RESUMO
During animal gastrulation, the specification of the embryonic axes is accompanied by epithelio-mesenchymal transition (EMT), the first major change in cell shape after fertilization. EMT takes place in disparate topographical arrangements, such as the circular blastopore of amphibians, the straight primitive streak of birds and mammals or in intermediate gastrulation forms of other amniotes such as reptiles. Planar cell movements are prime candidates to arrange specific modes of gastrulation but there is no consensus view on their role in different vertebrate classes. Here, we test the impact of interfering with Rho kinase-mediated cell movements on gastrulation topography in blastocysts of the rabbit, which has a flat embryonic disc typical for most mammals. Time-lapse video microscopy, electron microscopy, gene expression and morphometric analyses of the effect of inhibiting ROCK activity showed - besides normal specification of the organizer region - a dose-dependent disruption of primitive streak formation; this disruption resulted in circular, arc-shaped or intermediate forms, reminiscent of those found in amphibians, fishes and reptiles. Our results reveal a crucial role of ROCK-controlled directional cell movements during rabbit primitive streak formation and highlight the possibility that temporal and spatial modulation of cell movements were instrumental for the evolution of gastrulation forms.
Assuntos
Movimento Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/enzimologia , Linha Primitiva/citologia , Linha Primitiva/embriologia , Quinases Associadas a rho/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Evolução Biológica , Padronização Corporal/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Movimento Celular/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Gastrulação/efeitos dos fármacos , Organizadores Embrionários/citologia , Organizadores Embrionários/efeitos dos fármacos , Linha Primitiva/efeitos dos fármacos , Linha Primitiva/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Coelhos , Tiazolidinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
How embryos consistently orient asymmetries of the left-right (LR) axis is an intriguing question, as no macroscopic environmental cues reliably distinguish left from right. Especially unclear are the events coordinating LR patterning with the establishment of the dorsoventral (DV) axes and midline determination in early embryos. In frog embryos, consistent physiological and molecular asymmetries manifest by the second cell cleavage; however, models based on extracellular fluid flow at the node predict correct de novo asymmetry orientation during neurulation. We addressed these issues in Xenopus embryos by manipulating the timing and location of dorsal organizer induction: the primary dorsal organizer was ablated by UV irradiation, and a new organizer was induced at various locations, either early, by mechanical rotation, or late, by injection of lithium chloride (at 32 cells) or of the transcription factor XSiamois (which functions after mid-blastula transition). These embryos were then analyzed for the position of three asymmetric organs. Whereas organizers rescued before cleavage properly oriented the LR axis 90% of the time, organizers induced in any position at any time after the 32-cell stage exhibited randomized laterality. Late organizers were unable to correctly orient the LR axis even when placed back in their endogenous location. Strikingly, conjoined twins produced by late induction of ectopic organizers did have normal asymmetry. These data reveal that although correct LR orientation must occur no later than early cleavage stages in singleton embryos, a novel instructive influence from an early organizer can impose normal asymmetry upon late organizers in the same cell field.
Assuntos
Padronização Corporal/fisiologia , Organizadores Embrionários/fisiologia , Gêmeos Unidos/embriologia , Xenopus laevis , Animais , Linhagem da Célula , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/fisiologia , Embrião não Mamífero/efeitos da radiação , Indução Embrionária/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hibridização In Situ , Fatores de Determinação Direita-Esquerda/metabolismo , Cloreto de Lítio/farmacologia , Organizadores Embrionários/efeitos dos fármacos , Organizadores Embrionários/efeitos da radiação , Situs Inversus/genética , Situs Inversus/metabolismo , Situs Inversus/patologia , Raios Ultravioleta , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/anatomia & histologia , Xenopus laevis/embriologiaRESUMO
The organizer of the vertebrate gastrula is an important signalling centre that induces and patterns dorsal axial structures. Although a topic of long-standing interest, the evolutionary origin of the organizer remains unclear. Here we show that the gastrula of the cephalochordate amphioxus expresses dorsal/ventral (D/V) patterning genes (for example, bone morphogenetic proteins (BMPs), Nodal and their antagonists) in patterns reminiscent of those of their vertebrate orthlogues, and that amphioxus embryos, like those of vertebrates, are ventralized by exogenous BMP protein. In addition, Wnt-antagonists (for example, Dkks and sFRP2-like) are expressed anteriorly, whereas Wnt genes themselves are expressed posteriorly, consistent with a role for Wnt signalling in anterior/posterior (A/P) patterning. These results suggest evolutionary conservation of the mechanisms for both D/V and A/P patterning of the early gastrula. In light of recent phylogenetic analyses placing cephalochordates basally in the chordate lineage, we propose that separate signalling centres for patterning the D/V and A/P axes may be an ancestral chordate character.
Assuntos
Evolução Biológica , Padronização Corporal/fisiologia , Cordados/embriologia , Organizadores Embrionários/fisiologia , Animais , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Cordados/genética , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Organizadores Embrionários/efeitos dos fármacos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Gastrulation is a fundamental process that results in formation of the three germ layers in an embryo. It involves highly coordinated cell migration. Cell to cell communication through cell surface and the surrounding molecular environment governs cell migration. In the present work, cell surface features, which are indicative of the migratory status of a cell, of an early gastrulating chick embryo were studied using scanning electron microscopy. The distinct ultrastructural features of cells located in the various regions of the epiblast are described. Differences in the surface features of cells from distinct embryonic regions indicate differences in their migratory capacities. Further, the dynamic nature of these cell surface features by their response to altered fibroblast growth factor (FGF) signaling, experimentally created by using either excess FGF or inhibition of FGF signaling are demonstrated.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Gastrulação/efeitos dos fármacos , Placa Neural/ultraestrutura , Organizadores Embrionários/ultraestrutura , Linha Primitiva/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Animais , Embrião de Galinha , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Placa Neural/efeitos dos fármacos , Placa Neural/embriologia , Placa Neural/metabolismo , Organizadores Embrionários/efeitos dos fármacos , Organizadores Embrionários/embriologia , Organizadores Embrionários/metabolismo , Linha Primitiva/efeitos dos fármacos , Linha Primitiva/embriologia , Linha Primitiva/metabolismo , Proteínas Recombinantes/farmacologia , Suramina/farmacologiaRESUMO
Dorsal-ventral patterning is specified by signaling centers secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen gradient is translated intracellularly into fate decisions remains largely unknown. Here, we report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high levels of p38 activator, MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal organizing center. p38 MAPK and CREB function downstream of maternal Wnt/beta-catenin and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E induces expression of lateral genes without inducing the expression of dorsal genes. Loss of CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB activities along the axis.
Assuntos
Padronização Corporal/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Mesoderma/embriologia , Xenopus laevis/embriologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Reporter , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Mesoderma/citologia , Mesoderma/metabolismo , Microinjeções , Oligonucleotídeos Antissenso/farmacologia , Organizadores Embrionários/efeitos dos fármacos , Organizadores Embrionários/embriologia , Fosforilação/efeitos dos fármacos , RNA/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
We have explored the role of fibroblast growth factor (Fgf) signaling in regulating gene expression in the early zebrafish hindbrain primordium. We demonstrate that a dominant negative Fgf receptor (FgfR) construct disrupts gene expression along the entire rostrocaudal axis of the hindbrain primordium and, using an FgfR antagonist, we find that this Fgf signal is required at early gastrula stages. This effect cannot be mimicked by morpholino antisense oligos to Fgf3, Fgf8 or Fgf24--three Fgf family members known to be secreted from signaling centers at the midbrain-hindbrain boundary (MHB), in rhombomere 4 and in caudal mesoderm at gastrula stages. We propose that an Fgf signal is required in the early gastrula to initiate hindbrain gene expression and that this is distinct from the later roles of Fgfs in patterning the hindbrain during late gastrula/early segmentation stages. We also find that blocking either retinoic acid (RA) or Fgf signaling disrupts hindbrain gene expression at gastrula stages, suggesting that both pathways are essential at this stage. However, both pathways must be blocked simultaneously to disrupt hindbrain gene expression at segmentation stages, indicating that these signaling pathways become redundant at later stages. Furthermore, exogenous application of RA or Fgf alone is sufficient to induce hindbrain genes in gastrula stage tissues, suggesting that the two-signal requirement can be overcome under some conditions. Our results demonstrate an early role for Fgf signaling and reveal a dynamic relationship between the RA and Fgf signaling pathways during hindbrain development.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Rombencéfalo/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/fisiologia , Células Cultivadas , Cicloeximida/farmacologia , Proteínas de Ligação a DNA , Embrião não Mamífero , Fatores de Crescimento de Fibroblastos/química , Gástrula/efeitos dos fármacos , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Microinjeções/métodos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Organizadores Embrionários/efeitos dos fármacos , Organizadores Embrionários/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Pirróis/farmacologia , RNA Mensageiro/biossíntese , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores do Ácido Retinoico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rombencéfalo/efeitos dos fármacos , Rombencéfalo/embriologia , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/citologia , Medula Espinal/metabolismo , Fatores de Tempo , Tretinoína/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The organizer anchors the primary embryonic axis, and balance between dorsal (organizer) and ventral domains is fundamental to body patterning. LNX (ligand of Numb protein-X) is a RING finger and four PDZ domain-containing E3 ubiquitin ligase. LNX serves as a binding platform and may have a role in cell fate determination, but its in vivo functions are unknown. Here we show that Lnx-l (Lnx-like) functions as a critical regulator of dorso-ventral axis formation in zebrafish. Depletion of Lnx-l using specific antisense morpholinos (MOs) caused strong embryonic dorsalization. We identified Bozozok (Boz, also known as Dharma or Nieuwkoid) as a binding partner and substrate of Lnx-l. Boz is a homeodomain-containing transcriptional repressor induced by canonical Wnt signalling that is critical for dorsal organizer formation. Lnx-l induced K48-linked polyubiquitylation of Boz, leading to its proteasomal degradation in human 293T cells and in zebrafish embryos. Dorsalization induced by Boz overexpression was suppressed by raising the level of Lnx-l, but Lnx-l failed to counteract dorsalization caused by mutant Boz lacking a critical motif for Lnx-l binding. Furthermore, dorsalization induced by depletion of Lnx-l was alleviated by attenuation of Boz expression. We conclude that Lnx-l modulates Boz activity to prevent the invasion of ventral regions of the embryo by organizer tissue. These studies introduce a ubiquitin ligase, Lnx-l, as a balancing modulator of axial patterning in the zebrafish embryo.
Assuntos
Proteínas de Homeodomínio/metabolismo , Organizadores Embrionários/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Padronização Corporal/efeitos dos fármacos , Linhagem Celular , Proteína Goosecoid/metabolismo , Humanos , Oligonucleotídeos Antissenso/farmacologia , Organizadores Embrionários/efeitos dos fármacos , Poliubiquitina/metabolismo , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Proteínas de Peixe-Zebra/deficiênciaRESUMO
The role of microRNAs in embryonic cell fate specification is largely unknown. In vertebrates, the miR-430/427/302 family shows a unique expression signature and is exclusively expressed during early embryogenesis. Here, we comparatively address the embryonic function of miR-302 in human embryonic stem cells (hESCs) and its ortholog miR-427 in Xenopus laevis. Interestingly, we found that this miRNA family displays species-specific target selection among ligands of the Nodal pathway, with a striking conservation of the inhibitors, Lefties, but differential targeting of the activators, Nodals. The Nodal pathway plays a crucial role in germ layer specification. Accordingly, by gain and loss of function experiments in hESCs, we show that miR-302 promotes the mesendodermal lineage at the expense of neuroectoderm formation. Similarly, depletion of miR-427 in Xenopus embryos hinders the organizer formation and leads to severe dorsal mesodermal patterning defects. These findings suggest a crucial role for the miR-430/427/302 family in vertebrate embryogenesis by controlling germ layer specification.
Assuntos
Padronização Corporal , Linhagem da Célula , Endoderma/citologia , Mesoderma/citologia , MicroRNAs/metabolismo , Xenopus laevis/embriologia , Animais , Sequência de Bases , Padronização Corporal/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Endoderma/efeitos dos fármacos , Endoderma/embriologia , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Fatores de Determinação Direita-Esquerda/metabolismo , Mesoderma/efeitos dos fármacos , Mesoderma/embriologia , Mesoderma/metabolismo , MicroRNAs/genética , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Organizadores Embrionários/citologia , Organizadores Embrionários/efeitos dos fármacos , Organizadores Embrionários/embriologia , Especificidade da Espécie , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMO
Human embryos exposed to alcohol (ethanol) develop a complex developmental phenotype known as fetal alcohol spectrum disorder (FASD). In Xenopus embryos, ethanol reduces the levels of retinoic acid (RA) signaling during gastrulation. RA, a metabolite of vitamin A (retinol), is required for vertebrate embryogenesis, and deviation from its normal levels results in developmental malformations. Retinaldehyde dehydrogenase 2 (RALDH2) is required to activate RA signaling at the onset of gastrulation. We studied the effect of alcohol on embryogenesis by manipulating retinaldehyde dehydrogenase activity in ethanol-treated embryos. In alcohol-treated embryos, we analyzed RA signaling levels, phenotypes induced and changes in gene expression. Developmental defects that were characteristic of high ethanol concentrations were phenocopied by a low ethanol concentration combined with partial RALDH inhibition, whereas Raldh2 overexpression rescued the developmental malformations induced by high ethanol. RALDH2 knockdown resulted in similar RA signaling levels when carried out alone or in combination with ethanol treatment, suggesting that RALDH2 is the main target of ethanol. The biochemical evidence that we present shows that, at the onset of RA signaling during early gastrulation, the ethanol effect centers on the competition for the available retinaldehyde dehydrogenase activity. In light of the multiple regulatory roles of RA, continued embryogenesis in the presence of abnormally low RA levels provides an etiological explanation for the malformations observed in individuals with FASD.
Assuntos
Aldeído Oxidase/metabolismo , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Etanol/toxicidade , Gastrulação/efeitos dos fármacos , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Família Aldeído Desidrogenase 1 , Aldeído Oxidase/genética , Animais , Embrião não Mamífero/enzimologia , Gástrula/anormalidades , Gástrula/efeitos dos fármacos , Gástrula/enzimologia , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Organizadores Embrionários/efeitos dos fármacos , Organizadores Embrionários/enzimologia , Fenótipo , Retinal Desidrogenase , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologia , Xenopus/genética , Proteínas de Xenopus/genéticaRESUMO
Fetal alcohol spectrum disorder (FASD) is the combination of developmental, morphological, and neurological defects that result from exposing human embryos to ethanol (EtOH). Numerous embryonic structures are affected, leading to a complex viable phenotype affecting among others, the anterior/posterior axis, head, and eye formation. Recent studies have provided evidence suggesting that EtOH teratogenesis is mediated in part through a reduction in retinoic acid (RA) levels, targeting mainly the embryonic organizer (Spemann's organizer) and its subsequent functions. EtOH-treated Xenopus embryos were subjected to an analysis of gene expression patterns. Analysis of organizer-specific genes revealed a transient delay in the invagination of gsc- and chordin-positive cells that eventually reach their normal rostro-caudal position. Dorsal midline genes show defects along the rostro-caudal axis, lacking either their rostral (Xbra and Xnot2) or caudal (FoxA4b and Shh) expression domains. Head-specific markers like Otx2, en2, and Shh show abnormal expression patterns. Otx2 exhibits a reduction in expression levels, while en2 becomes restricted along the dorsal/ventral axis. During neurula stages, Shh becomes up-regulated in the rostral region and it is expressed in an abnormal pattern. These results and histological analysis suggest the existence of malformations in the brain region including a lack of the normal fore brain ventricle. An increase in the size of both the prechordal plate and the notochord was observed, while the spinal cord is narrower. The reduction in head and eye size was accompanied by changes in the eye markers, Pax6 and Tbx3. Our results provide evidence for the early molecular changes induced by EtOH exposure during embryogenesis, and may explain some of the structural changes that are part of the EtOH teratogenic phenotype also in FASD individuals.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário , Etanol/farmacologia , Organizadores Embrionários/efeitos dos fármacos , Xenopus laevis/embriologia , Animais , Feminino , Transtornos do Espectro Alcoólico Fetal/patologia , Gástrula/efeitos dos fármacos , Gástrula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Microftalmia/patologia , Modelos Animais , Morfogênese/efeitos dos fármacos , Tretinoína/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
In molluscs, the 3D vegetal blastomere acts as a developmental signaling center, or organizer, and is required to establish bilateral symmetry in the embryo. 3D is similar to organizing centers in other metazoans, but detailed comparisons are difficult, in part because its organizing function is poorly understood. To elucidate 3D function in a standardized fashion, we used monensin and brefeldin A (BFA) to rapidly and reversibly interfere with protein processing and secretion, thereby inhibiting the signaling interactions that underlie its specification and patterning. In the gastropods, Patella vulgata and Lymnaea stagnalis, the polyplacophoran, Mopalia muscosa, and the scaphopod, Antalis entalis, treatments initiated before the organizer-dependent onset of bilateral cleavage resulted in radialization of subsequent development. In radialized P. vulgata, L. stagnalis, and M. muscosa, organizer specification was blocked, and embryos failed to make the transition to bilateral cleavage. In all four species, the subsequent body plan was radially symmetric and was similarly organized about a novel aboral-oral axis. Our results demonstrate that brefeldin A (BFA) and monensin can be used to inhibit 3D's organizing function in a comparative fashion and that, at least in M. muscosa, the organizer-dependent developmental architecture of the embryo predicts subsequent patterns of morphogenetic movements in gastrulation and, ultimately, the layout of the adult body plan.
Assuntos
Brefeldina A/farmacologia , Moluscos/efeitos dos fármacos , Moluscos/embriologia , Monensin/farmacologia , Organizadores Embrionários/efeitos dos fármacos , Animais , Padronização Corporal/efeitos dos fármacos , Gastrópodes/efeitos dos fármacos , Gastrópodes/embriologia , Lymnaea/efeitos dos fármacos , Lymnaea/embriologia , Microscopia Eletrônica de Varredura , Moluscos/classificação , Moluscos/fisiologia , Organizadores Embrionários/embriologia , Poliplacóforos/efeitos dos fármacos , Poliplacóforos/embriologia , Inibidores da Síntese de Proteínas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Xenopus homeobox gene, PV.1 ventralizes activin-induced dorsal mesoderm and inhibits neuralization of ectoderm in animal cap when overexpressed. Here we generated PV.1/engrailed fusion construct (N-PV1-EnR) to perform loss-of-function study for this transcription factor. N-PV1-EnR showed an extremely antimorphic effect, causing a partial secondary embryonic axis when expressed at ventral marginal zone of blastula. In ventral marginal zone cells, this chimeric protein induced organizer genes and suppressed ventral markers mimicking those effects reported for dominant negative BMP-4 receptor (DNBR). Moreover, N-PV1-EnR rescued the ventralized embryos caused by the ectopic dorsal expression of PV.1 but not by that of Xvent-2. These results suggested that PV.1 functions at downstream of BMP-4 as a ventralizing effector which acts separately from Xvent-2 and the dominant negative effect gained by this specific mutant is applicable for the further studies of BMP-4 downstream pathway.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Homeodomínio/farmacologia , Organizadores Embrionários/metabolismo , Proteínas Serina-Treonina Quinases , Receptores de Fatores de Crescimento , Fatores de Transcrição , Proteínas de Xenopus , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Padronização Corporal/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/embriologia , Embrião não Mamífero/fisiologia , Indução Embrionária/efeitos dos fármacos , Genes Dominantes , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Técnicas In Vitro , Microinjeções , Organizadores Embrionários/efeitos dos fármacos , Fenótipo , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , XenopusRESUMO
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.