BMP signaling positively regulates Nodal expression during left right specification in the chick embryo

Exogenous application of BMP to the lateral plate mesoderm (LPM) of chick embryos at the early somite stage had a positive effect on Nodal expression. BMP applications into the right LPM were followed by a rapid activation of Nodal, while applications into the left LPM resulted in expansion of the normal domain of Nodal expression. Conversely, blocking of BMP signaling by Noggin in the left LPM interfered with the activation of Nodal expression. These results support a positive role for endogenous BMP on Nodal expression in the LPM. We also report that BMP positively regulates the expression of Caronte, Snail and Cfc in both the left and right LPM. BMP-treated embryos had molecular impairment of the midline with downregulation of Lefty1, Brachyury and Shh but we also show that the midline defect was not sufficient to induce ectopic Nodal expression. We discuss our findings in the context of the known molecular control of the specification of left-right asymmetry.     

BMP2 is a positive regulator of Nodal signaling during left-right axis formation in the chicken embryo

A model of left-right axis formation in the chick involves inhibition of bone morphogenetic proteins by the antagonist Car as a mechanism of upregulating Nodal in the left lateral plate mesoderm. By contrast, expression of CFC, a competence factor, which is absolutely required for Nodal signaling in the lateral plate mesoderm is dependent on a functional BMP signaling pathway. We have therefore investigated the relationship between BMP and Nodal in further detail. We implanted BMP2 and Noggin-expressing cells into the left lateral plate and paraxial mesoderm and observed a strong upregulation of Nodal and its target genes Pitx2 and Nkx3.2. In addition Cfc, the Nodal type II receptor ActrIIa and Snr were found to depend on BMP signaling for their expression. Comparison of the expression domains of Nodal, Bmp2, Car and Cfc revealed co-expression of Nodal, Cfc and Bmp2, while Car and Nodal only partially overlapped. Ectopic application of BMP2, Nodal, and Car as well as combinations of this signaling molecules to the right lateral plate mesoderm revealed that BMP2 and Car need to synergize in order to specify left identity. We propose a novel model of left-right axis formation, which involves BMP as a positive regulator of Nodal signaling in the chick embryo.     

Unveiling the establishment of left-right asymmetry in the chick embryo

Vertebrates display striking left-right asymmetries in the placement of internal organs, which are concealed by a seemingly bilaterally symmetric body plan. The establishment of asymmetries about the left-right axis occurs early during embryo development and requires the concerted and sequential action of several epigenetic, genetic and cellular mechanisms. Experiments in the chick embryo model have contributed crucially to our current understanding of such mechanisms and are reviewed here. Particular emphasis is given to the elucidation of a genetic network that conveys left-right information from Hensen's node to the organ primordia, characterized to a significant degree of detail in the chick embryo. We also point out a number of early and late events in the determination of left-right asymmetries that are currently poorly understood and for whose study the chick embryo model presents several advantages. We anticipate that the availability of the chick genome sequence will be combined with multidisciplinary approaches from experimental embryology, biophysics, live-cell imaging, and mathematical modeling to boost up our knowledge of left-right organ asymmetry in the near future.     

Direct and indirect control of oral ectoderm regulatory gene expression by Nodal signaling in the sea urchin embryo

The Nodal signaling pathway is known from earlier work to be an essential mediator of oral ectoderm specification in the sea urchin embryo, and indirectly, of aboral ectoderm specification as well. Following expression of the Nodal ligand in the future oral ectoderm during cleavage, a sequence of regulatory gene activations occur within this territory which depend directly or indirectly on nodal gene expression. Here we describe additional regulatory genes that contribute to the oral ectoderm regulatory state during specification in Strongylocentrotus purpuratus, and show how their spatial expression changes dynamically during development. By means of system wide perturbation analyses we have significantly improved current knowledge of the epistatic relations among the regulatory genes of the oral ectoderm. From these studies there emerge diverse circuitries relating downstream regulatory genes directly and indirectly to Nodal signaling. A key intermediary regulator, the role of which had not previously been discerned, is the not gene. In addition to activating several genes earlier described as targets of Nodal signaling, the not gene product acts to repress other oral ectoderm genes, contributing crucially to the bilateral spatial organization of the embryonic oral ectoderm.     

FGF signaling is required for determination of otic neuroblasts in the chick embryo

The interplay between intrinsic and extrinsic factors is essential for the transit into different cell states during development. We have analyzed the expression and function of FGF10 and FGF-signaling during the early stages of the development of otic neurons. FGF10 is expressed in a highly restricted domain overlapping the presumptive neurogenic region of the chick otic placode. A detailed study of the expression pattern of FGF10, proneural, and neurogenic genes revealed the following temporal sequence for the onset of gene expression: FGF10>Ngn1/Delta1/Hes5>NeuroD/NeuroM. FGF10 and FGF receptor inhibition cause opposed effects on cell determination and cell proliferation. Ectopic expression of FGF10 in vivo promotes an increase in NeuroD and NeuroM expression. BrdU incorporation experiments showed that the increase in NeuroD-expressing cells is not due to an increase in cell proliferation. Inhibition of FGF receptor signaling in otic explants causes a severe reduction in Neurogenin1, NeuroD, Delta1, and Hes5 expression with no change in non-neural genes like Lmx1. However, it does not interfere with NeuroD expression within the CVG or with neuroblast delamination. The loss of proneural gene expression caused by FGF inhibition is not caused by decreased cell proliferation or by increased cell death. We suggest that FGF signaling in the otic epithelium is required for neuronal precursors to withdraw from cell division and irreversibly commit to neuronal fate.     

Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination

During vertebrate development, signaling by the TGFbeta ligand Nodal is critical for mesoderm formation, correct positioning of the anterior-posterior axis, normal anterior and midline patterning, and left-right asymmetric development of the heart and viscera. Stimulation of Alk4/EGF-CFC receptor complexes by Nodal activates Smad2/3, leading to left-sided expression of target genes that promote asymmetric placement of certain internal organs. We identified Ttrap as a novel Alk4- and Smad3-interacting protein that controls gastrulation movements and left-right axis determination in zebrafish. Morpholino-mediated Ttrap knockdown increases Smad3 activity, leading to ectopic expression of snail1a and apparent repression of e-cadherin, thereby perturbing cell movements during convergent extension, epiboly and node formation. Thus, although the role of Smad proteins in mediating Nodal signaling is well-documented, the functional characterization of Ttrap provides insight into a novel Smad partner that plays an essential role in the fine-tuning of this signal transduction cascade.     

Wnt6 marks sites of epithelial transformations in the chick embryo

In a screen for Wnt genes executing the patterning function of the vertebrate surface ectoderm, we have isolated a novel chick Wnt gene, chick Wnt6. This gene encodes the first pan-epidermal Wnt signalling molecule. Further sites of expression are the boundary of the early neural plate and surface ectoderm, the roof of mesencephalon, pretectum and dorsal thalamus, the differentiating heart, and the otic vesicle. The precise sites of Wnt6 expression coincide with crucial changes in tissue architecture, namely epithelial remodelling and epithelial-mesenchymal transformation (EMT). Moreover, the expression of Wnt6 is closely associated with areas of Bmp signalling.     

beta-Hexosaminidase expression in chick embryo fibroblasts in vitro.

1. Two forms of beta-hexosaminidase, similar to hexosaminidase A and hexosaminidase C, were separated by DEAE-cellulose chromatography in chick embryo skin fibroblasts in vitro. 2. beta-Hexosaminidase specific activity increases during development in cultured chick embryo skin fibroblasts in vitro. 3. Concanavalin-A treatment determines the increase of the neutral form, hexosaminidase C, during development. 4. Concanavalin-A reduces the specific activity of beta-hexosaminidase during development.     

Retinoic acid is required in the mouse embryo for left-right asymmetry determination and heart morphogenesis.

Determination of the left-right position (situs) of visceral organs involves lefty, nodal and Pitx2 genes that are specifically expressed on the left side of the embryo. We demonstrate that the expression of these genes is prevented by the addition of a retinoic acid receptor pan-antagonist to cultured headfold stage mouse embryos, whereas addition of excess retinoic acid leads to their symmetrical expression. Interestingly, both treatments lead to randomization of heart looping and to defects in heart anteroposterior patterning. A time course analysis indicates that only the newly formed mesoderm at the headfold-presomite stage is competent for these retinoid effects. We conclude that retinoic acid, the active derivative of vitamin A, is essential for heart situs determination and morphogenesis.     

Nodal flow and the generation of left-right asymmetry

The establishment of left-right asymmetry in mammals is a good example of how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left-right axis. Nodal flow is autonomously generated by the rotation of cilia that are tilted toward the posterior on cells of the ventral node. These cilia are built by transport via the KIF3 motor complex. How nodal flow is interpreted to create left-right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of membrane-sheathed particles, called nodal vesicular parcels (NVPs), may result in the activation of the non-canonical Hedgehog signaling pathway, an asymmetric elevation in intracellular Ca(2+) and changes in gene expression.     

Moderate expression of Wnt signaling genes is essential for porcine parthenogenetic embryo development

Parthenogenetic embryos are invariably lost in mid-gestation, possibly due to the lack of the paternal genome and the consequent induction of aberrant gene expression. Wnt signaling is essential for embryonic development; however, the studies of this pathway in porcine parthenogenetic embryos have been limited. Here, the role of Wnt signaling in porcine parthenogenetic embryos was studied. In vivo embryos were used as controls. Single cell quantitative real-time PCR showed that Wnt signaling was down-regulated in porcine parthenogenetic embryos. Furthermore, immunofluorescence staining and real-time PCR demonstrated that porcine parthenogenetic embryo development was largely unaffected by the inhibition of Wnt signaling with IWP-2, but blastocyst hatching and trophectoderm development was blocked. In addition, parthenogenetic blastocyst hatching was improved by the activation of Wnt signaling by BIO. However, the developmental competency of porcine embryos, including blastocyst hatching, was impaired and apoptosis was induced upon the excessive activation of Wnt signaling. These findings constitute novel evidence that Wnt signaling is important for porcine pre-implantation development and that its down-regulation may lead to the low hatching rate of porcine parthenogenetic blastocysts.     

Wnt3a links left-right determination with segmentation and anteroposterior axis elongation

The alignment of the left-right (LR) body axis relative to the anteroposterior (AP) and dorsoventral (DV) axes is central to the organization of the vertebrate body plan and is controlled by the node/organizer. Somitogenesis plays a key role in embryo morphogenesis as a principal component of AP elongation. How morphogenesis is coupled to axis specification is not well understood. We demonstrate that Wnt3a is required for LR asymmetry. Wnt3a activates the Delta/Notch pathway to regulate perinodal expression of the left determinant Nodal, while simultaneously controlling the segmentation clock and the molecular oscillations of the Wnt/beta-catenin and Notch pathways. We provide evidence that Wnt3a, expressed in the primitive streak and dorsal posterior node, acts as a long-range signaling molecule, directly regulating target gene expression throughout the node and presomitic mesoderm. Wnt3a may also modulate the symmetry-breaking activity of mechanosensory cilia in the node. Thus, Wnt3a links the segmentation clock and AP axis elongation with key left-determining events, suggesting that Wnt3a is an integral component of the trunk organizer.     

Absence of Nodal signaling promotes precocious neural differentiation in the mouse embryo

After implantation, mouse embryos deficient for the activity of the transforming growth factor-beta member Nodal fail to form both the mesoderm and the definitive endoderm. They also fail to specify the anterior visceral endoderm, a specialized signaling center which has been shown to be required for the establishment of anterior identity in the epiblast. Our study reveals that Nodal-/- epiblast cells nevertheless express prematurely and ectopically molecular markers specific of anterior fate. Our analysis shows that neural specification occurs and regional identities characteristic of the forebrain are established precociously in the Nodal-/- mutant with a sequential progression equivalent to that of wild-type embryo. When explanted and cultured in vitro, Nodal-/- epiblast cells readily differentiate into neurons. Genes normally transcribed in organizer-derived tissues, such as Gsc and Foxa2, are also expressed in Nodal-/- epiblast. The analysis of Nodal-/-;Gsc-/- compound mutant embryos shows that Gsc activity plays no critical role in the acquisition of forebrain characters by Nodal-deficient cells. This study suggests that the initial steps of neural specification and forebrain development may take place well before gastrulation in the mouse and highlights a possible role for Nodal, at pregastrula stages, in the inhibition of anterior and neural fate determination.     

Differential deployment of paralogous Wnt genes in the mouse and chick embryo during development

Genes encoding Wnt ligands are crucial in body patterning and are highly conserved among metazoans. Given their conservation at the protein‐coding level, it is likely that changes in where and when these genes are active are important in generating evolutionary variations. However, we lack detailed knowledge about how their deployment has diverged. Here, we focus on four Wnt subfamilies (Wnt2, Wnt5, Wnt7, and Wnt8) in mammalian and avian species, consisting of a paralogous gene pair in each, believed to have duplicated in the last common ancestor of vertebrates. We use three‐dimensional imaging to capture expression patterns in detail and carry out systematic comparisons. We find evidence of greater divergence between these subgroup paralogues than the respective orthologues, consistent with some level of subfunctionalization/neofunctionalization in the common vertebrate ancestor that has been conserved. However, there were exceptions; in the case of chick Wnt2b, individual sites were shared with both mouse Wnt2 and Wnt2b. We also find greater divergence, between paralogues and orthologues, in some subfamilies (Wnt2 and Wnt8) compared to others (Wnt5 and Wnt7) with the more highly similar expression patterns showing more extensive expression in more structures in the embryo. Wnt8 genes were most restricted and most divergent. Major sites of expression for all subfamilies include CNS, limbs, and facial region, and in general there were more similarities in gene deployment in these territories with divergent patterns featuring more in organs such as heart and gut. A detailed comparison of gene expression patterns in the limb showed similarities in overall combined domains across species with notable differences that may relate to lineage‐specific morphogenesis.