RESUMEN
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.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Gastrulación/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Placa Neural/anomalías , Placa Neural/metabolismo , Animales , Diferenciación Celular , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/metabolismo , Factor de Crecimiento Epidérmico/deficiencia , Factor de Crecimiento Epidérmico/genética , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Placa Neural/citología , Transducción de SeñalRESUMEN
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.
Asunto(s)
Tipificación del Cuerpo/fisiología , Desarrollo Embrionario/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Animales , Animales Modificados Genéticamente , Quimera , Factor de Crecimiento Epidérmico/genética , Retroalimentación Fisiológica/genética , Femenino , Gástrula/embriología , Gástrula/metabolismo , Regulación de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Proteínas de Neoplasias/genética , Proteína Nodal , Transducción de Señal/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Embryonic stem (ES) cells have been suggested as candidate therapeutic tools for cell replacement therapy in neurodegenerative disorders. However, limitations for the use of these cells lie in our restricted knowledge of the molecular mechanisms involved in their specialized differentiation and in the risk of tumor formation. Recent findings suggest that the EGF-CFC protein Cripto is a key player in the signaling pathways controlling neural induction in ES cells. Here we show that in vitro differentiation of Cripto(-/-) ES cells results in increased dopaminergic differentiation and that, upon transplantation into Parkinsonian rats, they result in behavioral and anatomical recovery with no tumor formation. The use of knockout ES cells that can generate dopamine cells while eliminating tumor risk holds enormous potential for cell replacement therapy in Parkinson's disease.