Wnt/PCP) signaling polarizes the motile behaviors of cells with respect to the anteroposterior embryonic axis. Understanding how Wnt/PCP signaling mediates convergence and extension (C&E) movements requires analysis of the mechanisms employed to alter cell morphology and behavior with respect to embryonic polarity. Here, we examine the interactions between the microtubule cytoskeleton and Wnt/PCP signaling during zebrafish gastrulation. First, we assessed the location of the centrosome/microtubule organizing center (MTOC) relative to the cell nucleus and the body axes, as a marker of cell polarity. The intracellular position of MTOCs was polarized, perpendicular to the plane of the germ layers, independently of Wnt/PCP signaling. In addition, this position became biased posteriorly and medially within the plane of the germ layers at the transition from mid- to late gastrulation and from slow to fast C&E movements. This depends on intact Wnt/PCP signaling through Knypek (Glypican4/6) and Dishevelled components. Second, we tested whether microtubules are required for planar cell polarization. Once the planar cell polarity is established, microtubules are not required for accumulation of Prickle at the anterior cell edge. However, microtubules are needed for cell-cell contacts and initiation of its anterior localization. Reciprocal interactions occur between Wnt/PCP signaling and microtubule cytoskeleton during C&E gastrulation movements. Wnt/PCP signaling influences the polarity of the microtubule cytoskeleton and, conversely, microtubules are required for the asymmetric distribution of Wnt/PCP pathway components.

Prostaglandin Gbetagamma signaling stimulates gastrulation movements by limiting cell adhesion through Snai1a stabilization.

Gastrulation movements form the germ layers and shape them into the vertebrate body. Gastrulation entails a variety of cell behaviors, including directed cell migration and cell delamination, which are also involved in other physiological and pathological processes, such as cancer metastasis. Decreased Prostaglandin E(2) (PGE(2)) synthesis due to interference with the Cyclooxygenase (Cox) and Prostaglandin E synthase (Ptges) enzymes halts gastrulation and limits cancer cell invasiveness, but how PGE(2) regulates cell motility remains unclear. Here we show that PGE(2)-deficient zebrafish embryos, impaired in the epiboly, internalization, convergence and extension gastrulation movements, exhibit markedly increased cell-cell adhesion, which contributes to defective cell movements in the gastrula. Our analyses reveal that PGE(2) promotes cell protrusive activity and limits cell adhesion by modulating E-cadherin transcript and protein, in part through stabilization of the Snai1a (also known as Snail1) transcriptional repressor, an evolutionarily conserved regulator of cell delamination and directed migration. We delineate a pathway whereby PGE(2) potentiates interaction between the receptor-coupled G protein betagamma subunits and Gsk3beta to inhibit proteasomal degradation of Snai1a. However, overexpression of beta-catenin cannot stabilize Snai1a in PGE(2)-deficient gastrulae. Thus, the Gsk3beta-mediated and beta-catenin-independent inhibition of cell adhesion by Prostaglandins provides an additional mechanism for the functional interactions between the PGE(2) and Wnt signaling pathways during development and disease. We propose that ubiquitously expressed PGE(2) synthesizing enzymes, by promoting the stability of Snai1a, enable the precise and rapid regulation of cell adhesion that is required for the dynamic cell behaviors that drive various gastrulation movements.

Apelin and its receptor control heart field formation during zebrafish gastrulation.

The vertebrate heart arises during gastrulation as cardiac precursors converge from the lateral plate mesoderm territories toward the embryonic midline and extend rostrally to form bilateral heart fields. G protein-coupled receptors (GPCRs) mediate functions of the nervous and immune systems; however, their roles in gastrulation remain largely unexplored. Here, we show that the zebrafish homologs of the Agtrl1b receptor and its ligand, Apelin, implicated in physiology and angiogenesis, control heart field formation. Zebrafish gastrulae express agtrl1b in the lateral plate mesoderm, while apelin expression is confined to the midline. Reduced or excess Agtrl1b or Apelin function caused deficiency of cardiac precursors and, subsequently, the heart. In Apelin-deficient gastrulae, the cardiac precursors converged inefficiently to the heart fields and showed ectopic distribution, whereas cardiac precursors overexpressing Apelin exhibited abnormal morphology and rostral migration. Our results implicate GPCR signaling in movements of discrete cell populations that establish organ rudiments during vertebrate gastrulation.

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.

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.

MYH3-associated distal arthrogryposis zebrafish model is normalized with para-aminoblebbistatin.

Distal arthrogryposis (DA) is group of syndromes characterized by congenital joint contractures. Treatment development is hindered by the lack of vertebrate models. Here, we describe a zebrafish model in which a common MYH3 missense mutation (R672H) was introduced into the orthologous zebrafish gene smyhc1 (slow myosin heavy chain 1) (R673H). We simultaneously created a smyhc1 null allele (smyhc1- ), which allowed us to compare the effects of both mutant alleles on muscle and bone development, and model the closely related disorder, spondylocarpotarsal synostosis syndrome. Heterozygous smyhc1R673H/+ embryos developed notochord kinks that progressed to scoliosis with vertebral fusions; motor deficits accompanied the disorganized and shortened slow-twitch skeletal muscle myofibers. Increased dosage of the mutant allele in both homozygous smyhc1R673H/R673H and transheterozygous smyhc1R673H/- embryos exacerbated the notochord and muscle abnormalities, causing early lethality. Treatment of smyhc1R673H/R673H embryos with the myosin ATPase inhibitor, para-aminoblebbistatin, which decreases actin-myosin affinity, normalized the notochord phenotype. Our zebrafish model of MYH3-associated DA2A provides insight into pathogenic mechanisms and suggests a beneficial therapeutic role for myosin inhibitors in treating disabling contractures.

The Reissner Fiber Is Highly Dynamic In Vivo and Controls Morphogenesis of the Spine.

Cerebrospinal fluid (CSF) physiology is important for the development and homeostasis of the central nervous system, and its disruption has been linked to scoliosis in zebrafish [1, 2]. Suspended in the CSF is an extracellular structure called the Reissner fiber, which extends from the brain through the central canal of the spinal cord. Zebrafish scospondin-null mutants are unable to assemble a Reissner fiber and fail to form a straight body axis during embryonic development [3]. Here, we describe hypomorphic missense mutations of scospondin, which allow Reissner fiber assembly and extension of a straight axis. However, during larval development, these mutants display progressive Reissner fiber disassembly, which is concomitant with the emergence of axial curvatures and scoliosis in adult animals. Using a scospondin-GFP knockin zebrafish line, we demonstrate several dynamic properties of the Reissner fiber in vivo, including embryonic fiber assembly, the continuous rostral to caudal movement of the fiber within the brain and central canal, and subcommissural organ (SCO)-spondin-GFP protein secretion from the floor plate to merge with the fiber. Finally, we show that disassembly of the Reissner fiber is also associated with the progression of axial curvatures in distinct scoliosis mutant zebrafish models. Together, these data demonstrate a critical role for the Reissner fiber for the maintenance of a straight body axis and spine morphogenesis in adult zebrafish. Our study establishes a framework for future investigations to address the cellular effectors responsible for Reissner-fiber-dependent regulation of axial morphology. VIDEO ABSTRACT.

Convergence and extension in vertebrate gastrulae: cell movements according to or in search of identity?

During vertebrate gastrulation, convergence and extension cell movements both narrow and lengthen the forming embryonic axis. Concurrently, positional information established principally by the ventral-to-dorsal gradient of bone morphogenetic protein activity specifies cell fates within the gastrula. New data, primarily from zebrafish, have identified domains of distinct convergence and extension movements, and have established a role for the noncanonical Wnt signaling pathway in promoting the mediolateral cell polarization that underlies this morphogenetic process. Other observations suggest the intriguing possibility that positional information regulates convergence and extension movements in parallel with cell-fate specification.

Analysis of cell movements in zebrafish embryos: morphometrics and measuring movement of labeled cell populations in vivo.

Cell movements occur in all phases of animal life from embryogenesis, to maintaining adult organs, to comprising a critical component of pathology. During gastrulation, cells demonstrate a repertoire of morphogenetic movements coordinated with fate inductions to sculpt the embryonic body. The morphogenetic behaviors, underlying mechanisms, and their control, are the subject of much current study. External development of the transparent zebrafish embryo, the abundance of mutations influencing cell movements, as well as a range of observation and manipulation methods, make the zebrafish valuable for cell movement studies. This chapter offers a conceptual background for analysis of gastrulation cell movements by reviewing how region specific cell movements shape the wild-type zebrafish embryo, and how defective morphogenetic movements alone or in combination with altered cell fate specification distort the body plans of known zebrafish mutants. We furnish methods for the morphometric analysis of embryonic shape and organ rudiments in live and fixed embryos, and present data collected from live wild-type, dorsoventral patterning (somitabun and chordino) and convergence and extension (knypek and trilobite) classes of mutants. We provide a method for quantitative assessment of the movements of cell populations in vivo, and a method for determining whether cell fate and/or movement are disturbed.

Galpha12/13 regulate epiboly by inhibiting E-cadherin activity and modulating the actin cytoskeleton.

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation, and involves coordinated movements of several cell layers. Although recent studies have begun to elucidate the processes that underlie these epibolic movements, the cellular and molecular mechanisms involved remain to be fully defined. Here, we show that gastrulae with altered Galpha(12/13) signaling display delayed epibolic movement of the deep cells, abnormal movement of dorsal forerunner cells, and dissociation of cells from the blastoderm, phenocopying e-cadherin mutants. Biochemical and genetic studies indicate that Galpha(12/13) regulate epiboly, in part by associating with the cytoplasmic terminus of E-cadherin, and thereby inhibiting E-cadherin activity and cell adhesion. Furthermore, we demonstrate that Galpha(12/13) modulate epibolic movements of the enveloping layer by regulating actin cytoskeleton organization through a RhoGEF/Rho-dependent pathway. These results provide the first in vivo evidence that Galpha(12/13) regulate epiboly through two distinct mechanisms: limiting E-cadherin activity and modulating the organization of the actin cytoskeleton.

Initiation of convergence and extension movements of lateral mesoderm during zebrafish gastrulation.

Embryonic morphogenesis is accomplished by cellular movements, rearrangements, and cell fate inductions. Vertebrate gastrulation entails morphogenetic processes that generate three germ layers, endoderm, mesoderm, and ectoderm, shaped into head, trunk, and tail. To understand how cell migration mechanistically contributes to tissue shaping during gastrulation, we examined migration of lateral mesoderm in the zebrafish. Our results illustrate that cell behaviors, different from mediolaterally oriented cell intercalation, also promote convergence and extension (C&E). During early gastrulation, upon internalization, individually migrating mesendodermal cells contribute to the elongation of the mesoderm by moving animally, without dorsal movement. Convergence toward dorsal starts later, by 70% epiboly (7.7 hpf). Depending on location along the Animal-Vegetal axis, an animal or vegetal bias is added to the dorsalward movement, so that paths fan out and the lateral mesoderm both converges and extends. Onset of convergence is independent of noncanonical Wnt signaling but is delayed when Stat3 signaling is compromised. To understand which aspects of motility are controlled by guidance cues, we measured turning behavior of lateral mesodermal cells. We show that cells exhibit directional preference, directionally-regulated speed, and turn toward dorsal when off-course. We estimate that ectoderm could supply from a fraction to all the dorsalward displacement seen in mesoderm cells. Using mathematical modeling, we demonstrate that directional preference is sufficient to account for mesoderm convergence and extension, and that, at minimum, two sources of guidance cues could orient cell paths realistically if located in the dorsal midline.

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.