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PubMed1997

Frzb, a secreted protein expressed in the Spemann organizer, binds and inhibits Wnt-8.

We isolated a Xenopus homolog of Frzb, a newly described protein containing an amino-terminal Frizzled motif. It dorsalized Xenopus embryos and was expressed in the Spemann organizer during early gastrulation. Unlike Frizzled proteins, endogenous Frzb was soluble. Frzb was secretable and could act across cell boundaries. In several functional assays, Frzb antagonized Xwnt-8, a proposed ventralizing factor with an expression pattern complementary to that of Frzb. Furthermore, Frzb blocked induction of MyoD, an action reported recently for a dominant-negative Xwnt-8. Frzb coimmunoprecipitated with Wnt proteins, providing direct biochemical evidence for Frzb-Wnt interactions. These observations implicate Frzb in axial patterning and support the concept that Frzb binds and inactivates Xwnt-8 during gastrulation, preventing inappropriate ventral signaling in developing dorsal tissues.

Animals↗
PubMed1995

Mesoderm induction in Xenopus caused by activation of MAP kinase.

Mesoderm induction is a critical early step in vertebrate development, involving changes in gene expression and morphogenesis. In Xenopus, normal mesoderm formation depends on signalling through the fibroblast growth factor (FGF) tyrosine kinase receptor. One important signalling pathway from receptor tyrosine kinases involves p21ras (ref. 5). Ras associates with the serine kinase c-Raf-1 in a GTP-dependent manner, and this complex phosphorylates and activates MAPK/ERK kinase (MEK), a protein kinase with dual specificity. MEK then activates p42mapk and (at least in mammals) p44mapk, members of the mitogen-activated protein (MAP) kinase family. FGF activates MAP kinase during mesoderm induction, and the use of dominant-negative constructs suggests that mesoderm induction by FGF requires both Ras and Raf. However, these experiments do not reveal whether Ras and Raf do act through MAP kinase to induce mesoderm or whether another pathway, such as the phosphatidylinositol 3-kinase cascade, is involved. Here we show that expression of active forms of MEK or of MAP kinase induces ventral mesoderm of the kind elicited by FGF. Overexpression of a Xenopus MAP kinase phosphatase blocks mesoderm induction by FGF, and causes characteristic defects in mesoderm formation in intact embryos, whereas inhibition of the P13 kinase and p70 S6 kinase pathways has no effect on mesoderm induction by FGF. FGF induces different types of mesoderm in a dose-dependent manner; strikingly, this is mimicked by expressing different levels of activated MEK. Together, these experiments demonstrate that activation of MAP kinases is necessary and sufficient for mesoderm formation.

Actins↗
PubMed1999

FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression.

Beads containing recombinant FGF8 (FGF8-beads) were implanted in the prospective caudal diencephalon or midbrain of chick embryos at stages 9-12. This induced the neuroepithelium rostral and caudal to the FGF8-bead to form two ectopic, mirror-image midbrains. Furthermore, cells in direct contact with the bead formed an outgrowth that protruded laterally from the neural tube. Tissue within such lateral outgrowths developed proximally into isthmic nuclei and distally into a cerebellum-like structure. These morphogenetic effects were apparently due to FGF8-mediated changes in gene expression in the vicinity of the bead, including a repressive effect on Otx2 and an inductive effect on En1, Fgf8 and Wnt1 expression. The ectopic Fgf8 and Wnt1 expression domains formed nearly complete concentric rings around the FGF8-bead, with the Wnt1 ring outermost. These observations suggest that FGF8 induces the formation of a ring-like ectopic signaling center (organizer) in the lateral wall of the brain, similar to the one that normally encircles the neural tube at the isthmic constriction, which is located at the boundary between the prospective midbrain and hindbrain. This ectopic isthmic organizer apparently sends long-range patterning signals both rostrally and caudally, resulting in the development of the two ectopic midbrains. Interestingly, our data suggest that these inductive signals spread readily in a caudal direction, but are inhibited from spreading rostrally across diencephalic neuromere boundaries. These results provide insights into the mechanism by which FGF8 induces an ectopic organizer and suggest that a negative feedback loop between Fgf8 and Otx2 plays a key role in patterning the midbrain and anterior hindbrain.

Animals↗
PubMed1996

Direct and long-range action of a DPP morphogen gradient.

During development of the Drosophila wing, the decapentaplegic (dpp) gene is expressed in a stripe of cells along the anteroposterior compartment boundary and gives rise to a secreted protein that exerts a long-range organizing influence on both compartments. Using clones of cells that express DPP, or in which DPP receptor activity has been constitutively activated or abolished, we show that DPP acts directly and at long range on responding cells, rather than by proxy through the short-range induction of other signaling molecules. Further, we show that two genes, optomotor-blind and spalt are transcriptionally activated at different distances from DPP-secreting cells and provide evidence that these genes respond to different threshold concentrations of DPP protein. We propose that DPP acts as a gradient morphogen during wing development.

Animals↗
PubMed1993

Epithelial-mesenchymal interactions are required for msx 1 and msx 2 gene expression in the developing murine molar tooth.

Duplication of the msh-like homeobox gene of Drosophila may be related to the evolution of the vertebrate head. The murine homologues of this gene, msx 1 and msx 2 are expressed in the developing craniofacial complex including the branchial arches, especially in regions of epithelial-mesenchymal organogenesis including the developing tooth. By performing in vitro recombination experiments using homochronic dental and non-dental epithelial and mesenchymal tissues from E10 to E18 mouse embryos, we have found that the maintenance of homeobox gene expression in the tooth is dependent upon tissue interactions. In homotypic recombinants, dental-type tissue interactions occur, leading to expression of both genes in a manner similar to that seen during in vivo development. msx 1 is expressed exclusively in mesenchyme, both in the dental papilla and follicle. msx 2 is expressed in the dental epithelium and only in the mesenchyme of the dental papilla. In heterotypic recombinants, the dental epithelium is able to induce msx 1 expression in non-dental mesenchyme, this potential being lost at the bell stage. In these recombinants msx 2 was induced by presumptive dental epithelium prior to the bud stage but not thereafter. The expression of msx 1 and msx 2 in dental mesenchyme requires the presence of epithelium until the early bell stage. However, whereas non-dental, oral epithelium is capable of maintaining expression of msx 1 in dental mesenchyme throughout tooth development, induction of msx 2 was temporally restricted suggesting regulation by a specific epithelial-mesenchymal interaction related to the inductive events of tooth formation. msx 1 and msx 2, as putative transcription factors, may play a role in regulating the expression of other genes during tooth formation. We conclude that expression of msx 1 in jaw mesenchyme requires a non-specific epithelial signal, whereas msx 2 expression in either epithelium or mesenchyme requires reciprocal interactions between specialized dental cell populations.

Animals↗
PubMed1978

Transfilter induction of kidney tubules as a function of the extent and duration of intercellular contacts.

The kinetics of kidney tubule induction were examined in transfilter experiments by varying the time of transfilter apposition, the porosity and pore size of the filters, and the culture conditions. Transfilter contact between the interacting cells is established within an hour when cytoplasmic processes emerge through the interposed filter; then a further 16-24 h are needed for completion of induction. This lag is a function of thickness and pore size of the filter, and is not reduced by precultivation of the inductor on the filter. Material that accumulates on the far side of the filter during such cultivation displays no morphogenetic activity. The intensity of the mesenchymal response was roughly quantified and shown to be a function of pore size, pore density and duration of transfilter contact.

Animals↗
PubMed1999

Overexpression of ptc1 inhibits induction of Shh target genes and prevents normal patterning in the neural tube.

Patched (Ptc) is a human tumor suppressor protein and a candidate receptor for Hedgehog (Hh) proteins, which regulate growth and patterning in embryos. Ptc represses expression of Hh target genes such as Gli1 and ptc1 itself. Localized secretion of Hh appears to induce transcription of target genes in specific patterns by binding to Ptc and preventing it from functioning in recipient cells. People who are heterozygous for PTC1 exhibit a range of developmental defects, suggesting that some genes are inappropriately expressed when there is not enough Ptc protein. To test the idea that a balance between Hh and Ptc activities is essential for normal development, we overexpressed Ptc in the neural tube. We find that excess Ptc is sufficient to inhibit expression of Gli1 and ptc1, suggesting that Sonic hedgehog (Shh) cannot signal effectively. This leads to partial dorsalization of the neural tube and a wide spectrum of neural defects, ranging from embryonic lethality to hydrocephaly.

Animals↗
PubMed1997

A novel Xenopus homologue of bone morphogenetic protein-7 (BMP-7).

We identified a Xenopus gene closely related to mammalian bone morphogenetic protein (BMP)-7 (also termed osteogenic protein-1 or OP-1). It resembles the mammalian gene in primary structure and expression pattern much more closely than does a previously described Xenopus homologue, originally termed XBMP-7 [Nishimatsu, Suzuki, Shoda, Murakami and Ueno (1992) Biochem. Biophys. Res. Commun. 186, 1487-1495]. The novel gene has therefore been designated XBMP-7 and the gene described earlier has been renamed XBMP-7R (M. Moos and N. Ueno, unpublished work). It has a broad distribution, primarily in the anterior and posterior ventral regions during gastrulation, subsequently becoming prominent at different stages in a wide variety of structures (eyes, neural structures, heart, pronephros, posterior ventral region and other structures), paralleling the distribution of XBMP-4 closely. However, its expression begins later than that of XBMP-4 during gastrulation. Lithium treatment of embryos concentrates the XBMP-7 expression in the expanded eye and heart structures. Ventral overexpression of XBMP-7 produces large protrusions that ultimately develop colouration characteristic of haemoglobin, which is confirmed by markedly expanded expression of alpha-globin. Dorsal overexpression suppresses dorsal anterior structures. Molecular analysis of animal caps overexpressing XBMP-7 reveals induction of markers associated with ventral and haematopoietic tissue, which is consistent with whole-embryo overexpression results. Globin induction by XBMP-7 can be blocked by a truncated BMP receptor previously shown to interrupt BMP-4 signalling, indicating XBMP-7 also interacts with this receptor. Our data support the concept that XBMP-7 may play a variety of roles during embryogenesis, and suggest a possible role in haematogenesis.

Amino Acid Sequence↗
PubMed1997

Siamois is required for formation of Spemann's organizer.

Spemann's organizer develops in response to dorsal determinants that act via maternal components of the wnt pathway. The function of siamois, a wnt-inducible homeobox gene, in Spemann's organizer development was examined by fusion of defined transcriptional regulatory domains to the siamois homeodomain. Similar to native siamois, a VP16 activator fusion induced axis formation, indicating that siamois functions as a transcriptional activator in axis induction. Fusion of the engrailed repressor generated a dominant inhibitor that blocked axis induction by Xwnt8, beta-catenin, and siamois, and repressed wnt activation of the goosecoid promoter. Dorsal injection of the engrailed-siamois fusion resulted in complete inhibition of dorsal development and organizer gene expression, an effect rescued by siamois, but not by Xwnt8 or beta-catenin. Thus, as a zygotic mediator of maternal dorsal signals, siamois function is required for development of Spemann's organizer.

Animals↗
PubMed1995

The Caenorhabditis elegans lin-12 gene mediates induction of ventral uterine specialization by the anchor cell.

The anchor cell (AC) of the Caenorhabditis elegans gonad has a critical role in the development of a functional egg-laying system, which is accomplished through cell-cell interactions. Lateral inhibitory lin-12-mediated signaling among two bipotential cells causes one to adopt the ventral uterine precursor (VU) cell fate while the other becomes the AC. The AC then induces formation of vulval tissue. We find that the AC also induces a particular ventral uterine intermediate precursor fate (pi) by a mechanism that is genetically and temporally distinct from vulval induction. This process requires lin-12, but unlike previously described lin-12-mediated decisions, signaling is unidirectional, is between dissimilar cells and does not involve lateral inhibition. The pi fates are necessary for egg laying and appear to produce a distinct specialized cell type. Thus, patterning of the ventral uterus by the AC is crucial to the development of a functional egg-laying system.

Animals↗
PubMed2000

Assembling a functional tympanic membrane: signals from the external acoustic meatus coordinate development of the malleal manubrium.

In terrestrial mammals, hearing starts with the perception of acoustic pressure by the tympanic membrane. Vibrations in this membrane are then transduced into the inner ear by the ossicle chain of the middle ear, composed of the malleus, incus and stapes. The proper connection of the ossicle chain with the tympanic membrane, provided by the insertion of the manubrium of the malleus into the eardrum, is essential for the functionality of the hearing apparatus. We describe here the mechanisms regulating the development of the manubrium and its integration into the tympanic membrane. We show that the external acoustic meatus (EAM), which eventually forms the outer epithelium of the tympanic membrane, plays an essential role in this developmental process. Histological and expression analyses indicate that the manubrium develops close to the EAM with a similar temporal sequence. In addition, when the middle ear ossicles are allowed to develop in vitro under conditions that do not support further EAM development, the manubrium develops only up to the stage of its induction at the time of explantation. Moreover, genetically or teratogenically derived alterations in the EAM also have an effect on manubrial development. Finally, we show that the EAM is the source of two quite opposite activities, one that induces chondrogenesis and another that represses it. The combination of these two activities results in the proper positioning of the manubrium.

Animals↗
PubMed1991

Neural induction.

The study of neural induction in Xenopus can be approached from two broad perspectives. One can study the competence of the ectoderm to respond to neural induction signals and any potential prepattern within the ectoderm. The second area of study involves the neural induction signals, in terms of the chemical nature of the signals, their sources, and their method of delivery. The neural competence of ectoderm has been studied by either grafting the ectoderm into areas which normally form neural tissue or by grafting tissues which normally induce neural structures onto the ectoderm to be tested. In general, it appears that ectoderm loses neural competence by midgastrula. However, there is some experimental evidence from various amphibian studies that the loss of neural competence in ectoderm does not occur simultaneously throughout all regions. It is not yet known if this phenomenon is also true in Xenopus. There appear to be several signaling events involved in the process of neural induction. Molecular probes have made it possible to study early steps in the neural induction and patterning processes which were not possible to study using only the development of neural morphology as a marker for neural induction. Antibodies directed against early epidermal versus neural epithelium indicate that the dorsal animal blastomeres are biased toward a neural pathway during early cleavage. Another signaling event occurs at early gastrula and the resulting dorsal ectoderm now responds more readily to some neural induction events than does the ventral ectoderm. The source of the early gastrula signals has been studied by a variety of methods, including exogastrula embryos, Keller sandwiches, and grafting a blastopore lip to the edge of competent ectoderm. The blastopore lip can send signals through the plane of the ectoderm capable of inducing competent ectoderm to become neural tissue. There are several issues relative to the process of neural induction which are not yet resolved. The major issue involves the mechanism of establishing pattern within the neural plate. Ectoderm appears to lose neural competence prior to the time when involuted dorsal mesoderm comes to underlie the anterior neural plate region. Several investigators have shown that information for expression of spatially restricted neural-specific molecules can travel through the ectoderm, independent of underlying dorsal mesoderm.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Planar induction of convergence and extension of the neural plate by the organizer of Xenopus.

This paper demonstrates that convergence and extension within the neural plate of Xenopus laevis are regulated by planar inductive interactions with the adjacent Spemann organizer. The companion article (Keller et al.: Developmental Dynamics 193:199-217, 1992) showed that the prospective hindbrain and spinal cord occupy a very short and very wide area just above the Spemann organizer in the early gastrula and that these regions converge and extend greatly during gastrulation and neurulation, using a sequence of radial and mediolateral cell intercalations. In this article, we show that "planar" contact of these regions with the organizer at their vegetal edge until stage 11 is sufficient to induce convergence and extension, after which their convergence and extension become autonomous. Grafts of the organizer in planar contact with uninduced ectodermal tissues induce these ectodermal tissues to converge and extend by a planar inductive signal from the organizer. Labeling of the inducing or responding tissues confirms that only planar interactions occur. Neural convergence and extension are actually hindered in explants deliberately constructed so that vertical interactions occur. These results show unambiguously that the Spemann organizer induces the extraordinary and precocious convergence and extension movements of the Xenopus neural plate by planar interactions acting over short distances.

Animals↗
PubMed2004

Independent induction and formation of the dorsal and ventral fins in Xenopus laevis.

It has been known since the 1930s that the dorsal fin is induced by the underlying neural crest. The inducer of the ventral fin, however, has remained elusive. We have investigated the source of the inducer of the ventral fin in Xenopus and show that it is the ventral mesoderm and not the neural crest. This induction takes place during mid-neurula stages and is completed by late neurulation. In terms of cell composition, the dorsal fin mesenchyme core arises from neural crest cells, while the mesenchyme of the ventral fin has a dual origin. The ventral fin contains neural crest cells that migrate in from the dorsal side of the embryo, but a contribution is also made by cells from the ventral mesoderm.

Animals↗
PubMed1995

The SH2-containing protein-tyrosine phosphatase SH-PTP2 is required upstream of MAP kinase for early Xenopus development.

SH-PTP2, the vertebrate homolog of Drosophila corkscrew, associates with several activated growth factor receptors, but its biological function is unknown. We assayed the effects of injection of wild-type and mutant SH-PTP2 RNAs on Xenopus embryogenesis. An internal phosphatase domain deletion (delta P) acts as a dominant negative mutant, causing severe posterior truncations. This phenotype is rescued by SH-PTP2, but not by the closely related SH-PTP1. In ectodermal explants, delta P blocks fibroblast growth factor (FGF)- and activin-mediated induction of mesoderm and FGF-induced mitogen-activated protein (MAP) kinase activation. Our results indicate that SH-PTP2 is required for early vertebrate development, acting as a positive component in FGF signaling downstream of the FGF receptor and upstream of MAP kinase.

Activins↗
PubMed2006

Genome-wide transcriptional profiling of human embryonic stem cells differentiating to cardiomyocytes.

Mammals are unable to regenerate their heart after major cardiomyocyte loss caused by myocardial infarction. Human embryonic stem cells (hESCs) can give rise to functional cardiomyocytes and therefore have exciting potential as a source of cells for replacement therapy. Understanding the molecular regulation of cardiomyocyte differentiation from stem cells is crucial for the stepwise enhancement and scaling of cardiomyocyte production that will be necessary for transplantation therapy. Our novel hESC differentiation protocol is now efficient enough for meaningful genome-wide transcriptional profiling by microarray technology of hESCs, differentiating toward cardiomyocytes. Here, we have identified and validated time-dependent gene expression patterns and shown a reflection of early embryonic events; induction of genes of the primary mesoderm and endodermal lineages is followed by those of cardiac progenitor cells and fetal cardiomyocytes in consecutive waves of known and novel genes. Collectively, these results permit enhancement of stepwise differentiation and facilitate isolation and expansion of cardiac progenitor cells. Furthermore, these genes may provide new clinically relevant clues for identifying causes of congenital heart defects.

Cell Differentiation↗
PubMed2005

Genetic network during neural crest induction: from cell specification to cell survival.

The concerted action of extracellular signals such as BMP, Wnt, FGF, RA and Notch activate a genetic program required to transform a naïve ectodermal cell into a neural crest cell. In this review we will analyze the extracellular signals and the network of transcription factors that are required for this transformation. We will propose the division of this complex network of factors in two main steps: an initial cell specification step followed by a maintenance or cell survival step.

Animals↗
PubMed2003

The lateral somitic frontier: dorso-ventral aspects of anterio-posterior regionalization in avian embryos.

Patterning events along the anterior-posterior (AP) axis of vertebrate embryos result in the distribution of muscle and bone forming a highly effective functional system. A key aspect of regionalized AP patterning results from variation in the migratory pattern of somite cells along the dorsal-ventral (DV) axis of the body. This occurs as somite cell populations expand around the axis or migrate away from the dorsal midline and cross into the lateral plate. The fate of somitic cells has been intensely studied and many details have been reported about inductive signaling from other tissues that influence somite cell fate and behavior. We are interested in understanding the specific differences between somites in particular AP regions and how these differences contribute to the global pattern of the organism. Using orthotopic transplants of segmental plate between quail and chick embryos, we have mapped the interface of the somitic and lateral plate mesoderm during the formation of the body wall in cervical and thoracic regions. This interface does not change dramatically in the mid-cervical region, but undergoes extensive changes in the thoracic region. Based on this regional mapping and consistent with the extensive literature, we suggest a revised method of classifying regions of the body wall that relies on embryonic cell lineages rather than adult functional criteria.

Animals↗