Ret isoform function and marker gene expression in the enteric nervous system is conserved across diverse vertebrate species

The enteric nervous system (ENS) derives from migratory neural crest cells that colonize the developing gut tube, giving rise to an integrated network of neurons and glial cells, which together regulate important aspects of gut function, including coordinating the smooth muscle contractions of the gut wall. The absence of enteric neurons in portions of the gut (aganglionosis) is the defining feature of Hirschsprung’s disease (HSCR) and has been replicated in a number of mouse models. Mutations in the RET tyrosine kinase account for over half of familial cases of HSCR and mice mutant for Ret exhibit aganglionosis. RET exists in two main isoforms, RET9 and RET51 and studies in mouse have shown that RET9 is sufficient to allow normal development of the ENS. In the last several years, zebrafish has emerged as a model of vertebrate ENS development, having been supported by a number of demonstrations of conservation of gene function between zebrafish, mouse and human. In this study we further analyse the potential similarities and differences between ENS development in zebrafish, mouse and human. We demonstrate that zebrafish Ret is required in a dose-dependent manner to regulate colonization of the gut by neural crest derivatives, as in human. Additionally, we show that as in mouse and human, zebrafish ret is produced as two isoforms, ret9 and ret51. Moreover, we show that, as in mouse, the Ret9 isoform is sufficient to support colonization of the gut by enteric neurons. Finally, we identify zebrafish orthologues of genes previously identified to be expressed in the mouse ENS and demonstrate that these genes are expressed in the developing zebrafish ENS, thereby identifying useful ENS markers in this model organism. These studies reveal that the similarities between gene expression and gene function across vertebrate species is more extensive than previously appreciated, thus supporting the use of zebrafish as a general model for vertebrate ENS development and the use of zebrafish genetic screens as a way to identify candidate genes mutated in HSCR cases.     

Phox2b function in the enteric nervous system is conserved in zebrafish and is sox10-dependent

Zebrafish lacking functional sox10 have defects in non-ectomesenchymal neural crest derivatives including the enteric nervous system (ENS) and as such provide an animal model for human Waardenburg Syndrome IV. Here, we characterize zebrafish phox2b as a functionally conserved marker of the developing ENS. We show that morpholino-mediated knockdown of Phox2b generates fish modeling Hirschsprung disease. Using markers, including phox2b, we investigate the ontogeny of the sox10 ENS phenotype. As previously shown for melanophore development, ENS progenitor fate specification fails in these mutant fish. However, in addition, we trace back the sox10 mutant ENS defect to an even earlier time point, finding that most neural crest cells fail to migrate ventrally to the gut primordium.     

Photoconvertible fluorescent proteins: a versatile tool in zebrafish skeletal imaging

One of the most frequently applied techniques in zebrafish (Danio rerio) research is the visualisation or manipulation of specific cell populations using transgenic reporter lines. The generation of these transgenic zebrafish, displaying cell- or tissue-specific expression of frequently used fluorophores such as Green Fluorescent Protein (GFP) or mCherry, is relatively easy using modern techniques. Fluorophores with different emission wavelengths and driven by different promoters can be monitored simultaneously in the same animal. Photoconvertible fluorescent proteins (pcFPs) are different from these standard fluorophores because their emission spectrum is changed when exposed to UV light, a process called photoconversion. Here, the benefits and versatility of using pcFPs for both single and dual fluorochrome imaging in zebrafish skeletal research in a previously generated osx:Kaede transgenic line are illustrated. In this line, Kaede, which is expressed under control of the osterix, otherwise known as sp7, promoter thereby labelling immature osteoblasts, can switch from green to red fluorescence upon irradiation with UV light. First, this study demonstrates that osx:Kaede exhibits an expression pattern similar to a previously described osx:nuGFP transgenic line in both larval and adult stages, hereby validating the use of this line for the imaging of immature osteoblasts. More in-depth experiments highlight different applications for osx:Kaede, such as lineage tracing and its combined use with in vivo skeletal staining and other transgenic backgrounds. Mineral staining in combination with osx:Kaede confirms osteoblast-independent mineralisation of the notochord. Osteoblast lineage tracing reveals migration and dedifferentiation of scleroblasts during fin regeneration. Finally, this study shows that combining two transgenics, osx:Kaede and osc:GFP, with similar emission wavelengths is possible when using a pcFP such as Kaede.     

Targeted deletion of Hand2 in enteric neural precursor cells affects its functions in neurogenesis, neurotransmitter specification and gangliogenesis, causing functional aganglionosis

Targeted deletion of the bHLH DNA-binding protein Hand2 in the neural crest, impacts development of the enteric nervous system (ENS), possibly by regulating the transition from neural precursor cell to neuron. We tested this hypothesis by targeting Hand2 deletion in nestin-expressing neural precursor (NEP) cells. The mutant mice showed abnormal ENS development, resulting in lethal neurogenic pseudo-obstruction. Neurogenesis of neurons derived from NEP cells identified a second nestin non-expressing neural precursor (NNEP) cell in the ENS. There was substantial compensation for the loss of neurons derived from the NEP pool by the NNEP pool but this was insufficient to abrogate the negative impact of Hand2 deletion. Hand2-mediated regulation of proliferation affected both neural precursor and neuron numbers. Differentiation of glial cells derived from the NEP cells was significantly decreased with no compensation from the NNEP pool of cells. Our data indicate differential developmental potential of NEPs and NNEPs; NNEPs preferentially differentiate as neurons, whereas NEPs give rise to both neurons and glial cells. Deletion of Hand2 also resulted in complete loss of NOS and VIP and a significant decrease in expression of choline acetyltransferase and calretinin, demonstrating a role for Hand2 in neurotransmitter specification and/or expression. Loss of Hand2 resulted in a marked disruption of the developing neural network, exemplified by lack of a myenteric plexus and extensive overgrowth of fibers. Thus, Hand2 is essential for neurogenesis, neurotransmitter specification and neural network patterning in the developing ENS.     

HuC:Kaede, a useful tool to label neural morphologies in networks in vivo

A simple and efficient procedure for labeling neurons is a prerequisite for investigating the development of neural networks in zebrafish. To label neurons we used Kaede, a fluorescent protein with a photoconversion property allowing conversion from green to red fluorescence following irradiation with UV or violet light. We established a zebrafish stable transgenic line, Tg(HuC:Kaede), expressing Kaede in neurons under the control of the HuC promoter. This transgenic line was used to label a small number of neurons in the trigeminal ganglion. Also, using embryos injected with the transgene, we labeled peripheral axon arbors of a Rohon-Beard neuron at 4 days postfertilization and observed the dendrite development of a tectal neuron for 3 days. These data indicate that Kaede is a useful tool to selectively label neural networks in zebrafish.     

An enhancer trap zebrafish line for lateral line development and regulation of six2b expression

Zebrafish lateral line system which is derived from neurogenic placodes has become a popular model for developmental biology since its formation involves cell migration, pattern formation, organogenesis, and hair cell regeneration. Transgenic lines play a crucial role in lateral line system study. Here, we identified an enhancer trap transgenic zebrafish line Et(gata2a:EGFP)189b (ET189b for short), which expressed enhanced green fluorescent protein (EGFP) in the pituitary, otic, and lateral line placodes and their derivatives. Especially, in neuromast, the accessory cells rather than hair cells were labeled by EGFP. Furthermore, we found the Tol2 transposon construct is integrated at the proximal upstream region of six2b gene locus. And EGFP expression of ET189b closely reflects the expression of endogenous six2b during development and after dkk1b over-expression. Taken together, our results indicated that ET189b is an ideal line for research on lateral line development and regulation of six2b expression.     

New transgenic reporters identify somatosensory neuron subtypes in larval zebrafish

To analyze somatosensory neuron diversity in larval zebrafish, we identified several enhancers from the zebrafish and pufferfish genomes and used them to create five new reporter transgenes. Sequential deletions of three of these enhancers identified small sequence elements sufficient to drive expression in zebrafish trigeminal and Rohon‐Beard (RB) neurons. One of these reporters, using the Fru.p2x3‐2 enhancer, highlighted a somatosensory neuron subtype that expressed both the p2rx3a and pkcα genes. Comparison with a previously described trpA1b reporter revealed that it highlighted the same neurons as the Fru.p2x3‐2 reporter. To determine whether neurons of this subtype possess characteristic peripheral branching morphologies or central axon projection patterns, we analyzed the morphology of single neurons. Surprisingly, although these analyses revealed diversity in peripheral axon branching and central axon projection, PKCα/p2rx3a/trpA1b‐expressing RB cells did not possess obvious characteristic morphological features, suggesting that even within this molecularly defined subtype, individual neurons may possess distinct properties. The new transgenes created in this study will be powerful tools for further characterizing the molecular, morphological, and developmental diversity of larval somatosensory neurons. © 2012 Wiley Periodicals, Inc., 2013     

Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish

The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest cells (NCC). The developmental controls that govern the migration, proliferation and patterning of the ENS precursors are not well understood. We have investigated the roles of endoderm and Sonic hedgehog (SHH) in the development of the ENS. We show that endoderm is required for the migration of ENS NCC from the vagal region to the anterior end of the intestine. We show that the expression of shh and its receptor ptc-1 correlate with the development of the ENS and demonstrate that hedgehog (HH) signaling is required in two phases, a pre-enteric and an enteric phase, for normal ENS development. We show that HH signaling regulates the proliferation of vagal NCC and ENS precursors in vivo. We also show the zebrafish hand2 is required for the normal development of the intestinal smooth muscle and the ENS. Furthermore we show that endoderm and HH signaling, but not hand2, regulate gdnf expression in the intestine, highlighting a central role of endoderm and SHH in patterning the intestine and the ENS.     

Limited Impact of 6-Mercaptopurine on Inflammation-Induced Chemokines Expression Profile in Primary Cultures of Enteric Nervous System

Increasing evidences indicate that the enteric nervous system (ENS) and enteric glial cells (EGC) play important regulatory roles in intestinal inflammation. Mercaptopurine (6-MP) is a cytostatic compound clinically used for the treatment of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn’s disease. However, potential impacts of 6-MP on ENS response to inflammation have not been evaluated yet. In this study, we aimed to gain deeper insights into the profile of inflammatory mediators expressed by the ENS and on the potential anti-inflammatory impact of 6-MP in this context. Genome-wide expression analyses were performed on ENS primary cultures exposed to lipopolysaccharide (LPS) and 6-MP alone or in combination. Differential expression of main hits was validated by quantitative real-time PCR (qPCR) using a cell line for EGC. ENS cells expressed a broad spectrum of cytokines and chemokines of the C-X-C motif ligand (CXCL) family under inflammatory stress. Induction of Cxcl5 and Cxcl10 by inflammatory stimuli was confirmed in EGC. Inflammation-induced protein secretion of TNF-α and Cxcl5 was partly inhibited by 6-MP in ENS primary cultures but not in EGC. Further work is required to identify the cellular mechanisms involved in this regulation. These findings extend our knowledge of the anti-inflammatory properties of 6-MP related to the ENS and in particular of the EGC-response to inflammatory stimuli.

     

Functional analysis of zebrafish GDNF

We have identified zebrafish orthologues of glial cell line-derived neurotrophic factor (GDNF) and the ligand-binding component of its receptor GFRalpha1. We examined the mRNA expression pattern of these genes in the developing spinal cord primary motor neurons (PMN), kidney, and enteric nervous systems (ENS) and have identified areas of correlated expression of the ligand and the receptor that suggest functional significance. Many aspects of zebrafish GDNF expression appear conserved with those reported in mouse, rat, and avian systems. In the zebrafish PMN, GFRalpha1 is only expressed in the CaP motor neuron while GDNF is expressed in the ventral somitic muscle that it innervates. To test the functional significance of this correlated expression pattern, we ectopically overexpressed GDNF in somitic muscle during the period of motor axon outgrowth and found specific perturbations in the pattern of CaP axon growth. We also depleted GDNF protein in zebrafish embryos using morpholino antisense oligos and found that GDNF protein is critical for the development of the zebrafish ENS but appears dispensable for the development of the kidney and PMN.     

A zebrafish SKIV2L2-enhancer trap line provides a useful tool for the study of peripheral sensory circuit development

The zebrafish is an ideal model for elucidating the cellular and molecular mechanisms that underlie development of the peripheral nervous system. A transgenic line that selectively labels all the sensory circuits would be a valuable tool for such investigations. In this study, we describe such a line: the enhancer trap zebrafish line Tg(SKIV2L2:gfp)^j1775 which expresses green fluorescent protein (gfp) in the peripheral sensory ganglia. We show that this transgene marks all peripheral ganglia and sensory nerves, beginning at the time when the neurons are first extending their processes, but does not label the efferent nerves. The trapped reporter is inserted just upstream of a previously poorly described gene: lhfpl4 on LG6. The expression pattern of this gene by in situ hybridization reveals a different, but overlapping, pattern of expression compared to that of the transgene. This pattern also does not mimic that of the gene (skiv2l2), which provided the promoter element in the construct. These findings indicate that reporter expression is not dictated by an endogenous enhancer element, but instead arises through an unknown mechanism. Regardless, this reporter line should prove to be a valuable tool in the investigation of peripheral nervous system formation in the zebrafish.     

Cell death and the developing enteric nervous system

This review discusses current knowledge about cell death in the developing enteric nervous system (ENS). It also includes findings about the molecular mechanisms by which such death is mediated. Additional consideration is given to trophic factors that contribute to survival of the precursors and neurons and glia of the ENS, as well to genes that, when mutated or deleted, trigger their death. Although further confirmation is needed, present observations support the view that enteric neural crest-derived precursor cells en route to the gut undergo substantial levels of apoptotic death, but that once these cells colonize the gut, there is relatively little death of precursor cells or of neurons and glia during the fetal period. There are also indications that normal neuron loss occurs in the ENS, but at times beyond the perinatal stage. Taken together, these findings suggest that ENS development is similar is some ways, but different in others from extra-enteric areas of the vertebrate central and peripheral nervous systems, in which large-scale apoptotic death of precursor neurons and glia occurs during the fetal and perinatal periods. Potential reasons for these differences are discussed such as a fetal enteric microenvironment that is especially rich in trophic support. In addition to the cell death that occurs during normal ENS development, this review discusses mechanisms of experimentally-induced ENS cell death, such as those that are associated with defective glial cell-line derived neurotrophic factor/Ret signaling, which are an animal model of human congenital megacolon (aganglionosis; Hirschsprung’s disease). Such considerations underscore the importance of understanding cell death in the developing ENS, not just from a curiosity-driven point of view, but also because the pathophysiology behind many disorders of human gastrointestinal function may originate in abnormalities of the mechanisms that govern cell survival and death during ENS development.     

From neural crest to bowel: Development of the enteric nervous system

The ENS resembles the brain and differs both physiologically and structurally from any other region of the PNS. Recent experiments in which crest cell migration has been studied with DiI, a replication-deficient retrovirus, or antibodies that label cells of neural crest origin, have confirmed that both the avian and mammalian bowel are colonized by émigrés from the sacral as well as the vagal level of the neural crest. Components of the extracellular matrix, such as laminin, may play roles in enteric neural and glial development. The observation that an overabundance of laminin develops in the presumptive aganglionic region of the gut in Is/Is mutant mice and is associated with the inability of crest-derived cells to colonize this region of the bowel has led to the hypothesis that laminin promotes the development of crest-derived cells as enteric neurons. Premature expression of a neuronal phenotype would cause crest-derived cells to cease migrating before they complete the colonization of the gut. The acquisition by crest-derived cells of a nonintegrin, nervespecific, 110 kD laminin-binding protein when they enter the bowel may enable these cells to respond to laminin differently from their pre-enteric migrating predecessors. Crest-derived cells migrating along the vagal pathway to the mammalian gut are transiently catecholaminergic (TC). This phenotype appears to be lost rapidly as the cells enter the bowel and begin to follow their program of terminal differentiation. The appearance and disappearance of TC cells may thus be an example of the effects of the enteric microenvironment on the differentiation of crest-derived cells in situ. Crest-derived cells can be isolated from the enteric microenvironment by immunoselection, a method that takes advantage of the selective expression on the surfaces of crest-derived cells of certain antigens. One neurotrophin, NT-3, promotes the development of enteric neurons and glia in vitro. Because trkC is expressed in the developing and mature gut, it seems likely that NT-3 plays a critical role in the development of the ENS in situ. Although the factors that are responsible for the development of the unique properties of the ENS remain unknown, progress made in understanding enteric neuronal development has recently accelerated. The application of new techniques and recently developed probes suggest that the accelerated pace of discovery in this area can be expected to continue. © 1993 John Wiley & Sons, Inc.     

Distinct Neuroblastoma-associated Alterations of PHOX2B Impair Sympathetic Neuronal Differentiation in Zebrafish Models

Heterozygous germline mutations and deletions in PHOX2B, a key regulator of autonomic neuron development, predispose to neuroblastoma, a tumor of the peripheral sympathetic nervous system. To gain insight into the oncogenic mechanisms engaged by these changes, we used zebrafish models to study the functional consequences of aberrant PHOX2B expression in the cells of the developing sympathetic nervous system. Allelic deficiency, modeled by phox2b morpholino knockdown, led to a decrease in the terminal differentiation markers th and dbh in sympathetic ganglion cells. The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects. We demonstrate that Phox2b is capable of regulating itself as well as ascl1, and that phox2b deficiency uncouples this autoregulatory mechanism, leading to inhibition of sympathetic neuron differentiation. This effect on terminal differentiation is associated with an increased number of phox2b⁺, ascl1⁺, elavl3⁻ cells that respond poorly to retinoic acid. These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.     

Expression of PROKR1 and PROKR2 in human enteric neural precursor cells and identification of sequence variants suggest a role in HSCR

Background

The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung''s disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS.

Principal Findings

In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function.

Conclusions

Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.     

Establishment of a transgenic zebrafish EF1α:Kaede for monitoring cell proliferation during regeneration

Zebrafish is considered as a versatile experimental animal for various research models from development to diseases. In this study, we report the development of transgenic zebrafish line named as Tg(EF1α:Kaede) that expresses translation elongation factor 1 subunit alpha (EF1α) promoter linked to a fluorescent protein (FP), Kaede for monitoring proliferating cells in during regeneration. It was revealed that about 1.4 kb 5′-flanking region of the EF1α was sufficient for its promoter activity. Expression of Kaede with a property of photo-conversion from green to red was detected in different embryonic stages as well as various organs such as brain, heart, pancreas, intestine, ovary, and fins of adult fish. Cell proliferation pattern during fin regeneration was monitored after amputation of Tg(EF1α:Kaede) caudal fin and results shown that this system is simple and efficient method for detecting proliferating cells during tissue regeneration. Developed Tg(EF1α:Kaede) line has potential to investigate the cell proliferation, regeneration, wound healing capacities after tissue damage and evaluate the therapeutic power of wound healing drugs.     

GDNF availability determines enteric neuron number by controlling precursor proliferation

To clarify the role of Ret signaling components in enteric nervous system (ENS) development, we evaluated ENS anatomy and intestinal contractility in mice heterozygous for Ret, GFRalpha1 and Ret ligands. These analyses demonstrate that glial cell line-derived neurotrophic factor (GDNF) and neurturin are important for different aspects of ENS development. Neurturin is essential for maintaining the size of mature enteric neurons and the extent of neuronal projections, but does not influence enteric neuron number. GDNF availability determines enteric neuron number by controlling ENS precursor proliferation. However, we were unable to find evidence of programmed cell death in the wild type ENS by immunohistochemistry for activated caspase 3. In addition, enteric neuron number is normal in Bax(-/-) and Bid(-/-) mice, suggesting that, in contrast to most of the rest of the nervous system, programmed cell death is not important for determining enteric neuron numbers. Only mild reductions in neuron size and neuronal fiber counts occur in Ret(+/-) and Gfra1(+/-) mice. All of these heterozygous mice, however, have striking problems with intestinal contractility and neurotransmitter release, demonstrating that Ret signaling is critical for both ENS structure and function.