Summary Roles for GFR α 1 receptors in zebrafish enteric nervous system development

The vertebrate enteric nervous system (ENS) consists of an integrated network of neurons that controls gut motility and function. Enteric neurons are principally derived from neural crest cells that migrate ventrally just posterior to the pharyngeal arches, enter the anterior gut primordium and proceed posteriorly to populate the gut tube (Newgreen and Young, 2002b). As they migrate, ENS precursors proliferate and then differentiate to form a wide variety of neuronal subtypes necessary for intestinal function (Furness, 1987; Gershon, 1994). In avian and mammalian species, a second population of neural crest from sacral regions also contributes to the distal gut (Burns and Douarin, 1998; Le Douarin and Teillet, 1973; Pomeranz et al., 1991; Serbedzija et al., 1991). If migration or proliferation of the vagal neural crest is impaired, varying lengths of the gut become aganglionic (Newgreen and Young, 2002a; Newgreen and Young, 2002b). The intestinal motility disorder Hirschprung’s Disease results from this type of impairment. Individuals with this condition have aganglionosis of varying lengths of the distal portion of the colon (Kapur, 1999; Newgreen and Young, 2002a; Newgreen and Young, 2002b; Parisi and Kapur, 2000). The ENS therefore requires proper specification, migration and proliferative expansion of enteric precursors for correct development and function. A key regulatory factor for ENS development is glial cell line-derived neurotrophic factor (GDNF), a potent trophic factor for many different types of CNS and PNS neurons (Arenas et al., 1995; Ebendal et al., 1995; Henderson et al., 1994; Mount et al., 1995; Trupp et al., 1995). GDNF is the founding member of a subgroup of the TGF β superfamily, of which there are four members: GDNF, neurturin (Kotzbauer et al., 1996), persepehin (Milbrandt et al., 1998) and artemin (Baloh et al., 1998b). The biological activities of the GDNF family ligands are mediated through a multicomponent receptor complex that consists of a GPI-linked ligand binding component, the GDNF family receptor α (GFRα) subunit and a common transmembrane signaling component, the tyrosine kinase Ret (Jing et al., 1996; Treanor et al., 1996). There are four GFRα subunits; each preferentially binds to a different GDNF family member (Baloh et al., 1998a; Baloh et al., 1997; Baloh et al., 1998b; Buj-Bello et al., 1997; Jing et al., 1996; Jing et al., 1997; Klein et al., 1997; Masure et al., 2000; Naveilhan et al., 1998; Thompson et al., 1998; Trupp et al., 1998; Worby et al., 1998). GDNF preferentially binds to GFRα1 (Jing et al., 1996; Treanor et al., 1996) but can also bind to and mediate signaling in vitro through GFR α2, the α subunit that preferentially binds to neurturin (Baloh et al., 1997; Creedon et al., 1997; Sanicola et al., 1997). The role of GDNF in ENS development has been studied extensively in vitro and in vivo. In cell culture, GDNF has been shown to promote survival, proliferation, differentiation and neurite out growth of enteric precursors (Chalazonitis et al., 1998; Hearn et al., 1998; Heuckeroth et al., 1998; Taraviras et Components of the zebrafish GDNF receptor complex are expressed very early in the development of enteric nervous system precursors, and are already present as these cells begin to enter the gut and migrate caudally along its length. Both gfra1a and gfra1b as well as retare expressed at this time, while gfra2 expression, the receptor component that binds the GDNF-related ligand neurturin, is not detected until the precursors have migrated along the gut. Gfra genes are also expressed in regions of the zebrafish brain and peripheral ganglia, expression domains conserved with other species. Enteric neurons are eliminated after injection with antisense morpholino oligonucleotides against ret or against both Gfra1 orthologs, but are not affected by antisense oligonucleotides against gfra2. Blocking GDNF signaling prevents migration of enteric neuron precursors, which remain positioned at the anterior end of the gut. Phenotypes induced by injection of antisense morpholinos against both Gfra orthologs can be rescued by introduction of mRNA for gfra1a or for gfra2, suggesting that GFRα1 and GFRα2 are functionally equivalent.