Neurogenesis and synaptogenesis in the enteric nervous system: Molecules that generate neuronal phenotypic and synaptic diversity

The enteric nervous system (ENS), due to its highly complex organization and composition, requires tightly regulated mechanisms to achieve its normal development. Basic-Helix-Loop-Helix proteins are transcription factors that are extensively used throughout evolution to control developmental processes. Hand1 and Hand2 belong to this subfamily and are expressed in the ENS, where their functions are unknown. I found that Hand2 is restricted to neurons and glia in the gut, whereas, Hand1 is only expressed by enteric mesoderm and its derivatives. I investigated the role of Hand2 in enteric neurogenesis and found that Hand2 is required by ENS precursors to give rise to terminally differentiated neurons, without being necessary for some of them to express early neuronal markers; even, decreased expression of Hand2 resulted in ENS defects, including decreased HuC and HuD transcription and failure of subsets of nNOS neurons to form properly. In addition, these defects were functionally significant because a decrease of Hand2 expression led to slower intestinal motility. I also investigated the role played by the cell adhesion molecules, neurexins and neuroligins, which had not previously been explored in the ENS and have been shown previously to be essential for the establishment of CNS synapses. I found that neurexins and neuroligins are expressed during the time when enteric synapses form and neuroligins were expressed both in neurons and innervated neuroeffector cells. As in the CNS, both neurexin and neuroligin transcripts underwent extensive alternative splicing in the ENS. When expressed in transfected non-neuronal cells, neuroligin and neurexin induced developing enteric neurons to form presynaptic and postsynaptic structures respectively. Presynaptic components induced to form by neuroligins included recycling synaptic vesicles and junctional membrane specializations. Postsynaptic components recruited and clustered in response to contact with neurexin included neuroligins and neurotransmitter receptors. Engineered soluble neurexin, which interferes with endogenous neurexin-neuroligin binding, inhibited enteric synapse formation/stabilization and recruitment of neurotransmitter receptors. Finally, neurexin and neuroligin splice variants exerted differential effects, a finding that is compatible with the idea that the alternative splicing of these molecules contributes to ENS synaptic specificity.