Nodal signalling in the epiblast patterns the early mouse embryo.

Shortly after implantation the mouse embryo comprises three tissue layers. The founder tissue of the embryo proper, the epiblast, forms a radially symmetric cup of epithelial cells that grows in close apposition to the extra-embryonic ectoderm and the visceral endoderm. This simple cylindrical structure exhibits a distinct molecular pattern along its proximal-distal axis. The anterior-posterior axis of the embryo is positioned later by coordinated cell movements that rotate the pre-existing proximal-distal axis. The transforming growth factor-beta family member Nodal is known to be required for formation of the anterior-posterior axis. Here we show that signals from the epiblast are responsible for the initiation of proximal-distal polarity. Nodal acts to promote posterior cell fates in the epiblast and to maintain molecular pattern in the adjacent extra-embryonic ectoderm. Both of these functions are independent of Smad2. Moreover, Nodal signals from the epiblast also pattern the visceral endoderm by activating the Smad2-dependent pathway required for specification of anterior identity in overlying epiblast cells. Our experiments show that proximal-distal and subsequent anterior-posterior polarity of the pregastrulation embryo result from reciprocal cell-cell interactions between the epiblast and the two extra-embryonic tissues.     

Specification of limb development in the Drosophila embryo by positional cues from segmentation genes

Limb development in Drosophila requires the activity of a proximo-distal pattern-forming system, in addition to the antero-posterior and dorso-ventral pattern-forming systems that subdivide the embryo. Several lines of genetic evidence indicate that the Distal-less gene plays an important part in specifying proximo-distal positional information. The Distal-less locus encodes a homoeodomain-containing protein, which suggests that Distal-less may exert its activity through differential regulation of subordinate genes. The spatially restricted pattern of Distal-less expression allows direct visualization of the limb primordia during early embryogenesis. Here I report that from their inception, the leg primordia span the parasegment boundary. The segment polarity gene wingless seems to have a key part in defining the positions at which leg primordia will develop along the antero-posterior axis of the embryo. This analysis allows a direct molecular visualization of the compartments that subdivide the limb primordia into discrete developmental domains.     

Interaction between peptide growth factors and homoeobox genes in the establishment of antero-posterior polarity in frog embryos

The expression of the Xenopus homoeobox gene xhox3 is an early response to mesoderm induction by peptide growth factors and the level of xhox3 expression marks the antero-posterior character of the induced mesoderm. Different peptide growth factors specify different antero-posterior mesodermal cell fates as seen by the level of xhox3 expression and the capacity to induce specific secondary neural/epidermal structures. These factors and homoeobox genes thus form part of the mechanism necessary for establishing antero-posterior polarity in the frog embryo.     

The status of Wnt signalling regulates neural and epidermal fates in the chick embryo

The acquisition of neural fate by embryonic ectodermal cells is a fundamental step in the formation of the vertebrate nervous system. Neural induction seems to involve signalling by fibroblast growth factors (FGFs) and attenuation of the activity of bone morphogenetic protein (BMP). But FGFs, either alone or in combination with BMP antagonists, are not sufficient to induce neural fate in prospective epidermal ectoderm of amniote embryos. These findings suggest that additional signals are involved in the specification of neural fate. Here we show that the state of Wnt signalling is a critical determinant of neural and epidermal fates in the chick embryo. Continual Wnt signalling blocks the response of epiblast cells to FGF signals, permitting the expression and signalling of BMP to direct an epidermal fate. Conversely, a lack of exposure of epiblast cells to Wnt signals permits FGFs to induce a neural fate.     

The zebrafish Nodal signal Squint functions as a morphogen

Secreted morphogens induce distinct cellular responses in a concentration-dependent manner and act directly at a distance. The existence of morphogens during mesoderm induction and patterning in vertebrates has been highly controversial, and it remains unknown whether endogenous mesoderm inducers act directly as morphogens, function locally or act through relay mechanisms. Here we test the morphogen properties of Cyclops and Squint-two Nodal-related transforming growth factor-beta signals required for mesoderm formation and patterning in zebrafish. Whereas different levels of both Squint and Cyclops can induce different downstream genes, we find that only Squint can function directly at a distance. These results indicate that Squint acts as a secreted morphogen that does not require a relay mechanism.     

影响骨形态发育因素研究

骨骼的发育经历了极复杂的过程.众多因素调节了此过程,这些因素包括TGF-β、骨形态发生蛋白、核心结合因子A1、甲状旁腺激素、基质金属蛋白酶、FGF及其受体等.研究了这些因素在骨形成过程中的作用.     

Silencing of TGF-beta signalling by the pseudoreceptor BAMBI.

Members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta, bone morphogenetic proteins (BMPs), activins and nodals, are vital for regulating growth and differentiation. These growth factors transduce their signals through pairs of transmembrane type I and type II receptor kinases. Here, we have cloned a transmembrane protein, BAMBI, which is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. We show that BAMBI is co-expressed with the ventralizing morphogen BMP4 (refs 5, 6) during Xenopus embryogenesis and that it requires BMP signalling for its expression. The protein stably associates with TGF-beta-family receptors and inhibits BMP and activin as well as TGF-beta signalling. Finally, we provide evidence that BAMBI's inhibitory effects are mediated by its intracellular domain, which resembles the homodimerization interface of a type I receptor and prevents the formation of receptor complexes. The results indicate that BAMBI negatively regulates TGF-beta-family signalling by a regulatory mechanism involving the interaction of signalling receptors with a pseudoreceptor.     

Eomesodermin is required for mouse trophoblast development and mesoderm formation

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.     

Retinoic acid signalling links left-right asymmetric patterning and bilaterally symmetric somitogenesis in the zebrafish embryo

During embryogenesis, cells are spatially patterned as a result of highly coordinated and stereotyped morphogenetic events. In the vertebrate embryo, information on laterality is conveyed to the node, and subsequently to the lateral plate mesoderm, by a complex cascade of epigenetic and genetic events, eventually leading to a left-right asymmetric body plan. At the same time, the paraxial mesoderm is patterned along the anterior-posterior axis in metameric units, or somites, in a bilaterally symmetric fashion. Here we characterize a cascade of laterality information in the zebrafish embryo and show that blocking the early steps of this cascade (before it reaches the lateral plate mesoderm) results in random left-right asymmetric somitogenesis. We also uncover a mechanism mediated by retinoic acid signalling that is crucial in buffering the influence of the flow of laterality information on the left-right progression of somite formation, and thus in ensuring bilaterally symmetric somitogenesis.     

Planar cell polarity signalling controls cell division orientation during zebrafish gastrulation

Oriented cell division is an integral part of pattern development in processes ranging from asymmetric segregation of cell-fate determinants to the shaping of tissues. Despite proposals that it has an important function in tissue elongation, the mechanisms regulating division orientation have been little studied outside of the invertebrates Caenorhabditis elegans and Drosophila melanogaster. Here, we have analysed mitotic divisions during zebrafish gastrulation using in vivo confocal imaging and found that cells in dorsal tissues preferentially divide along the animal-vegetal axis of the embryo. Establishment of this animal-vegetal polarity requires the Wnt pathway components Silberblick/Wnt11, Dishevelled and Strabismus. Our findings demonstrate an important role for non-canonical Wnt signalling in oriented cell division during zebrafish gastrulation, and indicate that oriented cell division is a driving force for axis elongation. Furthermore, we propose that non-canonical Wnt signalling has a conserved role in vertebrate axis elongation, orienting both cell intercalation and mitotic division.     

cdx4 mutants fail to specify blood progenitors and can be rescued by multiple hox genes

Organogenesis is dependent on the formation of distinct cell types within the embryo. Important to this process are the hox genes, which are believed to confer positional identities to cells along the anteroposterior axis. Here, we have identified the caudal-related gene cdx4 as the locus mutated in kugelig (kgg), a zebrafish mutant with an early defect in haematopoiesis that is associated with abnormal anteroposterior patterning and aberrant hox gene expression. The blood deficiency in kgg embryos can be rescued by overexpressing hoxb7a or hoxa9a but not hoxb8a, indicating that the haematopoietic defect results from perturbations in specific hox genes. Furthermore, the haematopoietic defect in kgg mutants is not rescued by scl overexpression, suggesting that cdx4 and hox genes act to make the posterior mesoderm competent for blood development. Overexpression of cdx4 during zebrafish development or in mouse embryonic stem cells induces blood formation and alters hox gene expression. Taken together, these findings demonstrate that cdx4 regulates hox genes and is necessary for the specification of haematopoietic cell fate during vertebrate embryogenesis.     

Experimental manipulation of a contact guidance system in amphibian gastrulation by mechanical tension

Contact guidance has been implied in various morphogenetic movements including neural crest cell migration, primordial germ cell migration and guidance of axonal growth cone. In urodele gastrulae, we reported the presence of an aligned network of extracellular fibrils on the inside of the ectodermal layer and suggested that it guides the migration of the presumptive mesodermal cells from the blastopore towards the animal pole. We also reported in vitro experiments in which the fibril network of the ectodermal layer was transferred onto the surface of a coverslip. Dissociated mesodermal cells attach to and locomote actively on such conditioned surfaces in an oriented fashion along the blastopore-animal pole axis (bp-ap axis) of the ectodermal layer that conditioned the surface. Recent reports suggest that these fibrils contain fibronectin. We now report that the fibril network on the conditioned surface can be artificially aligned in any orientation by exerting mechanical tension on the ectodermal layer during the conditioning. Such prepared surfaces cause cell movements aligned along the tension axis, even when the tension axis is perpendicular to the natural axis of alignment along the bp-ap axis. These results suggest that the extracellular matrix fibrils aligned by the mechanical stress that arises in embryos during development can orient cell migration by the contact guidance, in a similar manner to that reported in the collagen gel and fibroblasts system.     

Protein kinase C mediates neural induction in Xenopus laevis

Inductive cell interactions are essential in early embryonic development, but virtually nothing is known about the molecular mechanisms involved. Recently factors resembling fibroblast growth factor and transforming growth factor-beta were shown to be involved in mesoderm induction in Xenopus laevis, suggesting that membrane receptor-mediated signal transduction is important in induction processes. Here we report direct measurements of protein kinase C (PKC) activity in uninduced ectoderm, and in neuroectoderm shortly after induction by the involuting mesoderm, in Xenopus laevis embryos. Membrane-bound PKC activity increased three to fourfold in the induced neuroectoderm while the cytosolic PKC activity was decreasing, indicating that PKC activity was translocated during neural induction. A similar time- and dose-dependent translocation of activity was seen after incubation with the PKC activator 12-O-tetradecanoyl phorbol-13-acetate, which also induced neural tissue in competent ectoderm, suggesting that PKC is involved in the response to the endogenous inducing signal during neural induction.     

Antero-posterior tissue polarity links mesoderm convergent extension to axial patterning

Remodelling its shape, or morphogenesis, is a fundamental property of living tissue. It underlies much of embryonic development and numerous pathologies. Convergent extension (CE) of the axial mesoderm of vertebrates is an intensively studied model for morphogenetic processes that rely on cell rearrangement. It involves the intercalation of polarized cells perpendicular to the antero-posterior (AP) axis, which narrows and lengthens the tissue. Several genes have been identified that regulate cell behaviour underlying CE in zebrafish and Xenopus. Many of these are homologues of genes that control epithelial planar cell polarity in Drosophila. However, elongation of axial mesoderm must be also coordinated with the pattern of AP tissue specification to generate a normal larval morphology. At present, the long-range control that orients CE with respect to embryonic axes is not understood. Here we show that the chordamesoderm of Xenopus possesses an intrinsic AP polarity that is necessary for CE, functions in parallel to Wnt/planar cell polarity signalling, and determines the direction of tissue elongation. The mechanism that establishes AP polarity involves graded activin-like signalling and directly links mesoderm AP patterning to CE.     

The molecular nature of the zebrafish tail organizer

Based on grafting experiments, Mangold and Spemann showed the dorsal blastopore lip of an amphibian gastrula to be able to induce a secondary body axis. The equivalent of this organizer region has been identified in different vertebrates including teleosts. However, whereas the graft can induce ectopic head and trunk, endogenous and ectopic axes fuse in the posterior part of the body, raising the question of whether a distinct organizer region is necessary for tail development. Here we reveal, by isochronic and heterochronic transplantation, the existence of a tail organizer deriving from the ventral margin of the zebrafish embryo, which is independent of the dorsal Spemann organizer. Loss-of-function experiments reveal that bone morphogenetic protein (BMP), Nodal and Wnt8 signalling pathways are required for tail development. Moreover, stimulation of naive cells by a combination of BMP, Nodal and Wnt8 mimics the tail-organizing activity of the ventral margin and induces surrounding tissues to become tail. In contrast to induction of the vertebrate head, known to result from the triple inhibition of BMP, Nodal and Wnt, here we show that induction of the tail results from the triple stimulation of BMP, Nodal and Wnt8 signalling pathways.     

Pre-existent pattern in Xenopus animal pole cells revealed by induction with activin

Activin, a peptide growth factor related to tumour growth factor-beta, has been implicated in early inductive interactions in vertebrates and can induce Xenopus blastula ectodermal explants to develop a rudimentary axial pattern with anteroposterior and dorsoventral polarity. Here we demonstrate that prospective dorsal and ventral regions of the ectoderm respond differently to the same concentration of activin. Thus, activin does not seem to endow ectodermal cells with polarity but rather reveals a pre-existent pattern. Our results suggest that patterning of mesoderm is determined not only by a localized inducer, but also by the differential competence of cells in the responding tissue.