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.     

Neural induction in Xenopus: requirement for ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin, and Cerberus

The origin of the signals that induce the differentiation of the central nervous system (CNS) is a long-standing question in vertebrate embryology. Here we show that Xenopus neural induction starts earlier than previously thought, at the blastula stage, and requires the combined activity of two distinct signaling centers. One is the well-known Nieuwkoop center, located in dorsal-vegetal cells, which expresses Nodal-related endomesodermal inducers. The other is a blastula Chordin- and Noggin-expressing (BCNE) center located in dorsal animal cells that contains both prospective neuroectoderm and Spemann organizer precursor cells. Both centers are downstream of the early beta-Catenin signal. Molecular analyses demonstrated that the BCNE center was distinct from the Nieuwkoop center, and that the Nieuwkoop center expressed the secreted protein Cerberus (Cer). We found that explanted blastula dorsal animal cap cells that have not yet contacted a mesodermal substratum can, when cultured in saline solution, express definitive neural markers and differentiate histologically into CNS tissue. Transplantation experiments showed that the BCNE region was required for brain formation, even though it lacked CNS-inducing activity when transplanted ventrally. Cell-lineage studies demonstrated that BCNE cells give rise to a large part of the brain and retina and, in more posterior regions of the embryo, to floor plate and notochord. Loss-of-function experiments with antisense morpholino oligos (MO) showed that the CNS that forms in mesoderm-less Xenopus embryos (generated by injection with Cerberus-Short [CerS] mRNA) required Chordin (Chd), Noggin (Nog), and their upstream regulator beta-Catenin. When mesoderm involution was prevented in dorsal marginal-zone explants, the anterior neural tissue formed in ectoderm was derived from BCNE cells and had a complete requirement for Chd. By injecting Chd morpholino oligos (Chd-MO) into prospective neuroectoderm and Cerberus morpholino oligos (Cer-MO) into prospective endomesoderm at the 8-cell stage, we showed that both layers cooperate in CNS formation. The results suggest a model for neural induction in Xenopus in which an early blastula beta-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.     

Shisa promotes head formation through the inhibition of receptor protein maturation for the caudalizing factors, Wnt and FGF

Head formation requires simultaneous inhibition of multiple caudalizing signals during early vertebrate embryogenesis. We identified a novel antagonist against Wnt and FGF signaling for head formation, Shisa, which functions cell autonomously in the endoplasmic reticulum (ER). Shisa is specifically expressed in the prospective head ectoderm and the Spemann organizer of Xenopus gastrulae. Overexpression of Shisa inhibited both Wnt and FGF signaling in Xenopus embryos and in a cell line. Loss of Shisa function sensitized the neuroectoderm to Wnt signaling and suppressed head formation during gastrulation. Shisa physically interacted with immature forms of the Wnt receptor Frizzled and the FGF receptor within the ER and inhibited their posttranslational maturation and trafficking to the cell surface. Taken together, these findings indicate that Shisa is a novel molecule that controls head formation by regulating the establishment of the receptors for caudalizing factors.     

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.     

Regulation of dharma/bozozok by the Wnt pathway

The zebrafish homeobox gene dharma/bozozok (boz) is required for the formation and/or function of the Nieuwkoop center and the subsequent induction of the Spemann organizer. dharma is expressed soon after the midblastula transition in the dorsal blastomeres and the dorsal yolk syncytial layer (YSL). We found that the expression of dharma was upregulated or ectopically induced by misexpression of a Wnt protein and cytoplasmic components of the Wnt signaling pathway and downregulated by the expression of dominant-negative Tcf3. A 1.4-kbp fragment of the dharma promoter region contains consensus sequences for Tcf/Lef binding sites. This promoter region recapitulated the Wnt-dependent and dorsal dharma expression pattern when it was fused to luciferase or GFP. Deletion and point mutant analyses revealed that the Tcf/Lef binding sites were required to drive this expression pattern. These data established that dharma/boz functions between the dorsal determinants-mediated Wnt signals and the formation of the Nieuwkoop center.     

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.     

Regulatory expression of Brachyury and Goosecoid in P19 embryonal carcinoma cells

Mouse P19 embryonal carcinoma cells can differentiate into various cell types depending on culture conditions. Here we show that the expression of the mesodermal genes Brachyury (Bra) and Goosecoid (Gsc) are under regulatory control in P19 cells. When P19 cells were cultured in a tissue culture dish in the presence of serum, Bra and Gsc were unexpectedly expressed. Expression of Bra and Gsc was greatly reduced with culture time, and expression levels at 144 h of culture were below 25% those at 48 h of culture. Members of the Tgf-beta family such as Activin and Nodal have been known to up-regulate expression of mesodermal genes. Treatment with SB431542, an Alk4/5/7 inhibitor, decreased Bra and Gsc in a dose-dependent manner, whereas it induced the expression of the neuroectodermal genes Mash-1 and Pax-6. Quantitative RT-PCR and dsRNAi transfection indicated Nodal as a possible ligand responsible for the regulation of Bra and Gsc. In addition, exogenous Nodal increased expression of Bra and Gsc in a dose-dependent manner. Serum concentration in culture medium positively related to expression of Nodal, Bra, Gsc, and Cripto, which encodes a membrane-tethered protein required for Nodal signaling. Addition of the culture supernatant of P19 cells at 144 h of culture to medium decreased expression of these genes. The present study reveals that stimulation and inhibition of the Nodal pathway increases mesodermal genes and neuroectodermal genes, respectively, indicating the importance of control of Nodal and Cripto expression for mesodermal formation and neurogenesis.