ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.

Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the absence of enteric nervous system (ENS) in the distal region of the intestine. Down Syndrome (DS) patients have a >50-fold higher risk of developing HSCR than the general population, suggesting that overexpression of human chromosome 21 (Hsa21) genes contribute to HSCR etiology. However, identification of responsible genes remains challenging. Here, we describe a genetic screening of potential candidate genes located on Hsa21, using the zebrafish. Candidate genes were located in the DS-HSCR susceptibility region, expressed in the human intestine, were known potential biomarkers for DS prenatal diagnosis, and were present in the zebrafish genome. With this approach, four genes were selected: RCAN1, ITSN1, ATP5PO and SUMO3. However, only overexpression of ATP5PO, coding for a component of the mitochondrial ATPase, led to significant reduction of ENS cells. Paradoxically, in vitro studies showed that overexpression of ATP5PO led to a reduction of ATP5PO protein levels. Impaired neuronal differentiation and reduced mitochondrial ATP production, were also detected in vitro, after overexpression of ATP5PO in a neuroblastoma cell line. Finally, epistasis was observed between ATP5PO and ret, the most important HSCR gene. Taken together, our results identify ATP5PO as the gene responsible for the increased risk of HSCR in DS patients in particular if RET variants are also present, and show that a balanced expression of ATP5PO is required for normal ENS development.

The zebrafish mutant lessen: an experimental model for congenital enteric neuropathies.

BACKGROUND: Congenital enteric neuropathies of the distal intestine (CEN) are characterized by the partial or complete absence of enteric neurons. Over the last decade, zebrafish has emerged as a leading model organism in experimental research. Our aim was to demonstrate that the mutant zebrafish, lessen, expressing CEN characteristics, is an equally valuable animal model alongside mammalian models for CEN, by studying its enteric phenotype. METHODS: The effect of the lessen mutation on the development of the enteric nervous system (ENS), interstitial cells of Cajal (ICC), and intestinal motility in each intestinal region of mutant and wild-type (wt) zebrafish embryos at 3-6 dpf, was analyzed by immunofluorescent detection of neurochemical markers and motility assays. KEY RESULTS: Development of intestinal motility in the mutant was delayed and the majority of the observed contractions were disturbed. A significant disturbance in ENS development resulted in a distal intestine that was almost free of neuronal elements, in reduced neuronal density in the proximal and mid-intestine, and in a defect in the expression of neurochemical markers. Furthermore, markedly disturbed development of ICC gave rise to a less dense network of ICC. CONCLUSIONS & INFERENCES: The observed alterations in intestinal motility, intrinsic innervation and ICC network of the mutant in comparison with the wt zebrafish, are similar to those seen in the oligo- and aganglionic regions of the intestine of CEN patients. It is concluded that the zebrafish mutant lessen is an appropriate animal model to investigate CEN.

Early development of efferent projections from the chick tectum.

The early development of the uncrossed tectobulbar and the crossed tectospinal tracts was studied. These two projections arise from the same structure, the mesencephalon, and develop during the same time period, but follow divergent courses. We have traced the pathways followed by these projections and identified the positions at which axon guidance decisions are made. The first neurons differentiate either side of the entire rostrocaudal extent of the dorsal midline and initiate axons that extend dorsoventrally across the surface of the tectum. At the ventral edge of the tectum these axons turn abruptly and fasciculate to form a caudal descending projection to the hindbrain. These axons extend to the caudal hindbrain and do not project to the periphery along cranial nerve roots. We therefore consider this tract to be the tectobular, rather than the mesencephalic division of the trigeminal. While the tectobulbar projection is still developing, a second wave of axons is initiated, which arises from only the rostral part of the tectum. These axons grow beyond the tectobulbar turn point and continue toward the ventral midline, where they cross the floor plate, before turning caudally at the lateral edge of the main descending hindbrain tract, the ventrolateral tract. We discuss the development of these tracts with reference to possible guidance cues mediating their course.

Choices choices: regulation of precursor differentiation during enteric nervous system development.

Background The enteric nervous system (ENS) is the largest subdivision of the peripheral nervous system and forms a complex circuit of neurons and glia that controls the function of the gastrointestinal (GI) tract. Within this circuit, there are multiple subtypes of neurons and glia. Appropriate differentiation of these various cell subtypes is vital for normal ENS and GI function. Studies of the pediatric disorder Hirschprung's Disease (HSCR) have provided a number of important insights into the mechanisms and molecules involved in ENS development; however, there are numerous other GI disorders that potentially may result from defects in development/differentiation of only a subset of ENS neurons or glia. Purpose Our understanding of the mechanisms and molecules involved in enteric nervous system differentiation is far from complete. Critically, it remains unclear at what point the fates of enteric neural crest cells (ENCCs) become committed to a specific subtype cell fate and how these cell fate choices are made. We will review our current understanding of ENS differentiation and highlight key questions that need to be addressed to gain a more complete understanding of this biological process.

Genetic model system studies of the development of the enteric nervous system, gut motility and Hirschsprung's disease.

The enteric nervous system (ENS) is the largest and most complicated subdivision of the peripheral nervous system. Its action is necessary to regulate many of the functions of the gastrointestinal tract including its motility. Whilst the ENS has been studied extensively by developmental biologists, neuroscientists and physiologists for several decades it has only been since the early 1990s that the molecular and genetic basis of ENS development has begun to emerge. Central to this understanding has been the use of genetic model organisms. In this article, we will discuss recent advances that have been achieved using both mouse and zebrafish model genetic systems that have led to new insights into ENS development and the genetic basis of Hirschsprung's disease.

Collapsin-1/semaphorin D is a repellent for chick ganglion of Remak axons.

Chick collapsin-1/human semaphorin III/mouse semaphorin D is believed to guide the extension of specific axons by a repellent mechanism. Here we examine its role in the guidance of axons of the ganglion of Remak (Remak) in the developing chick intestine. Early in embryogenesis Remak axons extend parallel to, but do not enter, the intestine when collapsin-1 is expressed in the adjacent rectal wall. Remak axons later penetrate the peripheral portions of the rectal wall when collapsin-1 expression retreats from the outer muscle layer to the more internal submucosal and mucosal layers of the rectum. Extension of Remak neurites is repelled in vitro by rectum explants and also by 293T cells expressing collapsin-1. The rectal chemorepellent activity is blocked by anti-collapsin-1 antibodies. Our results suggest that collapsin-1 may help prevent Remak axons from projecting into the intestinal wall at early developmental times and later restricts Remak axon trajectories to the outer part of the intestinal muscle layer.

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 sensory axon repellent secreted from ventral spinal cord explants is neutralized by antibodies raised against collapsin-1.

During embryogenesis, different subclasses of sensory neurons extend central projections to specific locations in the spinal cord. Muscle and cutaneous afferents initially project to the same location in the dorsal cord. Later, specific muscle afferents leave other afferents behind and project into the ventral cord. Previous studies have shown that ventral spinal cord explants secrete a repellent for sensory neurites. We now find that antibodies to collapsin-1 neutralize this repellent activity. Additional data suggest that all afferents respond to collapsin-1 when they are first confined to the dorsal cord, but that ventrally projecting muscle afferents become collapsin-1 insensitive as they project into the ventral cord. Our results suggest that the transient dorsal expression of collapsin-1 prevents all efferents from entering the cord early and sustained ventral expression prevents dorsally terminating afferents from entering the ventral cord later.