Development of enteric neuron diversity

Abstract

The mature enteric nervous system (ENS) is composed of many different neuron subtypes and enteric glia, which all arise from the neural crest. How this diversity is generated from neural crest-derived cells is a central question in neurogastroenterology, as defects in these processes are likely to underlie some paediatric motility disorders. Here we review the developmental appearance (the earliest age at which expression of specific markers can be localized) and birthdates (the age at which precursors exit the cell cycle) of different enteric neuron subtypes, and their projections to some targets. We then focus on what is known about the mechanisms underlying the generation of enteric neuron diversity and axon pathfinding. Finally, we review the development of the ENS in humans and the etiologies of a number of paediatric motility disorders.

Figure 1

(A1-3) Single optical section through a myenteric ganglion in wholemount preparation of colon from a 28-day-old mouse following immunostaining performed with the pan-neuronal marker, PGP9.5 (red) and the glial marker, S100β (blue). The nuclei had been stained using the nucleic acid stain, SYTO (green). The nuclei adjacent to the ganglion (asterisks) belong to fibroblasts, interstitial cells of Cajal or circular smooth muscle cells. (B) Wholemount preparation of colon from an E12.5 Ret^TGM mouse [219] in which the neural crest cells express GFP that had also been immunostained performed with the pan-neuronal marker, Tuj1. Tuj1⁺ cell bodies (open arrows) are intermingled with other crest-derived (GFP⁺) cells, and they project axon-like processes (arrows) in close association with migrating crest cells. (C) Wholemount preparation of colon from an E12.5 mouse following staining with the pan-neural crest cell marker, Phox2b (green), and NOS (red). Some of the NOS cell bodies (open arrow) give rise to axon-like processes (arrow) that project caudally. (D, E) Wholemount preparations of small intestine from E16.5 (D) and E18.5 (E) mice immunostained with the pan-neuronal marker, Hu (green), and the neuron subtype marker, NOS (red). NOS fibres (arrows) are present in the circular muscle of E18.5 mice (E), but not in E16.5 (D) mice. These fibres run perpendicular to the first nerve fibre tracts to form, which project longitudinally (see Fig. 2E). (F) Submucosal neurons in the small intestine of an E18.5 mouse immunostained for Hu (green) and NOS (red). Although only around 1% of submucosal neurons in adult mice express NOS [19], approximately 50% of submucosal neurons in late embryonic and early post-natal stages express NOS [78]. All scale bars except D, E = 25 μm; D, E = 50 μm.

Figure 2

(A1-4) A dividing immature neuron in a preparation of E11.5 midgut immunostained performed with the pan-neural crest cell marker, p75 (blue) and the pan-neuronal marker, neurofilament-M (red-NF). The nuclei had been stained using the nucleic acid stain, SYTO (green). The immature neuron is undergoing mitosis as the chromosomes are condensed (A3) Cells that do not show p75 immunostaining (asterisks) are mesenchymal cells. (B) Low magnification image of a cell body (open arrow) and axon (arrow) retrogradely labelled by the lipophilic dye, DiI, in the midgut from an E11.5 mouse. The neuron projects caudally. (C) Higher magnification image of a caudally projecting neuron in the E11.5 gut with a single axon (arrow). The neuron possesses several short, filamentous dendrites. (D) Preparation of midgut from an E11.5 mouse immunostained performed with antibodies to the pan-neuronal marker, Tuj1. Most of the neurites (arrows) are varicose and project longitudinally. Tuj1 cell bodies (open arrows) are scattered along the gut. (E) E11.5 gut immunostained with Ret (red) and Sox10 (green) antibodies. Most Ret⁺ cells also express Sox10. (F) E14.5 small intestine immunostained with PGP9.5 and Sox10. Sox10 is not expressed by PGP9.5⁺ cells. Scale bars: A = 5 μm; B = 100 μm; C = 10 μm; D, E, F = 25 μm.

Figure 3

Differentiation of enteric neurons and glial cells. At E10.5, the progeny of Sox10⁺/Phox2b⁺/p75⁺/Ret⁺ neural progenitors are further self-renewing cells plus neuron precursors, which express Hand2 ± Ascl1. From E11.5 onwards, glial precursors are also generated, and neuron subtype expression commences. GDNF promotes the proliferation of enteric neural crest-derived cells, but it is unclear whether it only promotes the proliferation of neuronal precursors, or all progenitors. It is likely that the competence of Sox10⁺/Phox2b⁺/p75⁺/Ret⁺ cells changes with age. TC, transiently catecholaminergic; TH, tyrosine hydroxylase. Data from [84, 109, 110, 115, 116, 143, 145, 147, 156].