Requirement for anti-dorsalizing morphogenetic protein in organizer patterning

The amphibian Spemann organizer is subdivided in trunk and head organizer and it is unclear how this division is regulated. The Xenopus trunk organizer expresses anti-dorsalizing morphogenetic protein (ADMP), a potent organizer antagonist. We show that ADMP represses head formation during gastrulation and that its expression is activated by BMP antagonists. A specifically acting dominant-negative ADMP anteriorizes embryos and its coexpression with BMP antagonists induces secondary embryonic axes with heads as well as expression of head inducers. Unlike other BMPs, ADMP is not inhibited by a dominant-negative BMP type I receptor, Noggin, Cerberus and Chordin but by Follistatin, suggesting that it utilizes a distinct TGF-β receptor pathway and displays differential sensitivity to BMP antagonists. The results indicate that ADMP functions in the trunk organizer to antagonize head formation, thereby regulating organizer patterning.     

Cooperative action of ADMP- and BMP-mediated pathways in regulating cell fates in the zebrafish gastrula.

It was shown in Xenopus and chick that Spemann's organizer activity is regulated through the negative action of Anti-Dorsalizing Morphogenetic Protein (ADMP). We report the characterization and functional properties of admp in zebrafish. admp expression profile is consistent with a role in the organizer, including the tail organizer. We studied admp function through overexpression experiments, with the use of a dominant-negative form (TR-ADMP) and of an antisense morpholino-modified oligonucleotide. Our results indicate that the ADMP pathway causes the restriction of anterior and axial fates and that ADMP, BMP2b, and BMP7 pathways have distinct actions but cooperate in establishing proper dorso-ventral regionalization. This is shown by partial rescue of the dorsalized mutant snailhouse and of the ventralized mutant chordino, upon admp and tr-admp RNA injection, respectively. Moreover, ADMP and BMP7 probably form heterodimers as shown by the ability of TR-ADMP and BMP7 to antagonize each other. We observed that a MYC-tagged ADMP was secreted and detected in the extracellular space, suggesting that admp could act at a distance. Simultaneous local inhibition of bmp function at the blastoderm margin and impairment of ADMP secretion led to the induction of secondary head structures, confirming that the two pathways cooperatively regulate organizer formation and activity.     

The role of the Spemann organizer in anterior-posterior patterning of the trunk

The formation of the vertebrate body axis during gastrulation strongly depends on a dorsal signaling centre, the Spemann organizer as it is called in amphibians. This organizer affects embryonic development by self-differentiation, regulation of morphogenesis and secretion of inducing signals. Whereas many molecular signals and mechanisms of the organizer have been clarified, its function in anterior-posterior pattern formation remains unclear. We dissected the organizer functions by generally blocking organizer formation and then restoring a single function. In experiments using a dominant inhibitory BMP receptor construct (tBr) we find evidence that neural activation by antagonism of the BMP pathway is the organizer function that enables the establishment of a detailed anterior-posterior pattern along the trunk. Conversely, the exclusive inhibition of neural activation by expressing a constitutive active BMP receptor (hAlk-6) in the ectoderm prohibits the establishment of an anterior-posterior pattern, even though the organizer itself is still intact. Thus, apart from the formerly described separation into a head and a trunk/tail organizer, the organizer does not deliver positional information for anterior-posterior patterning. Rather, by inducing neurectoderm, it makes ectodermal cells competent to receive patterning signals from the non-organizer mesoderm and thereby enable the formation of a complete and stable AP pattern along the trunk.     

The homeobox gene bozozok promotes anterior neuroectoderm formation in zebrafish through negative regulation of BMP2/4 and Wnt pathways

The neuroectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be regionalized into forebrain, midbrain, hindbrain and spinal cord by a two-step process. In the activation step, the Spemann gastrula organizer induces neuroectoderm with anterior character, followed by posteriorization by a transforming signal. Recently, simultaneous inhibition of BMP and Wnt signaling was shown to induce head formation in frog embryos. However, how the inhibition of BMP and Wnt signaling pathways specify a properly patterned head, and how they are regulated in vivo, is not understood. Here we demonstrate that the loss of anterior neural fates observed in zebrafish bozozok (boz) mutants occurs during gastrulation due to a reduction and subsequent posteriorization of neuroectoderm. The neural induction defect was correlated with decreased chordino expression and consequent increases in bmp2b/4 expression, and was suppressed by overexpression of BMP antagonists. Whereas expression of anterior neural markers was restored by ectopic BMP inhibition in early boz gastrulae, it was not maintained during later gastrulation. The posteriorization of neuroectoderm in boz was correlated with ectopic dorsal wnt8 expression. Overexpression of a Wnt antagonist rescued formation of the organizer and anterior neural fates in boz mutants. We propose that boz specifies formation of anterior neuroectoderm by regulating BMP and Wnt pathways in a fashion consistent with Nieuwkoop's two-step neural patterning model. boz promotes neural induction by positively regulating organizer-derived chordino and limiting the antineuralizing activity of BMP2b/4 morphogens. In addition, by negative regulation of Wnt signaling, boz promotes organizer formation and limits posteriorization of neuroectoderm in the late gastrula.     

Dickkopf1 and the Spemann-Mangold head organizer

Work in amphibians indicates that inhibition of Wnt and BMP signals is essential for head development and that head induction by the Spemann-Mangold organizer may be mediated by secreted Wnt antagonists. Wnts are potent posteriorizing factors and antagonize the Spemann-Mangold organizer. Dickkopf1 (dkk1) encodes a secreted effector expressed in head organizing centers of Xenopus, mouse and zebrafish. It acts as a Wnt inhibitor and is able together with BMP inhibitors to induce the formation of ectopic embryonic heads in Xenopus. It anteriorizes both mesendoderm and neuroectoderm, promoting prechordal plate and forebrain fates. Injection of inhibitory antibodies leads to microcephaly and cyclopia. Dkk1 thus is an essential mediator of the vertebrate head organizer.     

Roles of organizer factors and BMP antagonism in mammalian forebrain establishment

A critical question in mammalian development is how the forebrain is established. In amphibians, bone morphogenetic protein (BMP) antagonism emanating from the gastrula organizer is key. Roles of BMP antagonism and the organizer in mammals remain unclear. Anterior visceral endoderm (AVE) promotes early mouse head development, but its function is controversial. Here, we explore the timing and regulation of forebrain establishment in the mouse. Forebrain specification requires tissue interaction through the late streak stage of gastrulation. Foxa2(-/-) embryos lack both the organizer and its BMP antagonists, yet about 25% show weak forebrain gene expression. A similar percentage shows ectopic AVE gene expression distally. The distal VE may thus be a source of forebrain promoting signals in these embryos. In wild-type ectoderm explants, AVE promoted forebrain specification, while anterior mesendoderm provided maintenance signals. Embryological and molecular data suggest that the AVE is a source of active BMP antagonism in vivo. In prespecification ectoderm explants, exogenous BMP antagonists triggered forebrain gene expression and inhibited posterior gene expression. Conversely, BMP inhibited forebrain gene expression, an effect that could be antagonized by anterior mesendoderm, and promoted expression of some posterior genes. These results lead to a model in which BMP antagonism supplied by exogenous tissues promotes forebrain establishment and maintenance in the murine ectoderm.     

Chordin and noggin promote organizing centers of forebrain development in the mouse

In this study we investigate the roles of the organizer factors chordin and noggin, which are dedicated antagonists of the bone morphogenetic proteins (BMPs), in formation of the mammalian head. The mouse chordin and noggin genes (Chrd and Nog) are expressed in the organizer (the node) and its mesendodermal derivatives, including the prechordal plate, an organizing center for rostral development. They are also expressed at lower levels in and around the anterior neural ridge, another rostral organizing center. To elucidate roles of Chrd and Nog that are masked by the severe phenotype and early lethality of the double null, we have characterized embryos of the genotype Chrd(-/-);Nog(+/-). These animals display partially penetrant neonatal lethality, with defects restricted to the head. The variable phenotypes include cyclopia, holoprosencephaly, and rostral truncations of the brain and craniofacial skeleton. In situ hybridization reveals a loss of SHH expression and signaling by the prechordal plate, and a decrease in FGF8 expression and signaling by the anterior neural ridge at the five-somite stage. Defective Chrd(-/-);Nog(+/-) embryos exhibit reduced cell proliferation in the rostral neuroepithelium at 10 somites, followed by increased cell death 1 day later. Because these phenotypes result from reduced levels of BMP antagonists, we hypothesized that they are due to increased BMP activity. Ectopic application of BMP2 to wild-type cephalic explants results in decreased FGF8 and SHH expression in rostral tissue, suggesting that the decreased expression of FGF8 and SHH observed in vivo is due to ectopic BMP activity. Cephalic explants isolated from Chrd;Nog double mutant embryos show an increased sensitivity to ectopic BMP protein, further supporting the hypothesis that these mutants are deficient in BMP antagonism. These results indicate that the BMP antagonists chordin and noggin promote the inductive and trophic activities of rostral organizing centers in early development of the mammalian head.     

The FGFR pathway is required for the trunk-inducing functions of Spemann's organizer.

Xenopus laevis embryogenesis is controlled by the inducing activities of Spemann's organizer. These inducing activities are separated into two distinct suborganizers: a trunk organizer and a head organizer. The trunk organizer induces the formation of posterior structures by emitting signals and directing morphogenesis. Here, we report that the fibroblast growth factor receptor (FGFR) signaling pathway, also known to regulate posterior development, performs critical functions within the cells of Spemann's organizer. Specifically, the FGFR pathway was required in the organizer cells in order for those cells to induce the formation of somitic muscle and the pronephros. Since the organizer influences the differentiation of these tissues by emitting signals that pattern the mesodermal germ layer, our data indicate that the FGFR regulates the production of these signals. In addition, the FGFR pathway was required for the expression of chordin, an organizer-specific protein required for the trunk-inducing activities of Spemann's organizer. Significantly, the FGFR pathway had a minimal effect on the function of the head organizer. We propose that the FGFR pathway is a defining molecular component that distinguishes the trunk organizer from the head organizer by controlling the expression of organizer-specific genes required to induce the formation of posterior structures and somitic muscle in neighboring cells. The implications of our findings for the evolutionarily conserved role of the FGFR pathway in the functions of Spemann's organizer and other vertebrate-signaling centers are discussed.     

Spatially distinct head and heart inducers within the Xenopus organizer region.

BACKGROUND: The mouse anterior visceral endoderm, an extraembryonic tissue, expresses several genes essential for normal development of structures rostral to the anterior limit of the notochord and has been termed the head organizer. This tissue also has heart-inducing activity and expresses mCer1 which, like its Xenopus homolog cerberus, can induce markers of cardiac specification and anterior neural tissue when ectopically expressed. We investigated the relationship between head and heart induction in Xenopus embryos, which lack extraembryonic tissues. RESULTS: We found three regions of gene expression in the Xenopus organizer: deep endoderm, which expressed cerberus; prechordal mesoderm, which showed overlapping but non-identical expression of genes characteristic of the murine head organizer, such as XHex and XANF-1; and leading-edge dorsoanterior endoderm, which expressed both cerberus and a subset of the genes expressed by the prechordal mesoderm. Microsurgical ablation of the cerberus-expressing endoderm decreased the incidence of heart, but not head, formation. Removal of prechordal mesoderm, in contrast, caused deficits of anterior head structures. Finally, although misexpression of cerberus induced ectopic heads, it was unable to induce genes thought to participate in head induction. CONCLUSIONS: In Xenopus, the cerberus-expressing endoderm is required for heart, but not head, inducing activity. Therefore, this tissue is not the topological equivalent of the murine anterior visceral endoderm. We propose that, in Xenopus, cerberus is redundant to other bone morphogenetic protein (BMP) and Wnt antagonists located in prechordal mesoderm for head induction, but may be necessary for heart induction.     

Depletion of three BMP antagonists from Spemann's organizer leads to a catastrophic loss of dorsal structures

Transplanted Spemann's organizer induces dorsal embryonic cell fates such as the nervous system and somites, but in normal development, elimination of individual organizer signals (such as the bone morphogenetic protein [BMP] antagonists) has surprisingly modest effects on these tissues. Thus, the role of BMP antagonists may be limited to fine tuning the size of the dorsal domain. However, at least five BMP antagonists are specifically expressed in the organizer, and all can mimic aspects of organizer function, suggesting overlapping functions. Here, we deplete the function of three BMP antagonists, chordin, noggin, and follistatin, in Xenopus tropicalis. We demonstrate that this results in catastrophic failure of dorsal development and expansion of ventral and posterior fates. We conclude that BMP antagonists are required for formation of the neural plate and dorsal mesoderm. In addition, our results show that neural specification requires the continuous activity of BMP antagonists from blastula through gastrula stages.     

FGF signaling and the anterior neural induction in Xenopus

We previously showed that FGF was capable of inducing Xenopus gastrula ectoderm cells in culture to express position-specific neural markers along the anteroposterior axis in a dose-dependent manner. However, conflicting results have been obtained concerning involvement of FGF signaling in the anterior neural induction in vivo using the same dominant-negative construct of Xenopus FGF receptor type-1 (delta XFGFR-1 or XFD). We explored this issue by employing a similar construct of receptor type-4a (XFGFR-4a) in addition, since expression of XFGFR-4a was seen to peak between gastrula and neurula stages, when the neural induction and patterning take place, whereas expression of XFGFR-1 had not a distinct peak during that period. Further, these two FGFRs are most distantly related in amino acid sequence in the Xenopus FGFR family. When we injected mRNA of a dominant-negative version of XFGFR-4a (delta XFGFR-4a) into eight animal pole blastomeres at 32-cell stage, anterior defects including loss of normal structure in telencephalon and eye regions became prominent as examined morphologically or by in situ hybridization. Overexpression of delta XFGFR-1 appeared far less effective than that of delta XFGFR-4a. Requirement of FGF signaling in ectoderm for anterior neural development was further confirmed in culture: when ectoderm cells that were overexpressing delta XFGFR-4a were cocultured with intact organizer cells from either early or late gastrula embryos, expression of anterior and posterior neural markers was inhibited, respectively. We also showed that autonomous neuralization of the anterior-type observed in ectoderm cells that were subjected to prolonged dissociation was strongly suppressed by delta XFGFR-4a, but not as much by delta XFGFR-1. It is thus indicated that FGF signaling in ectoderm, mainly through XFGFR-4, is required for the anterior neural induction by organizer. We may reconcile our data to the current "neural default model," which features the central roles of BMP4 signaling in ectoderm and BMP4 antagonists from organizer, simply postulating that the neural default pathway in ectoderm includes constitutive FGF signaling step.     

Noggin and noggin-like genes control dorsoventral axis regeneration in planarians

Planarians regenerate a whole animal from a small body piece within a few days. Recent studies have shown that the bone morphogenetic protein (BMP) pathway is required to reestablish the dorsoventral (DV) axis. In vertebrates, the specification of the DV axis depends on the coordinated action of a dual organizer defined by BMP and antidorsalizing morphogenetic protein (ADMP) under the control of several factors, including the inhibitors chordin and noggin. Planarians have an expanded noggin family (up to ten members), which have been classified as canonical noggin (nog) and noggin-like (nlg) genes, the latter carrying an insertion within the noggin domain. Here we show that a BMP/ADMP organizer governs DV axis reestablishment during planarian regeneration, highlighting a greater-than-thought conservation of the mechanisms that establish this axis in protostomes and deuterostomes. Also, we report that whereas noggin genes function as canonical BMP inhibitors, the silencing of planarian nlg8 induces ectopic neurogenesis and enhances ventralizing bmp(RNAi) phenotypes. Finally, we show that noggin-like genes are conserved from cnidarian to vertebrates and that both planarian nlg8 and Xenopus nlg ventralize Xenopus embryos when overexpressed. Remarkably, this ventralization is not associated with an increase in SMAD1/5/8 phosphorylation.     

Neural and head induction by insulin-like growth factor signals.

Evidence is presented for a new pathway participating in anterior neural development. It was found that IGF binding protein 5 (IGFBP-5), as well as three IGFs expressed in early embryos, promoted anterior development by increasing the head region at the expense of the trunk in mRNA-injected Xenopus embryos. A secreted dominant-negative type I IGF receptor (DN-IGFR) had the opposite effect. IGF mRNAs led to the induction of ectopic eyes and ectopic head-like structures containing brain tissue. In ectodermal explants, IGF signals induced anterior neural markers in the absence of mesoderm formation and DN-IGFR inhibited neural induction by the BMP antagonist Chordin. Thus, active IGF signals appear to be both required and sufficient for anterior neural induction in Xenopus.     

Twisted gastrulation loss-of-function analyses support its role as a BMP inhibitor during early Xenopus embryogenesis

BMP signals play important roles in the regulation of diverse events in development and in the adult. In amniotes, like the amphibian Xenopus laevis, BMPs promote ventral specification, while chordin and other BMP inhibitors expressed dorsally in the Spemann's organizer play roles in establishment and/or maintenance of this region as dorsal endomesoderm. The activities of chordin are in turn regulated by the secreted proteolytic enzymes BMP1 and Xolloid. Recently, we and others have identified the protein twisted gastrulation (TSG) as a soluble BMP modulator that functions by modifying chordin activity. Overexpression and genetic analyses in Drosophila, Xenopus and zebrafish together with in vitro biochemical studies suggest that TSG might act as a BMP antagonist; but there is also evidence that TSG may promote BMP signaling. Here we report examination of the in vivo function of TSG in early Xenopus development using a loss-of-function approach. We show that reducing TSG expression using antisense TSG morpholino oligonucleotides (MOs) results in moderate head defects. These defects can be rescued both by a TSG that cannot be inhibited by the MO, and by the BMP antagonists chordin and noggin. Furthermore, while neither the onset of gastrulation nor the expression of marker genes are affected in early gastrulae, dorsal marker gene expression is reduced at the expense of expanded ventral marker gene expression beginning at mid to late gastrula stage. TSG-MO and Chd-MOs also cooperate to strongly repress head formation. Finally, we note that the loss of TSG function results in a shift in tissue responsiveness to the BMP inhibitory function of chordin in both animal caps and the ventral marginal zone, a result that implies that the activity of TSG may be required for chordin to efficiently inhibit BMPs in these developmental contexts. These data, taken together with the biochemistry and overexpression studies, argue that TSG plays an important role in regulating the potency of chordin's BMP inhibitory activity and TSG and chordin act together to regulate the extent of dorsoanterior development of early frog embryos.     

Animal-vegetal asymmetries influence the earliest steps in retina fate commitment in Xenopus.

An individual retina descends from a restricted and invariant group of nine animal blastomeres at the 32-cell stage. We tested which molecular signaling pathways are responsible for the competence of animal blastomeres to contribute to the retina. Inactivation of activin/Vg1 or fibroblast growth factor (FGF) signaling by expression of dominant-negative receptors does not prevent an animal blastomere from contributing to the retina. However, increasing bone morphogenetic protein (BMP) signaling in the retina-producing blastomeres significantly reduces their contribution. Conversely, reducing BMP signaling by expression of a dominant-negative BMP receptor or Noggin allows other animal blastomeres to contribute to the retina. Thus, the initial step in the retinal lineage is regulated by position within the BMP/Noggin field of epidermal versus neural induction. Vegetal tier blastomeres, in contrast, cannot contribute to the retina even when given access to the appropriate position and signaling fields by transplantation to the dorsal animal pole. We tested whether expression of molecules within the mesoderm inducing (activin, FGF), mesoderm-modifying (Wnt), or neural-inducing (BMP, Noggin) pathways impart a retinal fate on vegetal cell descendants. None of these, several of which induce secondary head structures, caused vegetal cells to contribute to retina. This was true even if the injected blastomeres were transplanted to the dorsal animal pole. Two pathways that specifically induce head tissues also were investigated. The simultaneous blockade of Wnt and BMP signaling, which results in the formation of a complete secondary axis with head and eyes, did not cause the vegetal clone to give rise to retina. However, Cerberus, a secreted protein that also induces an ectopic head with eyes, redirected vegetal progeny into the retina. These experiments indicate that vegetal blastomere incompetence to express a retinal fate is not due to a lack of components of known signaling pathways, but relies on a specific pathway of head induction.