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The tissues of a developing embryo are simultaneously patterned, moved and differentiated according to an exchange of information between their constituent cells. We argue that these complex self-organizing phenomena can only be fully understood with quantitative mathematical frameworks that allow specific hypotheses to be formulated and tested. The quantitative and dynamic imaging of growing embryos at the molecular, cellular and tissue level is the key experimental advance required to achieve this interaction between theory and experiment. Here we describe how mathematical modelling has become an invaluable method to integrate quantitative biological information across temporal and spatial scales, serving to connect the activity of regulatory molecules with the morphological development of organisms.

The migration of single cells and epithelial sheets is of great importance for gastrulation and organ formation in developing embryos and, if misregulated, can have dire consequences e.g. during cancer metastasis. A keystone of cell migration is the regulation of adhesive contacts, which are dynamically assembled and disassembled via endocytosis. Here, we discuss some of the basic concepts about the function of endocytic trafficking during cell migration: transport of integrins from the cell rear to the leading edge in fibroblasts; confinement of signalling to the front of single cells by endocytic transport of growth factors; regulation of movement coherence in multicellular sheets by cadherin turnover; and shaping of extracellular chemokine gradients. Taken together, endocytosis enables migrating cells and tissues to dynamically modulate their adhesion and signalling, allowing them to efficiently migrate through their extracellular environment.

Nuclear movements play an essential role in metazoan development. Although the intracellular transport mechanisms underlying nuclear movements have been studied in detail, relatively little is known about signals from surrounding cells and tissues controlling these movements. Here, we show that, in gastrulating zebrafish embryos, convergence movements of nuclei within the yolk syncytial layer (YSL) are guided by mesoderm and endoderm progenitors migrating along the surface of the yolk towards the dorsal side of the developing gastrula. Progenitor cells direct the convergence movements of internal yolk syncytial nuclei (iYSN) by modulating cortical flow within the YSL in which the iYSN are entrained. The effect of mesoderm and endoderm progenitors on the convergence movement of iYSN depends on the expression of E-cadherin, indicating that adhesive contact between the cells and the YSL is required for the mesendoderm-modulated YSL cortical flow mediating nuclear convergence. In summary, our data reveal a crucial function for cortical flow in the coordination of syncytial nuclear movements with surrounding cells and tissues during zebrafish gastrulation.

The zebrafish is a favorite model organism to study tissue morphogenesis during development at a subcellular level. This largely results from the fact that zebrafish embryos are transparent and thus accessible to various imaging techniques, such as confocal and two-photon excitation (2PE) microscopy. In particular, 2PE microscopy has been shown to be useful for imaging deep cell layers within the embryo and following tissue morphogenesis over long periods. This chapter describes how to use 2PE microscopy to study morphogenetic movements during early zebrafish embryonic development, providing a general blueprint for its use in zebrafish.

Apical cell contraction triggers tissue folding and invagination in epithelia. During Drosophila gastrulation, ventral furrow formation was thought to be driven by smooth, purse-string-like constriction of an acto-myosin belt underlying adherens junctions. Now Martin et al. report in Nature that ventral furrow formation is triggered by asynchronous pulsed contractions of the apical acto-myosin cortex in individual cells.

During vertebrate gastrulation, cells forming the prechordal plate undergo directed migration as a cohesive cluster. Recent studies revealed that E-cadherin-mediated coherence between these cells plays an important role in effective anterior migration, and that platelet-derived growth factor (Pdgf) appears to act as a guidance cue in this process. However, the mechanisms underlying this process at the individual cell level remain poorly understood. We have identified miles apart (mil) as a suppressor of defective anterior migration of the prospective prechordal plate in silberblick (slb)/wnt11 mutant embryos, in which E-cadherin-mediated coherence of cell movement is reduced. mil encodes Edg5, a sphingosine-1-phosphate (S1P) receptor belonging to a family of five G-protein-coupled receptors (S1PRs). S1P is a lipid signalling molecule that has been implicated in regulating cytoskeletal rearrangements, cell motility and cell adhesion in a variety of cell types. We examined the roles of Mil in anterior migration of prechordal plate progenitor cells and found that, in slb embryos injected with mil-MO, cells migrate with increased motility but decreased directionality, without restoring the coherence of cell migration. This indicates that prechordal plate progenitor cells can migrate effectively as individuals, as well as in a coherent cluster of cells. Moreover, we demonstrate that Mil regulates cell motility and polarisation through Pdgf and its intracellular effecter PI3K, but modulates cell coherence independently of the Pdgf/PI3K pathway, thus co-ordinating cell motility and coherence. These results suggest that the net migration of prechordal plate progenitors is determined by different parameters, including motility, persistence and coherence.

The zyxin-related LPP protein is localized at focal adhesions and cell-cell contacts and is involved in the regulation of smooth muscle cell migration. A known interaction partner of LPP in human is the tumor suppressor protein SCRIB. Knocking down scrib expression during zebrafish embryonic development results in defects of convergence and extension (C&E) movements, which occur during gastrulation and mediate elongation of the anterior-posterior body axis. Mediolateral cell polarization underlying C&E is regulated by a noncanonical Wnt signaling pathway constituting the vertebrate planar cell polarity (PCP) pathway. Here, we investigated the role of Lpp during early zebrafish development. We show that morpholino knockdown of lpp results in defects of C&E, phenocopying noncanonical Wnt signaling mutants. Time-lapse analysis associates the defective dorsal convergence movements with a reduced ability to migrate along straight paths. In addition, expression of Lpp is significantly reduced in Wnt11 morphants and in embryos overexpressing Wnt11 or a dominant-negative form of Rho kinase 2, which is a downstream effector of Wnt11, suggesting that Lpp expression is dependent on noncanonical Wnt signaling. Finally, we demonstrate that Lpp interacts with the PCP protein Scrib in zebrafish, and that Lpp and Scrib cooperate for the mediation of C&E.

Animal body plan arises during gastrulation and organogenesis by the coordination of inductive events and cell movements. Several signaling pathways, such as BMP, FGF, Hedgehog, Nodal, and Wnt have well-recognized instructive roles in cell fate specification during vertebrate embryogenesis. Growing evidence indicates that BMP, Nodal, and FGF signaling also regulate cell movements, and that they do so through mechanisms distinct from those that specify cell fates. Moreover, pathways controlling cell movements can also indirectly influence cell fate specification by regulating dimensions and relative positions of interacting tissues. The current challenge is to delineate the molecular mechanisms via which the major signaling pathways regulate cell fate specification and movements, and how these two processes are coordinated to ensure normal development.

Handedness of the vertebrate body plan critically depends on transient embryonic structures/organs that generate cilia-dependent leftward fluid flow within constrained extracellular environments. Although the function of ciliated organs in laterality determination has been extensively studied, how they are formed during embryogenesis is still poorly understood. Here we show that Kupffer's vesicle (KV), the zebrafish organ of laterality, arises from a surface epithelium previously thought to adopt exclusively extra-embryonic fates. Live multi-photon confocal imaging reveals that surface epithelial cells undergo Nodal/TGFbeta signalling-dependent ingression at the dorsal germ ring margin prior to gastrulation, to give rise to dorsal forerunner cells (DFCs), the precursors of KV. DFCs then migrate attached to the overlying surface epithelium and rearrange into rosette-like epithelial structures at the end of gastrulation. During early somitogenesis, these epithelial rosettes coalesce into a single rosette that differentiates into the KV with a ciliated lumen at its apical centre. Our results provide novel insights into the morphogenetic transformations that shape the laterality organ in zebrafish and suggest a conserved progenitor role of the surface epithelium during laterality organ formation in vertebrates.

The controlled adhesion of cells to each other and to the extracellular matrix is crucial for tissue development and maintenance. Numerous assays have been developed to quantify cell adhesion. Among these, the use of atomic force microscopy (AFM) for single-cell force spectroscopy (SCFS) has recently been established. This assay permits the adhesion of living cells to be studied in near-physiological conditions. This implementation of AFM allows unrivaled spatial and temporal control of cells, as well as highly quantitative force actuation and force measurement that is sufficiently sensitive to characterize the interaction of single molecules. Therefore, not only overall cell adhesion but also the properties of single adhesion-receptor-ligand interactions can be studied. Here we describe current implementations and applications of SCFS, as well as potential pitfalls, and outline how developments will provide insight into the forces, energetics and kinetics of cell-adhesion processes.

Understanding the factors that direct tissue organization during development is one of the most fundamental goals in developmental biology. Various hypotheses explain cell sorting and tissue organization on the basis of the adhesive and mechanical properties of the constituent cells. However, validating these hypotheses has been difficult due to the lack of appropriate tools to measure these parameters. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of individual ectoderm, mesoderm and endoderm progenitor cells from gastrulating zebrafish embryos. Combining these data with tissue self-assembly in vitro and the sorting behaviour of progenitors in vivo, we have shown that differential actomyosin-dependent cell-cortex tension, regulated by Nodal/TGFbeta-signalling (transforming growth factor beta), constitutes a key factor that directs progenitor-cell sorting. These results demonstrate a previously unrecognized role for Nodal-controlled cell-cortex tension in germ-layer organization during gastrulation.

This study provides direct functional evidence that differential adhesion, measurable as quantitative differences in tissue surface tension, influences spatial positioning between zebrafish germ layer tissues. We show that embryonic ectodermal and mesendodermal tissues generated by mRNA-overexpression behave on long-time scales like immiscible fluids. When mixed in hanging drop culture, their cells segregate into discrete phases with ectoderm adopting an internal position relative to the mesendoderm. The position adopted directly correlates with differences in tissue surface tension. We also show that germ layer tissues from untreated embryos, when extirpated and placed in culture, adopt a configuration similar to those of their mRNA-overexpressing counterparts. Down-regulating E-cadherin expression in the ectoderm leads to reduced surface tension and results in phase reversal with E-cadherin-depleted ectoderm cells now adopting an external position relative to the mesendoderm. These results show that in vitro cell sorting of zebrafish mesendoderm and ectoderm tissues is specified by tissue interfacial tensions. We perform a mathematical analysis indicating that tissue interfacial tension between actively motile cells contributes to the spatial organization and dynamics of these zebrafish germ layers in vivo.

The controlled internalization of membrane receptors and lipids is crucial for cells to control signaling pathways and interact with their environment. During clathrin-mediated endocytosis, membrane constituents are transported via endocytic vesicles into early endosomes, from which they are further distributed within the cell. The small guanosine triphosphatase (GTPase) Rab5 is both required and sufficient for the formation of these early endosomes and can be used to experimentally address endocytic processes. Recent evidence shows that endocytic turnover of E-cadherin regulates the migration of mesendodermal cells during zebrafish gastrulation by modulating their adhesive interactions with neighboring cells. This in turn leads to effective and synchronized movement within the embryo. In this review, we discuss techniques to manipulate E-cadherin endocytosis by morpholino-mediated knockdown of rab5 during zebrafish gastrulation. We describe the use of antibodies specifically directed against zebrafish E-cadherin to detect its intracellular localization and of in situ hybridization and primary cell culture to reveal patterns of cell migration and adhesion, respectively.

Background: Bone morphogenetic proteins (Bmps) are required for the specification of ventrolateral cell fates during embryonic dorsoventral patterning and for proper convergence and extension gastrulation movements, but the mechanisms underlying the latter role remained elusive., Results: Via bead implantations, we show that the Bmp gradient determines the direction of lateral mesodermal cell migration during dorsal convergence in the zebrafish gastrula. This effect is independent of its role during dorsoventral patterning and of noncanonical Wnt signaling. However, it requires Bmp signal transduction through Alk8 and Smad5 to negatively regulate Ca(2+)/Cadherin-dependent cell-cell adhesiveness. In vivo, converging mesodermal cells form lamellipodia that attach to adjacent cells. Bmp signaling diminishes the Cadherin-dependent stability of such contact points, thereby abrogating subsequent cell displacement during lamellipodial retraction., Conclusions: We propose that the ventral-to-dorsal Bmp gradient has an instructive role to establish a reverse gradient of cell-cell adhesiveness, thereby defining different migratory zones and directing lamellipodia-driven cell migrations during dorsal convergence in lateral regions of the zebrafish gastrula.

Gastrulation is a morphogenetic process that results in the formation of the embryonic germ layers. Here we detail the major cell movements that occur during zebrafish gastrulation: epiboly, internalization, and convergent extension. Although gastrulation is known to be regulated by signaling pathways such as the Wnt/planar cell polarity pathway, many questions remain about the underlying molecular and cellular mechanisms. Key factors that may play a role in gastrulation cell movements are cell adhesion and cytoskeletal rearrangement. In addition, some of the driving force for gastrulation may derive from tissue interactions such as those described between the enveloping layer and the yolk syncytial layer. Future exploration of gastrulation mechanisms relies on the development of sensitive and quantitative techniques to characterize embryonic germ-layer properties.

Wnt11 is a key signal, determining cell polarization and migration during vertebrate gastrulation. It is known that Wnt11 functionally interacts with several signaling components, the homologues of which control planar cell polarity in Drosophila melanogaster. Although in D. melanogaster these components are thought to polarize cells by asymmetrically localizing at the plasma membrane, it is not yet clear whether their subcellular localization plays a similarly important role in vertebrates. We show that in zebrafish embryonic cells, Wnt11 locally functions at the plasma membrane by accumulating its receptor, Frizzled 7, on adjacent sites of cell contacts. Wnt11-induced Frizzled 7 accumulations recruit the intracellular Wnt signaling mediator Dishevelled, as well as Wnt11 itself, and locally increase cell contact persistence. This increase in cell contact persistence is mediated by the local interaction of Wnt11, Frizzled 7, and the atypical cadherin Flamingo at the plasma membrane, and it does not require the activity of further downstream effectors of Wnt11 signaling, such as RhoA and Rok2. We propose that Wnt11, by interacting with Frizzled 7 and Flamingo, modulates local cell contact persistence to coordinate cell movements during gastrulation.

The detection of microRNAs (miRNAs) at single-cell resolution is important for studying the role of these posttranscriptional regulators. Here, we use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid, injected into zebrafish blastomeres or electroporated into defined tissues of mouse embryos in utero or ex utero, to monitor the dynamics of specific miRNAs in individual live cells. This approach reveals, for example, that in the developing mouse central nervous system, miR-124a is expressed not only in postmitotic neurons but also in neuronal progenitor cells. Collectively, our results demonstrate that acute administration of DFRS plasmids offers an alternative to previous in situ hybridization and transgenic approaches and allows the monitoring of miRNA appearance and disappearance in defined cell lineages during vertebrate development.

The molecular and cellular mechanisms governing cell motility and directed migration in response to the chemokine SDF-1 are largely unknown. Here, we demonstrate that zebrafish primordial germ cells whose migration is guided by SDF-1 generate bleb-like protrusions that are powered by cytoplasmic flow. Protrusions are formed at sites of higher levels of free calcium where activation of myosin contraction occurs. Separation of the acto-myosin cortex from the plasma membrane at these sites is followed by a flow of cytoplasm into the forming bleb. We propose that polarized activation of the receptor CXCR4 leads to a rise in free calcium that in turn activates myosin contraction in the part of the cell responding to higher levels of the ligand SDF-1. The biased formation of new protrusions in a particular region of the cell in response to SDF-1 defines the leading edge and the direction of cell migration.

Epithelial morphogenesis depends on coordinated changes in cell shape, a process that is still poorly understood. During zebrafish epiboly and Drosophila dorsal closure, cell-shape changes at the epithelial margin are of critical importance. Here evidence is provided for a conserved mechanism of local actin and myosin 2 recruitment during theses events. It was found that during epiboly of the zebrafish embryo, the movement of the outer epithelium (enveloping layer) over the yolk cell surface involves the constriction of marginal cells. This process depends on the recruitment of actin and myosin 2 within the yolk cytoplasm along the margin of the enveloping layer. Actin and myosin 2 recruitment within the yolk cytoplasm requires the Ste20-like kinase Msn1, an orthologue of Drosophila Misshapen. Similarly, in Drosophila, actin and myosin 2 localization and cell constriction at the margin of the epidermis mediate dorsal closure and are controlled by Misshapen. Thus, this study has characterized a conserved mechanism underlying coordinated cell-shape changes during epithelial morphogenesis.

During vertebrate gastrulation, a well-orchestrated series of morphogenetic changes leads to the formation of the three germ layers: the ectoderm, mesoderm and endoderm. The analysis of gene expression patterns during gastrulation has been central to the identification of genes involved in germ layer formation. However, many proteins are regulated on a translational or post-translational level and are thus undetectable by gene expression analysis. Therefore, we developed a 2D-gel-based comparative proteomic approach to target proteins involved in germ layer morphogenesis during zebrafish gastrulation. Proteomes of ectodermal and mesendodermal progenitor cells were compared and 35 significantly regulated proteins were identified by mass spectrometry, including several proteins with predicted functions in cytoskeletal organization. A comparison of our proteomic results with data obtained in an accompanying microarray-based gene expression analysis revealed no significant overlap, confirming the complementary nature of proteomics and transcriptomics. The regulation of ezrin2, which was identified based on a reduction in spot intensity in mesendodermal cells, was independently validated. Furthermore, we show that ezrin2 is activated by phosphorylation in mesendodermal cells and is required for proper germ layer morphogenesis. We demonstrate the feasibility of proteomics in zebrafish, concluding that proteomics is a valuable tool for analysis of early development.

Detailed reconstruction of the spatiotemporal history of embryonic cells is key to understanding tissue formation processes but is often complicated by the large number of cells involved, particularly so in vertebrates. Through a combination of high-resolution time-lapse lineage tracing and antibody staining, we have analyzed the movement of mesencephalic and metencephalic cell populations in the early zebrafish embryo. To facilitate the analysis of our cell tracking data, we have created TracePilot, a software tool that allows interactive manipulation and visualization of tracking data. We demonstrate its utility by showing novel visualizations of cell movement in the developing zebrafish brain. TracePilot (http://www.mpi-cbg.de/tracepilot) is Java-based, available free of charge, and has a program structure that allows the incorporation of additional analysis tools., ((c) 2006 Wiley-Liss, Inc.)

Background: Zebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed., Results: As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins., Conclusion: The provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis.

Wnt11 plays a central role in tissue morphogenesis during vertebrate gastrulation, but the molecular and cellular mechanisms by which Wnt11 exerts its effects remain poorly understood. Here, we show that Wnt11 functions during zebrafish gastrulation by regulating the cohesion of mesodermal and endodermal (mesendodermal) progenitor cells. Importantly, we demonstrate that Wnt11 activity in this process is mediated by the GTPase Rab5, a key regulator of early endocytosis, as blocking Rab5c activity in wild-type embryos phenocopies slb/wnt11 mutants, and enhancing Rab5c activity in slb/wnt11 mutant embryos rescues the mutant phenotype. In addition, we find that Wnt11 and Rab5c control the endocytosis of E-cadherin and are required in mesendodermal cells for E-cadherin-mediated cell cohesion. Together, our results suggest that Wnt11 controls tissue morphogenesis by modulating E-cadherin-mediated cell cohesion through Rab5c, a novel mechanism of Wnt signaling in gastrulation.

During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.

During vertebrate gastrulation, the three germ layers, ectoderm, mesoderm and endoderm are formed, and the resulting progenitor cells are brought into the positions from which they will later contribute more complex tissues and organs. A core element in this process is the internalization of mesodermal and endodermal progenitors at the onset of gastrulation. Although many of the molecules that induce mesendoderm have been identified, much less is known about the cellular mechanisms underlying mesendodermal cell internalization and germ layer formation. Here we show that at the onset of zebrafish gastrulation, mesendodermal progenitors in dorsal/axial regions of the germ ring internalize by single cell delamination. Once internalized, mesendodermal progenitors upregulate E-Cadherin (Cadherin 1) expression, become increasingly motile and eventually migrate along the overlying epiblast (ectodermal) cell layer towards the animal pole of the gastrula. When E-Cadherin function is compromised, mesendodermal progenitors still internalize, but, with gastrulation proceeding, fail to elongate and efficiently migrate along the epiblast, whereas epiblast cells themselves exhibit reduced radial cell intercalation movements. This indicates that cadherin-mediated cell-cell adhesion is needed within the forming shield for both epiblast cell intercalation, and mesendodermal progenitor cell elongation and migration during zebrafish gastrulation. Our data provide insight into the cellular mechanisms underlying mesendodermal progenitor cell internalization and subsequent migration during zebrafish gastrulation, and the role of cadherin-mediated cell-cell adhesion in these processes.

In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphenucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.

During vertebrate gastrulation, a relatively limited number of blastodermal cells undergoes a stereotypical set of cellular movements that leads to formation of the three germ layers: ectoderm, mesoderm and endoderm. Gastrulation, therefore, provides a unique developmental system in which to study cell movements in vivo in a fairly simple cellular context. Recent advances have been made in elucidating the cellular and molecular mechanisms that underlie cell movements during zebrafish gastrulation. These findings can be compared with observations made in other model systems to identify potential general mechanisms of cell migration during development.

During vertebrate gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. In zebrafish, silberblick (slb)/wnt11 regulates normal gastrulation movements by activating a signalling pathway similar to the Frizzled-signalling pathway, which establishes epithelial planar cell polarity (PCP) in Drosophila. However, the cellular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still unclear. Using high-resolution two-photon confocal imaging followed by computer-assisted reconstruction and motion analysis, we have analysed the movement and morphology of individual cells in three dimensions during the course of gastrulation. We show that in slb-mutant embryos, hypoblast cells within the forming germ ring have slower, less directed migratory movements at the onset of gastrulation. These aberrant cell movements are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. We conclude that slb/wnt11-mediated orientation of cellular processes plays a role in facilitating and stabilising movements of hypoblast cells in the germ ring, thereby pointing at a novel function of the slb/wnt11 signalling pathway for the regulation of migratory cell movements at early stages of gastrulation.

Background: During vertebrate gastrulation, cell polarization and migration are core components in the cellular rearrangements that lead to the formation of the three germ layers, ectoderm, mesoderm, and endoderm. Previous studies have implicated the Wnt/planar cell polarity (PCP) signaling pathway in controlling cell morphology and movement during gastrulation. However, cell polarization and directed cell migration are reduced but not completely abolished in the absence of Wnt/PCP signals; this observation indicates that other signaling pathways must be involved., Results: We show that Phosphoinositide 3-Kinases (PI3Ks) are required at the onset of zebrafish gastrulation in mesendodermal cells for process formation and cell polarization. Platelet Derived Growth Factor (PDGF) functions upstream of PI3K, while Protein Kinase B (PKB), a downstream effector of PI3K activity, localizes to the leading edge of migrating mesendodermal cells. In the absence of PI3K activity, PKB localization and cell polarization are strongly reduced in mesendodermal cells and are followed by slower but still highly coordinated and directed movements of these cells., Conclusions: We have identified a novel role of a signaling pathway comprised of PDGF, PI3K, and PKB in the control of morphogenetic cell movements during gastrulation. Furthermore, our findings provide insight into the relationship between cell polarization and directed cell migration at the onset of zebrafish gastrulation.

Recent studies show that signaling through integrin receptors is required for normal cell movements during Xenopus gastrulation. Integrins function in this process by modulating the activity of cadherin adhesion molecules within tissues undergoing convergence and extension movements.

Wnt genes play important roles in regulating patterning and morphogenesis during vertebrate gastrulation. In zebrafish, slb/wnt11 is required for convergence and extension movements, but not cell fate specification during gastrulation. To determine if other Wnt genes functionally interact with slb/wnt11, we analysed the role of ppt/wnt5 during zebrafish gastrulation. ppt/wnt5 is maternally provided and zygotically expressed at all stages during gastrulation. The analysis of ppt mutant embryos reveals that Ppt/Wnt5 regulates cell elongation and convergent extension movements in posterior regions of the gastrula, while its function in more anterior regions is largely redundant to that of Slb/Wnt11. Frizzled-2 functions downstream of ppt/wnt5, indicating that it might act as a receptor for Ppt/Wnt5 in this process. The characterisation of the role of Ppt/Wnt5 provides insight into the functional diversity of Wnt genes in regulating vertebrate gastrulation movements.

During vertebrate gastrulation, large cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. Zebrafish offer many genetic and experimental advantages for studying vertebrate gastrulation movements. For instance, several mutants, including silberblick, knypek and trilobite, exhibit defects in morphogenesis during gastrulation. The identification of the genes mutated in these lines together with the analysis of the mutant phenotypes has provided new insights into the molecular and cellular mechanisms that underlie vertebrate gastrulation movements.

Vertebrate homologues of the Strabismus/van Gogh (stbm/vang) gene have been implicated in patterning and morphogenesis during gastrulation. Recent work shows that stbm/vang is mutated in zebrafish trilobite mutants and that stbm/vang is required for morphogenesis but not patterning during zebrafish gastrulation.

We have identified widerborst (wdb), a B' regulatory subunit of PP2A, as a conserved component of planar cell polarization mechanisms in both Drosophila and in zebrafish. In Drosophila, wdb acts at two steps during planar polarization of wing epithelial cells. It is required to organize tissue polarity proteins into proximal and distal cortical domains, thus determining wing hair orientation. It is also needed to generate the polarized membrane outgrowth that becomes the wing hair. Widerborst activates the catalytic subunit of PP2A and localizes to the distal side of a planar microtubule web that lies at the level of apical cell junctions. This suggests that polarized PP2A activation along the planar microtubule web is important for planar polarization. In zebrafish, two wdb homologs are required for convergent extension during gastrulation, supporting the conjecture that Drosophila planar cell polarization and vertebrate gastrulation movements are regulated by similar mechanisms.

Members of the Wnt family have been implicated in a variety of developmental processes including axis formation, patterning of the central nervous system and tissue morphogenesis. Recent studies have shown that a Wnt signalling pathway similar to that involved in the establishment of planar cell polarity in Drosophila regulates convergent extension movements during zebrafish and Xenopus gastrulation. This finding provides a good starting point to dissect the complex cell biology and genetic regulation of vertebrate gastrulation movements.

Recent studies on vertebrate homologues of the van gogh/strabismus (vang/stbm) gene, a key player in planar cell polarity signalling in Drosophila, show that vang/stbm is involved in patterning and morphogenesis during vertebrate gastrulation where it modulates two distinct Wnt signals.

Zebrafish embryos homozygous for the masterblind (mbl) mutation exhibit a striking phenotype in which the eyes and telencephalon are reduced or absent and diencephalic fates expand to the front of the brain. Here we show that mbl(-/-) embryos carry an amino-acid change at a conserved site in the Wnt pathway scaffolding protein, Axin1. The amino-acid substitution present in the mbl allele abolishes the binding of Axin to Gsk3 and affects Tcf-dependent transcription. Therefore, Gsk3 activity may be decreased in mbl(-/-) embryos and in support of this possibility, overexpression of either wild-type Axin1 or Gsk3beta can restore eye and telencephalic fates to mbl(-/-) embryos. Our data reveal a crucial role for Axin1-dependent inhibition of the Wnt pathway in the early regional subdivision of the anterior neural plate into telencephalic, diencephalic, and eye-forming territories.

Vertebrate gastrulation involves the specification and coordinated movement of large populations of cells that give rise to the ectodermal, mesodermal and endodermal germ layers. Although many of the genes involved in the specification of cell identity during this process have been identified, little is known of the genes that coordinate cell movement. Here we show that the zebrafish silberblick (slb) locus encodes Wnt11 and that Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation. In the absence of Slb/Wnt11 function, abnormal extension of axial tissue results in cyclopia and other midline defects in the head. The requirement for Slb/Wnt11 is cell non-autonomous, and our results indicate that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue. We also show that the slb phenotype is rescued by a truncated form of Dishevelled that does not signal through the canonical Wnt pathway, suggesting that, as in flies, Wnt signalling might mediate morphogenetic events through a divergent signal transduction cascade. Our results provide genetic and experimental evidence that Wnt activity in lateral tissues has a crucial role in driving the convergent extension movements underlying vertebrate gastrulation.

During the development of the zebrafish nervous system both noi, a zebrafish pax2 homolog, and ace, a zebrafish fgf8 homolog, are required for development of the midbrain and cerebellum. Here we describe a dominant mutation, aussicht (aus), in which the expression of noi and ace is upregulated. In aus mutant embryos, ace is upregulated at many sites in the embryo, while noi expression is only upregulated in regions of the forebrain and midbrain which also express ace. Subsequent to the alterations in noi and ace expression, aus mutants exhibit defects in the differentiation of the forebrain, midbrain and eyes. Within the forebrain, the formation of the anterior and postoptic commissures is delayed and the expression of markers within the pretectal area is reduced. Within the midbrain, En and wnt1 expression is expanded. In heterozygous aus embryos, there is ectopic outgrowth of neural retina in the temporal half of the eyes, whereas in putative homozygous aus embryos, the ventral retina is reduced and the pigmented retinal epithelium is expanded towards the midline. The observation that aus mutant embryos exhibit widespread upregulation of ace raised the possibility that aus might represent an allele of the ace gene itself. However, by crossing carriers for both aus and ace, we were able to generate homozygous ace mutant embryos that also exhibited the aus phenotype. This indicated that aus is not tightly linked to ace and is unlikely to be a mutation directly affecting the ace locus. However, increased Ace activity may underly many aspects of the aus phenotype and we show that the upregulation of noi in the forebrain of aus mutants is partially dependent upon functional Ace activity. Conversely, increased ace expression in the forebrain of aus mutants is not dependent upon functional Noi activity. We conclude that aus represents a mutation involving a locus normally required for the regulation of ace expression during embryogenesis.

In zebrafish, as in other vertebrates, an initially singular eye field within the neural plate has to split during morphogenesis to allow the development of two separated eyes. It has been suggested that anterior progression of midline tissue within the neural plate is involved in the bilateralization of the eye field. Mutations in the recently identified silberblick (slb) gene cause an incomplete separation of the eyes. During gastrulation and early somitogenesis, the ventral midline of the central nervous system (CNS) together with the underlying axial mesendoderm is shortened and broadened in slb embryos. While in wild-type embryos the ventral CNS midline extends to the anterior limit of the neural plate at the end of gastrulation, there is a gap between the anterior tip of the ventral CNS midline and the anterior edge of the neural plate in slb. To investigate the cause for the shortening of the ventral CNS midline in slb we determined the fate of labeled ventral CNS midline cells in wild-type and slb embryos at different stages of development. In slb, anterior migration of ventral CNS midline cells is impaired, which indicates that migration of these cells is needed for elongation of the ventral CNS midline. The anterior shortening of the ventral CNS midline in slb leads to medial instead of bilateral induction of optic stalks followed by a partial fusion of the eyes at later developmental stages. The analysis of the slb phenotype indicates that anterior migration of midline cells within the neural plate is required for proper induction and subsequent bilateralization of an initially singular eye field. These findings may therefore provide a starting point in elucidating the role of neural plate morphogenesis in positioning of the eyes.

The epiphysial region of the dorsal diencephalon is the first site at which neurogenesis occurs in the roof of the zebrafish forebrain. We show that the homeobox containing gene floating head (flh) is required for neurogenesis to proceed in the epiphysis. In flh- embryos, the first few epiphysial neurons are generated, but beyond the 18 somite stage, neuronal production ceases. In contrast, in masterblind- (mbl-) embryos, epiphysial neurons are generated throughout the dorsal forebrain. We show that mbl is required to prevent the expression of flh in dorsal forebrain cells rostral to the epiphysis. Furthermore, epiphysial neurons are not ectopically induced in mbl-/flh- embryos, demonstrating that the epiphysial phenotype of mbl- embryos is mediated by ectopic Flh activity. We propose a role for Flh in linking the signaling pathways that regulate regional patterning to the signaling pathways that regulate neurogenesis.

Mutations giving rise to anatomical defects in the inner ear have been isolated in a large scale screen for mutations causing visible abnormalities in the zebrafish embryo (Haffter, P., Granato, M., Brand, M. et al. (1996) Development 123, 1-36). 58 mutants have been classified as having a primary ear phenotype; these fall into several phenotypic classes, affecting presence or size of the otoliths, size and shape of the otic vesicle and formation of the semicircular canals, and define at least 20 complementation groups. Mutations in seven genes cause loss of one or both otoliths, but do not appear to affect development of other structures within the ear. Mutations in seven genes affect morphology and patterning of the inner ear epithelium, including formation of the semicircular canals and, in some, development of sensory patches (maculae and cristae). Within this class, dog-eared mutants show abnormal development of semicircular canals and lack cristae within the ear, while in van gogh, semicircular canals fail to form altogether, resulting in a tiny otic vesicle containing a single sensory patch. Both these mutants show defects in the expression of homeobox genes within the otic vesicle. In a further class of mutants, ear size is affected while patterning appears to be relatively normal; mutations in three genes cause expansion of the otic vesicle, while in little ears and microtic, the ear is abnormally small, but still contains all five sensory patches, as in the wild type. Many of the ear and otolith mutants show an expected behavioural phenotype: embryos fail to balance correctly, and may swim on their sides, upside down, or in circles. Several mutants with similar balance defects have also been isolated that have no obvious structural ear defect, but that may include mutants with vestibular dysfunction of the inner ear (Granato, M., van Eeden, F. J. M., Schach, U. et al. (1996) Development, 123, 399-413,). Mutations in 19 genes causing primary defects in other structures also show an ear defect. In particular, ear phenotypes are often found in conjunction with defects of neural crest derivatives (pigment cells and/or cartilaginous elements of the jaw). At least one mutant, dog-eared, shows defects in both the ear and another placodally derived sensory system, the lateral line, while hypersensitive mutants have additional trunk lateral line organs.

Somitogenesis is the basis of segmentation of the mesoderm in the trunk and tail of vertebrate embryos. Two groups of mutants with defects in this patterning process have been isolated in our screen for zygotic mutations affecting the embryonic development of the zebrafish (Danio rerio). In mutants of the first group, boundaries between individual somites are invisible early on, although the paraxial mesoderm is present. Later, irregular boundaries between somites are present. Mutations in fused somites (fss) and beamter (bea) affect all somites, whereas mutations in deadly seven (des), after eight (aei) and white tail (wit) only affect the more posterior somites. Mutants of all genes but wit are homozygous viable and fertile. Skeletal stainings and the expression pattern of myoD and snail1 suggest that anteroposterior patterning within individual somites is abnormal. In the second group of mutants, formation of the horizontal myoseptum, which separates the dorsal and ventral part of the myotome, is reduced. Six genes have been defined in this group (you-type genes). you-too mutants show the most severe phenotype; in these the adaxial cells, muscle pioneers and the primary motoneurons are affected, in addition to the horizontal myoseptum. The horizontal myoseptum is also missing in mutants that lack a notochord. The similarity of the somite phenotype in mutants lacking the notochord and in the you-type mutants suggests that the genes mutated in these two groups are involved in a signaling pathway from the notochord, important for patterning of the somites.

In the zebrafish, Danio rerio, a caudal and pectoral fin fold develop during embryogenesis. At larval stages the caudal fin fold is replaced by four different fins, the unpaired anal, dorsal and tail fins. In addition the paired pelvic fins are formed. We have identified a total of 118 mutations affecting larval fin formation. Mutations in 11 genes lead to abnormal morphology or degeneration of both caudal and pectoral fin folds. Most mutants survive to adulthood and form a surprisingly normal complement of adult fins. Mutations in nine genes result in an increased or reduced size of the pectoral fins. Interestingly, in mutants of one of these genes, dackel (dak), pectoral fin buds form initially, but later the fin epithelium fails to expand. Expression of sonic hedgehog mRNA in the posterior mesenchyme of the pectoral fin bud is initiated in dak embryos, but not maintained. Mutations in five other genes affect adult fin but not larval fin development. Two mutants, longfin (lof) and another longfin (alf) have generally longer fins. Stein und bein (sub) has reduced dorsal and pelvic fins, whereas finless (fls) and wanda (wan) mutants affect all adult fins. Finally, mutations in four genes causing defects in embryonic skin formation will be briefly reported.

In a large-scale screen, we isolated mutants displaying a specific visible phenotype in embryos or early larvae of the zebrafish, Danio rerio. Males were mutagenized with ethylnitrosourea (ENU) and F2 families of single pair matings between sibling F1 fish, heterozygous for a mutagenized genome, were raised. Egg lays were obtained from several crosses between F2 siblings, resulting in scoring of 3857 mutagenized genomes. F3 progeny were scored at the second, third and sixth day of development, using a stereomicroscope. In a subsequent screen, fixed embryos were analyzed for correct retinotectal projection. A total of 4264 mutants were identified. Two thirds of the mutants displaying rather general abnormalities were eventually discarded. We kept and characterized 1163 mutants. In complementation crosses performed between mutants with similar phenotypes, 894 mutants have been assigned to 372 genes. The average allele frequency is 2.4. We identified genes involved in early development, notochord, brain, spinal cord, somites, muscles, heart, circulation, blood, skin, fin, eye, otic vesicle, jaw and branchial arches, pigment pattern, pigment formation, gut, liver, motility and touch response. Our collection contains alleles of almost all previously described zebrafish mutants. From the allele frequencies and other considerations we estimate that the 372 genes defined by the mutants probably represent more than half of all genes that could have been discovered using the criteria of our screen. Here we give an overview of the spectrum of mutant phenotypes obtained, and discuss the limits and the potentials of a genetic saturation screen in the zebrafish.

In a screen for early developmental mutants of the zebrafish, we have identified mutations specifically affecting the internal organs. We identified 53 mutations affecting the cardiovascular system. Nine of them affect specific landmarks of heart morphogenesis. Mutations in four genes cause a failure in the fusion of the bilateral heart primordia, resulting in cardia bifida. In lonely atrium, no heart venticle is visible and the atrium is directly fused to the outflow tract. In the overlooped mutant, the relative position of the two heart chambers is distorted. The heart is enormously enlarged in the santa mutant. In two mutants, scotch tape and superglue, the cardiac jelly between the two layers of the heart is significantly reduced. We also identified a number of mutations affecting the function of the heart. The mutations affecting heart function can be subdivided into two groups, one affecting heart contraction and another affecting the rhythm of the heart beat. Among the contractility group of mutants are 5 with no heart beat at all and 15 with a reduced heart beat of one or both chambers. 6 mutations are in the rhythmicity group and specifically affect the beating pattern of the heart. Mutations in two genes, bypass and kurzschluss, cause specific defects in the circulatory system. In addition to the heart mutants, we identified 23 mutations affecting the integrity of the liver, the intestine or the kidney. In this report, we demonstrate that it is feasible to screen for genes specific for the patterning or function of certain internal organs in the zebrafish. The mutations presented here could serve as an entry point to the establishment of a genetic hierarchy underlying organogenesis.

We identified four zebrafish mutants with defects in forebrain induction and patterning during embryogenesis. The four mutants define three genes: masterblind (mbl), silberblick (slb), and knollnase (kas). In mbl embryos, the anterior forebrain acquires posterior forebrain characteristics: anterior structures such as the eyes, olfactory placodes and the telencephalon are missing, whereas the epiphysis located in the posterior forebrain is expanded. In slb embryos, the extension of the embryonic axis is initially delayed and eventually followed by a partial fusion of the eyes. Finally, in kas embryos, separation of the telencephalic primordia is incomplete and dorsal midline cells fail to form a differentiated roof plate. Analysis of the mutant phenotypes indicates that we have identified genes essential for the specification of the anterior forebrain (mbl), positioning of the eyes (slb) and differentiation of the roof plate (kas). In an appendix to this study we list mutants showing alterations in the size of the eyes and abnormal differentiation of the lenses.

In a large scale screen for mutants that affect the early development of the zebrafish, 109 mutants were found that cause defects in the formation of the jaw and the more posterior pharyngeal arches. Here we present the phenotypic description and results of the complementation analysis of mutants belonging to two major classes: (1) mutants with defects in the mandibular and hyoid arches and (2) mutants with defects in cartilage differentiation and growth in all arches. Mutations in four of the genes identified during the screen show specific defects in the first two arches and leave the more posterior pharyngeal arches largely unaffected (schmerle, sucker, hoover and sturgeon). In these mutants ventral components of the mandibular and hyoid arches are reduced (Meckel's cartilage and ceratohyal cartilage) whereas dorsal structures (palatoquadrate and hyosymplectic cartilages) are of normal size or enlarged. Thus, mutations in single genes cause defects in the formation of first and second arch structures but also differentially affect development of the dorsal and ventral structures within one arch. In 27 mutants that define at least 8 genes, the differentiation of cartilage and growth is affected. In hammerhead mutants particularly the mesodermally derived cartilages are reduced, whereas jellyfish mutant larvae are characterized by a severe reduction of all cartilaginous elements, leaving only two pieces in the position of the ceratohyal cartilages. In all other mutant larvae all skeletal elements are present, but consist of smaller and disorganized chondrocytes. These mutants also exhibit shortened heads and reduced pectoral fins. In homozygous knorrig embryos, tumor-like outgrowths of chondrocytes occur along the edges of all cartilaginous elements. The mutants presented here may be valuable tools for elucidating the genetic mechanisms that underlie the development of the mandibular and the hyoid arches, as well as the process of cartilage differentiation.

Tissues of the dorsal midline of vertebrate embryos, such as notochord and floor plate, have been implicated in inductive interactions that pattern the neural tube and somites. In our screen for embryonic visible mutations in the zebrafish we found 113 mutations in more than 27 genes with altered body shape, often with additional defects in CNS development. We concentrated on a subgroup of mutations in ten genes (the midline-group) that cause defective development of the floor plate. By using floor plate markers, such as the signaling molecule sonic hedgehog, we show that the schmalspur (sur) gene is needed for early floor plate development, similar to one-eyed-pinhead (oep) and the previously described cyclops (cyc) gene. In contrast to oep and cyc, sur embryos show deletions of ventral CNS tissue restricted to the mid- and hindbrain, whereas the forebrain appears largely unaffected. In the underlying mesendodermal tissue of the head, sur is needed only for development of the posterior prechordal plate, whereas oep and cyc are required for both anterior and posterior prechordal plate development. Our analysis of sur mutants suggests that defects within the posterior prechordal plate may cause aberrant development of ventral CNS structures in the mid- and hindbrain. Later development of the floor plate is affected in mutant chameleon, you-too, sonic-you, iguana, detour, schmalhans and monorail embryos; these mutants often show additional defects in tissues that are known to depend on signals from notochord and floor plate. For example, sur, con and yot mutants show reduction of motor neurons; median deletions of brain tissue are seen in sur, con and yot embryos; and cyc, con, yot, igu and dtr mutants often show no or abnormal formation of the optic chiasm. We also find fusions of the ventral neurocranium for all midline mutants tested, which may reveal a hitherto unrecognized function of the midline in influencing differentiation of neural crest cells at their destination. As a working hypothesis, we propose that midline-group genes may act to maintain proper structure and inductive function of zebrafish midline tissues.