Laranjeira, C.S.T.; (2010) In vivo identification of neural stem cells in the enteric nervous system. Doctoral thesis , UCL (University College London).

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Abstract

The enteric nervous system (ENS) in vertebrates is derived from neural crest cells which emerge during embryogenesis from the hindbrain and, following stereotypical migratory pathways, colonize the entire gastrointestinal tract. Assembly of enteric ganglia and formation of functional neuronal circuits throughout the gut depends on the highly regulated differentiation of enteric neural crest stem cells (eNCSCs) into a plethora of neuronal subtypes and glia. The identification of eNCSCs and the lineages they generate is fundamental to understand ENS organogenesis. However, the study of the properties of eNCSCs has been hindered by the lack of specific markers and genetic tools to efficiently identify and follow these cells in vivo. Although previous in vitro studies have suggested that Sox10-expressing cells of the mammalian gut generate both enteric neurons and glia, the differentiation potential of these Sox10+ cells in vivo is currently unclear. Here, we have developed a genetic marking system which allows us to identify Sox10+ cells and follow their fate in vivo. Using this system we demonstrate that Sox10+ cells of the gut generate both enteric neurons and glia in vivo, thus representing multilineage ENS progenitors. To examine whether the neurogenic potential of Sox10+ eNCSCs is temporally regulated over the course of gut organogenesis, we generated additional transgenic mouse lines expressing a tamoxifen-inducible Cre recombinase (iCreERT2) under the control of the Sox10 locus (Sox10iCreERT2). Activation of iCreERT2 in Sox10iCreERT2 transgenic mice at specific developmental stages and analysis of enteric ganglia from adult animals showed that the pool of Sox10+ cells progressively lose their neurogenic potential. These findings raise the question of the origin of multilineage ENS progenitors isolated from cultures of post-neurogenic gut. By combining genetic fate mapping in mice, cultures of enteric ganglia and an ENS injury model, we demonstrate that glial cells in the adult ENS retain neurogenic potential which can be activated both in vitro and in vivo, in response to injury. The signals that lead Sox10+ progenitor cells to become either neurons or glial cells remain unclear. We hypothesized that the receptor tyrosine kinase RET may be part of the molecular fate switch between the two lineages being able to divert differentiation of eNCSCs away from the glial lineage and towards the neuronal fate. Here, we describe a genetic strategy to attain persistent expression of RET in vivo, in a temporally and spatially controlled manner. Such a strategy will allow us to assess the role of RET in ENS differentiation during development. Taken together, our data provide a framework for exploring the molecular mechanisms that control enteric neurogenesis in vivo and identify glial cells as a potential target for cell replacement therapies in cases associated with congenital absence or acquired loss of enteric neurons.

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