A role for N-cadherin in mesodermal morphogenesis during gastrulation

Cell adhesion molecules mediate numerous developmental processes necessary for the segregation and organization of tissues. Here we show that the zebrafish biber (bib) mutant encodes a dominant allele at the N-cadherin locus. When knocked down with antisense oligonucleotides, bib mutants phenocopy parachute (pac) null alleles, demonstrating that bib is a gain-of-function mutation. The mutant phenotype disrupts normal cell-cell contacts throughout the mesoderm as well as the ectoderm. During gastrulation stages, cells of the mesodermal germ layer converge slowly; during segmentation stages, the borders between paraxial and axial tissues are irregular and somite borders do not form; later, myotomes are fused. During neurulation, the neural tube is disorganized. Although weaker, all traits present in bib mutants were found in pac mutants. When the distribution of N-cadherin mRNA was analyzed to distinguish mesodermal from neuroectodermal expression, we found that N-cadherin is strongly expressed in the yolk cell and hypoblast in the early gastrula, just preceding the appearance of the bib mesodermal defects. Only later is N-cadherin expressed in the anlage of the CNS, where it is found as a radial gradient in the forming neural plate. Hence, besides a well-established role in neural and somite morphogenesis, N-cadherin is essential for morphogenesis of the mesodermal germ layer during gastrulation.     

A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation

Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF-beta family protein, induces mesodermal/endodermal tissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI (Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal (NLS), and two other NLSs in the C-terminal domain. BENI mRNA was localized to the animal hemisphere at the gastrula stages and to ectoderm except for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects and inhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealed reduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholino oligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animal caps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, and that this regulation is crucial for Xbra expression.     

Distinct functions of Rho and Rac are required for convergent extension during Xenopus gastrulation

We have undertaken the first detailed analysis of Rho GTPase function during vertebrate development by analyzing how RhoA and Rac1 control convergent extension of axial mesoderm during Xenopus gastrulation. Monitoring of a number of parameters in time-lapse recordings of mesoderm explants revealed that Rac and Rho have both distinct and overlapping roles in regulating the motility of axial mesoderm cells. The cell behaviors revealed by activated or inhibitory versions of these GTPases in native tissue were clearly distinct from those previously documented in cultured fibroblasts. The dynamic properties and polarity of protrusive activity, along with lamellipodia formation, were controlled by the two GTPases operating in a partially redundant manner, while Rho and Rac contributed separately to cell shape and filopodia formation. We propose that Rho and Rac operate in distinct signaling pathways that are integrated to control cell motility during convergent extension.     

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.     

An essential role for Fgfs in endodermal pouch formation influences later craniofacial skeletal patterning

Fibroblast growth factor (Fgf) proteins are important regulators of pharyngeal arch development. Analyses of Fgf8 function in chick and mouse and Fgf3 function in zebrafish have demonstrated a role for Fgfs in the differentiation and survival of postmigratory neural crest cells (NCC) that give rise to the pharyngeal skeleton. Here we describe, in zebrafish, an earlier, essential function for Fgf8 and Fgf3 in regulating the segmentation of the pharyngeal endoderm into pouches. Using time-lapse microscopy, we show that pharyngeal pouches form by the directed lateral migration of discrete clusters of endodermal cells. In animals doubly reduced for Fgf8 and Fgf3, the migration of pharyngeal endodermal cells is disorganized and pouches fail to form. Transplantation and pharmacological experiments show that Fgf8 and Fgf3 are required in the neural keel and cranial mesoderm during early somite stages to promote first pouch formation. In addition, we show that animals doubly reduced for Fgf8 and Fgf3 have severe reductions in hyoid cartilages and the more posterior branchial cartilages. By examining early pouch and later cartilage phenotypes in individual animals hypomorphic for Fgf function, we find that alterations in pouch structure correlate with later cartilage defects. We present a model in which Fgf signaling in the mesoderm and segmented hindbrain organizes the segmentation of the pharyngeal endoderm into pouches. Moreover, we argue that the Fgf-dependent morphogenesis of the pharyngeal endoderm into pouches is critical for the later patterning of pharyngeal cartilages.     

The Drosophila trio plays an essential role in patterning of axons by regulating their directional extension

We identified the Drosophila trio gene, which encodes a Dbl family protein carrying two Dbl homology (DH) domains, each of which potentially activates Rho family GTPases. Trio was distributed along axons in the central nervous system (CNS) of embryos and was strongly expressed in subsets of brain regions, including the mushroom body (MB). Loss-of-function trio mutations resulted in the misdirection or stall of axons in embryos and also caused malformation of the MB. The MB phenotypes were attributed to alteration in the intrinsic nature of neurites, as revealed by clonal analyses. Thus, Trio is essential in order for neurites to faithfully extend on the correct pathways. In addition, the localization of Trio in the adult brain suggests its postdevelopmental role in neurite terminals.     

An abundant, highly conserved tonoplast protein in seeds

We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with M_r 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds.     

A kinesin,invA, plays an essential role in volvox morphogenesis

In Volvox carteri adults, reproductive cells called gonidia are enclosed within a spherical monolayer of biflagellate somatic cells. Embryos must "invert" (turn inside out) to achieve this configuration, however, because at the end of cleavage the gonidia are on the outside and the flagellar ends of all somatic cells point inward. Generation of a bend region adequate to turn the embryo inside out involves a dramatic change in cell shape, plus cell movements. Here, we cloned a gene called invA that is essential for inversion and found that it codes for a kinesin localized in the cytoplasmic bridges that link all cells to their neighbors. In invA null mutants, cells change shape normally, but are unable to move relative to the cytoplasmic bridges. A normal bend region cannot be formed and inversion stops. We conclude that the InvA kinesin provides the motile force that normally drives inversion to completion.     

Essential role for beta-arrestin 2 in the regulation of Xenopus convergent extension movements

beta-Arrestin 2 (betaarr2) is a multifunctional protein that regulates numerous aspects of G-protein-coupled receptor function. However, its possible involvement in developmental processes is poorly understood. In this work, we examined the potential role of betaarr2 during Xenopus early development. Gain- and loss-of-function studies showed that Xenopus betaarr2 (xbetaarr2) is required for proper convergent extension (CE) movements, and normal cell polarization and intercalation without affecting cell fate. Moreover, for CE movements, betaarr2 acts as an essential regulator of dishevelled-mediated PCP (planar cell polarity) signaling, but not G-protein-mediated Ca(2+) signaling. Notably, xbetaarr2 is localized with the same distribution as the dishevelled protein, which is reasonable, as xbetaarr2 is required for dishevelled activation of RhoA. Furthermore, xbetaarr2 interacts with the N-terminal quarter of Daam1 and RhoA proteins, but not Rac1, and regulates RhoA activation through Daam1 activation for CE movements. We provide evidence that the endocytic activity of xbetaarr2 is essential for control of CE movements. Taken together, our results suggest that betaarr2 has a pivotal role in the regulation of Xenopus CE movements.     

Six3 inactivation reveals its essential role for the formation and patterning of the vertebrate eye

The establishment of retinal identity and the subsequent patterning of the optic vesicle are the key steps in early vertebrate eye development. To date little is known about the nature and interaction of the genes controlling these steps. So far few genes have been identified that, when over-expressed, can initiate ectopic eye formation. Of note is Six3, which is expressed exclusively in the anterior neural plate. However, 'loss of function' analysis has not been reported. Using medaka fish, we show that vertebrate Six3 is necessary for patterning of the anterior neuroectoderm including the retina anlage. Inactivation of Six3 function by morpholino knock-down results in the lack of forebrain and eyes. Corroborated by gain-of-function experiments, graded interference reveals an additional role of Six3 in the proximodistal patterning of the optic vesicle. During both processes of vertebrate eye formation, Six3 cooperates with Pax6.     

Syndecan-4 regulates non-canonical Wnt signalling and is essential for convergent and extension movements in Xenopus embryos

Early shaping of Xenopus laevis embryos occurs through convergent and extension movements, a process that is driven by intercalation of polarized dorsal mesodermal cells and regulated by non-canonical Wnt signalling. Here, we have identified Xenopus syndecan-4 (xSyn4), a cell-surface transmembrane heparan sulphate proteoglycan. At the gastrula stage, xSyn4 is expressed in the involuting dorsal mesoderm and the anterior neuroectoderm. Later, it is found in the pronephros, branchial arches, brain and tailbud. Both gain- and loss-of-function of xSyn4 impaired convergent extension movements in Xenopus embryos and in activin-treated ectodermal explants. xSyn4 interacts functionally and biochemically with the Wnt receptor Frizzled7 (xFz7) and its signal transducer Dishevelled (xDsh). Furthermore, xSyn4 is necessary and sufficient for translocation of xDsh to the plasma membrane - a landmark in the activation of non-canonical Wnt signalling. Our results suggest that the ability of xSyn4 to translocate xDsh is regulated by fibronectin, a component of the extracellular matrix required for proper convergent extension movements. We propose a model where xSyn4 and fibronectin cooperate with xFz7 and Wnt in the specific activation of the non-canonical Wnt pathway.     

A positive role for Short gastrulation in modulating BMP signaling during dorsoventral patterning in the Drosophila embryo

Positional information in the dorsoventral axis of the Drosophila embryo is encoded by a BMP activity gradient formed by synergistic signaling between the BMP family members Decapentaplegic (DPP) and Screw (SCW). short gastrulation (sog), which is functionally homologous to Xenopus Chordin, is expressed in the ventrolateral regions of the embryo and has been shown to act as a local antagonist of BMP signaling. Here we demonstrate that SOG has a second function, which is to promote BMP signaling on the dorsal side of the embryo. We show that a weak, homozygous-viable sog mutant is enhanced to lethality by reduction in the activities of the Smad family members Mad or Medea, and that the lethality is caused by defects in the molecular specification and subsequent cellular differentiation of the dorsal-most cell type, the amnioserosa. While previous data had suggested that the negative function of SOG is directed against SCW, we present data that suggests that the positive activity of SOG is directed towards DPP. We demonstrate that Chordin shares the same apparent ligand specificity as does SOG, preferentially inhibiting SCW but not DPP activity. However, in Drosophila assays Chordin does not have the same capacity to elevate BMP signaling as does SOG, identifying a functional difference in the otherwise well conserved process of dorsoventral pattern formation in arthropods and chordates.     

ALK7, a receptor for nodal, is dispensable for embryogenesis and left-right patterning in the mouse

Mesendoderm formation and left-right patterning during vertebrate development depend upon selected members of the transforming growth factor beta superfamily, particularly Nodal and Nodal-related ligands. Two type I serine/threonine kinase receptors have been identified for Nodal, ALK4 and ALK7. Mouse embryos lacking ALK4 fail to produce mesendoderm and die shortly after gastrulation, resembling the phenotype of Nodal knockout mice. Whether ALK4 contributes to left-right patterning is still unknown. Here we report the generation and initial characterization of mice lacking ALK7. Homozygous mutant mice were born at the expected frequency and remained viable and fertile. Viability at weaning was not different from that of the wild type in ALK7(-/-); Nodal(+/-) and ALK7(-/-); ALK4(+/-) compound mutants. ALK7 and ALK4 were highly expressed in interdigital regions of the developing limb bud. However, ALK7 mutant mice displayed no skeletal abnormalities or limb malformations. None of the left-right patterning abnormalities and organogenesis defects identified in mice carrying mutations in Nodal or in genes encoding ActRIIA and ActRIIB coreceptors, including heart malformations, pulmonary isomerism, right-sided gut, and spleen hypoplasia, were observed in mice lacking ALK7. Finally, the histological organization of the cerebellum, cortex, and hippocampus, all sites of significant ALK7 expression in the rodent brain, appeared normal in ALK7 mutant mice. We conclude that ALK7 is not an essential mediator of Nodal signaling during mesendoderm formation and left-right patterning in the mouse but may instead mediate other activities of Nodal and related ligands in the development or function of particular tissues and organs.     

An essential role for DNA methyltransferase 3a in melanoma tumorigenesis

Abnormal DNA methylation and associated silencing of tumor suppressor genes are common to many types of cancers. Among the three coordinate DNA methyltransferases (Dnmts), Dnmt1 and Dnmt3b were both shown to be important for cancer cell survival and tumorigenesis. However, the relationship between Dnmt3a and tumorigenesis is still largely unknown. Here, we show that inhibition of Dnmt3a expression, by stable transfection of a Dnmt3a-RNA interference (RNAi) construct dramatically inhibited melanoma growth and metastasis in mouse melanoma models. Microarray analysis revealed that genes critical for the tumor immune response, were implicated in the inhibition of melanoma growth. Expression of a cluster of class I and class II MHC genes, class II transactivator (Ciita), as well as a subset of 5 chemokines (Cxcl9, Cxcl16, Ccl12, Ccl4, and Ccl2) were up-regulated. Furthermore, we determined that the promoter IV of Ciita was significantly demethylated in Dnmt3a-depleted tumors. In addition, several known tumor-related genes, which are critical for developmental processes and cell cycle, were confirmed to be misregulated, including TgfB1, Socs1, Socs2, E2F6, Ccne1, and Cyr61. The results presented in this report strongly suggest that Dnmt3a plays an essential role in melanoma tumorigenesis, and that the underlying mechanisms include the modulation of the tumor immune response, as well as other processes.