Marlene M. Hao

Development of enteric neuron diversity.

•  Neuronal diversity in the adult enteric nervous system (ENS) •  Developmental appearance and birthdating of enteric neuron subtypes and glial cells ‐  Developmental appearance of pan‐neuronal markers and enteric neuron subtypes ‐  Time of exit from cell cycle of different neuron types •  Morphological development of enteric neurons •  Axon guidance in the developing ENS •  Development of connectivity •  Mechanisms controlling enteric neuronal differentiation and the generation of neuron diversity ‐  Transcriptional control of enteric neuronal differentiation and the generation of neuron diversity ‐  Role of glial cell line‐derived neurotrophic factor (GDNF) family members in enteric neuronal differentiation and the generation of neuron diversity ‐  Role of endothelin‐3/Ednrb signalling ‐  Role of other signalling pathways ‐  Neurotrophin‐3 (NT‐3) ‐  Bone morphogenetic proteins (BMPs) ‐  L1 ‐  Sonic hedgehog ‐  Role of electrical activity •  Development of enteric glia •  Development of neurons and neuronal subtypes in the human ENS and clinical relevance ‐  Hirschsprungs disease ‐  Is the ganglionic segment of Hirschsprungs patients ‘normal’? ‐  Other paediatric motility disorders ‐  Defects in the development of subtypes of enteric neurons ‐  Defects in the number of enteric neurons •  Conclusions

Hirschsprung disease: a developmental disorder of the enteric nervous system.

Hirschsprung disease (HSCR), which is also called congenital megacolon or intestinal aganglionosis, is characterized by an absence of enteric (intrinsic) neurons from variable lengths of the most distal bowel. Because enteric neurons are essential for propulsive intestinal motility, infants with HSCR suffer from severe constipation and have a distended abdomen. Currently the only treatment is surgical removal of the affected bowel. HSCR has an incidence of around 1:5,000 live births, with a 4:1 male:female gender bias. Most enteric neurons arise from neural crest cells that emigrate from the caudal hindbrain and then migrate caudally along the entire gut. The absence of enteric neurons from variable lengths of the bowel in HSCR results from a failure of neural crest‐derived cells to colonize the affected gut regions. HSCR is therefore regarded as a neurocristopathy. HSCR is a multigenic disorder and has become a paradigm for understanding complex factorial disorders. The major HSCR susceptibility gene is RET. The penetrance of several mutations in HSCR susceptibility genes is sex‐dependent. HSCR can occur as an isolated disorder or as part of syndromes; for example, Type IV Waardenburg syndrome is characterized by deafness and pigmentation defects as well as intestinal aganglionosis. Studies using animal models have shown that HSCR genes regulate multiple processes including survival, proliferation, differentiation, and migration. Research into HSCR and the development of enteric neurons is an excellent example of the cross fertilization of ideas that can occur between human molecular geneticists and researchers using animal models. WIREs Dev Biol 2013, 2:113–129. doi: 10.1002/wdev.57

Colonizing while migrating: how do individual enteric neural crest cells behave?

BackgroundDirected cell migration is essential for normal development. In most of the migratory cell populations that have been analyzed in detail to date, all of the cells migrate as a collective from one location to another. However, there are also migratory cell populations that must populate the areas through which they migrate, and thus some cells get left behind while others advance. Very little is known about how individual cells behave to achieve concomitant directional migration and population of the migratory route. We examined the behavior of enteric neural crest-derived cells (ENCCs), which must both advance caudally to reach the anal end and populate each gut region.ResultsThe behavior of individual ENCCs was examined using live imaging and mice in which ENCCs express a photoconvertible protein. We show that individual ENCCs exhibit very variable directionalities and speed; as the migratory wavefront of ENCCs advances caudally, each gut region is populated primarily by some ENCCs migrating non-directionally. After populating each region, ENCCs remain migratory for at least 24 hours. Endothelin receptor type B (EDNRB) signaling is known to be essential for the normal advance of the ENCC population. We now show that perturbation of EDNRB principally affects individual ENCC speed rather than directionality. The trajectories of solitary ENCCs, which occur transiently at the wavefront, were consistent with an unbiased random walk and so cell-cell contact is essential for directional migration. ENCCs migrate in close association with neurites. We showed that although ENCCs often use neurites as substrates, ENCCs lead the way, neurites are not required for chain formation and neurite growth is more directional than the migration of ENCCs as a whole.ConclusionsEach gut region is initially populated by sub-populations of ENCCs migrating non-directionally, rather than stopping. This might provide a mechanism for ensuring a uniform density of ENCCs along the growing gut.

The role of neural activity in the migration and differentiation of enteric neuron precursors.

Background  As they migrate through the developing gut, a sub‐population of enteric neural crest‐derived cells (ENCCs) begins to differentiate into neurons. The early appearance of neurons raises the possibility that electrical activity and neurotransmitter release could influence the migration or differentiation of ENNCs.

Imaging neuron-glia interactions in the enteric nervous system

The enteric nervous system (ENS) is a network of neurons and glia within the wall of the gastrointestinal tract that is able to control many aspects of digestive function independently from the central nervous system. Enteric glial cells share several features with astrocytes and are closely associated with enteric neurons and their processes both within enteric ganglia, and along interconnecting fiber bundles. Similar to other parts of the nervous system, there is communication between enteric neurons and glia; enteric glial cells can detect neuronal activity and have the machinery to intermediate neurotransmission. However, due to the close contact between these two cell types and the particular characteristics of the gut wall, the recording of enteric glial cell activity in live imaging experiments, especially in the context of their interaction with neurons, is not straightforward. Most studies have used calcium imaging approaches to examine enteric glial cell activity but in many cases, it is difficult to distinguish whether observed transients arise from glial cells, or neuronal processes or varicosities in their vicinity. In this technical report, we describe a number of approaches to unravel the complex neuron-glia crosstalk in the ENS, focusing on the challenges and possibilities of live microscopic imaging in both animal models and human tissue samples.

The migratory behavior of immature enteric neurons

While they are migrating caudally along the developing gut, around 10%–20% of enteric neural crest‐derived cells start to express pan‐neuronal markers and tyrosine hydroxylase (TH). We used explants of gut from embryonic TH‐green fluorescence protein (GFP) mice and time‐lapse microscopy to examine whether these immature enteric neurons migrate and their mode of migration. In the gut of E10.5 and E11.5 TH‐GFP mice, around 50% of immature enteric neurons (GFP+ cells) migrated, with an average speed of around 15 μm/h. This is slower than the speed at which the population of enteric neural crest‐derived cells advances along the developing gut, and hence neuronal differentiation seems to slow, but not necessarily halt, the caudal migration of enteric neural crest cells. Most migrating immature enteric neurons migrated caudally by extending a long‐leading process followed by translocation of the cell body. This mode of migration is different from that of non‐neuronal enteric neural crest‐derived cells and neural crest cells in other locations, but resembles that of migrating neurons in many regions of the developing central nervous system (CNS). In migrating immature enteric neurons, a swelling often preceded the movement of the nucleus in the direction of the leading process. However, the centrosomal marker, pericentrin, was not localized to either the leading process or swelling. This seems to be the first detailed report of neuronal migration in the developing mammalian peripheral nervous system.

Early Emergence of Neural Activity in the Developing Mouse Enteric Nervous System

Neurons of the enteric nervous system (ENS) arise from neural crest cells that migrate into and along the developing gastrointestinal tract. A subpopulation of these neural-crest derived cells express pan-neuronal markers early in development, shortly after they first enter the gut. However, it is unknown whether these early enteric “neurons” are electrically active. In this study we used live Ca2+ imaging to examine the activity of enteric neurons from mice at embryonic day 11.5 (E11.5), E12.5, E15.5, and E18.5 that were dissociated and cultured overnight. PGP9.5-immunoreactive neurons from E11.5 gut cultures responded to electrical field stimulation with fast [Ca2+]i transients that were sensitive to TTX and ω-conotoxin GVIA, suggesting roles for voltage-gated Na+ channels and N-type voltage-gated Ca2+ channels. E11.5 neurons were also responsive to the nicotinic cholinergic agonist, dimethylphenylpiperazinium, and to ATP. In addition, spontaneous [Ca2+]i transients were present. Similar responses were observed in neurons from older embryonic gut. Whole-cell patch-clamp recordings performed on E12.5 enteric neurons after 2–10 h in culture revealed that these neurons fired both spontaneous and evoked action potentials. Together, our results show that enteric neurons exhibit mature forms of activity at early stages of ENS development. This is the first investigation to directly examine the presence of neural activity during enteric neuron development. Along with the spinal cord and hindbrain, the ENS appears to be one of the earliest parts of the nervous system to exhibit electrical activity.

Development of myenteric cholinergic neurons in ChAT-Cre;R26R-YFP mice

Cholinergic neurons are the major excitatory neurons of the enteric nervous system (ENS), and include intrinsic sensory neurons, interneurons, and excitatory motor neurons. Cholinergic neurons have been detected in the embryonic ENS; however, the development of these neurons has been difficult to study as they are difficult to detect prior to birth using conventional immunohistochemistry. In this study we used ChAT‐Cre;R26R‐YFP mice to examine the development of cholinergic neurons in the gut of embryonic and postnatal mice. Cholinergic (YFP+) neurons were first detected at embryonic day (E)11.5, and the proportion of cholinergic neurons gradually increased during pre‐ and postnatal development. At birth, myenteric cholinergic neurons comprised less than half of their adult proportions in the small intestine (25% of myenteric neurons were YFP+ at P0 compared to 62% in adults). The earliest cholinergic neurons appear to mainly project anally. Projections into the presumptive circular muscle were first observed at E14.5. A subpopulation of cholinergic neurons coexpress calbindin through embryonic and postnatal development, but only a small proportion coexpressed neuronal nitric oxide synthase. Our study shows that cholinergic neurons in the ENS develop over a protracted period of time. J. Comp. Neurol. 521:3358–3370, 2013.

The emergence of neural activity and its role in the development of the enteric nervous system

The enteric nervous system (ENS) is a vital part of the autonomic nervous system that regulates many gastrointestinal functions, including motility and secretion. All neurons and glia of the ENS arise from neural crest-derived cells that migrate into the gastrointestinal tract during embryonic development. It has been known for many years that a subpopulation of the enteric neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development. Recent studies have demonstrated that some enteric neurons exhibit electrical activity from as early as E11.5 in the mouse, with further maturation of activity during embryonic and postnatal development. This article discusses the maturation of electrophysiological and morphological properties of enteric neurons, the formation of synapses and synaptic activity, and the influence of neural activity on ENS development.

Early Development of Electrical Excitability in the Mouse Enteric Nervous System

Neural activity is integral to the development of the enteric nervous system (ENS). A subpopulation of neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development (at E10.5 in the mouse). However, the electrical activity of these cells has not been previously characterized, and it is not known whether all cells expressing neuronal markers are capable of firing action potentials (APs). In this study, we examined the activity of “neuron”-like cells (expressing pan-neuronal markers or with neuronal morphology) in the gut of E11.5 and E12.5 mice using whole-cell patch-clamp electrophysiology and compared them to the activity of neonatal and adult enteric neurons. Around 30–40% of neuron-like cells at E11.5 and E12.5 fired APs, some of which were very similar to those of adult enteric neurons. All APs were sensitive to tetrodotoxin (TTX), indicating that they were driven by voltage-gated Na+ currents. Expression of mRNA encoding several voltage-gated Na+ channels by the E11.5 gut was detected using RT-PCR. The density of voltage-gated Na+ currents increased from E11.5 to neonates. Immature active responses, mediated in part by TTX- and lidocaine-insensitive channels, were observed in most cells at E11.5 and E12.5, but not in P0/P1 or adult neurons. However, some cells expressing neuronal markers at E11.5 or E12.5 did not exhibit an active response to depolarization. Spontaneous depolarizations resembling excitatory postsynaptic potentials were observed at E12.5. The ENS is one of the earliest parts of the developing nervous system to exhibit mature forms of electrical activity.