RESUMO
Alternative splicing of pre-mRNAs is an important means of regulating developmental processes, yet the molecular mechanisms governing alternative splicing in embryonic contexts are just beginning to emerge. Polyglutamine-binding protein 1 (PQBP1) is an RNA-splicing factor that, when mutated, in humans causes Renpenning syndrome, an X-linked intellectual disability disease characterized by severe cognitive impairment, but also by physical defects that suggest PQBP1 has broader functions in embryonic development. Here, we reveal essential roles for PQBP1 and a binding partner, WBP11, in early development of Xenopus embryos. Both genes are expressed in the nascent mesoderm and neurectoderm, and morpholino knockdown of either causes defects in differentiation and morphogenesis of the mesoderm and neural plate. At the molecular level, knockdown of PQBP1 in Xenopus animal cap explants inhibits target gene induction by FGF but not by BMP, Nodal or Wnt ligands, and knockdown of either PQBP1 or WBP11 in embryos inhibits expression of fgf4 and FGF4-responsive cdx4 genes. Furthermore, PQBP1 knockdown changes the alternative splicing of FGF receptor-2 (FGFR2) transcripts, altering the incorporation of cassette exons that generate receptor variants (FGFR2 IIIb or IIIc) with different ligand specificities. Our findings may inform studies into the mechanisms underlying Renpenning syndrome.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesoderma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Imunoprecipitação , Hibridização In Situ , Morfolinos/genética , Proteínas do Tecido Nervoso/genética , Plasmídeos/genética , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas de Xenopus/genéticaRESUMO
BACKGROUND: Canonical Wnt signals, transduced by stabilized ß-catenin, play similar roles across animals in maintaining stem cell pluripotency, regulating cell differentiation, and instructing normal embryonic development. Dysregulated Wnt/ß-catenin signaling causes diseases and birth defects, and a variety of regulatory processes control this pathway to ensure its proper function and integration with other signaling systems. We previously identified GTP-binding protein 2 (Gtpbp2) as a novel regulator of BMP signaling, however further exploration revealed that Gtpbp2 can also affect Wnt signaling, which is a novel finding reported here. RESULTS: Knockdown of Gtpbp2 in Xenopus embryos causes severe axial defects and reduces expression of Spemann-Mangold organizer genes. Gtpbp2 knockdown blocks responses to ectopic Wnt8 ligand, such as organizer gene induction in ectodermal tissue explants and induction of secondary axes in whole embryos. However, organizer gene induction by ectopic Nodal2 is unaffected by Gtpbp2 knockdown. Epistasis tests, conducted by activating Wnt signal transduction at sequential points in the canonical pathway, demonstrate that Gtpbp2 is required downstream of Dishevelled and Gsk3ß but upstream of ß-catenin, which is similar to the previously reported effects of Axin1 overexpression in Xenopus embryos. Focusing on Axin in Xenopus embryos, we find that knockdown of Gtpbp2 elevates endogenous or exogenous Axin protein levels. Furthermore, Gtpbp2 fusion proteins co-localize with Dishevelled and co-immunoprecipitate with Axin and Gsk3b. CONCLUSIONS: We conclude that Gtpbp2 is required for canonical Wnt/ß-catenin signaling in Xenopus embryos. Our data suggest a model in which Gtpbp2 suppresses the accumulation of Axin protein, a rate-limiting component of the ß-catenin destruction complex, such that Axin protein levels negatively correlate with Gtpbp2 levels. This model is supported by the similarity of our Gtpbp2-Wnt epistasis results and previously reported effects of Axin overexpression, the physical interactions of Gtpbp2 with Axin, and the correlation between elevated Axin protein levels and lost Wnt responsiveness upon Gtpbp2 knockdown. A wide variety of cancer-causing Wnt pathway mutations require low Axin levels, so development of Gtpbp2 inhibitors may provide a new therapeutic strategy to elevate Axin and suppress aberrant ß-catenin signaling in cancer and other Wnt-related diseases.
Assuntos
Proteína Axina/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteína Axina/genética , Proteínas Desgrenhadas/metabolismo , Epistasia Genética , GTP Fosfo-Hidrolases/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Ligação Proteica , Via de Sinalização Wnt , Xenopus , Proteínas de Xenopus/genéticaRESUMO
Smad proteins convey canonical intracellular signals for activated receptors in the TGFß superfamily, but the activity of Smads and their impact on target genes are further regulated by a wide variety of cofactors and partner proteins. We have identified a new Smad1 partner, a GTPase named Gtpbp2 that is a distant relative of the translation factor eEf1a. Gtpbp2 affects canonical signaling in the BMP branch of the TGFß superfamily, as morpholino knockdown of Gtpbp2 decreases, and overexpression of Gtpbp2 enhances, animal cap responses to BMP4. During Xenopus development, gtpbp2 transcripts are maternally expressed and localized to the egg animal pole, and partitioned into the nascent ectodermal and mesodermal cells during cleavage and early gastrulation stages. Subsequently, gtpbp2 is expressed in the neural folds, and in early tadpoles undergoing organogenesis gtpbp2 is expressed prominently in the brain, eyes, somites, ventral blood island and branchial arches. Consistent with its expression, morpholino knockdown of Gtpbp2 causes defects in ventral-posterior germ layer patterning, gastrulation and tadpole morphology. Overexpressed Gtpbp2 can induce ventral-posterior marker genes and localize to cell nuclei in Xenopus animal caps, highlighting its role in regulating BMP signaling in the early embryo. Here, we introduce this large GTPase as a novel factor in BMP signaling and ventral-posterior patterning.
Assuntos
Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Transdução de Sinais/genética , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Animais , Western Blotting , DNA Complementar/genética , GTP Fosfo-Hidrolases/genética , Técnicas de Silenciamento de Genes , Imunoprecipitação , Hibridização In Situ , Luciferases , Microscopia de Fluorescência , Morfolinos/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Proteína Smad1/metabolismo , Proteínas de Xenopus/genéticaRESUMO
BACKGROUND: As the sea anemone Nematostella vectensis emerges as a model for studying regeneration, new tools will be needed to assess its regenerative processes and describe perturbations resulting from experimental investigation. Chief among these is the need for a universal set of staging criteria to establish morphological landmarks that will provide a common format for discussion among investigators. RESULTS: We have established morphological criteria to describe stages for rapidly assessing regeneration of the aboral end (physa) of Nematostella. Using this staging system, we observed rates of regeneration that are temperature independent during wound healing and temperature dependent afterward. Treatment with 25 µM lipoic acid delays the progression through wound healing without significantly affecting the subsequent rate of regeneration. Also, while an 11-day starvation before amputation causes only a minimal delay in regeneration, this delay is exacerbated by lipoic acid treatment. CONCLUSIONS: A system for staging the progression of regeneration in amputated Nematostella physa based on easily discernible morphological features provides a standard for the field. This system has allowed us to identify both temperature-dependent and -independent phases of regeneration, as well as a nutritional requirement for normal regenerative progression that is exacerbated by lipoic acid.
Assuntos
Regeneração/fisiologia , Anêmonas-do-Mar/embriologia , Anêmonas-do-Mar/fisiologia , AnimaisRESUMO
We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Xenopus/embriologia , Xenopus/genética , Animais , Encéfalo/metabolismo , Embrião não Mamífero/metabolismo , Olho/metabolismo , Proteínas do Olho/metabolismo , Loci Gênicos , Genoma , Células Germinativas/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteína Homeobox SIX3RESUMO
T-box family transcription factors play many roles in Metazoan development. Here we characterise Tbx6r, a unique Tbx6 paralogue isolated from the amphibian Xenopus. The evolution and developmental integration of this divergent T-box gene within the vertebrates reveals an unexpected level of plasticity within this conserved family of developmental regulators. We show that despite their co-expression, Tbx6 and Tbx6r have dissimilar transcriptional responses to ligand treatment, and their ability to activate ligand expression is also very different. The two paralogues have distinct inductive properties: Tbx6 induces mesoderm whereas Tbx6r induces anterior neural markers. We use hybrid proteins in an effort to understand this difference, and implicate the C-terminal regions of the proteins in their inductive specificities. Through loss-of-function analyses using antisense morpholino oligonucleotides we show that both Tbx6 paralogues perform essential functions in the development of the paraxial and intermediate mesoderm and the neural crest in Xenopus. We demonstrate that Tbx6 and Tbx6r both induce FGF8 expression as well as that of pre-placodal markers, and that Tbx6 can also induce neural crest markers via a ligand-dependent mechanism involving FGF8 and Wnt8. Our data thus identify an important new function for this key developmental regulator.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Crista Neural/metabolismo , Proteínas com Domínio T/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Padronização Corporal , Embrião não Mamífero/metabolismo , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Ligantes , Mesoderma/citologia , Dados de Sequência Molecular , Filogenia , Proteínas com Domínio T/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMO
Hedgehog signaling is an important component of cell-cell communication during bilaterian development, and abnormal Hedgehog signaling contributes to disease and birth defects. Hedgehog genes are composed of a ligand ("hedge") domain and an autocatalytic intein ("hog") domain. Hedgehog (hh) ligands bind to a conserved set of receptors and activate downstream signal transduction pathways terminating with Gli/Ci transcription factors. We have identified five intein-containing genes in the anthozoan cnidarian Nematostella vectensis, two of which (NvHh1 and NvHh2) contain definitive hedgehog ligand domains, suggesting that to date, cnidarians are the earliest branching metazoan phylum to possess definitive Hh orthologs. Expression analysis of NvHh1 and NvHh2, the receptor NvPatched, and a downstream transcription factor NvGli (a Gli3/Ci ortholog) indicate that these genes may have conserved roles in planar and trans-epithelial signaling during gut and germline development, while the three remaining intein-containing genes (NvHint1,2,3) are expressed in a cell-type-specific manner in putative neural precursors. Metazoan intein-containing genes that lack a hh ligand domain have previously only been identified within nematodes. However, we have identified intein-containing genes from both Nematostella and in two newly annotated lophotrochozoan genomes. Phylogenetic analyses suggest that while nematode inteins may be derived from an ancestral true hedgehog gene, the newly identified cnidarian and lophotrochozoan inteins may be orthologous, suggesting that both true hedgehog and hint genes may have been present in the cnidarian-bilaterian ancestor. Genomic surveys of N. vectensis suggest that most of the components of both protostome and deuterostome Hh signaling pathways are present in anthozoans and that some appear to have been lost in ecdysozoan lineages. Cnidarians possess many bilaterian cell-cell signaling pathways (Wnt, TGFbeta, FGF, and Hh) that appear to act in concert to pattern tissues along the oral-aboral axis of the polyp. Cnidarians represent a diverse group of animals with a predominantly epithelial body plan, and perhaps selective pressures to pattern epithelia resulted in the ontogeny of the hedgehog pathway in the common ancestor of the Cnidaria and Bilateria.
Assuntos
Cnidários/embriologia , Cnidários/genética , Evolução Molecular , Proteínas Hedgehog/genética , Transdução de Sinais , Animais , Teorema de Bayes , DNA Complementar , Embrião não Mamífero , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Hibridização In Situ , Inteínas/genética , Íntrons , Ligantes , Funções Verossimilhança , Modelos Biológicos , Técnicas de Amplificação de Ácido Nucleico , Filogenia , Estrutura Terciária de ProteínaRESUMO
Cnidarians (corals, sea anemones, hydroids, and jellyfish) are a basal taxon closely related to bilaterally symmetrical animals and have been characterized as diploblastic and as radially symmetrical around their longitudinal axis. We show that some orthologs of key bilaterian dorso/ventral (D/V) patterning genes, including the TGFbeta signaling molecules NvDpp and NvBMP5-8 and their antagonist NvChordin, are initially expressed asymmetrically at the onset of gastrulation in the anthozoan sea anemone Nematostella vectensis. Surprisingly, unlike flies and vertebrates, the TGFbeta ligands and their antagonist are colocalized at the onset of gastrulation but then segregate by germ layer as gastrulation proceeds. TGFbeta ligands, their extracellular enhancer, NvTolloid, and components of their downstream signaling pathway (NvSmad1/5 and NvSmad4) are all coexpressed in presumptive endoderm, indicating that only planar TGFbeta signaling operates at these stages. NvChordin expression forms a boundary between TGFbeta-expressing endodermal cells and aboral ectoderm. Manipulation of nuclear beta-catenin localization affects TGFbeta ligand and antagonist expression, suggesting that the ancestral role of the dpp/chordin antagonism during gastrulation may have been in germ-layer segregation and/or epithelial patterning rather than dorsal/ventral patterning.
Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Anêmonas-do-Mar/embriologia , Anêmonas-do-Mar/genética , Animais , Evolução Molecular , Perfilação da Expressão Gênica , Ligantes , Modelos Genéticos , Dados de Sequência Molecular , Proteínas/genética , Proteínas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Anêmonas-do-Mar/metabolismo , Transdução de SinaisRESUMO
Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both "classic" regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.
Assuntos
Regeneração , Anêmonas-do-Mar/crescimento & desenvolvimento , AnimaisRESUMO
BACKGROUND: Activity of the Transforming growth factor-ß (TGFß) pathway is essential to the establishment of body axes and tissue differentiation in bilaterians. Orthologs for core pathway members have been found in all metazoans, but uncertain homology of the body axes and tissues patterned by these signals raises questions about the activities of these molecules across the metazoan tree. We focus on the principal canonical transduction proteins (R-Smads) of the TGFß pathway, which instruct both axial patterning and tissue differentiation in the developing embryo. We compare the activity of R-Smads from a cnidarian (Nematostella vectensis), an arthropod (Drosophila melanogaster), and a vertebrate (Xenopus laevis) in Xenopus embryonic assays. RESULTS: Overexpressing NvSmad1/5 ventralized Xenopus embryos when expressed in dorsal blastomeres, similar to the effects of Xenopus Smad1. However, NvSmad1/5 was less potent than XSmad1 in its ability to activate downstream target genes in Xenopus animal cap assays. NvSmad2/3 strongly induced general mesendodermal marker genes, but weakly induced ones involved in specifying the Spemann organizer. NvSmad2/3 was unable to induce a secondary trunk axis in Xenopus embryos, whereas the orthologs from Xenopus (XSmad2 and XSmad3) and Drosophila (dSmad2) were capable of doing so. Replacement of the NvSmad2/3 MH2 domain with the Xenopus XSmad2 MH2 slightly increased its inductive capability, but did not confer an ability to generate a secondary body axis. CONCLUSIONS: Vertebrate and cnidarian Smad1/5 have similar axial patterning and induction activities, although NvSmad1/5 is less efficient than the vertebrate gene. We conclude that the activities of Smad1/5 orthologs have been largely conserved across Metazoa. NvSmad2/3 efficiently activates general mesendoderm markers, but is unable to induce vertebrate organizer-specific genes or to produce a secondary body axis in Xenopus. Orthologs dSmad2 and XSmad3 generate a secondary body axis, but activate only low expression of organizer-specific genes that are strongly induced by XSmad2. We suggest that in the vertebrate lineage, Smad2 has evolved a specialized role in the induction of the embryonic organizer. Given the high level of sequence identity between Smad orthologs, this work underscores the functional importance of the emergence and fixation of a few divergent amino acids among orthologs during evolution.
RESUMO
Mustn1 is a vertebrate-specific protein that, in vitro, was showed to be essential for prechondrocyte function and thus it has the potential to regulate chondrogenesis during embryonic development. We use Xenopus laevis as a model to examine Mustn1 involvement in chondrogenesis. Previous work suggests that Mustn1 is necessary but not sufficient for chondrogenic proliferation and differentiation, as well as myogenic differentiation in vitro. Mustn1 was quantified and localized in developing Xenopus embryos using RT-PCR and whole mount in situ hybridization. Xenopus embryos were injected with either control morpholinos (Co-MO) or one designed against Mustn1 (Mustn1-MO) at the four cell stage. Embryos were scored for morphological defects and Sox9 was visualized via in situ hybridization. Finally, Mustn1-MO-injected embryos were co-injected with Mustn1-MO resistant mRNA to confirm the specificity of the observed phenotype. Mustn1 is expressed from the mid-neurula stage to the swimming tadpole stages, predominantly in anterior structures including the pharyngeal arches and associated craniofacial tissues, and the developing somites. Targeted knockdown of Mustn1 in craniofacial and dorsal axial tissues resulted in phenotypes characterized by small or absent eye(s), a shortened body axis, and tail kinks. Further, Mustn1 knockdown reduced cranial Sox9 mRNA expression and resulted in the loss of differentiated cartilaginous head structures (e.g. ceratohyal and pharyngeal arches). Reintroduction of MO-resistant Mustn1 mRNA rescued these effects. We conclude that Mustn1 is necessary for normal craniofacial cartilage development in vivo, although the exact molecular mechanism remains unknown.
Assuntos
Condrogênese/genética , Proteínas Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Região Branquial/embriologia , Região Branquial/metabolismo , Diferenciação Celular/genética , Regulação para Baixo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Dados de Sequência Molecular , Proteínas Nucleares/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteínas de Xenopus/genéticaRESUMO
Transforming growth factor ß superfamily members signal through Smad transcription factors. Bone morphogenetic proteins (BMPs) act via Smads 1, 5 and 8 and TGF-ßs signal through Smads 2 and 3. The endocytic adaptor protein Eps15R, or 'epidermal growth factor (EGF) receptor pathway substrate 15-related protein' is a component of EGF signal transduction, mediating internalization of the EGF receptor. We show that it interacts with Smad proteins, is required for BMP signalling in animal caps and stimulates Smad1 transcriptional activity. This function resides in the Asp-Pro-Phe motif-enriched 'DPF domain' of Eps15R, which activates transcription and antagonizes Smad2 signalling. In living cells, Eps15R segregates into spatially distinct regions with different Smads, indicating an unrecognized level of Smad compartmentalization.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Receptores ErbB/metabolismo , Proteínas Smad/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Geneticamente Modificados , Compartimento Celular , Regulação da Expressão Gênica no Desenvolvimento , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Proteína Smad1/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Xenopus laevis/genéticaRESUMO
The transforming growth factor (TGF)-beta superfamily regulates cell proliferation, apoptosis, differentiation, migration, and development. Canonical TGFbeta signals are transduced to the nucleus via Smads in both major signaling branches, bone morphogenetic protein (BMP) or Activin/Nodal/TGFbeta. Smurf ubiquitin (Ub) ligases attenuate these pathways by targeting Smads and other signaling components for degradation by the 26S proteasome. Here, we identify tumor necrosis factor (TNF)-receptor-associated factor-4 (TRAF4) as a new target of Smurf1, which polyubiquitylates TRAF4 to trigger its proteasomal destruction. Unlike other TRAF family members, which mediate signal transduction by TNF, interleukin, or Toll-like receptors, we find that TRAF4 potentiates BMP and Nodal signaling. In the frog Xenopus laevis, TRAF4 mRNA is stored maternally in the egg animal pole, and in the embryo it is expressed in the gastrula marginal zone, neural plate, and cranial and trunk neural crest. Knockdown of embryonic TRAF4 impairs signaling, neural crest development and neural folding, whereas TRAF4 overexpression boosts signaling and expands the neural crest. In human embryonic kidney 293 cells, small interfering RNA knockdown of Smurf1 elevates TRAF4 levels, indicating endogenous regulation of TRAF4 by Smurf1. Our results uncover new functions for TRAF4 as a Smurf1-regulated mediator of BMP and Nodal signaling that are essential for neural crest development and neural plate morphogenesis.
Assuntos
Crista Neural/embriologia , Crista Neural/metabolismo , Transdução de Sinais , Fator 4 Associado a Receptor de TNF/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Xenopus/embriologia , Sequência de Aminoácidos , Animais , Padronização Corporal/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Dados de Sequência Molecular , Crista Neural/efeitos dos fármacos , Placa Neural/efeitos dos fármacos , Placa Neural/embriologia , Placa Neural/metabolismo , Tubo Neural/efeitos dos fármacos , Tubo Neural/metabolismo , Tubo Neural/patologia , Proteína Nodal/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Transporte Proteico/efeitos dos fármacos , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Fator 4 Associado a Receptor de TNF/química , Fator 4 Associado a Receptor de TNF/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Wnt/metabolismo , Xenopus/genéticaRESUMO
Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase-anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Repressoras/metabolismo , Fuso Acromático/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Anáfase , Catálise , Linhagem Celular , Centrossomo/enzimologia , Posicionamento Cromossômico , Segregação de Cromossomos , Citocinese , Ativação Enzimática , Inativação Gênica , Humanos , Proteínas Mad2 , Metáfase , Modelos Biológicos , Transporte Proteico , TermodinâmicaRESUMO
The fibroblast growth factor (FGF) signal transduction pathway serves as one of the key regulators of early metazoan development, displaying conserved roles in the specification of endodermal, mesodermal, and neural fates during vertebrate development. FGF signals also regulate gastrulation, in part, by triggering epithelial to mesenchymal transitions in embryos of both vertebrates and invertebrates. Thus, FGF signals coordinate gastrulation movements across many different phyla. To help understand the breadth of FGF signaling deployment across the animal kingdom, we have examined the presence and expression of genes encoding FGF pathway components in the anthozoan cnidarian Nematostella vectensis. We isolated three FGF ligands (NvFGF8A, NvFGF8B, and NvFGF1A), two FGF receptors (NvFGFRa and NvFGFRb), and two orthologs of vertebrate FGF responsive genes, Sprouty (NvSprouty), an inhibitor of FGF signaling, and Churchill (NvChurchill), a Zn finger transcription factor. We found these FGF ligands, receptors, and response gene expressed asymmetrically along the oral/aboral axis during gastrulation and in a developing chemosensory structure of planula stages known as the apical tuft. These results suggest a conserved role for FGF signaling molecules in coordinating both gastrulation and neural induction that predates the Cambrian explosion and the origins of the Bilateria.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Anêmonas-do-Mar/embriologia , Anêmonas-do-Mar/fisiologia , Animais , Indução Embrionária , Evolução Molecular , Fatores de Crescimento de Fibroblastos/genética , Gástrula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Ligação Genética , Ligantes , Sistema Nervoso/embriologia , Filogenia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Anêmonas-do-Mar/genética , Transdução de SinaisRESUMO
The ubiquitin ligase Smurf1 can target a handful of signaling proteins for ubiquitin-mediated proteasomal destruction or functional modification, including TGF-beta receptors, Smads, transcription factors, RhoA and MEKK2. Smurf1 was initially implicated in BMP pathway regulation in embryonic development, but its potential role in vertebrate embryogenesis has yet to be clarified. Here we demonstrate that inhibition of Smurf1 in Xenopus laevis embryos with an antisense morpholino oligonucleotide or a dominant-negative protein disrupts early development, with the nervous system being the principal target. Smurf1 is enriched on the dorsal side of gastrula stage embryos, and blocking Smurf1 disturbs neural folding and neural, but not mesoderm differentiation, enhances BMP/Smad1 signaling, and elevates phospho-Smad1 levels in the dorsal ectoderm. We conclude that in Xenopus embryos, the BMP pathway is a major physiological target of Smurf1, and we propose that in normal development Smurf1 cooperates with secreted BMP antagonists to limit BMP signaling in dorsal ectoderm. Our data also reveal a novel role for Smurf1 and Smad1 in neural plate morphogenesis.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Gástrula/metabolismo , Sistema Nervoso/embriologia , Proteína Smad1/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Xenopus laevis/embriologia , Animais , Sequência de Bases , Regulação da Expressão Gênica no Desenvolvimento , Sistema Nervoso/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Xenopus laevis/metabolismoRESUMO
Nearly all metazoans show signs of bilaterality, yet it is believed the bilaterians arose from radially symmetric forms hundreds of millions of years ago. Cnidarians (corals, sea anemones, and "jellyfish") diverged from other animals before the radiation of the Bilateria. They are diploblastic and are often characterized as being radially symmetrical around their longitudinal (oral-aboral) axis. We have studied the deployment of orthologs of a number of family members of developmental regulatory genes that are expressed asymmetrically during bilaterian embryogenesis from the sea anemone, Nematostella vectensis. The secreted TGF-beta genes Nv-dpp, Nv-BMP5-8, six TGF-beta antagonists (NvChordin, NvNoggin1, NvNoggin2, NvGremlin, NvFollistatin, and NvFollistatin-like), the homeodomain proteins NvGoosecoid (NvGsc) and NvGbx, and the secreted guidance factor, NvNetrin, were studied. NvDpp, NvChordin, NvNoggin1, NvGsc, and NvNetrin are expressed asymmetrically along the axis perpendicular to the oral-aboral axis, the directive axis. Furthermore, NvGbx, and NvChordin are expressed in restricted domains on the left and right sides of the body, suggesting that the directive axis is homologous with the bilaterian dorsal-ventral axis. The asymmetric expression of NvNoggin1 and NvGsc appear to be maintained by the canonical Wnt signaling pathway. The asymmetric expression of NvNoggin1, NvNetrin, and Hox orthologs NvAnthox7, NvAnthox8, NvAnthox1a, and NvAnthox6, in conjunction with the observation that NvNoggin1 is able to induce a secondary axis in Xenopus embryos argues that N. vectensis could possess antecedents of the organization of the bilaterian central nervous system.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Anêmonas-do-Mar/genética , Animais , Padronização Corporal , Sistema Nervoso Central/embriologia , Evolução Molecular , Genoma , Hibridização In Situ , Cloreto de Lítio/farmacologia , Modelos Anatômicos , Modelos Biológicos , Dados de Sequência Molecular , Filogenia , Anêmonas-do-Mar/embriologia , XenopusRESUMO
ARID domain proteins are members of a highly conserved family involved in chromatin remodeling and cell-fate determination. Dril1 is the founding member of the ARID family and is involved in developmental processes in both Drosophila and Caenorhabditis elegans. We describe the first embryological characterization of this gene in chordates. Dril1 mRNA expression is spatiotemporally regulated and is detected in the involuting mesoderm during gastrulation. Inhibition of dril1 by either a morpholino or an engrailed repressor-dril1 DNA binding domain fusion construct inhibits gastrulation and perturbs induction of the zygotic mesodermal marker Xbra and the organizer markers chordin, noggin, and Xlim1. Xenopus tropicalis dril1 morphants also exhibit impaired gastrulation and axial deficiencies, which can be rescued by coinjection of Xenopus laevis dril1 mRNA. Loss of dril1 inhibits the response of animal caps to activin and secondary axis induction by smad2. Dril1 depletion in animal caps prevents both the smad2-mediated induction of dorsal mesodermal and endodermal markers and the induction of ventral mesoderm by smad1. Mesoderm induction by eFGF is uninhibited in dril1 morphant caps, reflecting pathway specificity for dril1. These experiments identify dril1 as a novel regulator of TGF(beta) signaling and a vital component of mesodermal patterning and embryonic morphogenesis.
Assuntos
Drosophila/embriologia , Drosophila/genética , Embrião não Mamífero/fisiologia , Mesoderma/fisiologia , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/fisiologia , Proteínas de Xenopus/genética , Animais , Sequência de Bases , Padronização Corporal/genética , Primers do DNA , Proteínas de Ligação a DNA , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Morfogênese/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
The Rho family of small guanosine triphosphatases regulates actin cytoskeleton dynamics that underlie cellular functions such as cell shape changes, migration, and polarity. We found that Smurf1, a HECT domain E3 ubiquitin ligase, regulated cell polarity and protrusive activity and was required to maintain the transformed morphology and motility of a tumor cell. Atypical protein kinase C zeta (PKCzeta), an effector of the Cdc42/Rac1-PAR6 polarity complex, recruited Smurf1 to cellular protrusions, where it controlled the local level of RhoA. Smurf1 thus links the polarity complex to degradation of RhoA in lamellipodia and filopodia to prevent RhoA signaling during dynamic membrane movements.