RESUMO
During development of the mammalian embryo, there is a complex relation between formation of the mesoderm and the neuroectoderm. In mouse, for example, the role of the node and its mesendoderm derivatives in anterior neural specification is still debated. Mouse Cripto(-/-) embryos could potentially help settle this debate because they lack almost all embryonic endoderm and mesoderm, including the node and its derivatives. In the present paper, we show that Cripto(-/-) embryos can still form functional neural stem cells that are able to differentiate and maintain a neural phenotype both in vivo and in vitro. These data suggest that signals emanating from the mesoderm and endoderm might not be essential for the formation and differentiation of neural stem cells. However, we use grafting experiments to show that the Cripto(-/-) isthmus (the secondary organizer located at the midbrain-hindbrain boundary) loses its inductive ability. We further show that the Cripto(-/-)isthmus expresses lower amounts of the isthmic signalling molecule, Fgf8. Since nearby tissues remain competent to respond to exogenously added Fgf8, this reduction in Fgf8 levels in the Cripto(-/-) isthmus is the potential cause of the loss of patterning ability in graft experiments. Overall, we interpret our data to suggest that the mammalian node and primitive streak are essential for the development of the regional identities that control the specification and formation of the secondary organizers within the developing brain.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Gastrulação/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Placa Neural/anormalidades , Placa Neural/metabolismo , Animais , Diferenciação Celular , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/metabolismo , Fator de Crescimento Epidérmico/deficiência , Fator de Crescimento Epidérmico/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Placa Neural/citologia , Transdução de SinaisRESUMO
The EGF-CFC gene cripto governs anterior-posterior (A-P) axis specification in the vertebrate embryo. Existing models suggest that Cripto facilitates binding of Nodal to an ActRII-activin-like kinase (ALK) 4 receptor complex. Cripto also has a crucial function in cellular transformation that is independent of Nodal and ALK4. However, how ALK4-independent Cripto pathways function in vivo has remained unclear. We have generated cripto mutants carrying the amino acid substitution F78A, which blocks the Nodal-ALK4-Smad2 signaling both in embryonic stem cells and cell-based assays. In cripto(F78A/F78A) mouse embryos, Nodal fails to expand its own expression domain and that of cripto, indicating that F78 is essential in vivo to stimulate Smad-dependent Nodal autoinduction. In sharp contrast to cripto-null mutants, cripto(F78A/F78A) embryos establish an A-P axis and initiate gastrulation movements. Our findings provide in vivo evidence that Cripto is required in the Nodal-Smad2 pathway to activate an autoinductive feedback loop, whereas it can promote A-P axis formation and initiate gastrulation movements independently of its stimulatory effect on the canonical Nodal-ALK4-Smad2 signaling pathway.
Assuntos
Padronização Corporal/fisiologia , Desenvolvimento Embrionário/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Animais Geneticamente Modificados , Quimera , Fator de Crescimento Epidérmico/genética , Retroalimentação Fisiológica/genética , Feminino , Gástrula/embriologia , Gástrula/metabolismo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Proteínas de Neoplasias/genética , Proteína Nodal , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
During early mouse development, the TGFbeta-related protein Nodal specifies the organizing centers that control the formation of the anterior-posterior (A-P) axis. EGF-CFC proteins are important components of the Nodal signaling pathway, most likely by acting as Nodal coreceptors. However, the extent to which Nodal activity depends on EGF-CFC proteins is still debated. Cripto is the earliest EGF-CFC gene expressed during mouse embryogenesis and is involved in both A-P axis orientation and mesoderm formation. To investigate the relation between Cripto and Nodal in the early mouse embryo, we removed the Nodal antagonist Cerberus 1 (Cer1) and simultaneously Cripto, by generating Cer1;Cripto double mouse mutants. We observed that two thirds of the Cer1;Cripto double mutants are rescued in processes that are severely compromised in Cripto(-/-) embryos, namely A-P axis orientation, anterior mesendoderm and posterior neuroectoderm formation. The observed rescue is strongly reduced in Cer1;Cripto;Nodal triple mutants, suggesting that Nodal can signal extensively in the absence of Cripto, if Cer1 is also inhibited. This signaling activity drives A-P axis positioning. Our results provide evidence for the existence of Cripto-independent signaling mechanisms, by which Nodal controls axis specification in the early mouse embryo.
Assuntos
Padronização Corporal/fisiologia , Fator de Crescimento Epidérmico/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/fisiologia , Animais , Padronização Corporal/genética , Citocinas , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Fator de Crescimento Epidérmico/deficiência , Fator de Crescimento Epidérmico/genética , Feminino , Gastrulação/genética , Gastrulação/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteína Nodal , Organizadores Embrionários/fisiologia , Gravidez , Proteínas/genética , Proteínas/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genéticaRESUMO
The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.
Assuntos
Diferenciação Celular/genética , Fator de Crescimento Epidérmico , Glicoproteínas de Membrana , Miocárdio/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II/metabolismo , Sequência de Aminoácidos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Indução Embrionária , Proteínas Ligadas por GPI , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Mutação , Miocárdio/citologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/farmacologia , Proteína Nodal , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/farmacologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/genética , Proteína Smad2 , Células-Tronco/citologia , Transativadores/metabolismo , Proteínas de XenopusRESUMO
The EGF-CFC proteins have been recently recognized as a novel family of extracellular factors required during early vertebrate development. Cripto is the founder member of the EGF-CFC family initially related to the epidermal growth factor (EGF); its expression is increased in human colon, gastric, pancreatic and lung carcinoma and in different types of both mouse and human breast carcinomas. Genetic studies in the mouse have established an essential role of cripto in the formation and correct positioning of the anterior-posterior axis. Furthermore, the absence of cripto results in a defective precardiac mesoderm, unable to differentiate into functional cardiomyocytes. Although mouse and human Cripto have been shown to activate the ras/raf/MAP kinase pathway in mammary epithelial cell lines, genetic evidence in Zebrafish has been provided for a relationship between the EGF-CFC proteins and Nodal, a member of the transforming growth factor family. Here we review the biological role of cripto in development and differentiation, pointing out recent findings on the biochemical interactions of Cripto, Nodal and Activin-like receptors.