Developmental appearance of pan-neuronal markers and enteric neuron subtypes
In the mouse, neural crest cells emigrate from the caudal hind-brain around E8.5, migrate ventrally and enter the foregut around E9.5-E10 . They then migrate caudally along the gut and reach the end of the hindgut around E14.5 [49, 50]. Pan-neuronal markers (molecular characteristics shared by all neurons) are not expressed by vagal neural crest cells en route to the foregut . But, by E10-10.5, around 10–20% of neural crest-derived cells in the stomach and rostral small intestine have already begun to express pan-neuronal markers including Hu, neuron class III p-tubulin (Tuj1), neurofilament-M and PGP9.5 [51, 52]. In both mice and chick embryos, cells expressing pan-neuronal markers are present close to the migratory wavefront [52–55] (Fig. 1B). A recent time-lapse imaging study showed that some cells expressing pan-neuronal markers continue to migrate caudally along the gut in close association with undifferentiated crest-derived cells . This is not surprising as it is well established that immature neurons in many parts of the CNS migrate, some for considerable distances .
As in most other parts of the nervous system, different enteric neuronal subtypes begin to differentiate at different developmental stages [58–60]. Some enteric neuron subtype-specific markers are expressed early during the development of the mouse ENS. For example, in the ENS of E11.5 mice, some neurons show calbindin or NOS immunoreactivity, or express Cart (cocaine- and amphetamine-regulated transcript), and some of the neurites show immunoreactivity to the intermediate conductance potassium channel IKCa- [61, 62] (Fig. 1C). In the mature ENS, VIP is co-localized with NOS in enteric neurons, but during development, VIP immunoreactivity cannot be detected until several days after the appearance of NOS . Thus there is a staggered appearance of markers of neuronal subtypes during development.
Acetylcholine is a major excitatory neurotransmitter at neuroneuronal and neuromuscular junctions in the mature ENS. Using the synthesis of 3H-acetylcholine from 3H-choline as a marker, cholinergic neurons were first detected from E10 to E12 , although immunoreactivity to ChAT and vesicular acetylcholine transporter (VAChT) cannot be detected until around E18.5, possibly because these antisera are not very sensitive. ChAT-immunoreactive neurons are already present in the myenteric plexus of the small intestine of E18 rats, but earlier stages were not examined . In embryonic mice, neuropeptide Y (NPY) can be first detected at E13–13.5 , substance P at E14–14.5  and calcitonin gene-related peptide (CGRP), which is expressed by intrinsic sensory neurons , can be first detected at E17–17.5 . In both mice and rats, some enteric neuron subtypes do not appear until after birth, and thus maturation of the ENS is thought to continue for several weeks after birth [60, 64, 67, 68].
In the zebrafish gut, cells expressing pan-neuronal markers appear between 24 and 48 hrs post-fertilisation (d.p.f.), beginning with the appearance of a few Hu-immunoreactive cells in the proximal gut [30, 69]. By 3 d.p.f, neurons are present along the entire length of the gut [30, 69, 70]. As in mice, it appears that the earliest developing neuron subtype is nitrergic neurons, with NOS immunoreactivity identified in the proximal midgut at 2 d.p.f. [28, 71, 72]. In situ hybridization studies also show the expression of nNOS mRNA in the gut between 2 and 3 d.p.f. [73, 74]. By 3 d.p.f., NOS neurons are present along most of the gut [71, 72]. VIP, pituitary adenylate cyclase activating peptide (PACAP), calbindin, calretinin and neurokinin A-immunoreactive neurons are also present along the entire gut at 3 d.p.f., and 5-HT and CGRP-immunoreactive neurons can be identified at 3–4 d.p.f. [70, 72]. ChAT-immunoreactivity could not be detected during zebrafish development, even though it is readily identifiable in the adult zebrafish ENS . This is similar to the situation in embryonic mice, where ChAT-immunoreactive neurons cannot be detected until late embryonic stages (see above).
The development of nerve terminals within various target tissues innervated by enteric neurons has been examined in some species. In the mouse, NOS-containing nerve fibres are present in the circular muscle 2 days prior to birth (Fig. 1D, E), but cholinergic (VAChT-immunoreactive) nerve terminals are relatively sparse in the colon at birth; the densities of both NOS and VAChT terminals in the colonic circular muscle increase dramatically between P0 and P10 . However, there is some functional evidence for a cholinergic innervation of the circular muscle in the small intestine of embryonic mice as excitatory junction potentials can be evoked by electrical field stimulation of E17 mice . The innervation of the mucosa in the small and large intestine of the pig develops around birth , whereas in the mouse, nerve terminals are present in the mucosa in the small intestine at E18.5, just prior to birth .
In the developing nervous system, some neurotransmitters or their synthesizing enzymes, or combinations of neurotransmitters, which are not expressed in the mature nervous system, are expressed transiently [79, 80]. In the developing ENS, the catecholamine synthetic enzyme, tyrosine hydroxylase (TH), is transiently expressed by all developing enteric neurons from E9.5 to E12.5 [51, 52, 56, 81, 82]. Signals arising from the gut appear to be important in the down-regulation of TH expression by immature enteric neurons . A very small population of TH neurons (<0.5% of myenteric neurons) is present in the adult mouse , but these do not appear to arise from the transiently TH cells that are present early in embryonic development . Although NOS is only expressed by a very small percentage of submucosal neurons in the small intestine , and the mucosa is not innervated by NOS nerve fibres in most mammals , in late embryonic and early post-natal mice, nearly 50% of submucosal neurons transiently express NOS (Fig. 1F), and NOS nerve fibres are present transiently in the mucosa .
Time of exit from cell cycle of different neuron types
The stage at which a neuronal precursor exits the cell cycle is defined as the birthdate of the neuron. In the CNS, neuronal precursors are generally thought to exit the cell cycle prior to expressing pan-neuronal markers. However, like developing sympathetic neurons, neural crest-derived cells continue to divide after expressing pan-neuronal markers during enteric neurogenesis [81, 86] (Fig. 2A). To date, there are no reports of mammalian enteric neurons undergoing cell division after the expression of neuron subtype-specific markers, so exit from the cell cycle appears to occur after the expression of pan-neuronal markers but prior to the expression of subtype-specific markers .
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.
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Only two studies have examined the birthdates of enteric neurons [88, 89]. In these studies in mouse, radiolabelled thymidine or thymidine analogues were injected into pregnant mice carrying embryos at different development stages [88, 89]. The 5-HT and cholinergic neurons are born first at very early ages, from E8 to E14, and E8 to E15, respectively, and myenteric VIP, NPY enkephalin and CGRP neurons begin to be born at E10 . A subsequent study showed that NOS, calbindin and γ-amino butyric acid (GABA)ergic neurons were born from E12.5 to P1, although younger birthdates were not investigated in this study . Submucosal neurons tend to be born later than myenteric plexus neurons [88, 89]. In chick embryos, VIP neurons are born between E3 and E10, with a peak at E7, and VIP immunoreactivity is first detectable at E5.5–E6.5 .
Overall, there does not appear to be any obvious correlation between the birthdates of different types of neurons and the developmental stage at which they express specific markers at detectable levels for the first time (Table 1). For example, although the birth of 5-HT neurons peaks at E10 , endogenous production of 5-HT is not detectable until E18 . Conversely, NOS- and calbindin-immunoreactive neurons can be identified at E11.5, and the peak of their births occurs at E14.5 . Thus, the length of time between cell cycle exit and detectable expression of neuron subtype-specific markers differs for different enteric neuron subtypes.
Table 1. The earliest detection of subtype-specific markers, birthdates and the peak of births of myenteric neuron subtypes in the mouse small intestine
|Enteric neuron subtype||Earliest detection||Birthdates (myenteric plexus)||Peak of birth (myenteric)||References|
|CART (cocaine- and amphetamine-regulated transcript)||E11.5||?||?|||