Age-specific mechanisms regulate neural crest and mesodermal contribution to the 1 enteric nervous system in health and disease

24 The enteric nervous system (ENS), a collection of neurons contained in the wall of the gut, is of 25 fundamental importance to gastrointestinal and systemic health. According to the prevailing 26 paradigm, the ENS arises from progenitor cells migrating from the embryonic neural crest and 27 remains largely unchanged thereafter. Here, we show that the lineage composition of maturing 28 ENS changes with time, with a decline in the canonical lineage of neural-crest derived neurons 29 and their replacement by a newly identified lineage of mesoderm-derived neurons. Single cell 30 transcriptomics and immunochemical approaches establish a distinct expression profile of 31 mesoderm-derived neurons. The dynamic balance between the proportions of neurons from these 32 two different lineages in the post-natal gut is dependent on the availability of their respective 33 trophic signals, GDNF-RET and HGF-MET. With increasing age, the mesoderm-derived neurons 34 become the dominant form of neurons in the ENS, a change associated with significant functional 35 effects on intestinal motility which can be reversed by GDNF supplementation. Transcriptomic 36 analyses of human gut tissues show reduced GDNF-RET signaling in patients with intestinal 37 dysmotility which is associated with reduction in neural crest-derived neuronal markers and 38 concomitant increase in transcriptional patterns specific to mesoderm-derived neurons. Normal 39 intestinal function in the adult gastrointestinal tract therefore appears to require an optimal 40 balance between these two distinct lineages within the ENS. 41