Secreted noggin protein mimics the Spemann organizer in dorsalizing Xenopus mesoderm

A dorsalizing signal acts during gastrulation to change the specification of lateral mesodermal tissues from ventral (blood, mesenchyme) to more dorsal fates (muscle, heart, pronephros). This signal, from Spemann's organizer, cannot be mimicked by the mesoderm inducers activin and fibroblast growth factor. The gene noggin is expressed in the organizer, and could be the dorsalizing signal. Here we show that soluble noggin protein added to ventral marginal zones during gastrulation induces muscle, but that activin does not. Dorsal pattern can be partially rescued in ventralized embryos by injection of a plasmid that expresses noggin during gastrulation. The results suggest that the noggin product may be the dorsalizing signal from the organizer.     

Zebrafish organizer development and germ-layer formation require nodal-related signals

The vertebrate body plan is established during gastrulation, when cells move inwards to form the mesodermal and endodermal germ layers. Signals from a region of dorsal mesoderm, which is termed the organizer, pattern the body axis by specifying the fates of neighbouring cells. The organizer is itself induced by earlier signals. Although members of the transforming growth factor-beta (TGF-beta) and Wnt families have been implicated in the formation of the organizer, no endogenous signalling molecule is known to be required for this process. Here we report that the zebrafish squint (sqt) and cyclops (cyc) genes have essential, although partly redundant, functions in organizer development and also in the formation of mesoderm and endoderm. We show that the sqt gene encodes a member of the TGF-beta superfamily that is related to mouse nodal. cyc encodes another nodal-related proteins, which is consistent with our genetic evidence that sqt and cyc have overlapping functions. The sqt gene is expressed in a dorsal region of the blastula that includes the extraembryonic yolk syncytial layer (YSL). The YSL has been implicated as a source of signals that induce organizer development and mesendoderm formation. Misexpression of sqt RNA within the embryo or specifically in the YSL induces expanded or ectopic dorsal mesoderm. These results establish an essential role for nodal-related signals in organizer development and mesendoderm formation.     

Bridging the GAP in inositol 1,3,4,5-tetrakisphosphate signalling

Recent advances in analyzing wnt signaling have provided evidence that frizzled proteins can function as wnt receptors. We have identified Xfz3, a Xenopus frizzled family member. The amino acid sequence is 89% identical to the product of the murine gene Mfz3, and is predicted to be a serpentine receptor with seven transmembrane domains. Xfz3 is a maternal mRNA with low levels of expression until the end of gastrulation. The expression level increases significantly from neurulation onward. Whole-mount in situ hybridization analysis shows that expression of Xfz3 is highly restricted to the central nervous system. High levels of expression are detected in the anterior neural folds. Low levels of expression are also detected in the optic and otic vesicles, as well as in the pronephros anlage. In addition, Xfz3 mRNA is concentrated in a large band in the midbrain. Overexpression of Xfz3 blocks neural tube closure, resulting in embryos with either bent and strongly reduced anteroposterior axis in a dose-dependent manner. However, it does not affect gastrulation, the expression and localization of organizer-specific genes such as goosecoid, chordin and noggin. Therefore, Xfz3 is not involved in early mesodermal patterning. Injection of RNA encoding GFP-tagged Xfz3 shows that overexpressed proteins can be detected on the cell surface until at least late neurula stage, suggesting that they can exert an effect after gastrulation. Our expression data and functional analyses suggest that the Xfz3 gene product has an antagonizing activity in the morphogenesis during Xenopus development.     

Laminar development of the mouse barrel cortex: effects of neurotoxins against monoamines

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.     

Characterization of CMIX, a chicken homeobox gene related to the Xenopus gene mix.1

Members of the TGFbeta, Wnt and FGF families act in concert to induce and pattern the mesoderm of gastrulating embryos. Downstream effectors for these growth factors include homeobox proteins, which also feed back to activate and repress upstream signaling pathways (e.g. Fainsod, A., Steinbeisser, H., De Robertis, E.M. 1994. On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo. EMBO J. 13, 5015-5025; Carnac, G., Kodjabachian, L., Gurdon, J.B., Lemaire, P. 1996. The homeobox gene Siamois is a target of the Wnt dorsalization pathway and triggers organizer activity in the absence of mesoderm. Development 122, 3055-3065). As well as having interwoven upstream and downstream regulatory pathways Mix.1, siamois and goosecoid, all paired-type homeobox genes, may physically interact with each other as heterodimers to regulate dorsal-ventral polarity (Mead, P.E., Brivanlou, I.H., Kelley, C.M., Zon, L.I. 1996. BMP-4 responsive regulation of dorsal-ventral patterning by the homeobox protein Mix.1. Nature 382, 357-360). We report here a chicken paired-type homeobox gene, CMIX, with a homeodomain having 72% aa identity to its nearest homolog, Xenopus Mix.1. CMIX is expressed in the epiblast of the posterior marginal zone of early chick embryos, and later along the entire anterior-posterior axis of the primitive streak in cells of the medial ectoderm, in nascent mesoderm, but not in endoderm. Coincident with formation of prechordal mesoderm, CMIX mRNA levels rapidly decline throughout the embryo.     

Spatially distinct domains of cell behavior in the zebrafish organizer region

To determine the sequence of cell behaviors that is involved in the morphogenesis of the zebrafish organizer region, we have examined the dorsal marginal zone of vitally stained zebrafish embryos using time-lapse confocal microscopy. During the late-blastula stage, the zebrafish dorsal marginal zone segregates into several cellular domains, including a group of noninvoluting, highly endocytic marginal (NEM) cells. The NEM cell cluster, which lies in a superficial location of the dorsal marginal zone, is composed of both enveloping layer cells and one or two layers of underlying deep cells. The longitudinal position of this cellular domain accurately predicts the site of embryonic shield formation and occupies a homologous location to the organizer epithelium in Xenopus laevis. At the onset of gastrulation, deep cells underneath the superficial NEM cell domain undergo involution to form the nascent hypoblast of the embryonic shield. Deep cells within the NEM cell cluster, however, do not involute during early shield formation, but instead move in front of the blastoderm margin to form a loose mass of cells called forerunner cells. Forerunner cells coalesce into a wedge-shaped mass during late gastrulation and eventually become overlapped by the converging lateral lips of the germ ring. During early zebrafish tail elongation, most forerunner cells are incorporated into the epithelial lining of Kupffer's vesicle, a transient teleostean organ rudiment long thought to be an evolutionary vestige of the neurenteric canal. Owing to the location of NEM cells at the dorsal margin of blastula-stage embryos, as well as their early segregation from other deep cells, we hypothesized that NEM cells are specified by an early-acting dorsalizing signal. To test this possibility, we briefly treated early-blastula stage embryos with LiCl, an agent known to produce hyperdorsalized zebrafish embryos with varying degrees of expanded organizer tissue. In Li(+)-treated embryos, NEM cells appear either within expanded spatial domains or in ectopic locations, primarily within the marginal zone of the blastoderm. These results suggest that NEM cells represent a specific cell type that is specified by an early dorsal patterning pathway.     

The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle

Embryos with no dorsal axis were obtained when more than 15% of the egg surface was deleted from the vegetal pole of the early 1-cell embryo of Xenopus laevis. The timing of the deletion in the first cell cycle was critical: dorsal-deficient embryos were obtained when the deletion began before time 0.5 (50% of the first cell cycle) whereas normal dorsal axis usually formed when the deletion was done later than time 0.8. The axis deficiency could be restored by lithium treatment and the injection of vegetal but not animal cytoplasm. Bisection of the embryo at the 2-cell stage, which is known to restore the dorsal structures in the UV-ventralized embryos, had no effect on the vegetal-deleted embryos. These results show clearly that, in Xenopus, (1) the dorsal determinants (DDs) localized in the vegetal pole region at the onset of development are necessary for dorsal axis development and (2) the DDs move from the vegetal pole to a subequatorial region where they are incorporated into gastrulating cells to form the future organizing center. A model for the early axis formation process in Xenopus is proposed.     

Hematopoietic induction and respecification of A-P identity by visceral endoderm signaling in the mouse embryo

The anteroposterior axis of the developing embryo becomes morphologically apparent at the onset of gastrulation with the formation of the primitive streak. This structure, where the first mesodermal cells arise, marks the posterior aspect of the embryo. To examine the potential role of non-mesodermal signals in specifying posterior (hematopoietic and endothelial) cell fates in the mouse embryo, we have devised a transgenic explant culture system. We show that interactions between primitive endoderm and adjacent embryonic ectoderm or nascent mesoderm are required early in gastrulation for initiation of hematopoiesis and vasculogenesis. Surprisingly, primitive endoderm signals can respecify anterior (prospective neural) ectoderm to a posterior mesodermal fate, resulting in formation of blood and activation of endothelial markers. Reprogramming of anterior ectoderm does not require cell contact and is effected by stage-dependent, short-range, diffusible signal(s). Therefore, primitive endoderm signaling is a critical early determinant of hematopoietic and vascular development and plays a decisive role in anterior-posterior patterning during mouse embryogenesis.     

The Xcad-2 gene can provide a ventral signal independent of BMP-4

Patterning of the marginal zone in the Xenopus embryo has been attributed to interactions between dorsal genes expressed in the organizer and ventral-specific genes. In this antagonistic interplay of activities, BMP-4, a gene that is not expressed in the organizer, provides a strong ventralizing signal. The Xenopus caudal type homeobox gene, Xcad-2, which is expressed around the blastopore with a gap over the dorsal lip, was analyzed as part of the ventral signal. Xcad-2 was shown to efficiently repress during early gastrula stages the dorsal genes gsc, Xnot-2, Otx-2, XFKH1 and Xlim-1, while it positively regulates the ventral genes, Xvent-1 and Xvent-2, with Xpo exhibiting a strong positive response to Xcad-2 overexpression. Xcad-2 was also capable of inducing BMP-4 expression in the organizer region. Support for a ventralizing role for Xcad-2 was obtained from co-injection experiments with the dominant negative BMP receptor which was used to block BMP-4 signaling. Under lack-of-BMP-signaling conditions Xcad-2 could still regulate dorsal and ventral gene expression and restore normal development, suggesting that it can act downstream of BMP-4 signaling or independently of it. Xcad-2 could also inhibit secondary axis formation and dorsalization induced by the dominant negative BMP receptor. Xcad-2 was also shown to efficiently reverse the dorsalizing effects of LiCl. These results place Xcad-2 as part of the ventralizing gene program which acts during early gastrula stages and can execute its ventralizing function in the absence of BMP signaling.     

A role for FGF-8 in the dorsoventral patterning of the zebrafish gastrula

Signals released from Spemann's organizer, together with ventralizing factors such as BMPs, are necessary to pattern the dorsoventral axis of the vertebrate embryo. We report that a member of the FGF family, fgf-8, not secreted by the axial mesoderm but expressed in a dorsoventral gradient at the margin of the zebrafish gastrula, also contributes to the establishment of the dorsoventral axis of the embryo. Ectopic expression of FGF-8 leads to the expansion of dorsolateral derivatives at the expense of ventral and posterior domains. Moreover, FGF-8 displays some organizer properties as it induces the formation of a partial secondary axis in the absence of factors released from Spemann's organizer territory. Analysis of its interaction with the ventralizing factors, BMPs, reveals that overexpression of FGF-8 inhibits the expression of these factors in the ventral part of the embryo as early as blastula stage, suggesting that FGF-8 acts upstream of BMP2 and BMP4. We conclude that FGF-8 is involved in defining dorsoventral identity and is an important organizing factor responsible for specification of mesodermal and ectodermal dorsolateral territories of the zebrafish gastrula.     

The nieuwkoid gene characterizes and mediates a Nieuwkoop-center-like activity in the zebrafish

BACKGROUND: In amphibians, the Nieuwkoop center--a primary inducing region--has a central role in the induction of dorsal mesodermal cells to form the Spemann organizer. In teleosts, such as the zebrafish, Danio rerio, the functional equivalent of the amphibian Spemann organizer is the dorsal shield. Historically, a small region of the teleost yolk syncytial layer (YSL), an extraembryonic tissue that underlies the entire blastoderm, has been implicated in dorsal shield specification. Difficulties in transplanting discrete regions of the YSL and the previous lack of localized expression patterns unique to the YSL have, however, hindered efforts to prove definitively that the YSL possesses Nieuwkoop-center-like activities. RESULTS: Here, we describe the isolation and analysis of a new homeobox gene, called nieuwkoid, which is first expressed immediately following the mid-blastula transition on the dorsal side of the zebrafish pregastrula embryo. We found that, by the onset of gastrulation, nieuwkoid expression becomes localized to a restricted region of the YSL, directly underlying the future dorsal shield. Mis-expression of nieuwkoid in early zebrafish embryos was found to be sufficient for the induction of ectopic organizer regions and secondary axes. Mis-expression of nieuwkoid by cell transplantation or by direct injection into the YSL led to the non-autonomous induction of ectopic organizer gene expression. CONCLUSIONS: The dynamic and restricted expression of the nieuwkoid gene, combined with its potent dorsalizing activity, suggests that nieuwkoid is an important component in the regionalization of the gastrula organizer, possibly characterizing and mediating an organizer-inducing/Nieuwkoop-center-like activity.     

Attenuation of kindling-induced decreases in NT-3 mRNA by thyroid hormone depletion

Tat is one of the regulatory proteins of the HIV-1 virus. To date, besides the transactivation activity, a myriad of effects exerted by HIV-1 Tat on cellular and viral genes have been observed. The present study investigated the in vivo effects of HIV-1 Tat protein in the Xenopus embryo. We adopted the Xenopus system since expression of putative regulatory factors in the embryo has been widely used as a quick and effective first screen for protein function. Xenopus' early development is well characterized by stage-specific phenotypes, therefore, an in vivo HIV-1 Tat-mediated aberrant phenotype can easily be detected and analyzed. HIV-1 Tat protein expression through injection of synthetic mRNA into zygotes produced a marked delay in gastrulation leading to altered specification of the anterior-posterior axis and to partial or total loss of anterior structures. HIV-1 Tat effects resulted in a general suppression of gene expression, including that of Xbra and gsc, two early genes whose expression is required for proper gastrulation. The specificity of Tat effects was demonstrated by injecting a 'loss of function' mutant (Tat-C37S), lacking a single cysteine residue, which did not yield any effect. Both Tat and Tat-C37S were found to be localized mainly in the nucleus. The importance of subcellular targeting for the effects caused by HIV-1 Tat was demonstrated by injecting a second mutant (Tat-BDM), carrying an altered nuclear localization signal sequence. The Tat-BDM protein localized in the cytoplasm and accumulated at the cell membrane. Embryos injected with Tat-BDM mRNA did not develop beyond gastrulation. The importance of proper protein conformation and subcellular localization in determining Tat effects is discussed.     

Neurulation in amniote vertebrates: a novel view deduced from the use of quail-chick chimeras

Two apparently different mechanisms successively contribute to the formation of the neural tube in the avian embryo: bending of the neural plate during the primary neurulation in the cephalo-cervico-thoracic region and cavitation of the medullary cord during the secondary neurulation in the lumbo-sacral region. During both these processes, gastrulation continues by the caudal regression of Hensen's node--also called cordoneural hinge in the secondary neurulation. Labeling of Hensen's node or cordoneural hinge by the quail chick marker system revealed that this structure, which is the equivalent of the dorsal blastoporal lip of the Amphibian embryo, i.e., of the Spemann's organizer, gives rise to the midline cells of the three germ layers: the floor plate of the neural tube, the notocord and the dorsal cells of the intestinal endoderm. Caudally to the organizer, both in primary and secondary neurulation, the presumptive territory of the alar plates of the future neural tube overlies the precursors of the paraxial mesoderm. Regression of Hensen's node bisects the ectoderm in two bilateral neural plates leaving in its wake the floor plate, the notocord and the dorsal endoderm.     

A novel homeobox gene, dharma, can induce the organizer in a non-cell-autonomous manner

The formation of Spemann organizer is one of the most important steps in dorsoventral axis determination in vertebrate development. However, whether the organizer forms autonomously or is induced non-cell-autonomously is controversial. In this report we have isolated a novel zebrafish homeobox gene, dharma, capable of inducing the organizer ectopically. The expression of dharma was first detected in several blastomeres at one side of the margin soon after the mid-blastula transition and continued in the dorsal side of the yolk syncytial layer (YSL) under the embryonic shield, the zebrafish organizer, until the onset of gastrulation. Furthermore, dharma expressed in the YSL induced the organizer in a non-cell-autonomous manner. These results provided the first identification of a zygotic gene to be implicated in the formation of an organizer-inducing center.     

Determination of the zebrafish forebrain: induction and patterning

We report an analysis of forebrain determination and patterning in the zebrafish Danio rerio. In order to study these events, we isolated zebrafish homologs of two neural markers, odd-paired-like (opl), which encodes a zinc finger protein, and fkh5, which encodes a forkhead domain protein. At mid-gastrula, expression of these genes defines a very early pattern in the presumptive neurectoderm, with opl later expressed in the telencephalon, and fkh5 in the diencephalon and more posterior neurectoderm. Using in vitro explant assays, we show that forebrain induction has occurred even earlier, by the onset of gastrulation (shield stage). Signaling from the early gastrula shield, previously shown to be an organizing center, is sufficient for activation of opl expression in vitro. In order to determine whether the organizer is required for opl regulation, we removed from late blastula stage embryos either the presumptive prechordal plate, marked by goosecoid (gsc) expression, or the entire organizer, marked by chordin (chd) expression. opl was correctly expressed after removal of the presumptive prechordal plate and consistently, opl was correctly expressed in one-eyed pinhead (oep) mutant embryos, where the prechordal plate fails to form. However, after removal of the entire organizer, no opl expression was observed, indicating that this region is crucial for forebrain induction. We further show that continued organizer function is required for forebrain induction, since beads of BMP4, which promotes ventral fates, also prevented opl expression when implanted during gastrulation. Our data show that forebrain specification begins early during gastrulation, and that a wide area of dorsal mesendoderm is required for its patterning.