Hensen's Node Research Articles

Anteroposterior patterning in the zebrafish, Danio rerio: an explant assay reveals inductive and suppressive cell interactions.

We report the first extended culture system for analysing zebrafish (Danio rerio) embryogenesis with which we demonstrate neural induction and anteroposterior patterning. Explants from the animal pole region of blastula embryos ('animal caps') survived for at least two days and increased in cell number. Mesodermal and neural-specific genes were not expressed in cultured animal caps, although low levels of the dorsoanterior marker otx2 were seen. In contrast, we observed strong expression of gta3, a ventral marker and cyt1, a novel type I cytokeratin expressed in the outer enveloping layer. Isolated 'embryonic shield', that corresponds to the amphibian organizer and amniote node, went on to express the mesodermal genes gsc and ntl, otx2, the anterior neural marker pax6, and posterior neural markers eng3 and krx20. The expression of these genes defined a precise anteroposterior axis in shield explants. When conjugated to animal caps, the shield frequently induced expression of anterior neural markers. More posterior markers were rarely induced, suggesting that anterior and posterior neural induction are separable events. Mesodermal genes were also seldom activated in animal caps by the shield, demonstrating that neural induction did not require co-induction of mesoderm in the caps. Strikingly, ventral marginal zone explants suppressed the low levels of otx2 in animal caps, indicating that ventral tissues may play an active role in axial patterning. These data suggest that anteroposterior patterning in the zebrafish is a multi-step process.

Apoptosis removes chick embryo tail gut and remnant of the primitive streak

Removal of transient features in morphogenesis of chick embryo tail is by programmed cell death. We used ApopTagTM (Oncor, Gaithersburg, MD) with the peroxidase/diaminobenzidine (DAB) procedure to correlate apoptosis with earlier reports of patterns of cell death in stage HH17-25 embryos, and our results suggest that the cell death inferred with supravital staining and appearance of cells in morphogenesis of the tail bud is programmed cell death called apoptosis. Apoptosis markers in tail bud are most abundant in the median cell cord of occluded degenerating tail gut. Tail bud mesenchyme marks for apoptosis most frequently in the ventrum of older stages, where cell death has been reported. Cells of the remnant of the primitive streak (Hensen's node) mark for apoptosis, suggesting that programmed cell death is a stop signal for axial organization at the caudal terminus. Apoptosis markers in postmembrane cloacal endoderm anticipate the transient cloacal fenestra. Lack of apoptosis markers in neural tube, notochord, and somites supports the suggestion of Schoenwolf ([1981] Anat, Embryol. (Beri.) 162:183-197) that cells of those areas in the tail bud are assimilated into the growing rump of the chick embryo. Lack of markers in neural tube of tail bud formed by secondary neurulation suggests that apoptosis is not involved in cavitation of medullary cord, but further investigation is necessary. A limited investigation of pharyngeal membranes and midgut, where cell death has not been reported to be as important in morphogenesis, did not show apoptosis markers in those tissues (Miller and Briglin [1994] "Cell Death in Development and Cancer," Houston: University of Texas MD Anderson Cancer Center, pp, 82-83). Absence of apoptosis markers in roof of gut tube suggests that the lower frequency of thymidine labeling reported for those cells (Miller [1986] Anat. Rec. 214: 87A) is not a result of apoptosis. Clearly marked cells correlated with expected locations of migrating neural crest and primordial germ cells in these stages, but distribution of apoptosis markers was not abundant or general for either cell type.

Immortalized Hensen's Node Cells Secrete a Factor That Regulates Avian Neural Crest Cell Fatesin Vitro

The derivatives of the neural crest are regionally specified with respect to the anterior–posterior axis of the avian embryo. We have shown previously that young Hensen's node can actin vitroto regulate the expression of certain region-specific phenotypes in trunk neural crest cells. To study potential factors acting on the neural crest, we have generated an immortalized cell line from young Hensen's node. Here we show that a factor produced by these cells stimulates the expression of two cranial-specific phenotypes (fibronectin and smooth muscle actin) in trunk neural crest cells and decreases their expression of a trunk-specific phenotype (melanin). The active factor is a secreted protein with a molecular weight >30 kDa. Clonal studies suggest that the factor acts by changing the phenotypic fates of individual neural crest cells, rather than by selective effects on cell proliferation or survival. Previous work has shown that TGF-βs can mimic the effects of Hensen's node cells on neural crest differentiation. Results from the present study suggest that the factor in the conditioned medium of the immortalized node cell line is not a TGF-β isoform. However, the cranial phenotype-inducing activity of the conditioned medium factor requires the presence of neural crest cell-derived TGF-βs.

Characterizing the zebrafish organizer: microsurgical analysis at the early-shield stage.

The appearance of the embryonic shield, a slight thickening at the leading edge of the blastoderm during the formation of the germ ring, is one of the first signs of dorsoventral polarity in the zebrafish embryo. It has been proposed that the shield plays a role in fish embryo patterning similar to that attributed to the amphibian dorsal lip. In a recent study, we fate mapped many of the cells in the region of the forming embryonic shield, and found that neural and mesodermal progenitors are intermingled (Shih, J. and Fraser, S.E. (1995) Development 121, 2755-2765), in contrast to the coherent region of mesodermal progenitors found at the amphibian dorsal lip. Here, we examine the fate and the inductive potential of the embryonic shield to determine if the intermingling reflects a different mode of embryonic patterning than that found in amphibians. Using the microsurgical techniques commonly used in amphibian and avian experimental embryology, we either grafted or deleted the region of the embryonic shield. Homotopic grafting experiments confirmed the fates of cells within the embryonic shield region, showing descendants in the hatching gland, head mesoderm, notochord, somitic mesoderm, endoderm and ventral aspect of the neuraxis. Heterotopic grafting experiments demonstrated that the embryonic shield can organize a second embryonic axis; however, contrary to our expectations based on amphibian research, the graft contributes extensively to the ectopic neuraxis. Microsurgical deletion of the embryonic shield region at the onset of germ ring formation has little effect on neural development: embryos with a well-formed and well-patterned neuraxis develop in the complete absence of notochord cells. While these results show that the embryonic shield is sufficient for ectopic axis formation, they also raise questions concerning the necessity of the shield region for neural induction and embryonic patterning after the formation of the germ ring.

Cell proliferation in mammalian gastrulation: The ventral node and notochord are relatively quiescent

During gastrulation, the node of the mammalian embryo appears to be an organising centre, homologous to Hensen's node in the chick and the dorsal lip of the amphibian blastopore. In addition, the node serves as a precursor population for the head process, notochord and foregut endoderm. We have studied node architecture and cell morphology by electron microscopy, and cell proliferation using bromodeoxyuridine incorporation and mitotic counts. The dorsal (ectodermal) and ventral (endodermal) components of the node are two distinct populations, separated by a basement membrane. The ventral node, contiguous with the head process, is characterised by a relatively low proliferation rate, with only approximately 10% of cells incorporating BrdU over 4 hr, compared to > 95% in surrounding mesodermal and ectodermal tissues. This is the case from the beginning of node formation, at the no-allantoic-bud stage, until the 7 somite stage, and is not compatible with the idea that the ventral node is a stem cell population. The dorsal node is highly proliferative, its rate of division being indistinguishable from the neurectoderm, with which it is contiguous. In the ventral node, two regions can be recognised: cells in the "pit" are columnar and all monociliated; around them lies a "crown" of cells arranged radially in a horseshoe shape and less often ciliated. Node derivatives share common features with the ventral node; the head process and the notochord are relatively quiescent; and some head process cells are also monociliated. Node and head process monocilia are immotile and appear to be associated with non-proliferation. We suggest that the ventral node contains all the properties of the organiser, while the dorsal node is indistinct from the surrounding epiblast. The cranial end of the foregut pouch, the thyroid diverticulum, and the promyocardium of early somite stage embryos are also areas of low cell division. All the described regions of relative quiescence are sites of expression of members of the TGF beta family, which may be involved in maintaining non-proliferation.

Patterning of the neural primordium in the avian embryo

Abstract The quail-chick chimera system has been used to study two problems related to the patterning of the neural primordium. We first report our analysis of the secondary neurulation as it proceeds in the avian tail bud caudally to the posterior neuropore. We show that the territory located caudally to the primary neural tube and joining it to the caudal end of the notochord, designated as the cordoneural hinge (CNH), can be assimilated to the remainder of the Hensen's node. The CNH undergoes a craniocaudal progression during tail bud development and lays down the notocord and floor plate which thus originate from closely related progenitor cells. We show that the floor plate material becomes inserted within the neurectoderm thus forming the neural plate. The second part of this article is devoted to the hindbrain development. We show that transposition of the presumptive territory of anterior rhombomeres (r1 to r6) at the level of r7–r8 results in their phenotypic posteriorization which is preceded by their expression of posterior Hox genes characteristic to the AP level at which they are transplanted and hence that there are inductive signals within the neural epithelium itself.

A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers.

We have examined the role of fibroblast growth factor (FGF) signalling in neural induction. The approach takes advantage of the fact that both noggin and the dominant negative mutant activin receptor (delta1XAR1) directly induce neural tissues in the absence of dorsal mesoderm. A truncated FGF receptor (XFD) is co-expressed with noggin or delta1XAR1 in both whole embryos and isolated animal caps. We demonstrate that inhibition of FGF signalling prevents neural induction by both factors. Furthermore, neural induction by organizers (the dorsal lip of blastopore and Hensen's node) is also blocked by inhibiting FGF signalling in ectoderm. It has been proposed that the specification of anterior neuroectoderm, including the cement gland, occurs in a sequential manner as gastrulation proceeds. We show that the specification of the most anterior neuroectoderm by noggin may occur before gastrulation and does not require FGF signalling, since both the cement gland marker XCG-1 and the anterior neural marker Otx-2 are normally expressed in ectodermal explants co-injected with noggin and XFD RNAs, but the cement gland cells are poorly differentiated. In contrast, the expression of both genes induced by CSKA.noggin, which is expressed after the mid-blastula transition, is strongly inhibited by the presence of XFD. Therefore the noggin-mediated neural induction that takes place at gastrula stages is abolished in the absence of FGF signalling. Since inhibition of FGF signalling blocks the neuralizing effect of different neural inducers that function through independent mechanisms, we propose that FGF receptor-related-signalling is required for the response to inducing signals of ectodermal cells from gastrula.

Progression of trophoblast into the endometrium during implantation in the western spotted skunk.

In mustelid carnivores, several blastocysts become implanted either approximately 12 days following fertilization or after a delay of implantation. In the western spotted skunk, implantation occurs following a long period of delayed implantation and a brief activation stage. Within each implantation chamber, a large number of trophoblastic plaques form, and the syncytial trophoblast of these plaques adheres to and penetrates into the uterine luminal epithelium. The presence of multiple attachment sites was used to analyse the way in which trophoblast adheres to, penetrates, and removes uterine epithelium and its subsequent association with the subepithelial vascular plexus. Implantation chambers from 18 western spotted skunks were collected during the first week postimplantation and the tissue prepared for light microscopy and scanning and transmission electron microscopy. A series of trophoblastic plaques, which form a ring peripheral to the embryonic shield, attach to and penetrate the uterine epithelium. As new trophoblastic plaques are forming, the initial plaques enlarge and spread along the basal lamina of the luminal epithelium, and trophoblastic processes project through this basal lamina. Subsequently there is a stage of consolidation in which cytotrophoblast increases greatly in amount, attachment sites coalesce, and the luminal epithelium is eliminated. Syncytial trophoblast intrudes into endometrial gland lumina and surrounds subepithelial capillaries. It is suggested that the affinity of syncytial trophoblast for apical junctional complexes of uterine epithelial cells facilitates intrusion of syncytial trophoblast between cells, possibly guided by the marginal ridges of the uterine cells. The trophoblast shows no tendency to adhere to or invade maternal capillaries. This lack of adhesion to endothelial cells suggests either a change in adhesive properties of trophoblast following epithelial penetration or differences in adhesive properties between surface epithelial cells and endothelial cells. Although trophoblast differentiation appears to be chronologically regulated, it could be responding to maternally derived factors.

Hensen's node regulates avian neural crest differentiation in vitro.

Different anteroposterior (AP) regions of the neural crest normally produce different cell types, both in vivo and in vitro. AP differences in neural crest cell fates appear to be specified in part by mechanisms that act prior to neural crest cell migration. We, therefore, examined the possibility that the fates of neural crest cells, like those of neural tube cells, can be regulated by interactions with Hensen's node. Using a transfilter co-culture system, we found that young (stage 3+ to 4) Hensen's node up-regulates the expression of two cranial-specific phenotypes (fibronectin and smooth muscle actin immunoreactivities) in mass cultures of trunk neural crest cells, and down-regulates the expression of a trunk-specific phenotype (melanin synthesis). The changes in phenotype produced by exposure to young Hensen's node were not accompanied by changes in the proliferation of either fibronectin immunoreactive cells or melanocytes. The capacity of Hensen's node to elicit changes in trunk neural crest cell phenotype decreased as the developmental age of the node increased and was lost by stage 6. In addition, old Hensen's node did not stimulate the expression of trunk-specific phenotypes in cranial neural crest cells, suggesting that cranial- and trunk-specific phenotypes are induced by different mechanisms.

Secondary axis induction by heterospecific organizers in zebrafish.

To investigate the inductive activities of the vertebrate organizer, we transplanted the chicken organizer (Hensen's node) into zebrafish gastrula and analyzed resulting secondary axes. Grafted Hensen's node did not differentiate or participate in the secondary axis. It also did not induce a secondary notochord or expression of the genes normally expressed by the fish organizer including no tail, axial, goosecoid. Nevertheless, it recruited fish cells to organize a variety of tissues: the dorsal portion of the central nervous system including Rohon-Beard sensory neurons, otic vesicles, dorsal pigment stripe, dorsal fin, somites, heart, and pronephric ducts. Enlarged neural plate induced by the organizer was shown by the expression pattern of dlx3 and msxB genes, which demarcates the early presumptive neural tissue. In addition, Hensen's node of an earlier stage chicken embryo displayed differential movement in zebrafish from that of a later stage. This might reflect unknown differences in properties between the organizer at two different developmental stages related to its normal organizer activity. To create a model system to study the molecular mechanisms of the organizer, we next transplanted genetically modified mouse cells into zebrafish embryos. We found that Wnt3A-transfected NIH3T3 cells are much more potent in inducing a secondary axis than NIH3T3 cells alone. These results suggest that formation of a variety of tissues are controlled by signalling from the organizer itself with no requirement of participation of the organizer-derived tissues. Additionally, the activities of the organizer may involve a function of Wnt-family genes.

Expression of neurotrophin trk and p75 receptors in quail embryos undergoing gastrulation and neurulation.

We have previously demonstrated the presence of mRNA for the full-length neurotrophin receptors trkA, trkB and trkC in quail embryos from stages 1 through 6 using reverse transcription followed by the polymerase chain reaction (RT-PCR; Yao et al. [1994] Dev. Biol. 165: 727-730). Furthermore, we showed that mRNA for the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 was present from stage 1 onward, while nerve growth factor mRNA began to be expressed at stage 5. In the present study, wholemount in situ hybridization was used to localize full-length trk mRNA in embryos from stages 3 through 10. Structures expressing trkC mRNA included the primitive streak and Hensen's node, the neural plate or notochord, somites and the rostral neural tube. trkA and trkB mRNA were expressed at much lower levels than trkC mRNA; however, staining was detected on the primitive streak and Hensen's node. In addition to trk mRNA, we have also demonstrated the presence of full-length Trk protein in embryos from stages 3 through 11, suggesting that the trk mRNA detected at these early stages is translated into functional cell surface receptors. To support this hypothesis, we have shown that neurotrophins can induce phosphorylation of Trk on tyrosine residues, at least at stage 11. We also detected mRNA and protein for the nontyrosine kinase neurotrophin receptor, p75, at similar stages. The presence of neurotrophin receptors, as well as neurotrophin mRNA, in embryos undergoing gastrulation and neurulation leads to speculation that neurotrophins may be playing a role in these processes.

Mapping vertebrate embryos

We thank Richard Behringer, Cynthia Faust, Scott Fraser, Terry Magnuson, Andy McMahon and Peter Rowe for helpful discussions.

Hensen's Node from Vitamin A-Deficient Quail Embryo Induces Chick Limb Bud Duplication and Retains Its Normal Asymmetric Expression ofSonic hedgehog(Shh)

Both Hensen's node, the organizer center in chick embryo, and exogenous retinoic acid are known to induce limb duplication when grafted or applied to the host chick limb bud. Retinoic acid is known to be present in the node and has been proposed as the putative morphogen for chick limb development. Here, we report that Hensen's node from vitamin A-deficient quail embryo induces limb duplication in the host chick embryo similar to that induced by the node from vitamin A-sufficient control embryos. We also demonstrate that the expression ofSonic hedgehog(Shh), recently shown to be the mediator of polarizing activity in the chick limb bud, is not affected by the endogenous vitamin A status of the embryo. Furthermore, whole-mountin situhybridization revealed asymmetry ofShhexpression in the Hensen's node of both vitamin A-sufficient and -deficient quail embryos. Retinoids were not detectable in the eggs from which vitamin A-deficient embryos were obtained. Extracts from normal embryos induced a level of expression of reporter gene equivalent to the presence of 3.4 pg of active retinoids per embryo, while those from vitamin A-deficient embryos induced a baseline level of reporter gene expression similar to that of the controls. Our studies suggest that endogenous retinoic acid is not involved inShhexpression nor in regulating its asymmetry during normal early avian embryogenesis and support the current view that endogenous retinoic acid may not be a direct morphogen for limb bud duplication.

Digit Induction by Hensen's Node and Notochord Involves the Expression ofshhbut NotRAR-β2

It is well established that Hensen's nodes can induce the formation of supernumerary digits after grafting into the anterior margin of the developing limb bud. The recent finding that distinct mesodermal cell populations are segregated within the node has made it possible to isolate different prospective cell types in an attempt to correlate digit-inducing ability with cell fate. We find that the prospective notochord cells contained within Hensen's node are able to induce supernumerary digits, whereas presumptive somite cells cannot. This early difference in inducing ability persists into later stages of development: epithelial somites are unable to induce while notochord from all lengths of the neuraxis continues to induce. Using probes to retinoic acid receptor-β2 and sonic hedgehog (shh) we find no evidence to support the idea that inducing tissues generate extra digits by releasing retinoic acid into adjacent limb tissue but find that the inducing ability of a tissue correlates with its expression ofshh.

Identification of Inducing, Responding, and Suppressing Regions in an Experimental Model of Notochord Formation in Avian Embryos

The notochord normally arises from committed cells in the rostral tip of the primitive streak. After removal of these cells from the avian gastrula, embryos with notochords nevertheless develop in the majority of cases. A region required for the formation of this reconstituted notochord lies lateral to the primitive streak. In the present study we have determined that this region acts as an inducer for more lateral cells in the epiblast, which actually give rise to the reconstituted notochord. The strongest inducing region lies between 0–250 μm lateral to the streak and 500–750 μm caudal to the rostral end of the streak and chiefly contains cells normally fated to form lateral plate and somitic mesoderm. The responding region is located 250–500 μm lateral to the streak and 0–750 μm caudal to the rostral end of the streak. This area chiefly contains cells normally fated to form neural ectoderm, although cells normally fated to form lateral plate and somitic mesoderm are also within this area. The inducing and responding areas interact to form reconstituted notochord either when the primitive streak, including its rostral end (Hensen's node), is removed from the cultured blastoderm or when the inducer and responder are grafted together into an ectopic site. Grafting Hensen's node into isolates containing both inducer and responder blocks formation of reconstituted notochord, suggesting that Hensen's node suppresses formation of lateral notochords during normal development. These findings increase our understanding of the early interactions between mesoderm and ectoderm and provide a novel model system that is well defined and accessible for studying inductive events in higher vertebrates.

Transformations in null mutants of hox genes: Do they represent intercalary regenerates?

In the minds of many, Hox gene null mutant phenotypes have confirmed the direct role that these genes play in specifying the pattern of vertebrate embryos. The genes are envisaged as defining discrete spatial domains and, subsequently, conferring specific segmental identities on cells undergoing differentiation along the antero-posterior axis. However, several aspects of the observed mutant phenotypes are inconsistent with this view. These include: the appearance of other, unexpected transformations along the dorsal axis; the occurrence of mirror-image duplications; and the development of anomalies outside the established domains of normal Hox gene expression. In this paper, Hox gene disruptions are shown to elicit regeneration-like responses in tissues confronted with discontinuities in axial identity. The polarities and orientations of transformed segments which emerge as a consequence of this response obey the rules of distal transformation and intercalary regeneration. In addition, the incidence of periodic anomalies suggests that the initial steps of Hox-mediated patterning occurs in Hensen's node. As gastrulation proceeds, mesoderm cell cycle kinetics impose constraints upon subsequent cellular differentiation. This results in the delayed manifestation of transformations along the antero-posterior axis. Finally, a paradigm is sketched in which temporal, rather than spatial axial determinants direct differentiation. Specific, testable predictions are made about the role of Hox genes in the establishment of segmental identity.

Promotion of gastrulation by maternal growth factor in cultured rabbit blastocysts

Rabbit blastocysts of day 6 post coitus were cultured in a chemically defined, protein-free medium for 24 h. Although the trophoblast continued to grow, the embryonic disc degenerated. Addition of basic fibroblast growth factor (FGF-2, of human recombinant or bovine origin, 10 ng/ml) to the culture medium resulted in significant developmental progress. The embryonic disc became pear-shaped showing a round anterior edge and a posterior node. The primitive streak and Hensen's node indicated that gastrulation had begun. Mesoderm formation was confirmed from histological sections and by localization of the expression of T-gene transcripts in whole-mount preparations. FGF-2 mRNA was detected in both day-6 endometrium and day 6-blastocysts using in vitro translation followed by immunoprecipitation with a monoclonal antibody to FGF-2. In the uterine secretions of day-6 pregnant and pseudopregnant animals, several proteins exhibiting FGF-2 antigenicity were detected on Western blots following two-dimensional gel electrophoresis. As day-6 blastocysts required exogenous FGF-2 in vitro and as FGF-2 of uterine origin is present in the uterine secretion, the maternal growth factor can promote gastrulation in vivo.

The postnodal piece (PNP) of the chick embryo as a model system for studying organ differentiation

The postnodal piece of the chick embryo is prepared by cutting the blastoderm 0.6 mm behind Hensen's node at the primitive streak stage. The expiants with their area opaca can be maintained in flat culture (New's method) for 24–30 hr. With this method the polarity and the connections between various sheets are maintained, but the expiants are stretched and difficult to handle for histology and immunostaining. Several PNPs from which the area opaca had been trimmed were cultured on one vitelline membrane (Niu and Deshpande's method) for up to 4 days without stretching effects. The polarity and connections between the embryonic sheets are hard to recognize, but expiants can be easily processed for histology and immunofluorescence. In both culture types the expiants can be easily treated, even with high molecular weight substances. Although the flat culture was useful for the induction of somites and of neural plates, we describe the advantages of culture without the area opaca of neural plates induced by tubulin mRNA or by TPA, which can differentiate into neural tubes. We also demonstrated that TPA is a powerful neural inducer in the chick embryo and stimulates cell proliferation in ectoderm and endoderm.

The Organizer-Associated Chick Homeobox Gene, Gnot1, Is Expressed before Gastrulation and Regulated Synergistically by Activin and Retinoic Acid

Gnot1, a Not (for notochord) family homeobox gene, is expressed in the chick pregastrulation blastoderm. Gnot1 expression in the epiblast is upregulated as the posteriorly derived hypoblast moves forward anteriorly to form a layer beneath it, which is of particular interest considering the known inductive role of the hypoblast in axis formation in the chick. Both activin and retinoic acid are able to activate Gnot1 expression in cultured blastodermal cells and show a strong synergistic effect when applied in combination. Strong superinduction of Gnot1 transcripts in the presence of cycloheximide also indicates the presence of a potent and labile intracellular inhibitor capable of modulating Gnot1 expression. During gastrulation, Gnot1 transcripts become localized specifically to tissues associated with "organizer" function (Hensen's node, head process, notochord). The expression data and the response to mesoderm inducing factors and axial "caudalizing" signals suggest that Gnot1 may be involved in specification of the embryonic body axis and could play a part in regulating features of the trunk/tail organizer in the chick embryo.

Induction of a secondary embryonic axis in zebrafish occurs following the overexpression of β- catenin

Formation of the vertebrate axis may involve a Wnt signaling cascade similar to the Drosophila wingless pathway. Zebrafish wnt8 is a candidate for involvement in axis specification insofar as it is expressed maternally and when overexpressed it can induce goosecoid, a transcription factor normally expressed in the embryonic shield. In this study we demonstrate that β- catenin, a cadherin associated protein in the Wnt pathway, is expressed maternally in zebrafish and is widely distributed in the early embryo. Overexpressing β- catenin in early zebrafish embryos induces goosecoid and ntl, ultimately leading to a duplication of a complete secondary axis. These data are consistent with the involvement of β- catenin in a Wnt signaling pathway which is involved in mesoderm induction in zebrafish.