The Integrator Complex Subunit 6 (Ints6) Confines the Dorsal Organizer in Vertebrate Embryogenesis

Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.     

Sequential antagonism of early and late Wnt-signaling by zebrafish colgate promotes dorsal and anterior fates

The establishment of the vertebrate body plan involves patterning of the ectoderm, mesoderm, and endoderm along the dorsoventral and antero-posterior axes. Interactions among numerous signaling molecules from several multigene families, including Wnts, have been implicated in regulating these processes. Here we provide evidence that the zebrafish colgate(b382) (col) mutation results in increased Wnt signaling that leads to defects in dorsal and anterior development. col mutants display early defects in dorsoventral patterning manifested by a decrease in the expression of dorsal shield-specific markers and ectopic expression of ventrolaterally expressed genes during gastrulation. In addition to these early patterning defects, col mutants display a striking regional posteriorization within the neuroectoderm, resulting in a reduction in anterior fates and an expansion of posterior fates within the forebrain and midbrain-hindbrain regions. We are able to correlate these phenotypes to the overactivation of Wnt signaling in col mutants. The early dorsal and anterior patterning phenotypes of the col mutant embryos are selectively rescued by inactivation of Wnt8 function by morpholino translational interference. In contrast, the regionalized neuroectoderm posterioriorization phenotype is selectively rescued by morpholino-mediated inactivation of Wnt8b. These results suggest that col-mediated antagonism of early and late Wnt-signaling activity during gastrulation is normally required sequentially for both early dorsoventral patterning and the specification and patterning of regional fates within the anterior neuroectoderm.     

The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice

Recent work has identified LDL receptor-related family members, Lrp5 and Lrp6, as co-receptors for the transduction of Wnt signals. Our analysis of mice carrying mutations in both Lrp5 and Lrp6 demonstrates that the functions of these genes are redundant and are essential for gastrulation. Lrp5;Lrp6 double homozygous mutants fail to establish a primitive streak, although the anterior visceral endoderm and anterior epiblast fates are specified. Thus, Lrp5 and Lrp6 are required for posterior patterning of the epiblast, consistent with a role in transducing Wnt signals in the early embryo. Interestingly, Lrp5(+/-);Lrp6(-/-) embryos die shortly after gastrulation and exhibit an accumulation of cells at the primitive streak and a selective loss of paraxial mesoderm. A similar phenotype is observed in Fgf8 and Fgfr1 mutant embryos and provides genetic evidence in support of a molecular link between the Fgf and Wnt signaling pathways in patterning nascent mesoderm. Lrp5(+/-);Lrp6(-/-) embryos also display an expansion of anterior primitive streak derivatives and anterior neurectoderm that correlates with increased Nodal expression in these embryos. The effect of reducing, but not eliminating, Wnt signaling in Lrp5(+/-);Lrp6(-/-) mutant embryos provides important insight into the interplay between Wnt, Fgf and Nodal signals in patterning the early mouse embryo.     

Maternal xNorrin, a canonical Wnt signaling agonist and TGF-β antagonist, controls early neuroectoderm specification in Xenopus

Dorsal-ventral specification in the amphibian embryo is controlled by β-catenin, whose activation in all dorsal cells is dependent on maternal Wnt11. However, it remains unknown whether other maternally secreted factors contribute to β-catenin activation in the dorsal ectoderm. Here, we show that maternal Xenopus Norrin (xNorrin) promotes anterior neural tissue formation in ventralized embryos. Conversely, when xNorrin function is inhibited, early canonical Wnt signaling in the dorsal ectoderm and the early expression of the zygotic neural inducers Chordin, Noggin, and Xnr3 are severely suppressed, causing the loss of anterior structures. In addition, xNorrin potently inhibits BMP- and Nodal/Activin-related functions through direct binding to the ligands. Moreover, a subset of Norrin mutants identified in humans with Norrie disease retain Wnt activation but show defective inhibition of Nodal/Activin-related signaling in mesoderm induction, suggesting that this disinhibition causes Norrie disease. Thus, xNorrin is an unusual molecule that acts on two major signaling pathways, Wnt and TGF-β, in opposite ways and is essential for early neuroectoderm specification.     

Oral-aboral patterning and gastrulation of sea urchin embryos depend on sulfated glycosaminoglycans

Glycosaminoglycans (GAGs) are a heavily sulfated component of the extracellular matrix (ECM) implicated in a variety of cell signaling events involved in patterning of embryos. Embryos of the sea urchin Strongylocentrotus purpuratus were exposed to several inhibitors that disrupt GAG function during development. Treatment with chlorate, a general inhibitor of sulfation that leads to undersulfated GAGs, reduced sulfation of the urchin blastocoelar ECM. It also prevented correct specification of the oral-aboral axis and mouth formation, resulting in a radialized phenotype characterized by the lack of an oral field, incomplete gastrulation and formation of multiple skeletal spicule rudiments. Oral markers were initially expressed in most of the prospective ectoderm of chlorate-treated early blastulae, but then declined as aboral markers became expressed throughout most of the ectoderm. Nodal expression in the presumptive oral field is necessary and sufficient to specify the oral-aboral axis in urchins. Several lines of evidence suggest a deregulation of Nodal signaling is involved in the radialization caused by chlorate: (1) Radial embryos resemble those in which Nodal expression was knocked down. (2) Chlorate disrupted localized nodal expression in oral ectoderm, even when applied after the oral-aboral axis is specified and expression of other oral markers is resistant to treatment. (3) Inhibition with SB-431542 of ALK-4/5/7 receptors that mediate Nodal signaling causes defects in ectodermal patterning similar to those caused by chlorate. (4) Intriguingly, treatment of embryos with a sub-threshold dose of SB-431542 rescued the radialization caused by low concentrations of chlorate. Our results indicate important roles for sulfated GAGs in Nodal signaling and oral-aboral axial patterning, and in the cellular processes necessary for archenteron extension and mouth formation during gastrulation. We propose that interaction of the Nodal ligand with sulfated GAGs limits its diffusion, and is required to specify an oral field in the urchin embryo and organize the oral-aboral axis.     

A conserved role for the nodal signaling pathway in the establishment of dorso-ventral and left-right axes in deuterostomes

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.     

Back and forth between cell fate specification and movement during vertebrate gastrulation

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.     

Direct and indirect control of oral ectoderm regulatory gene expression by Nodal signaling in the sea urchin embryo

The Nodal signaling pathway is known from earlier work to be an essential mediator of oral ectoderm specification in the sea urchin embryo, and indirectly, of aboral ectoderm specification as well. Following expression of the Nodal ligand in the future oral ectoderm during cleavage, a sequence of regulatory gene activations occur within this territory which depend directly or indirectly on nodal gene expression. Here we describe additional regulatory genes that contribute to the oral ectoderm regulatory state during specification in Strongylocentrotus purpuratus, and show how their spatial expression changes dynamically during development. By means of system wide perturbation analyses we have significantly improved current knowledge of the epistatic relations among the regulatory genes of the oral ectoderm. From these studies there emerge diverse circuitries relating downstream regulatory genes directly and indirectly to Nodal signaling. A key intermediary regulator, the role of which had not previously been discerned, is the not gene. In addition to activating several genes earlier described as targets of Nodal signaling, the not gene product acts to repress other oral ectoderm genes, contributing crucially to the bilateral spatial organization of the embryonic oral ectoderm.     

Regional requirements for Dishevelled signaling during Xenopus gastrulation: separable effects on blastopore closure, mesendoderm internalization and archenteron formation

During amphibian gastrulation, the embryo is transformed by the combined actions of several different tissues. Paradoxically, many of these morphogenetic processes can occur autonomously in tissue explants, yet the tissues in intact embryos must interact and be coordinated with one another in order to accomplish the major goals of gastrulation: closure of the blastopore to bring the endoderm and mesoderm fully inside the ectoderm, and generation of the archenteron. Here, we present high-resolution 3D digital datasets of frog gastrulae, and morphometrics that allow simultaneous assessment of the progress of convergent extension, blastopore closure and archenteron formation in a single embryo. To examine how the diverse morphogenetic engines work together to accomplish gastrulation, we combined these tools with time-lapse analysis of gastrulation, and examined both wild-type embryos and embryos in which gastrulation was disrupted by the manipulation of Dishevelled (Xdsh) signaling. Remarkably, although inhibition of Xdsh signaling disrupted both convergent extension and blastopore closure, mesendoderm internalization proceeded very effectively in these embryos. In addition, much of archenteron elongation was found to be independent of Xdsh signaling, especially during the second half of gastrulation. Finally, even in normal embryos, we found a surprising degree of dissociability between the various morphogenetic processes that occur during gastrulation. Together, these data highlight the central role of PCP signaling in governing distinct events of Xenopus gastrulation, and suggest that the loose relationship between morphogenetic processes may have facilitated the evolution of the wide variety of gastrulation mechanisms seen in different amphibian species.     

p38 MAPK is essential for secondary axis specification and patterning in sea urchin embryos

Most eggs in the animal kingdom establish a primary, animal-vegetal axis maternally, and specify the remaining two axes during development. In sea urchin embryos, the expression of Nodal on the oral (ventral) side of the embryo is the first known molecular determinant of the oral-aboral axis (the embryonic dorsoventral axis), and is crucial for specification of the oral territory. We show that p38 MAPK acts upstream of Nodal and is required for Nodal expression in the oral territory. p38 is uniformly activated early in development, but, for a short interval at late blastula stage, is asymmetrically inactivated in future aboral nuclei. Experiments show that this transient asymmetry of p38 activation corresponds temporally to both oral specification and the onset of oral Nodal expression. Uniform inhibition of p38 prevents Nodal expression and axis specification, resulting in aboralized embryos. Nodal and its target Gsc each rescue oral-aboral specification and patterning when expressed asymmetrically in p38-inhibited embryos. Thus, our results indicate that p38 is required for oral specification through its promotion of Nodal expression in the oral territory.     

Conserved patterns of cell movements during vertebrate gastrulation

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

Lefty-dependent inhibition of Nodal- and Wnt-responsive organizer gene expression is essential for normal gastrulation

During gastrulation, diffusible "organizer" signals, including members of the TGFbeta Nodal subfamily, pattern dorsal mesoderm and the embryonic axes. Simultaneously, negative regulators of these signals, including the Nodal inhibitor Lefty, an atypical TGFbeta factor, are induced by Nodal. This suggests that Lefty-dependent modulation of organizer signaling might regulate dorsal mesoderm patterning and axial morphogenesis. Here, Xenopus Lefty (Xlefty) function was blocked by injection of anti-Xlefty morpholino oligonucleotides (MO). Xlefty-deficient embryos underwent exogastrulation, an aberrant morphogenetic process not predicted from deregulation of the Nodal pathway alone. In the absence of Xlefty, both Nodal- (Xnr2, gsc, cer, Xbra) and Wnt-responsive (gsc, Xnr3) organizer gene expression expanded away from the dorsal blastopore lip. Conversely, coexpression of Xlefty with Nodal or Wnt reduced the ectopic expression of Nodal- (Xbra) and Wnt-responsive (Xnr3) genes in a dose-dependent manner. Furthermore, Xlefty expression in the ectodermal animal pole inhibited endogenous Nodal- and Wnt-responsive gene expression in distant mesoderm cells, indicating that Xlefty inhibition can spread from its source. We hypothesize that Xlefty negatively regulates the spatial extent of Nodal- and Wnt-responsive gene expression in the organizer and that this Xlefty-dependent inhibition is essential for normal organizer patterning and gastrulation.     

Nodal cis-regulatory elements reveal epiblast and primitive endoderm heterogeneity in the peri-implantation mouse embryo

Nodal, a secreted factor known for its conserved functions in cell-fate specification and the establishment of embryonic axes, is also required in mammals to maintain the pluripotency of the epiblast, the tissue that gives rise to all fetal lineages. Although Nodal is expressed as early as E3.5 in the mouse embryo, its regulation and functions at pre- and peri-implantation stages are currently unknown. Sensitive reporter transgenes for two Nodal cis-regulatory regions, the PEE and the ASE, exhibit specific expression profiles before implantation. Mutant and inhibitor studies find them respectively regulated by Wnt/β-catenin signaling and Activin/Nodal signaling, and provide evidence for localized and heterogeneous activities of these pathways in the inner cell mass, the epiblast and the primitive endoderm. These studies also show that Nodal and its prime effector, FoxH1, are not essential to preimplantation Activin/Nodal signaling. Finally, a strong upregulation of the ASE reporter in implanting blastocysts correlates with a downregulation of the pluripotency factor Nanog in the maturing epiblast. This study uncovers conservation in the mouse blastocyst of Wnt/β-catenin and Activin/Nodal-dependent activities known to govern Nodal expression and the establishment of polarity in the blastula of other deuterostomes. Our results indicate that these pathways act early on to initiate distinct cell-specification processes in the ICM derivatives. Our data also suggest that the activity of the Activin/Nodal pathway is dampened by interactions with the molecular machinery of pluripotency until just before implantation, possibly delaying cell-fate decisions in the mouse embryo.     

Specification of ectoderm restricts the size of the animal plate and patterns neurogenesis in sea urchin embryos

The animal plate of the sea urchin embryo becomes the apical organ, a sensory structure of the larva. In the absence of vegetal signaling, an expanded and unpatterned apical organ forms. To investigate the signaling that restricts the size of the animal plate and patterns neurogenesis, we have expressed molecules that regulate specification of ectoderm in embryos and chimeras. Enhancing oral ectoderm suppresses serotonergic neuron differentiation, whereas enhancing aboral or ciliary band ectoderm increases differentiation of serotonergic neurons. In embryos in which vegetal signaling is blocked, Nodal expression does not reduce the size of the thickened animal plate; however, almost no neurons form. Expression of BMP in the absence of vegetal signaling also does not restrict the size of the animal plate, but abundant serotonergic neurons form. In chimeras in which vegetal signaling is blocked in the entire embryo, and one half of the embryo expresses Nodal, serotonergic neuron formation is suppressed in both halves. In similar chimeras in which vegetal signaling is blocked and one half of the embryo expresses Goosecoid (Gsc), serotonergic neurons form only in the half of the embryo not expressing Gsc. We propose that neurogenesis is specified by a maternal program that is restricted to the animal pole by signaling that is dependent on nuclearization of beta-catenin and specifies ciliary band ectoderm. Subsequently, neurogenesis in the animal plate is patterned by suppression of serotonergic neuron formation by Nodal. Like other metazoans, echinoderms appear to have a phase of neural development during which the specification of ectoderm restricts and patterns neurogenesis.     

Nodal-related signals establish mesendodermal fate and trunk neural identity in zebrafish

The vertebrate body plan arises during gastrulation, when morphogenetic movements form the ectoderm, mesoderm, and endoderm. In zebrafish, mesoderm and endoderm derive from the marginal region of the late blastula, and cells located nearer the animal pole form the ectoderm [1]. Analysis in mouse, Xenopus, and zebrafish has demonstrated that Nodal-related proteins, a subclass of the TGF-beta superfamily, are essential for mesendoderm development [2], but previous mutational studies have not established whether Nodal-related signals control fate specification, morphogenetic movements, or survival of mesendodermal precursors. Here, we report that Nodal-related signals are required to allocate marginal cells to mesendodermal fates in the zebrafish embryo. In double mutants for the zebrafish nodal-related genes squint (sqt) and cyclops (cyc) [3] [4] [5], dorsal marginal cells adopt neural fates, whereas in wild-type embryos, cells at this position form endoderm and axial mesoderm. Involution movements characteristic of developing mesendoderm are also blocked in the absence of Nodal signaling. Because it has been proposed [6] that inhibition of Nodal-related signals promotes the development of anterior neural fates, we also examined anteroposterior organization of the neural tube in sqt;cyc mutants. Anterior trunk spinal cord is absent in sqt;cyc mutants, despite the presence of more anterior and posterior neural fates. These results demonstrate that nodal-related genes are required for the allocation of dorsal marginal cells to mesendodermal fates and for anteroposterior patterning of the neural tube.