RESUMEN
RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.
Asunto(s)
Proteínas de Drosophila , Sistema Nervioso Entérico/embriología , Sistema Nervioso Entérico/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Muerte Celular/genética , Diferenciación Celular/genética , Supervivencia Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Factor Neurotrófico Ciliar , Sistema Digestivo/embriología , Sistema Digestivo/inervación , Dopamina/metabolismo , Embrión de Mamíferos/citología , Edad Gestacional , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Homocigoto , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Ratones , Ratones Transgénicos , Mutación , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/farmacología , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , Neurotrofina 3 , Neurturina , Proteínas Proto-Oncogénicas c-ret , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
The enteric nervous system of vertebrates is derived from neural crest cells that invade the gut wall and generate a highly organised network of enteric ganglia. Among the genes that play an important role in ENS development is c-Ret, mutations of which result in failure of formation of enteric ganglia (intestinal aganglionosis). To further understand the development of the mammalian ENS in general and the mechanism of action of the RET RTK in particular, we have developed and used an organotypic culture system of mouse fetal gut. At the stage of culture initiation, the gut is partially populated by undifferentiated ENS progenitors, but culture for several days results in extensive neuronal and glial differentiation. Using this organ culture system, we have compared the development of the ENS in wild-type and RET-deficient gut and showed that the aganglionic phenotype observed in vivo is consistently reproduced under the in vitro culture conditions. Microinjection of RET+ cells isolated from E11.5 mouse bowel into wild-type or RET-deficient aganglionic gut in organ culture, results in extensive repopulation of their wall by exogenously derived neurons and glia. Finally, using a similar approach, we demonstrate that single RET+ cells introduced into the wall of wild-type gut generate both cell lineages of the ENS, i.e. neurons and glia. Our data show the NC-derived RET+ population of fetal gut in mammalian embryos consists of multipotential progenitors capable of colonising efficiently both wild-type and RET-deficient aganglionic bowel in organ culture.
Asunto(s)
Sistema Digestivo/embriología , Proteínas de Drosophila , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/embriología , Esófago/embriología , Técnicas de Cultivo de Órganos/métodos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Diferenciación Celular , Sistema Digestivo/inervación , Esófago/inervación , Ratones , Mutación , Neuroglía , Neuronas , Proteínas Proto-Oncogénicas c-ret , Células MadreRESUMEN
The c-ret proto-oncogene, a member of the receptor tyrosine kinase gene superfamily, plays a critical role in the development of the excretory system and the enteric and autonomic nervous systems of mammalian embryos. To study the potential function of the c-ret locus in lower vertebrates, we have isolated its zebrafish homologue, ret1 and established its expression pattern during embryogenesis. Ret1 mRNA first appears during early somitogenesis in the presumptive brain, spinal cord and excretory system. Within the CNS, expression of ret1 is detected in primary motor and sensory (Rohon-Beard) neurons. Ret1 transcripts are also expressed in subsets of neural crest cells and cranial ganglia as well as in the enteric nervous system. In the excretory system, expression is detected in the developing nephric duct and the pronephros. Our findings reveal a remarkable similarity in the expression pattern of c-ret between higher and lower vertebrates, suggesting that the function of this locus has been conserved throughout vertebrate evolution. Furthermore, the conservation of ret1 expression in cell types which remain unaffected by the mammalian c-ret mutations, such as motor and sensory neurons, suggests a function of this receptor in these cell lineages.
Asunto(s)
Proteínas de Drosophila , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Sistema Nervioso Central/embriología , Pollos , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Nervios Periféricos/embriología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret , Proto-Oncogenes , Mapeo Restrictivo , Pez Cebra/embriología , Proteínas de Pez CebraRESUMEN
Mutational analysis in humans and mice has demonstrated that the Ret, the product of the c-ret proto-oncogene, a member of the receptor tyrosine kinase (RTK) superfamily, is essential for development of the enteric nervous system and kidney. Despite the established role of Ret in mammalian embryogenesis, its cognate ligand(s) is currently unknown. Here we demonstrate, by using a Xenopus embryo bioassay, that glial-cell-line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor (TGF)-beta superfamily, signals through the Ret RTK. Furthermore, using explant cultures from wild-type and Ret-deficient mouse embryos, we show that normal c-ret function is necessary for GDNF signalling in the peripheral nervous system. Our data strongly suggest that Ret is a functional receptor for GDNF, and that GDNF, in addition to its potential role in the differentiation and survival of central nervous system neurons, has profound effects on kidney organogenesis and the development of the peripheral nervous system.