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The embryonic hindbrain is subdivided into a series of metameric units termed rhombomeres, which display features that strongly suggest they are autonomous developmental units. However, some aspects of their phenotype develop nonautonomously. Here we have analyzed the possibility that interrhombomere interactions generate the pattern of segmental neuronal differentiation. The differentiation of both projection interneurons and motor neurons in the hindbrain is retarded in rhombomeres 3 and 5. We demonstrate here that if either rhombomere 3 or 5 is isolated from the influence of their neighbours, either in vitro or in vivo, then these segments no longer display delayed neuronal diufferentiation. We further show that the retardation of motor neurons differentiation in rhombomeres 3 and 5 is, at least in part, mediated by Bmp-4. If this molecule is inhibited, by grafting cells expressing chordin, then the motor neurons of these rhombomeres develop ahead of their normal schedule., (Copyright 2001 Academic Press.)

To elucidate the contribution of the extracellular microfibril-elastic fiber network to vertebrate organogenesis, we generated fibrillin 2 (Fbn2)-null mice by gene targeting and identified a limb-patterning defect in the form of bilateral syndactyly. Digit fusion involves both soft and hard tissues, and is associated with reduced apoptosis at affected sites. Two lines of evidence suggest that syndactily is primarily due to defective mesenchyme differentiation, rather than reduced apoptosis of interdigital tissue. First, fusion occurs before appearance of interdigital cell death; second, interdigital tissues having incomplete separation fail to respond to apoptotic clues from implanted BMP-4 beads. Syndactyly is associated with a disorganized matrix, but with normal BMP gene expression. On the other hand, mice double heterozygous for null Fbn2 and Bmp7 alleles display the combined digit phenotype of both nullizygotes. Together, these results imply functional interaction between Fbn2-rich microfibrils and BMP-7 signaling. As such, they uncover an unexpected relationship between the insoluble matrix and soluble factors during limb patterning. We also demonstrate that the Fbn2- null mutation is allelic to the recessive shaker-with-syndactyly (sy) locus on chromosome 18.

Morphogens are in the front line just now. Here I trace how the concept of a morphogen has evolved over the past 100 years and step a little beyond what we already know.

In the chick metencephalon, oligodendrocyte precursors arise in distinct domains of the ventricular zone. During development, the earliest oligodendrocyte precursors appear in the metencephalic ventral ventricular zone adjacent to the midline, consistent with their location in the spinal cord. In contrast to spinal cord, however, distinct domains in the lateral and dorsal metencephalic ventricular zone subsequently generate oligodendrocyte precursors. All oligodendrogenic domains of the metencephalon appear in close apposition to regions that transiently express sonic hedgehog (Shh). Inhibition studies demonstrate a functional requirement for Shh signaling in the early appearance of metencephalic oligodendrocyte precursors, while in vitro studies suggest a dose-dependent increase in the number of oligodendrocyte precursors in response to Shh. In purified cultures of oligodendrocyte precursors, Shh promotes cell survival and proliferation, suggesting that Shh can act directly on these cells. These data suggest that Shh may be responsible for the localized appearance of oligodendrocyte precursors throughout the CNS, irrespective of the dorso-ventral neural axis., (Copyright 2001 Academic Press.)

Differentiation and apoptosis are precisely regulated events in early embryogenesis. Retinoic acid-induced differentiation in the embryonal carcinoma (EC) cell line NCR-G3 triggers concurrent induction of apoptosis. Using this system, which serves as a model of early embryogenesis, the expression of various bcl-2-related genes was analyzed as these genes display either positive or negative regulatory effects on apoptosis. EAT/mcl-1, an antiapoptotic bcl-2-related gene and immediate early gene, was dramatically expressed at an early stage of NCR-G3 differentiation. Bcl-xL, another antiapoptotic gene, was induced at a middle stage of differentiation and then gradually decreased to basal level. Expression of Bax, a proapoptotic molecule, was detected at a high level and remained relatively constant. Meanwhile, Bcl-2 and Bcl-xS were below detectable levels throughout the various stages of differentiation. As the balance of bcl-2 genes is a crucial regulatory step in apoptosis, the results suggest that EAT and Bax likely regulate apoptosis in the early stages of differentiation. In later stages of differentiation, down-regulation of EAT was found to coincide with a gradual increase in apoptosis of NCR-G3 cells. Furthermore, use of the monoclonal antibody (3A2) specific to EAT revealed that EAT is localized to the outer mitochondrial membrane in human EC cells. In addition, EAT immunoreactivity was not detected in apoptotic NCR-G3 cells while it was observed in nearly all viable cells. The findings suggest that rapid induction of EAT may prevent NCR-G3 cells from undergoing apoptosis, thereby supporting viability at the early stage of differentiation., (Copyright 2001 Academic Press.)

Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dorsalizing the Xenopus embryo. A novel secreted protein, CHL (for chordin-like), with significant homology to chordin, was isolated from mouse bone marrow stromal cells. Injection of CHL RNA into Xenopus embryos induced a secondary axis. Recombinant CHL protein inhibited the BMP4-dependent differentiation of embryonic stem cells in vitro and interacted directly with BMPs, similar to chordin. However, CHL also weakly bound to TGFbetas. In situ hybridization revealed that the mouse CHL gene, located on the X chromosome, was expressed predominantly in mesenchyme-derived cell types: (1) the dermatome and limb bud mesenchyme and, later, the subdermal mesenchyme and the chondrocytes of the developing skeleton during embryogenesis and (2) a layer of fibroblasts/connective tissue cells in the gastrointestinal tract, the thick straight segments of kidney tubules, and the marrow stromal cells in adults. An exception was expression in the neural cells of the olfactory bulb and cerebellum. Interestingly, the spatiotemporal expression patterns of CHL were distinct from those of chordin in many areas examined. Thus, CHL may serve as an important BMP regulator for differentiating mesenchymal cells, especially during skeletogenesis, and for developing specific neurons., (Copyright 2001 Academic Press.)

Frizzled-1 (FZD1)-FZD10 are seven-transmembrane-type WNT receptors, and SFRP1-SFRP5 are soluble-type WNT antagonists. These molecules are encoded by mutually distinct genes. We have previously isolated and characterized the 7.7-kb FZD4 mRNA, encoding a seven-transmembrane receptor with the extracellular cysteine-rich domain (CRD). Here, we have cloned and characterized FZD4S, a splicing variant of the FZD4 gene. FZD4S, corresponding to the 10.0-kb FZD4 mRNA, consisted of exon 1, intron 1, and exon 2 of the FZD4 gene. FZD4S encoded a soluble-type polypeptide with the N-terminal part of CRD, and was expressed in human fetal kidney. Injection of synthetic FZD4S mRNA into the ventral marginal zone of Xenopus embryos at the 4-cell stage did not induce axis duplication by itself, but augmented the axis duplication potential of coinjected Xwnt-8 mRNA. These results indicate that the FZD4 gene gives rise to soluble-type FZD4S as well as seven-transmembrane-type FZD4 due to alternative splicing, and strongly suggest that FZD4S plays a role as a positive regulator of the WNT signaling pathway., (Copyright 2001 Academic Press.)

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.

Cortical interneurons arise from the proliferative zone of the ventral telencephalon, the ganglionic eminence, and migrate into the developing neocortex. The spatial patterns of migratory interneurons reflect the complementary expression of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, MET, in the forebrain. Scatter assays on forebrain explants demonstrate regionally specific motogenic activity due to HGF/SF. In addition, exogenous ligand disrupts normal cell migration. Mice lacking the urokinase-type plasminogen activator receptor (u-PAR), a key component of HGF/SF activation, exhibit deficient scatter activity in the forebrain, abnormal interneuron migration from the ganglionic eminence, and reduced interneurons in the frontal and parietal cortex. The data suggest that HGF/SF motogenic activity, which is essential for normal development of other organ systems, is a conserved mechanism that regulates trans-telencephalic migration of interneurons.

Perineuronal nets (PNs), consisting of extracellular matrix proteoglycans, complexed with hyaluronan and colocalized with tenascins, are associated with distinct neuronal populations in mature mammalian brain. PNs have been shown to appear postnatally during the period of synaptic refinement and myelination, indicating the commencement of mature physiological properties of neurons. Here we show that the developmental patterns of formation of PNs are well preserved in organotypic slice cultures prepared from rats on postnatal day 3-5 and maintained in vitro for 3-10 weeks. Staining of cultures with Wisteria floribunda agglutinin and immunocytochemical detection of chondroitin sulfate proteoglycans revealed developing PNs in the basal forebrain, mesencephalic regions, and the cerebellum after 2 weeks in vitro, and later in the neocortical areas and hippocampus. In contrast, neurons known to be devoid of PNs in the adult rat brain such as cholinergic basal forebrain neurons and catecholaminergic tegmental neurons differentiate without any formation of PNs in slice cultures. We show further that environmental factors influence the development of PNs around the net-associated types of neurons. Notably, chronic depolarization of the cultures, imposed by an elevated concentration of external potassium ions, enhanced the development of PNs. Blocking of calcium channels with magnesium chloride or with the L-type calcium channel blocker nifedipine, suppressed the development of PNs, while a block of voltage-gated sodium channels by tetrodotoxin had no obvious effects. The results show that extracellular matrix components specifically contribute to the organotypic patterns that develop in brain slice cultures. Evidence is provided that the differentiation of PNs is regulated by calcium-dependent signaling.

During normal cortical development, individual pyramidal neurons form intracortical axonal arbors that are specific for particular cortical layers. Pyramidal neurons within layer 6 are able to develop layer-specific projections in cultured slices of ferret visual cortex, indicating that extrinsic influences, including patterned visual activity, are not required (Dantzker and Callaway [1998] J Neurosci 18:4145-4154). However, when spontaneous activity is blocked in cultures with tetrodotoxin, layer 6 pyramidal neurons fail to preferentially target their axons to layer 4. To determine whether mechanisms that regulate the development of layer 6 pyramidal neuron arbors can be generalized to pyramidal neurons in other layers, we examined the development of layer 5 and layer 2/3 pyramidal neurons in cultured slices of ferret visual cortex prepared on postnatal day 14 or 15. Layer 5 pyramidal neurons developed layer-specific axonal arbors during 5-7 days in vitro. However, unlike layer 6 pyramidal neurons, layer 5 pyramidal neurons formed layer-specific axonal arbors in the presence of tetrodotoxin. In contrast to layer 5 and layer 6 pyramidal neurons, layer 2/3 pyramidal neurons did not form appropriate layer-specific projections during 5-7 days in vitro. Taken together, these data suggest that the development of layer-specific axons is regulated by different mechanisms for neurons in different layers and cannot be categorically classified as either activity-dependent or independent. Instead, the type of pyramidal neuron, the layers targeted, and the type of activity must be considered., (Copyright 2001 Wiley-Liss, Inc.)

During the course of a systematic screening of peptide signaling molecules in Hydra magnipapillata, a novel peptide, Hym-323, which enhances foot regeneration was identified. The peptide is 16 amino acids long, and is encoded in the precursor protein as a single copy. Northern blot analysis, in situ hybridization analysis and immunohistochemistry showed that it was expressed in both ectodermal and endodermal epithelial cells throughout the body, except for the basal disk and the head region. The peptide enhanced foot regeneration by acting on epithelial cells. Lateral transplantation experiments indicated that the foot activation potential was increased in the peptide-treated tissue. These results suggest that Hym-323 is a peptide involved in a foot-patterning process in Hydra.

Previous studies suggest that Fgf8 has a key role in regulating vertebrate development. In the rostral head of the embryonic chicken, there are increasing numbers of separate Fgf8 domains; these are present in tissues that appear to have previously expressed Otx2. As Fgf8 expression becomes established, Otx2 expression weakens, but remains in cells abutting the Fgf8 expression domain. These Fgf8 expression domains are closely associated with tissues expressing Bmp4 and Shh. Based on analogy with the embryonic limb, we suggest that Fgf8, Bmp4 and Shh function together in patterning regions of the embryonic head. Gene expression changes are particularly prominent in 14-21 somite stage embryos in the rostral forebrain, during early morphogenesis of the telencephalic and optic vesicles, when several new interfaces of Fgf8, Bmp4 and Shh are generated. To gain insights into the functions of fibroblast growth factor 8 (FGF8) in the embryonic forebrain, we studied the effects of implanting beads containing this protein in the dorsal prosencephalon of embryonic day 2 chicken embryos. Ectopic FGF8 had profound effects on morphogenesis of the telencephalic and optic vesicles. It disrupted formation of the optic stalk and caused a transformation of the pigment epithelium into neural retina. Within the telencephalon, FGF8 beads frequently induced a sulcus that had features of an ectopic rostral midline. The sulcus separated the telencephalon into rostral and caudal vesicles. Furthermore, we present evidence that FGF8 can regulate regionalization of the prosencephalon through inhibition of Otx2 and Emx2 expression. Thus, these experiments provide evidence that FGF8 can regulate both morphogenesis and patterning of the rostral prosencephalon (telencephalic and optic vesicles). FGF8 beads can induce midline properties (e.g. a sulcus) and can modulate the specification and differentiation of adjacent tissues. We suggest that some of these effects are through regulating the expression of homeobox genes (Otx2 and Emx2) that are known to participate in forebrain patterning.

Hormonal regulation in development and maintenance of synaptic transmission involves examination of both the presynaptic and postsynaptic components and a system in which the hormones can be controlled. We used the ecdysoneless heat-sensitive mutation (l(3)ecd(1)/l(3)ecd(1)) of Drosophila to provide the ability to regulate endogenous ecdysone production at various larval stages. In conjunction, we used the neuromuscular junctions of Drosophila since they offer the advantage of assessable preparations for both morphological and physiological measures. The growth in the Ib and Is motor nerve terminals and the corresponding muscle 6 in segment 4 of the larval Drosophila throughout the third instar stage in the presence of normal and a much reduced endogenous ecdysone level was investigated. Muscle 6 and the motor nerve terminals parallel in growth throughout the third instar. The nerve terminals increase in length and varicosity number, thus providing an increase in the number of synaptic release sites. The ecdysoneless larvae also show an increase in muscle size, however the Is and Ib motor nerve terminals do not mature to the extent of the wild-type ecdysone producing flies. The motor nerve terminal length is shorter with fewer numbers of varicosities per terminal. In spite of a shorter nerve terminal and fewer varicosities, with an increasing muscle fiber, the compound excitatory junctional potentials of Ib and Is in the ecdysoneless flies are larger, which is suggestive of synaptic structural modification. This study demonstrates ecdysone's role in modifying nerve terminal development and neuromuscular junction function.

A mesenchymal factor(s) is believed to modulate the development of the embryonic pancreas. However, the identity of this factor(s) remains elusive. Its importance in the development of the endocrine cells of the pancreas, particularly insulin cells, is considered. Our studies seek to identify factors that may influence insulin cell proliferation and differentiation, and hence may have positive implications for patients suffering from diabetes. Our experiments are based on the in vitro model which utilised the 5-day dorsal pancreatic bud of the chick embryo. These buds, devoid of most of their mesenchyme, are cultured on Matrigel in a serum-free medium for 7 days. We have investigated the effects of retinoic acid, retinoic acid in combination with IGF-1, nicotinamide and activin in this system. While most of these factors increased the proportion of insulin cells over that of their respective controls, as yet none have matched or exceeded the proportion of insulin cells demonstrated in vivo.

The most rostral cephalic crest cells in the chick embryo first populate ubiquitously in the rostroventral head. Before the influx of crest cells, the ventral head ectoderm expresses Fgf8 in two domains that correspond to the future mandibular arch. Bmp4 is expressed rostral and caudal to these domains. The rostral part of the Bmp4 domain develops into the rostral end of the maxillary process that corresponds to the transition between the maxillomandibular and premandibular regions. Thus, the distribution patterns of FGF8 and BMP4 appear to foreshadow the maxillomandibular region in the head ectoderm. In the ectomesenchyme of the pharyngula embryo, expression patterns of some homeobox genes overlap the distribution of their upstream growth factors. Dlx1 and Barx1, the targets of FGF8, are expressed in the mandibular ectomesenchyme, and Msx1, the target of BMP4, in its distal regions. Ectopic applications of FGF8 lead to shifted expression of the target genes as well as repatterning of the craniofacial primordia and of the trigeminal nerve branches. Focal injection of a lipophilic dye, DiI, showed that this shift was at least in part due to the posterior transformation of the original premandibular ectomesenchyme into the mandible, caused by the changed distribution of FGF8 that defines the mandibular region. We conclude that FGF8 in the early ectoderm defines the maxillomandibular region of the prepharyngula embryo, through epithelial-mesenchymal interactions and subsequent upregulation of homeobox genes in the local mesenchyme. BMP4 in the ventral ectoderm appears to limit the anterior expression of Fgf8. Ectopic application of BMP4 consistently diminished part of the mandibular arch., (Copyright 2000 Academic Press.)

Manipulation of cortical serotonin (5-HT) levels in perinatal rodents produces significant alterations in the development of the layer IV cortical representation of the mystacial vibrissae. Monoamine oxidase A (MAO(A)) knockout mice have highly elevated cortical 5-HT and completely lack barrels in somatosensory cortex (S-I). The present study was undertaken to determine whether the effects on thalamocortical development seen in MAO(A) knockout mice can be replicated in perinatal rats treated with an MAO(A) inhibitor and, second, to determine whether these effects persist with continued treatment or after discontinuation of the drug. Littermates were injected with either clorgyline (5 mg/kg) or sterile saline five times daily. Clorgyline administration from birth to postnatal day (P) 6, 8, or 10 produced increases of 1,589.4 +/- 53.3%, 1660.2 +/- 43.1% and 1,700.5 +/- 84.5 %, respectively, in cortical 5-HT as compared with controls. Serotonin immunocytochemistry, 1,1;-dioctadecyl-3,3,3", 3;-tetramethylindocarbocyanine perchlorate (DiI) labeling of thalamocortical afferents and Nissl and cytochrome oxidase staining of layer IV cellular aggregates demonstrated that clorgyline treatment from P0 to P6 produced a complete absence of any segmentation of vibrissae-related patches in S-I. However, continued treatment until P8 or P10 did not prevent the appearance of these patches. Animals treated with clorgyline from birth to P6 and killed on P8 or P10 had increases of 546.8 +/- 33.2% and 268.8 +/- 6.3% in cortical 5-HT and they had qualitatively normal vibrissae-related patterns in S-I. These results indicate that clorgyline treatment produces a transient disruption of vibrissae-related patterns, despite the continued presence of elevated cortical 5-HT., (Copyright 2000 Wiley-Liss, Inc.)

In the olfactory pathway, as in the limbs, branchial arches, and heart, mesenchymal/epithelial induction, mediated by retinoic acid (RA), FGF8, sonic hedgehog (shh), and the BMPs, defines patterning, morphogenesis, and differentiation. Neuronal differentiation in the olfactory epithelium and directed growth of axons in the nascent olfactory nerve depend critically upon this inductive interaction. When RA, FGF8, shh, or BMP signaling is disrupted, distinct aspects of olfactory pathway patterning and differentiation are compromised. Thus, a cellular and molecular mechanism that facilitates musculoskeletal and vascular development elsewhere in the embryo has been adapted to guide the differentiation of the olfactory pathway in the developing forebrain.

The secretion of Sonic hedgehog (Shh) from the notochord and floor plate appears to generate a ventral-to-dorsal gradient of Shh activity that directs progenitor cell identity and neuronal fate in the ventral neural tube. In principle, the establishment of this Shh activity gradient could be achieved through the graded distribution of the Shh protein itself, or could depend on additional cell surface or secreted proteins that modify the response of neural cells to Shh. Cells of the neural plate differentiate from a region of the ectoderm that has recently expressed high levels of BMPs, raising the possibility that prospective ventral neural cells are exposed to residual levels of BMP activity. We have examined whether modulation of the level of BMP signaling regulates neural cell responses to Shh, and thus might contribute to the patterning of cell types in the ventral neural tube. Using an in vitro assay of neural cell differentiation we show that BMP signaling markedly alters neural cell responses to Shh signals, eliciting a ventral-to-dorsal switch in progenitor cell identity and neuronal fate. BMP signaling is regulated by secreted inhibitory factors, including noggin and follistatin, both of which are expressed in or adjacent to the neural plate. Conversely, follistatin but not noggin produces a dorsal-to-ventral switch in progenitor cell identity and neuronal fate in response to Shh both in vitro and in vivo. These results suggest that the specification of ventral neural cell types depends on the integration of Shh and BMP signaling activities. The net level of BMP signaling within neural tissue may be regulated by follistatin and perhaps other BMP inhibitors secreted by mesodermal cell types that flank the ventral neural tube.

Krox20 and mafB/kr are regulatory genes involved in hindbrain segmentation and anteroposterior (AP) patterning. They are expressed in rhombomeres (r) r3/r5 and r5/r6 respectively, as well as in the r5/r6 neural crest. Since several members of the fibroblast growth factor (FGF) family are expressed in the otic/preotic region (r2-r6), we investigated their possible involvement in the regulation of Krox20 and mafB/kr. Application of exogenous FGFs to the neural tube of 4- to 7-somite chick embryos led to ectopic expression in the neural crest of the somitic hindbrain (r7 and r8) and to the extension of the Krox20- or mafB/kr-positive areas in the neuroepithelium. Application of an inhibitor of FGF signalling led to severe and specific downregulation of Krox20 and mafB/kr in the hindbrain neuroepithelium and neural crest. These data indicate that FGFs are involved in the control of regional induction and/or maintenance of Krox20 and mafB/kr expression, thus identifying a novel function for these factors in hindbrain development, besides their proposed more general role in early neural caudalisation.

The Pax6 gene encodes a transcription factor with a restricted expression in the ventricular zone of the pallium and subpallium. We tested whether the function of Pax6 is necessary for the correct patterning and morphogenesis of the vertebrate telencephalon. Homozygous embryos of the Pax6/Small eye mutant lack functional PAX6 protein because of a point mutation of the gene. In the mutant Small eye embryos we detected a ventralization of the molecular patterning of the telencephalon at two borders, the pallium/subpallium and the lateral/medial ganglionic eminence. The results indicate that Pax6 controls the lateral limit of the expression of Nkx2.1, Shh, and Lhx6 in the prechordal neural tube, the telencephalon. This finding is in agreement with previous studies and supports a model for a common genetic mechanism for modulation of the dorsoventral patterning of the prechordal and epichordal CNS. The pattern defects caused by the loss of Pax6 function result in multiple morphological abnormalities in the Small eye brain: dysgenesis of the piriform, insular, and lateral cortices, the claustrum-endopiriform nucleus, and a failure in the differentiation of a subpopulation of the cortical precursors. Together the results demonstrate that Pax6 has an essential role for the modulation of the dorsoventral patterning of the embryonic telencephalon, influencing thereby the forebrain morphogenesis.

A central feature of the tetrapod body plan is that two pairs of limbs develop at specific positions along the head-to-tail axis. However, the potential to form limbs in chick embryos is more widespread. This could have implications for understanding the basis of limb abnormalities. Here we extend the analysis to mouse embryos and examine systematically the potential of tissues in different regions outside the limbs to contribute to limb structures. We show that the ability of ectoderm to form an apical ridge in response to FGF4 in both mouse and chick embryos exists throughout the flank as does ability of mesenchyme to provide a polarizing region signal. In addition, neck tissue has weak polarizing activity. We show, in chick embryos, that polarizing activity of tissues correlates with the ability either to express Shh or to induce Shh expression. We also show that cells from chick tail can give rise to limb structures. Taken together these observations suggest that naturally occurring polydactyly could involve recruitment of cells from regions adjacent to the limb buds. We show that cells from neck, flank and tail can migrate into limb buds in response to FGF4, which mimics extension of the apical ectodermal ridge. Furthermore, when we apply simultaneously a polarizing signal and a limb induction signal to early chick flank, this leads to limb duplications.

To contribute to the development of the transcription map of human chromosome 21 (HC21), we isolated a new transcript, C21orf5 (chromosome 21 open reading frame 5), encoding a predicted 2298-amino-acid protein. Analysis of the genomic DNA sequence revealed that C21orf5 consists of 37 exons that extend over 130 kb and maps between the CBR3 (carbonyl reductase 3) and the KIAA0136 genes. Northern blot analyses showed a ubiquitously expressed RNA species of 8.5 kb. RNA in situ hybridization on brain sections of normal human embryos revealed a strong labeling in restricted areas of the cerebral cortex. In silico analysis of the deduced C21orf5 protein revealed several highly probable transmembrane segments but no known protein domains or homology with known proteins. However, there were significant homologies to several hypothetical Caenorhabditis elegans proteins and Drosophila melanogaster genomic sequences. To investigate the function of C21orf5, we isolated the cDNA of the C. elegans ortholog and performed double-stranded RNA-mediated genetic interference experiments. The major phenotype observed in the progeny of injected animals was embryonic lethality. Most of the tissues of the embryo failed to undergo proper patterning during gastrulation, and morphogenesis did not occur; thus we termed the ortholog pad-1, for patterning defective 1. These results indicated that pad-1 is essential for the development and the survival of C. elegans. This study provides the first example of the use of C. elegans as a model to study the function of genes on human chromosome 21 that might be involved in Down syndrome., (Copyright 2000 Academic Press.)

Wnt signaling has an important role in both embryonic development and tumorigenesis. beta-Catenin, a key component of the Wnt signaling pathway, interacts with the TCF/LEF family of transcription factors and activates transcription of Wnt target genes. Here, we identify a novel beta-catenin-interacting protein, ICAT, that was found to inhibit the interaction of beta-catenin with TCF-4 and represses beta-catenin-TCF-4-mediated transactivation. Furthermore, ICAT inhibited Xenopus axis formation by interfering with Wnt signaling. These results suggest that ICAT negatively regulates Wnt signaling via inhibition of the interaction between beta-catenin and TCF and is integral in development and cell proliferation.

The direct-developing frog, Eleutherodactylus coqui, has eliminated the tadpole stage from its ontogeny, and lacks many larval characters. We demonstrate that the dermal folds of E. coqui are homologous with the opercular folds of metamorphosing frogs. In both E. coqui and its metamorphic counterparts the opercular folds grow over the developing forelimb before perforating to free the entrapped limb. Opercular perforation in E. coqui occurs even in the absence of the forelimb but shows no signs of thyroid hormone dependence. The condensation of E. coqui development appears due to the excision of the extended larval period of developmental stasis. Analysis of opercular development, when viewed in conjunction with other developmental characters, suggests the ontogenetic period in the ancestral Eleutherodactylus life-history from which the tadpole was likely eliminated.

Dynamic expression patterns of four retinoid-metabolizing enzymes create rapidly changing retinoic acid (RA) patterns in the emerging eye anlage of the mouse. First, a RA-rich ventral zone is set up, then a RA-poor dorsal zone, and finally a tripartite organization consisting of dorsal and ventral RA-rich zones separated by a horizontal RA-poor stripe. This subdivision of the retina into three RA concentration zones is directly visible as beta-galactosidase labeling patterns in retinas of RA-reporter mice. Because the axons of retinal ganglion cells transport the reporter product anterogradely, the central projections from dorsal and ventral retina can be visualized as two heavily labeled axon bundles. Comparisons of the axonal labeling with physiologic recordings of visual topography in the adult mouse show that the labeled axons represent the upper and the lower visual fields. The RA-poor stripe develops into a broad horizontal zone of higher visual acuity. Comparisons of the retina labeling with eye-muscle insertions show that the axis of the RA pattern lines up with the dorsoventral axis of the oculomotor system. These observations indicate that the dorsoventral axis of the embryonic eye anlage determines the functional coordinates of both vision and eye movements in the adult., (Copyright 2000 Academic Press.)

Diethylstilbestrol (DES) was widely used to treat pregnant women through 1971. The reproductive tracts of their female offspring exposed to DES in utero are characterized by anatomic abnormalities. Here we show that DES administered to mice in utero produces changes in the expression pattern of several Hox genes that are involved in patterning of the reproductive tract. DES produces posterior shifts in Hox gene expression and homeotic anterior transformations of the reproductive tract. In human uterine or cervical cell cultures, DES induces HOXA9 or HOXA10 gene expression, respectively, to levels approximately twofold that induced by estradiol. The DES-induced expression is not inhibited by cyclohexamide. Estrogens are novel morphogens that directly regulate the expression pattern of posterior Hox genes in a manner analogous to retinoic acid regulation of anterior Hox genes. Alterations in HOX gene expression are a molecular mechanism by which DES affects reproductive tract development. Changes in Hox gene expression are a potential marker for the effects of in utero drug use that may become apparent only at late stages of development.

The embryonic vasculature forms by the segregation, migration, and assembly of angioblasts from mesoderm, a process termed vasculogenesis. The initial role of fibroblast growth factor 2 (FGF-2) in vascular development appears to be in the induction of endothelial precursors, angioblasts. Quail somites transplanted into chick embryos will give rise to angioblasts of quail origin. The number of angioblasts present within the chimera is dependent on the host environment. Angioblast induction can be demonstrated in vitro by the addition of FGF-2 to cultures of dissociated somitic mesoderm, as assessed by QH-1 epitope expression. Manipulation of FGF-2 concentration in the quail/chick chimeras by FGF-2 peptide or neutralizing antibody injections increases or decreases angioblast induction in the predicted manner. To better control growth factor release in vivo we have implanted beads that release FGF-2 into the embryonic environment. FGF-2 beads implanted into the somite induce angioblast differentiation in the epithelial somite; whereas, beads lateral to the somitic mesoderm induce the formation of ectopic vessels. These studies suggest that FGF-2 is important for both the induction of angioblasts and the assembly of angioblasts into the initial vasculature pattern., (Copyright 2000 Wiley-Liss, Inc.)

In recent years, genes that show left-right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula-neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus neurula embryos. After right-side injection of 10-250 pg activin protein, both early neurulae just after gastrulation movement (stage 13-14) and late neurulae just before neural tube closure (stage 17-18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1-50 pg) was microinjected into both sides of neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the neurula stage. Activin not only acts on the neurulae to cancel the original L-R specification up to the late neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of neurulae have equal potential for L-R differentiation., (Copyright 2000 Academic Press.)

beta-catenin plays an integral role in cell-cell adhesion by linking the cadherin complex of the adherens junction to the underlying actin cytoskeleton. In addition, beta-catenin transduces intracellular signals within the Wnt developmental pathway that are crucial to the proper establishment of embryonic axes and pattern formation of early mesoderm and ectoderm. For example, in the context of a defined dorsal 'organizer' region of early Xenopus embryos, beta-catenin enters the nucleus and associates with transcription factors of the HMG (High Mobility Group) Lef/Tcf protein family. Consequently, genes such as siamois, a homeobox gene contributing to the specification of the dorsoanterior axis, are activated. To further examine the role that beta-catenin plays in Wnt signaling, we generated a chimeric protein, beta-Engrailed (beta-Eng), in which the C-terminal trans-activation domain of beta-catenin is replaced with the transcriptional repression domain of Drosophila Engrailed. Dorsal overexpression of this mRNA in early Xenopus embryos leads to suppression of organizer-specific molecular markers such as siamois, Xnr-3 and goosecoid, corresponding with the dramatic morphological ventralization of embryos. Ventralized embryos further exhibit reduced activity of the Wnt pathway, as indicated by the loss of the notochord/organizer marker, chordin. Importantly, beta-Eng associates and functions normally with the known components of the cadherin complex, providing the experimental opportunity to repress beta-catenin's signaling function apart from its role in cadherin-mediated cell-cell adhesion.

Members of the fibroblast growth factor (FGF) family of peptide growth factors are widely expressed in the germ layer derivatives during gastrulation and early organogenesis of the mouse. We have investigated the effect of administering recombinant FGF-4 in the late-primitive streak stage embryo to test if the patterning of the body plan may be influenced by this growth factor. Shortly after FGF treatment the embryonic tissues up-regulated the expression of Brachyury and the RTK signaling regulator Spry2, suggesting that FGF signaling was activated as an immediate response to exogenous FGF. Concomitantly, Hesx1 expression was suppressed in the prospective anterior region of the embryo. After 24 h of in vitro development, embryos displayed a dosage-related suppression of forebrain morphogenesis, disruption of the midbrain-hindbrain partition, and inhibition of the differentiation of the embryonic mesoderm. Overall, development of the anterior-posterior axis in the late gastrula is sensitive to the delivery of exogenous FGF-4. The early response associated with the expression of Spry2 suggests that the later phenotype observed could be primarily related to an inhibition of the FGF signaling pathway., (Copyright 2000 Academic Press.)

XHNF1(&bgr;) is a homeobox-containing gene initially expressed at the blastula stage in the vegetal part of the Xenopus embryo. We investigated its early role by functional ablation, through mRNA injection of an XHNF1(beta)/engrailed repressor fusion construct (XHNF1(beta)/EngR). Dorsal injections of XHNF1(beta)/EngR mRNA abolish dorsal mesoderm formation, leading to axial deficiencies; ventral injections disrupt ventral mesoderm formation without affecting axial development. XHNF1(beta)/EngR phenotypic effects specifically depend on the DNA-binding activity of its homeodomain and are fully rescued by coinjection of XHNF1(beta) mRNA. Vegetal injection of XHNF1(beta)/EngR mRNA blocks the mesoderm-inducing ability of vegetal explants. Both B-Vg1 and VegT maternal determinants trigger XHNF1(beta) expression in animal caps. XHNF1(beta)/EngR mRNA blocks B-Vg1-mediated, but not by eFGF-mediated, mesoderm induction in animals caps. However, wild-type XHNF1(beta) mRNA does not trigger Xbra expression in animal caps. We conclude that XHNF1(beta) function is essential, though not sufficient, for mesoderm induction in the Xenopus embryo.

Netrin 1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. Here we used tissue explants to study the action of netrin 1 in the migration of several cerebellar and precerebellar cell progenitors. We show that netrin 1 exerts a strong chemoattractive effect on migrating neurons from the embryonic lower rhombic lip at E12-E14, which give rise to precerebellar nuclei. Netrin 1 promotes the exit of postmitotic migrating neurons from the embryonic lower rhombic lip and upregulates the expression of TAG-1 in these neurons. In addition, in the presence of netrin 1, the migrating neurons are not isolated but are associated with thick fascicles of neurites, typical of the neurophilic way of migration. In contrast, the embryonic upper rhombic lip, which contains tangentially migrating granule cell progenitors, did not respond to netrin 1. Finally, in the postnatal cerebellum, netrin 1 repels both the parallel fibres and migrating granule cells growing out from explants taken from the external germinal layer. The developmental patterns of expression in vivo of netrin 1 and its receptors are consistent with the notion that netrin 1 secreted in the midline acts as chemoattractive cue for precerebellar neurons migrating circumferentially along the extramural stream. Similarly, the pattern of expression in the postnatal cerebellum suggests that netrin 1 could regulate the tangential migration of postmitotic premigratory granule cells. Thus, molecular mechanisms considered as primarily involved in axonal guidance appear also to steer neuronal cell migration.

It has been proposed that digit identity in chick limb bud is specified in a dose-dependent fashion by a long-range morphogen, produced by the polarising region. One candidate is Sonic hedgehog (Shh) protein, but it is not clear whether Shh acts long or short range or via Bmps. Here we dissect the relationship between Shh and Bmp signalling. We show that Shh is necessary not only for initiating bmp2 expression but also for sustaining its expression during the period when additional digits are being specified. We also show that we can reproduce much of the effect of Shh during this period by applying only Bmp2. We further demonstrate that it is Bmps that are responsible for digit specification by transiently adding Noggin or Bmp antibodies to limbs treated with Shh. In such limbs, multiple additional digits still form but they all have the same identity. We also explored time dependency and range of Shh signalling by examining ptc expression. We show that high-level ptc expression is induced rapidly when either Shh beads or polarising regions are grafted to a host limb. Furthermore, we find that high-level ptc expression is first widespread but later more restricted. All these data lead us to propose a new model for digit patterning. We suggest that Shh initially acts long range to prime the region of the limb competent to form digits and thus control digit number. Then later, Shh acts short range to induce expression of Bmps, whose morphogenetic action specifies digit identity.

Much of what we currently know about digit morphogenesis during limb development is deduced from embryonic studies in the chick. In this study, we used ex utero surgical procedures to study digit morphogenesis during mouse embryogenesis. Our studies reveal some similarities; however, we have found considerable differences in how the chick and the mouse autopods respond to experimentation. First, we are not able to induce ectopic digit formation from interdigital cells as a result of wounding or TGFbeta-1 application in the mouse, in contrast to what is observed in the chick. Second, FGF4, which inhibits the formation of ectopic digits in the chick, induces a digit bifurcation response in the mouse. We demonstrate with cell marking studies that this bifurcation response results from a reorganization of the prechondrogenic tip of the digit rudiment. The FGF4 effect on digit morphogenesis correlates with changes in the expression of a number of genes, including Msx1, Igf2, and the posterior members of the HoxD cluster. In addition, the bifurcation response is digit-specific, being restricted to digit IV. We propose that FGF4 is an endogenous signal essential for skeletal branching morphogenesis in the mouse. This work stresses the existence of major differences between the chick and the mouse in how digit morphogenesis is regulated and is thus consistent with the view that vertebrate digit evolution is a relatively recent event. Finally, we discuss the relationship between the digit IV bifurcation restriction and the placement of the metapterygial axis in the evolution of the tetrapod limb., (Copyright 2000 Academic Press.)

The molecular motors dynein and kinesin are large protein complexes that convert the energy generated by ATP hydrolysis into directional movement along the microtubule cytoskeleton. They are required for a myriad of cellular processes, including mitotic spindle movement, axonal and vesicular transport, and ciliary beating. Recently, it has been shown that, in addition, they have a unique role during embryonic patterning: they are required to orient and establish the left-right axis in early vertebrate development.

In Xenopus and zebrafish certain bone morphognetic proteins (BMPs), and proteins that antagonise these by preventing their interaction with receptors, constitute a morphogen system in primary dorso-ventral patterning. This system may be directly involved in the parallel processes, within mesoderm and ectoderm, whereby the boundaries of the dorsal (paraxial) mesoderm and the neural plate are established. The bird blastoderm, amenable to grafting techniques and to direct exposure to specific proteins, has provided an opportunity to explore the phylogenetic conservation of such antagonistic system. We have grafted the gastrular organiser (node) into hosts, testing the effects of prior exposure of either grafted or host tissue to Follistatin (a known antagonist of TGFbeta superfamily ligands including BMP4) or to BMP4 protein. Strong, converse effects are seen from the two agents, the most consistent being on the sizes of new dorsalised areas (second neural plates) induced in host epiblast. Follistatin also enhances extension movements due to grafts, though without clear effect upon the rostro-caudal completeness of new patterns. Neural induction in chick epiblast by grafted mouse nodes are also more extensive, after their pre-incubation in Follistatin. Follistatin potentiates other, unknown but distinctive signals coming from the node, being unable to convert other non-inducing pieces of blastoderm into organisers on grafting. Pre-incubation of early blastoderms in BMP4 has such profound effects on normal dorsal axial development that host responsiveness of these blastoderms as hosts to node grafts is difficult to assess. Follistatin has no such overt effect on host development, but greatly enhances the competence of host epiblast to grafts of untreated nodes. Early chick BMP4 and BMP7 expressions are consistent with the proposed roles, though Follistatin is probably an experimental tool only in the present study.

Background: Animal melanin patterns are involved in diverse aspects of their ecology, from thermoregulation to mimicry. Many theoretical models have simulated pigment patterning, but little is known about the developmental mechanisms of color pattern formation. In Drosophila melanogaster, several genes are known to be necessary for cuticular melanization, but the involvement of these genes in melanin pattern evolution is unknown. We have taken a genetic approach to elucidate the developmental mechanisms underlying melanin pattern formation in various drosophilids., Results: We show that, in D. melanogaster, tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) are required for melanin synthesis. Ectopic expression of TH, but not DDC, alone was sufficient to cause ectopic melanin patterns in the wing. Thus, changes in the level of expression of a single gene can result in a new level of melanization. The ontogeny of this ectopic melanization resembled that found in Drosophila species bearing wing melanin patterns and in D. melanogaster ebony mutants. Importantly, we discovered that in D. melanogaster and three other Drosophila species these wing melanin patterns are dependent upon and shaped by the circulation patterns of hemolymph in the wing veins., Conclusions: Complex wing melanin patterns are determined by two distinct developmental mechanisms. Spatial prepatterns of enzymatic activity are established late in wing development. Then, in newly eclosed adults, melanin precursors gradually diffuse out from wing veins and are oxidized into dark brown or black melanin. Both the prepatterning and hemolymph-supplied components of this system can change during evolution to produce color pattern diversity.

The activity of bone morphogenetic protein (BMP) heterodimers has been shown to be more potent than that of homodimers in a number of contexts, including mesoderm induction. Although BMP-2/7 and -4/7 heterodimers are potent inducers of ventral mesoderm in ectodermal explants, we show that they are not a necessary component of the primary mesoderm-inducing signal in intact Xenopus embryos. The secreted BMP antagonists noggin and gremlin both efficiently block mesoderm induction by BMP homo- and heterodimers in animal caps. When these antagonists are ectopically expressed in the ventral marginal zone of early embryos the initial formation of mesoderm as indicated by panmesodermal markers remains unaffected. Only the subsequent dorsal/ventral patterning of this mesoderm appears to be altered, with expression of a number of organizer-specific transcripts observed in the marginal zone where BMP signaling has been abolished. Thus, we conclude that BMPs do not contribute an essential signal to mesodermal induction or patterning until gastrulation. The activities of noggin and gremlin are strikingly different from that of the multifunctional antagonist cerberus, which completely abolishes mesoderm induction when misexpressed during early development., (Copyright 1999 Academic Press.)

Invariant patterning of left-right asymmetry during embryogenesis depends upon a cascade of inductive and repressive interactions between asymmetrically expressed genes. Different cascades of asymmetric genes distinguish the left and right sides of the embryo and are maintained by a midline barrier. As such, the left and right sides of an embryo can be viewed as distinct and autonomous fields. Here we describe a series of experiments that indicate that the initiation of these programs requires communication between the two sides of the blastoderm. When deprived of either the left or the right lateral halves of the blastoderm, embryos are incapable of patterning normal left-right gene expression at Hensen's node. Not only are both flanks required, suggesting that there is no single signaling source for LR pattern, but the blastoderm must be intact. These results are consistent with our previously proposed model in which the orientation of LR asymmetry in the frog, Xenopus laevis, depends on large-scale partitioning of LR determinants through intercellular gap junction channels (M. Levin and M. Mercola (1998) Developmental Biology 203, 90-105). Here we evaluate whether gap junctional communication is required for the LR asymmetry in the chick, where it is possible to order early events relative to the well-characterized left and right hierarchies of gene expression. Treatment of cultured chick embryos with lindane, which diminishes gap junctional communication, frequently unbiased normal LR asymmetry of Shh and Nodal gene expression, causing the normally left-sided program to be recapitulated symmetrically on the right side of the embryo. A survey of early expression of connexin mRNAs revealed that Cx43 is present throughout the blastoderm at Hamburger-Hamilton stage 2-3, prior to known asymmetric gene expression. Application of antisense oligodeoxynucleotides or blocking antibody to cultured embryos also resulted in bilateral expression of Shh and Nodal transcripts. Importantly, the node and primitive streak at these stages lack Cx43 mRNA. This result, together with the requirement for an intact blastoderm, suggests that the path of communication through gap junction channels circumvents the node and streak. We propose that left-right information is transferred unidirectionally throughout the epiblast by gap junction channels in order to pattern left-sided Shh expression at Hensen's node.

Mesenchymal cells from different stages of chick limb buds sort out in monolayer culture, suggesting the presence of different cell affinities dependent on their positions along the proximodistal axis. However, it is still not clear which molecules are responsible for the sorting-out. Here, we propose that N-cadherin, a cell-adhesion molecule, is involved in the sorting-out and is likely to be a component of the mechanism of proximodistal patterning in the developing limb. N-cadherin proteins accumulate in the distal region of the chick limb bud as limb development proceeds. In monolayer culture of distal mesenchymal cells, the stage-dependent levels of N-cadherin proteins are maintained during cell sorting. The results of this study have also demonstrated that an anti-N-cadherin monoclonal antibody, NCD-2, clearly inhibits the cell sorting. Moreover, removal of the apical ectodermal ridge or retinoic-acid treatment of distal cells, which results in a change in the pattern of sorting-out, inhibits the accumulation of N-cadherin proteins, suggesting that the distribution of these proteins is related to the positional identity that gives rise to the different shape and number of cartilage elements along the proximodistal axis.

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.

Axin is a recently identified protein encoded by the fused locus in mice that is required for normal vertebrate axis formation. We have defined a 25-amino-acid sequence in axin that comprises the glycogen synthase kinase 3beta (GSK-3beta) interaction domain (GID). In contrast to full-length axin, which has been shown to antagonize Wnt signaling, the GID inhibits GSK-3beta in vivo and activates Wnt signaling. Similarly, mutants of axin lacking key regulatory domains such as the RGS domain, which is required for interaction with the adenomatous polyposis coli protein, bind and inhibit GSK-3beta in vivo, suggesting that these domains are critical for proper regulation of GSK-3beta activity. We have identified a novel self-interaction domain in axin and have shown that formation of an axin regulatory complex in vivo is critical for axis formation and GSK-3beta activity. Based on these data, we propose that the axin complex may directly regulate GSK-3beta enzymatic activity in vivo. These observations also demonstrate that alternative inhibitors of GSK-3beta can mimic the effect of lithium in developing Xenopus embryos.

Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrated that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules towards the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Finally, we demonstrate that overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus.

Nuclear receptors play key roles in anterior/posterior (A/P) axis formation during vertebrate embryogenesis. Within this gene family, retinoic acid receptors and retinoic acid itself have profound influences on the establishment of the A/P axis. Thyroid hormone receptors are expressed during early periods of development, long before the establishment of the thyroid gland, and are able to interact with retinoic acid receptors. Here we examined the ability of the thyroid hormone receptor alpha 1 to affect early embryonic development by mRNA injection of either repressor or activator forms of the thyroid hormone receptor. Overexpression of either the thyroid hormone receptor alpha 1 or a constitutive repressor form, v-erbA, caused a swelling in the rostral hindbrain. These defects were associated with disorganization and loss of rhombomere borders as well as an increase in the number of acetylcholine esterase positive cells. This phenotype correlated with a reduction in hoxa1 expression during gastrulation. Furthermore, injection of either thyroid hormone receptor alpha 1 or v-erbA mRNA repressed a reporter gene that contained a retinoic acid response element, demonstrating the ability of the thyroid hormone receptor alpha 1 to repress retinoic acid signaling during gastrulation. In contrast, embryos treated with retinoic acid alone or embryos injected with thyroid hormone receptor alpha 1 and treated with the thyroid hormone analog TRIAC displayed a similar set of defects, including loss of the midbrain-hindbrain border and severe disruption of the rostral hindbrain. These studies support the involvement of retinoic acid and its receptors in the direct control of Hox gene expression and the early patterning of the zebrafish central nervous system.

Mesoderm induction is one of the major events of early vertebrate embryonic patterning. It appears to be controlled by sequential and combinatorial actions of several kinds of peptide growth factors. These include activin, fibroblast growth factor (FGF), and transforming growth factor-beta (TGF-beta), among others. In the present study, the function of platelet-derived growth factor (PDGF) in early Xenopus laevis embryogenesis was investigated. In the animal-cap assay, PDGF caused pre-ectodermal tissue to develop a mesoderm specific morphology (elongation) and to express the mesoderm marker genes, MyoD family and alpha-cardiac actin. In addition, two other genes were expressed -related serum response factor SL1 (a dorsal mesodermal marker) and myosin light chain (MLC2-heart marker). A role for PDGF in normal (in vivo) mesoderm induction is implicated because injection of PDGF receptor alpha antisense RNA into 2-cell embryos erased the animal cap's mesoderm marker expression. Those injected embryos also exhibited morphological abnormalities including incomplete gastrulation, failure of neural fold closing, and abnormal somitogenesis.

BMP-4 is an extracellular signalling molecule belonging to the TGF-beta superfamily that plays a central role in dorsoventral patterning in vertebrate gastrulae. We review the evidence indicating that BMP-4 acts as a morphogen, specifying dorsoventral positional values in a concentration-dependent manner. An activity gradient of BMP-4 is established not by simple diffusion from a localised source, but by diffusion of inhibitory binding proteins that act on a uniform level of BMP-4 protein. These in turn are regulated by the activity of tolloid-related metalloproteases, such as Xenopus xolloid and zebrafish tolloid., (Copyright 1999 Academic Press.)

Proper dorsal-ventral patterning in the developing central nervous system requires signals from both the dorsal and ventral portions of the neural tube. Data from multiple studies have demonstrated that bone morphogenetic proteins (BMPs) and Sonic hedgehog protein are secreted factors that regulate dorsal and ventral specification, respectively, within the caudal neural tube. In the developing rostral central nervous system Sonic hedgehog protein also participates in ventral regionalization; however, the roles of BMPs in the developing brain are less clear. We hypothesized that BMPs also play a role in dorsal specification of the vertebrate forebrain. To test our hypothesis we implanted beads soaked in recombinant BMP5 or BMP4 into the neural tube of the chicken forebrain. Experimental embryos showed a loss of the basal telencephalon that resulted in holoprosencephaly (a single cerebral hemisphere), cyclopia (a single midline eye), and loss of ventral midline structures. In situ hybridization using a panel of probes to genes expressed in the dorsal and ventral forebrain revealed the loss of ventral markers with the maintenance of dorsal markers. Furthermore, we found that the loss of the basal telencephalon was the result of excessive cell death and not a change in cell fates. These data provide evidence that BMP signaling participates in dorsal-ventral patterning of the developing brain in vivo, and disturbances in dorsal-ventral signaling result in specific malformations of the forebrain.

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.