Control of microtubule dynamics is crucial for cell migration. We analyzed regulation of microtubule network dynamics in the zebrafish yolk cell during epiboly, the earliest coordinated gastrulation movement. We labeled microtubules with EMTB-3GFP and EB3-mCherry to visualize and measure microtubule dynamics by TIRF microscopy live imaging. Yolk cell microtubules dynamics is temporally modulated during epiboly progression. We used maternal zygotic Pou5f3 mutant (MZspg) embryos, which develop strong distortions of microtubule network organization and epiboly retardation, to investigate genetic control of microtubule dynamics. In MZspg embryos, microtubule plus-end growth tracks move slower and are less straight compared to wild-type. MZspg embryos have altered steroidogenic enzyme expression, resulting in increased pregnenolone and reduced progesterone levels. We show that progesterone positively affects microtubule plus-end growth and track straightness. Progesterone may thus act as a non-cell-autonomous regulator of microtubule dynamics across the large yolk cell, and may adjust differing demands on microtubule dynamics and stability during initiation and progression phases of epiboly.

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

During mouse gastrulation, cells in the primitive streak undergo epithelial-mesenchymal transformation and the resulting mesenchymal cells migrate out laterally to form mesoderm and definitive endoderm across the entire embryonic cylinder. The mechanisms underlying mesoderm and endoderm specification, migration, and allocation are poorly understood. In this study, we focused on the function of mouse Cripto, a member of the EGF-CFC gene family that is highly expressed in the primitive streak and migrating mesoderm cells on embryonic day 6.5. Conditional inactivation of Cripto during gastrulation leads to varied defects in mesoderm and endoderm development. Mutant embryos display accumulation of mesenchymal cells around the shortened primitive streak indicating a functional requirement of Cripto during the formation of mesoderm layer in gastrulation. In addition, some mutant embryos showed poor formation and abnormal allocation of definitive endoderm cells on embryonic day 7.5. Consistently, many mutant embryos that survived to embryonic day 8.5 displayed defects in ventral closure of the gut endoderm causing cardia bifida. Detailed analyses revealed that both the Fgf8-Fgfr1 pathway and p38 MAP kinase activation are partially affected by the loss of Cripto function. These results demonstrate a critical role for Cripto during mouse gastrulation, especially in mesoderm and endoderm formation and allocation.

We have undertaken the first detailed analysis of Rho GTPase function during vertebrate development by analyzing how RhoA and Rac1 control convergent extension of axial mesoderm during Xenopus gastrulation. Monitoring of a number of parameters in time-lapse recordings of mesoderm explants revealed that Rac and Rho have both distinct and overlapping roles in regulating the motility of axial mesoderm cells. The cell behaviors revealed by activated or inhibitory versions of these GTPases in native tissue were clearly distinct from those previously documented in cultured fibroblasts. The dynamic properties and polarity of protrusive activity, along with lamellipodia formation, were controlled by the two GTPases operating in a partially redundant manner, while Rho and Rac contributed separately to cell shape and filopodia formation. We propose that Rho and Rac operate in distinct signaling pathways that are integrated to control cell motility during convergent extension.

As the primary driving forces of gastrulation, convergence and extension (C&E) movements lead to a medio-lateral narrowing and an anterior-posterior elongation of the embryonic body axis. Histone methylation as a post-translational modification plays a critical role in early embryonic development, but its functions in C&E movements remain largely unknown. Here, we show that the setdb2-dvr1 transcriptional cascade plays a critical role in C&E movements during zebrafish gastrulation. Knockdown of Setdb2, a SET domain-containing protein possessing a potential histone H3K9 methyltransferase activity, induced abnormal C&E movements, resulting in anterior-posterior shortening and medio-lateral expansion of the embryonic axis, as well as abnormal notochord cell polarity. Furthermore, we found that Setdb2 functions through fine-tuning the expression of dvr1, a ligand of the TGF-β superfamily, to an appropriate level to ensure proper C&E movements in a non-cell-autonomous manner. In addition, both overexpression and knockdown of Dvr1 at the one-cell stage resulted in defects at epiboly and C&E. These data demonstrate that Setdb2 is a novel regulator for C&E movements and acts by modulating the expression level of dvr1, suggesting that Dvr1 acts as a direct and essential mediator for C&E cell movements.

Intricate interactions between the Wnt and Bmp signaling pathways pattern the gastrulating vertebrate embryo using a network of secreted protein ligands and inhibitors. While many of these proteins are expressed post-gastrula, their later roles have typically remained unclear, obscured by the effects of early perturbation. We find that Bmp signaling continues during somitogenesis in zebrafish embryos, with high activity in a small region of the mesodermal progenitor zone at the posterior end of the embryo. To test the hypothesis that Bmp inhibitors expressed just anterior to the tailbud are important to restrain Bmp signaling we produced a new zebrafish transgenic line, allowing temporal cell-autonomous activation of Bmp signaling and thereby bypassing the effects of the Bmp inhibitors. Ectopic activation of Bmp signaling during somitogenesis results in severe defects in the tailbud, including altered morphogenesis and gene expression. We show that these defects are due to non-autonomous effects on the tailbud, and present evidence that the tailbud defects are caused by alterations in Wnt signaling. We present a model in which the posteriorly expressed Bmp inhibitors function during somitogenesis to constrain Bmp signaling in the tailbud in order to allow normal expression of Wnt inhibitors in the presomitic mesoderm, which in turn constrain the levels of canonical and non-canonical Wnt signaling in the tailbud.

Establishment of the dorsal-ventral (DV) axis of the Drosophila embryo depends on ventral activation of the maternal Toll pathway, which creates a gradient of the NFkB/c-rel-related transcription factor dorsal. Signaling through the maternal BMP pathway also alters the dorsal gradient, probably by regulating degradation of the IkB homologue Cactus. The BMP4 homologue decapentaplegic (dpp) and the BMP antagonist short gastrulation (sog) are expressed by follicle cells during mid-oogenesis, but it is unknown how they affect embryonic patterning following fertilization. Here, we provide evidence that maternal Sog and Dpp proteins are secreted into the perivitelline space where they remain until early embryogenesis to modulate Cactus degradation, enabling their dual function in patterning the eggshell and embryo. We find that metalloproteases encoded by tolloid (tld) and tolkin (tok), which cleave Sog, are expressed by follicle cells and are required to generate DV asymmetry in the Dpp signal. Expression of tld and tok is ventrally restricted by the TGF-alpha ligand encoded by gurken, suggesting that signaling via the EGF receptor pathway may regulate embryonic patterning through two independent mechanisms: by restricting the expression of pipe and thereby activation of Toll signaling and by spatially regulating BMP activity.

Cell migration is the main driver of the evolutionarily conserved process of gastrulation, which shapes metazoan embryo morphology. The molecular and cellular mechanisms of cell migration during gastrulation though well researched lacks an understanding of the contribution of cell sizes to collective cell migration. This is especially important during the early phase of metazoan development, which is dominated by constantly changing cell sizes in the background of which cells migrate en mass to shape the embryo. Here we investigate this phenomenon in zebrafish embryos, a model system in which early cell divisions causes cell sizes to decrease naturally over time as cells migrate collectively to sculpt the embryonic body plan. Because mutations that can perturb cell sizes so early in development do not exist, we generate haploid and tetraploid zebrafish embryos and show that cell sizes in such embryos are smaller and larger than the diploid norm, respectively. Cells in embryos made of smaller or larger than normal cells migrate sub-optimally, leading to gastrulation defects. Gene expression analysis suggests that the observed defects originate from altered cell size, and not from pleiotropic effects of altered ploidy. This interpretation is strengthened when gastrulation defects are rescued by increasing cell sizes in embryos wherein cell sizes are smaller than normal. We show that the migration defects are cell-autonomous by live imaging migrating haploid and tetraploid cells during gastrulation in chimeric diploid embryos. Analysis of membrane protrusion dynamics in single cells shows that cells normally extend protrusions non-uniformly during migration, a phenomenon which is perturbed when cell sizes deviate from the norm. Thus, an optimal range of developmental stage-specific cell sizes appears necessary for collective cell migration to correctly position cells in space and time to shape an amorphous ball of blastoderm into an embryo.

In Cnidaria, modes of gastrulation to produce the two body layers vary greatly between species. In the hydrozoan species Clytia hemisphaerica gastrulation involves unipolar ingression of presumptive endoderm cells from an oral domain of the blastula, followed by migration of these cells to fill the blastocoel with concomitant narrowing of the gastrula and elongation along the oral-aboral axis. We developed a 2D computational boundary model capable of simulating the morphogenetic changes during embryonic development from early blastula stage to the end of gastrulation. Cells are modeled as polygons with elastic membranes and cytoplasm, colliding and adhering to other cells, and capable of forming filopodia. With this model we could simulate compaction of the embryo preceding gastrulation, bottle cell formation, ingression, and intercalation between cells of the ingressing presumptive endoderm. We show that embryo elongation is dependent on the number of endodermal cells, low endodermal cell-cell adhesion, and planar cell polarity (PCP). When the strength of PCP is reduced in our model, resultant embryo morphologies closely resemble those reported previously following morpholino-mediated knockdown of the core PCP proteins Strabismus and Frizzled. Based on our results, we postulate that cellular processes of apical constriction, compaction, ingression, and then reduced cell-cell adhesion and mediolateral intercalation in the presumptive endoderm, are required and when combined, sufficient for Clytia gastrulation.

Cells are the principal component of tissues and can drive morphogenesis through dynamic changes in structure and interaction. During gastrulation, the primary morphogenetic event of early development, cells change shape, exchange neighbors, and migrate long distances to establish cell layers that will form the tissues of the adult animal. Outside of Drosophila, little is known about how changes in cell behavior might drive gastrulation among arthropods. Here, we focus on three cell populations that form two aggregations during early gastrulation in the crustacean Parhyale hawaiensis. Using cytoskeletal markers and lineage tracing we observe bottle cells in anterior and visceral mesoderm precursors as gastrulation commences, and find that both Cytochalasin D, an inhibitor of actin polymerization, and ROCKOUT, an inhibitor of Rho-kinase activity, prevent gastrulation. Furthermore, by ablating specific cells, we show that each of the three populations acts independently during gastrulation, confirming previous hypotheses that cell behavior during Parhyale gastrulation relies on intrinsic signals instead of an inductive mechanism.

Netrin is a remarkably conserved midline landmark, serving as a chemotactic factor that organizes the bilateral neural architecture in the post-gastrula bilaterian embryos. Netrin signal also guides cell migration in many other neural and non-neural organogenesis events in later developmental stages but has never been found to participate in gastrulation - the earliest cell migration in metazoan embryogenesis. Here, we found that the netrin signaling molecules and their receptors are expressed during gastrulation of the leech Helobdella. Intriguingly, Hau-netrin-1 was expressed in the N lineage, which gives rise in part to the ventral midline of ectoderm, at the onset of gastrulation. We demonstrated that the N lineage is required for the entrance of mesoderm into the germinal band and that misexpression of Hau-netrin-1 in early gastrulation prevented mesoderm from entering the germinal band. Together, these results suggested that Hau-netrin-1 secreted by the N lineage guides mesoderm migration during germinal band assembly. Furthermore, ectopic expression of Hau-netrin-1 after the completion of germinal band assembly disrupted the epibolic migration of the germinal bands in a later stage of gastrulation. Thus, Hau-netrin-1 is likely involved in two distinct events in sequential stages of leech gastrulation: the assembly of germinal bands in early gastrulation and their epibolic migration in mid-gastrulation. Given that the leech netrin is expressed in the precursor cells of the ventral midline during gastrulation, we propose that a heterochronic change from the midline netrin expression had taken place in the evolution of a novel mode of gastrulation in the directly developing leech embryos.

We previously identified the protein Lbh as necessary for cranial neural crest (CNC) cell migration in Xenopus through the use of morpholinos. However, Lbh is a maternally deposited protein and morpholinos achieve knockdowns through prevention of translation. In order to investigate the role of Lbh in earlier embryonic events, we employed the new technique "Trim-Away" to degrade this maternally deposited protein. Trim-Away utilizes the E3 ubiquitin ligase trim21 to degrade proteins targeted with an antibody and was developed in mammalian systems. Our results show that Xenopus is amenable to the Trim-Away technique. We also show that early knockdown of Lbh in Xenopus results in defects in gastrulation that present with a decrease in fibronectin matrix assembly, an increased in mesodermal cell migration and decrease in endodermal cell cohesion. We further show that the technique is also effective on a second abundant maternal protein PACSIN2. We discuss potential advantages and limit of the technique in Xenopus embryos as well as the mechanism of gastrulation inhibition.

Endomesodermal cell fate specification and archenteron formation during gastrulation are tightly linked developmental processes in most metazoans. However, studies have shown that in the anthozoan cnidarian Nematostella vectensis, Wnt/β-catenin (cWnt) signalling-mediated endomesodermal cell fate specification can be experimentally uncoupled from Wnt/Planar Cell Polarity (PCP) signalling-mediated primary archenteron invagination. The upstream signalling mechanisms regulating cWnt signalling-dependent endomesoderm cell fate specification and Wnt/PCP signalling-mediated primary archenteron invagination in Nematostella embryos are not well understood. By screening for potential upstream mediators of cWnt and Wnt/PCP signalling, we identified two Nematostella Frizzled homologs that are expressed early in development. NvFzd1 is expressed maternally and in a broad pattern during early development while NvFzd10 is zygotically expressed at the animal pole in blastula stage embryos and is restricted to the invaginating cells of the presumptive endomesoderm. Molecular and morphological characterization of NvFzd1 and NvFzd10 knock-down phenotypes provide evidence for distinct regulatory roles for the two receptors in endomesoderm cell fate specification and primary archenteron invagination. These results provide further experimental evidence for the independent regulation of endomesodermal cell fate specification and primary archenteron invagination during gastrulation in Nematostella. Moreover, these results provide additional support for the previously proposed two-step model for the independent evolution of cWnt-mediated cell fate specification and Wnt/PCP-mediated primary archenteron invagination.

The ancestral state of animal gastrulation and its bearing for our understanding of bilaterian evolution still is one of the most controversially discussed topics in the field of evolutionary and developmental biology. One hypothesis, the so-called amphistomy scenario, suggests the presence of a slit-like blastopore in the last common ancestor of Bilateria. Onychophoran ontogeny at least superficially appears to support this scenario since a ventral groove clearly forms during gastrulation. The origin and nature of this groove, however, is another matter of ongoing controversy; i.e. the question of whether this structure actually represents the blastopore, or at least part of it. Recent research using genetic markers argued against the furrow representing a blastoporal structure. Here we investigate the origin of endoderm, which usually originates from the blastopore. We find conserved expression patterns of the endoderm- and gut-marker genes GATA456, GATA123, Hnf4 and fkh during gut development, and discuss the formation of the onychophoran gut in comparison with that in a range of arthropods. Despite expression of endodermal markers in and around the furrow we do not find convincing evidence that the furrow may be part of the blastopore, and thus we suggest that onychophoran development does not yield support for the amphistomy scenario.

The formation of the anteroposterior axis in mice requires a Wnt3-dependent symmetry-breaking event that leads to the formation of the primitive streak and gastrulation. Wnt3 is expressed sequentially in two distinct areas of the mouse embryo before the appearance of the primitive streak; first in the posterior visceral endoderm and soon after in the adjacent posterior epiblast. Hence, although an axial requirement for Wnt3 is well established, its temporal and tissue specific requirements remain an open question. Here, we report the conditional inactivation of Wnt3 in the epiblast of developing mouse embryos. Contrary to previous studies, our data shows that embryos lacking Wnt3 specifically in the epiblast are able to initiate gastrulation and advance to late primitive streak stages but fail to thrive and are resorbed by E9.5. At the molecular level, we provide evidence that Wnt3 regulates its own expression and that of other primitive streak markers via activation of the canonical Wnt signaling pathway.

The ubiquitously expressed Polycomb Group protein Yin-Yang1 (YY1) is believed to regulate gene expression through direct binding to DNA elements found in promoters or enhancers of target loci. Additionally, YY1 contains diverse domains that enable a plethora of protein-protein interactions, including association with the Oct4/Sox2 pluripotency complex and Polycomb Group silencing complexes. To elucidate the in vivo role of YY1 during gastrulation, we generated embryos with an epiblast specific deletion of Yy1. Yy1 conditional knockout (cKO) embryos initiate gastrulation, but both primitive streak formation and ingression through the streak is severely impaired. These streak descendants fail to repress E-Cadherin and are unable to undergo an appropriate epithelial to mesenchymal transition (EMT). Intriguingly, overexpression of Nodal and concomitant reduction of Lefty2 are observed in Yy1 cKO embryos, suggesting that YY1 is normally required for proper Nodal regulation during gastrulation. Furthermore, definitive endoderm is specified but fails to properly integrate into the outer layer. Although anterior neuroectoderm is specified, mesoderm production is severely restricted. We show that YY1 directly binds to the Lefty2 locus in E7.5 embryos and that pharmacological inhibition of Nodal signaling partially restores mesoderm production in Yy1 cKO mutant embryos. Our results reveal critical requirements for YY1 during several important developmental processes, including EMT and regulation of Nodal signaling. These results are the first to elucidate the diverse role of YY1 during gastrulation in vivo.

Formation of the nervous system initially requires the acquisition of neural identity, which is achieved through the inhibition of epidermalizing factors. A regional patterning then takes place within the neural plate through the activity of caudalizing factors. These two processes are tightly regulated early in development by the dorsal organizer. Here, we show that, in zebrafish embryos, two transcription factors, FoxA3 and Goosecoid, coexpressed at the dorsal blastula margin, are required for the definition of anterior neural fate. Their inactivation results in deletions of anterior head structures associated with an increase of Wnt8 activity at the dorsal blastula margin. These phenotypes can be fully rescued by overexpression of Wnt inhibitors or by inactivation of wnt8a. Altogether, foxA3 and goosecoid cooperate to promote formation of anterior neural tissue by protecting, as early as blastula stage, presumptive anterior neural cells from an irreversible caudalization by the posteriorizing factor Wnt8a.

The severity of numerous developmental abnormalities can vary widely despite shared genetic causes. Mice deficient in Twisted gastrulation (Twsg1(-/-)) display such phenotypic variation, developing a wide range of craniofacial malformations on an isogenic C57BL/6 strain background. To examine the molecular basis for this reduced penetrance and variable expressivity, we used exon microarrays to analyze gene expression in mandibular arches from several distinct, morphologically defined classes of Twsg1(-/-) and wild type (WT) embryos. Hierarchical clustering analysis of transcript levels identified numerous differentially expressed genes, clearly distinguishing severely affected and unaffected Twsg1(-/-) mutants from WT embryos. Several genes that play well-known roles in craniofacial development were upregulated in unaffected Twsg1(-/-) mutant embryos, suggesting that they may compensate for the loss of TWSG1. Imprinted genes were overrepresented among genes that were differentially expressed particularly between affected and unaffected mutants. The most severely affected embryos demonstrated increased p53 signaling and increased expression of its target, Trp53inp1. The frequency of craniofacial defects significantly decreased with a reduction of p53 gene dosage from 44% in Twsg1(-/-)p53(+/+) pups (N=675) to 30% in Twsg1(-/-)p53(+/-) (N=47, p=0.04) and 15% in Twsg1(-/-)p53(-/-) littermates (N=39, p=0.001). In summary, these results demonstrate that phenotypic variability in Twsg1(-/-) mice is associated with differential expression of certain developmentally regulated genes, and that craniofacial defects can be partially rescued by reduced p53 levels. We postulate that variable responses to stress may contribute to variable craniofacial phenotypes by triggering differential expression of genes and variable cellular apoptosis.

Knowledge of the molecular mechanisms regulating cell ingression, epithelial-mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.

PTEN phosphatase mediates several developmental cues involving cell proliferation, growth, death, and migration. We investigated the function of the PTEN gene at the transition from the cell proliferation state to morphogenesis around the midblastula transition (MBT) and gastrulation in Xenopus embryos. An immunoblotting analysis indicated that PTEN expresses constantly through embryogenesis. By up- or down-regulating PTEN activity using overexpression of the active form or C terminus of PTEN before MBT, we induced elongation of the cell cycle time just before MBT or maintained its speed even after MBT, respectively. The disruption of the cell cycle time by changing the activity of PTEN delayed gastrulation after MBT. In addition, PTEN began to localize to the plasma membranes and nuclei at MBT. Overexpression of a membrane-localizing mutant of PTEN caused dephosphorylation of Akt, whereas overexpression of the C terminus of PTEN caused phosphorylation of Akt and inhibited the localization of EGFP-PTEN to the plasma membranes and nuclei. These results indicate that an appropriate PTEN activity, probably regulated by its differential localization, is necessary for coordinating cell proliferation and early morphogenesis.