Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo

The dorsal ectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be specified towards an anterior neural fate by an activation signal, with its subsequent regionalization along the anteroposterior (AP) axis regulated by a graded transforming activity, leading to a properly patterned forebrain, midbrain, hindbrain and spinal cord. The activation phase involves inhibition of BMP signals by dorsal antagonists, but the later caudalization process is much more poorly characterized. Explant and overexpression studies in chick, Xenopus, mouse and zebrafish implicate lateral/paraxial mesoderm in supplying the transforming influence, which is largely speculated to be a Wnt family member. We have analyzed the requirement for the specific ventrolaterally expressed Wnt8 ligand in the posteriorization of neural tissue in zebrafish wild-type and Nodal-deficient embryos (Antivin overexpressing or cyclops;squint double mutants), which show extensive AP brain patterning in the absence of dorsal mesoderm. In different genetic situations that vary the extent of mesodermal precursor formation, the presence of lateral wnt8-expressing cells correlates with the establishment of AP brain pattern. Cell tracing experiments show that the neuroectoderm of Nodal-deficient embryos undergoes a rapid anterior-to-posterior transformation in vivo during a short period at the end of the gastrula stage. Moreover, in both wild-type and Nodal-deficient embryos, inactivation of Wnt8 function by morpholino (MO(wnt8)) translational interference dose-dependently abrogates formation of spinal cord and posterior brain fates, without blocking ventrolateral mesoderm formation. MO(wnt8) also suppresses the forebrain deficiency in bozozok mutants, in which inactivation of a homeobox gene causes ectopic wnt8 expression. In addition, the bozozok forebrain reduction is suppressed in bozozok;squint;cyclops triple mutants, and is associated with reduced wnt8 expression, as seen in cyclops;squint mutants. Hence, whereas boz and Nodal signaling largely cooperate in gastrula organizer formation, they have opposing roles in regulating wnt8 expression and forebrain specification. Our findings provide strong support for a model of neural transformation in which a planar gastrula-stage Wnt8 signal, promoted by Nodal signaling and dorsally limited by Bozozok, acts on anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the CNS.     

Cooperative roles of Bozozok/Dharma and Nodal-related proteins in the formation of the dorsal organizer in zebrafish

In vertebrates, specification of the dorso-ventral axis requires Wnt signaling, which leads to formation of the Nieuwkoop center and the Spemann organizer (dorsal organizer), through the nuclear accumulation of beta-catenin. Zebrafish bozozok/dharma (boz) and squint (sqt), which encode a homeodomain protein and a Nodal-related protein, respectively, are required for the formation of the dorsal organizer. The zygotic expression of boz and sqt in the dorsal blastoderm and dorsal yolk syncytial layer (YSL) was dependent on the maternally derived Wnt signal, and their expression at the late blastula and early gastrula stages was dependent on the zygotic expression of their own genes. The dorsal organizer genes, goosecoid (gsc) and chordin (din), were ectopically expressed in wild-type embryos injected with boz or sqt RNA. The expression of gsc strictly depended on both boz and sqt while the expression of din strongly depended on boz but only partially depended on sqt and cyclops (cyc, another nodal-related gene). Overexpression of boz in embryos defective in Nodal signaling elicited the ectopic expression of din but not gsc and resulted in dorsalization, implying that boz could induce part of the organizer, independent of the Nodal proteins. Furthermore, boz; sqt and boz;cyc double mutants displayed a severely ventralized phenotype with anterior truncation, compared with the single mutants, and boz;sqt;cyc triple mutant embryos exhibited an even more severe phenotype, lacking the anterior neuroectoderm and notochord, suggesting that Boz/Dharma and the Nodal-related proteins cooperatively regulate the formation of the dorsal organizer.     

Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling

The canonical Wnt/beta-catenin signaling has remarkably diverse roles in embryonic development, stem cell self-renewal and cancer progression. Here, we show that stabilized expression of beta-catenin perturbed human embryonic stem (hES)-cell self-renewal, such that up to 80% of the hES cells developed into the primitive streak (PS)/mesoderm progenitors, reminiscent of early mammalian embryogenesis. The formation of the PS/mesoderm progenitors essentially depended on the cooperative action of beta-catenin together with Activin/Nodal and BMP signaling pathways. Intriguingly, blockade of BMP signaling completely abolished mesoderm generation, and induced a cell fate change towards the anterior PS progenitors. The PI3-kinase/Akt, but not MAPK, signaling pathway had a crucial role in the anterior PS specification, at least in part, by enhancing beta-catenin stability. In addition, Activin/Nodal and Wnt/beta-catenin signaling synergistically induced the generation and specification of the anterior PS/endoderm. Taken together, our findings clearly demonstrate that the orchestrated balance of Activin/Nodal and BMP signaling defines the cell fate of the nascent PS induced by canonical Wnt/beta-catenin signaling in hES cells.     

The secreted Frizzled-related protein Sizzled functions as a negative feedback regulator of extreme ventral mesoderm

The prevailing model of dorsal ventral patterning of the amphibian embryo predicts that the prospective mesoderm is regionalized at gastrulation in response to a gradient of signals. This gradient is established by diffusible BMP and Wnt inhibitors secreted dorsally in the Spemann organizer. An interesting question is whether ventrolateral tissue passively reads graded levels of ventralizing signals, or whether local self-organizing regulatory circuits may exist on the ventral side to control cell behavior and differentiation at a distance from the Organizer. We provide evidence that sizzled, a secreted Frizzled-related protein expressed ventrally during and after gastrulation, functions in a negative feedback loop that limits allocation of mesodermal cells to the extreme ventral fate, with direct consequences for morphogenesis and formation of the blood islands. Morpholino-mediated knockdown of Sizzled protein results in expansion of ventral posterior mesoderm and the ventral blood islands, indicating that this negative regulation is required for proper patterning of the ventral mesoderm. The biochemical activity of sizzled is apparently very different from that of other secreted Frizzled-related proteins, and does not involve inhibition of Wnt8. Our data are consistent with the existence of some limited self-organizing properties of the extreme ventral mesoderm.     

Twisted gastrulation promotes BMP signaling in zebrafish dorsal-ventral axial patterning

In vertebrates and invertebrates, the bone morphogenetic protein (BMP) signaling pathway patterns cell fates along the dorsoventral (DV) axis. In vertebrates, BMP signaling specifies ventral cell fates, whereas restriction of BMP signaling by extracellular antagonists allows specification of dorsal fates. In misexpression assays, the conserved extracellular factor Twisted gastrulation (Tsg) is reported to both promote and antagonize BMP signaling in DV patterning. To investigate the role of endogenous Tsg in early DV patterning, we performed morpholino (MO)-based knockdown studies of Tsg1 in zebrafish. We found that loss of tsg1 results in a moderately strong dorsalization of the embryonic axis, suggesting that Tsg1 promotes ventral fates. Knockdown of tsg1 combined with loss of function of the BMP agonist tolloid (mini fin) or heterozygosity for the ligand bmp2b (swirl) enhanced dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppressed the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). Our results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning.     

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.     

Axial patterning interactions in the sea urchin embryo: suppression of nodal by Wnt1 signaling

Wnt and Nodal signaling pathways are required for initial patterning of cell fates along anterior-posterior (AP) and dorsal-ventral (DV) axes, respectively, of sea urchin embryos during cleavage and early blastula stages. These mechanisms are connected because expression of nodal depends on early Wnt/β-catenin signaling. Here, we show that an important subsequent function of Wnt signaling is to control the shape of the nodal expression domain and maintain correct specification of different cell types along the axes of the embryo. In the absence of Wnt1, the posterior-ventral region of the embryo is severely altered during early gastrulation. Strikingly, at this time, nodal and its downstream target genes gsc and bra are expressed ectopically, extending posteriorly to the blastopore. They override the initial specification of posterior-ventral ectoderm and endoderm fates, eliminating the ventral contribution to the gut and displacing the ciliary band dorsally towards, and occasionally beyond, the blastopore. Consequently, in Wnt1 morphants, the blastopore is located at the border of the re-specified posterior-ventral oral ectoderm and by larval stages it is in the same plane near the stomodeum on the ventral side. In normal embryos, a Nodal-dependent process downregulates wnt1 expression in dorsal posterior cells during early gastrulation, focusing Wnt1 signaling to the posterior-ventral region where it suppresses nodal expression. These subsequent interactions between Wnt and Nodal signaling are thus mutually antagonistic, each limiting the range of the other's activity, in order to maintain and stabilize the body plan initially established by those same signaling pathways in the early embryo.     

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.     

Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus

The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.     

Disrupted dorsal neural tube BMP signaling in the cilia mutant Arl13b hnn stems from abnormal Shh signaling

In the embryonic neural tube, multiple signaling pathways work in concert to create functional neuronal circuits in the adult spinal cord. In the ventral neural tube, Sonic hedgehog (Shh) acts as a graded morphogen to specify neurons necessary for movement. In the dorsal neural tube, bone morphogenetic protein (BMP) and Wnt signals cooperate to specify neurons involved in sensation. Several signaling pathways, including Shh, rely on primary cilia in vertebrates. In this study, we used a mouse mutant with abnormal cilia, Arl13b(hnn), to study the relationship between cilia, cell signaling, and neural tube patterning. Arl13b(hnn) mutants have abnormal ventral neural tube patterning due to disrupted Shh signaling; in addition, dorsal patterning defects occur, but the cause of these is unknown. Here we show that the Arl13b(hnn) dorsal patterning defects result from abnormal BMP signaling. In addition, we find that Wnt ligands are abnormally expressed in Arl13b(hnn) mutants; surprisingly, however, downstream Wnt signaling is normal. We demonstrate that Arl13b is required non-autonomously for BMP signaling and Wnt ligand expression, indicating that the abnormal Shh signaling environment in Arl13b(hnn) embryos indirectly causes dorsal defects.     

Maternally controlled (beta)-catenin-mediated signaling is required for organizer formation in the zebrafish

We have identified and characterized a zebrafish recessive maternal effect mutant, ichabod, that results in severe anterior and dorsal defects during early development. The ichabod mutation is almost completely penetrant, but exhibits variable expressivity. All mutant embryos fail to form a normal embryonic shield; most fail to form a head and notochord and have excessive development of ventral tail fin tissue and blood. Abnormal dorsal patterning can first be observed at 3.5 hpf by the lack of nuclear accumulation of (beta)-catenin in the dorsal yolk syncytial layer, which also fails to express bozozok/dharma/nieuwkoid and znr2/ndr1/squint. At the onset of gastrulation, deficiencies in expression of dorsal markers and expansion of expression of markers of ventral tissues indicate a dramatic alteration of dorsoventral identity. Injection of (beta)-catenin RNA markedly dorsalized ichabod embryos and often completely rescued the phenotype, but no measurable dorsalization was obtained with RNAs encoding upstream Wnt pathway components. In contrast, dorsalization was obtained when RNAs encoding either Bozozok/Dharma/Nieuwkoid or Znr2/Ndr1/Squint were injected. Moreover, injection of (beta)-catenin RNA into ichabod embryos resulted in activation of expression of these two genes, which could also activate each other. RNA injection experiments strongly suggest that the component affected by the ichabod mutation acts on a step affecting (beta)-catenin nuclear localization that is independent of regulation of (beta)-catenin stability. This work demonstrates that a maternal gene controlling localization of (beta)-catenin in dorsal nuclei is necessary for dorsal yolk syncytial layer gene activity and formation of the organizer in the zebrafish.     

SNW1 is a critical regulator of spatial BMP activity, neural plate border formation, and neural crest specification in vertebrate embryos

Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and thus neural crest specification.     

Wnt Signaling Interacts with Bmp and Edn1 to Regulate Dorsal-Ventral Patterning and Growth of the Craniofacial Skeleton

Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.     

Wnt, activin, and BMP signaling regulate distinct stages in the developmental pathway from embryonic stem cells to blood

The embryonic stem cell differentiation system was used to define the roles of the Activin/Nodal, BMP, and canonical Wnt signaling pathways at three distinct developmental stages during hematopoietic ontogeny: induction of a primitive streak-like population, formation of Flk1(+) mesoderm, and induction of hematopoietic progenitors. Activin/Nodal and Wnt, but not BMP, signaling are required for the induction of the primitive streak. Although BMP is not required for primitive streak induction, it displays a strong posteriorizing effect on this population. All three signaling pathways regulate induction of Flk1(+) mesoderm. The specification of Flk1(+) mesoderm to the hematopoietic lineages requires VEGF and Wnt, but not BMP or Activin/Nodal signaling. Specifically, Wnt signaling is essential for commitment of the primitive erythroid, but not the definitive lineages. These findings highlight dynamic changes in signaling requirements during blood cell development and identify a role for Wnt signaling in the establishment of the primitive erythroid lineage.     

A p38 MAPK-CREB pathway functions to pattern mesoderm in Xenopus

Dorsal-ventral patterning is specified by signaling centers secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen gradient is translated intracellularly into fate decisions remains largely unknown. Here, we report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high levels of p38 activator, MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal organizing center. p38 MAPK and CREB function downstream of maternal Wnt/β-catenin and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E induces expression of lateral genes without inducing the expression of dorsal genes. Loss of CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB activities along the axis.