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Acta histochemica 110 (2008) 163—171

0065-1281/$ - sdoi:10.1016/j.

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The development of avian enteric nervous system:Distribution of artemin immunoreactivity

Lucianna Maruccio�, Carla Lucini, Finizia Russo,Rosanna Antonucci, Luciana Castaldo

Dipartimento di Strutture, Funzioni e Tecnologie Biologiche, Universita degli Studi di Napoli ‘‘Federico II’’,Via Veterinaria 1, 80137 Naples, Italy

Received 29 May 2007; received in revised form 14 September 2007; accepted 1 October 2007

KEYWORDSBirds;Ducks;Gut;Intestine;Enteric nervoussystem;GDNF family

ee front matter & 2007acthis.2007.10.001

ing author. Tel.: +39 816097.ess: lmaruccio@virgilio

SummaryAmong the factors that control neural crest cell precursors within the entericnervous system, the ligands of the glial cell line-derived neurotrophic factor family(GFL) seem to be the most influential. Artemin, a member of the GFLs, waspreviously described only in the oesophagus and stomach of mouse embryos. In thisstudy, the presence and distribution of artemin is reported in duck embryos andadults. Artemin immunoreactivity was apparent in the intestinal tract at embryonicday 7 (d7), firstly in the myenteric plexus and then in the submucous plexus. Later,artemin immunoreactive nerve fibres were also seen in the longitudinal muscleplexus, the circular muscle plexus, the plexus of the muscularis mucosa and in themucosal plexus. Furthermore, at d7, weak labeling of artemin was detected inneurons and glial cells in the oesophagus, gastric region and duodenum.Subsequently, artemin was also detected in all other intestinal segments. Moreover,during development of the gut in the domestic duck, artemin immunoreactivitydecreased in neuronal cell bodies, whilst it increased in neuronal fibres and glialcells. These findings suggest an involvement of artemin in the development andbiology of the gut of the domestic duck.& 2007 Elsevier GmbH. All rights reserved.

Introduction

The enteric nervous system (ENS) derives fromthe vagal and the sacral regions of the neural crest

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2536129;

.it (L. Maruccio).

derived cells (NCC). The former contributes themajority of ENS precursors along the entire lengthof the gut, and the latter contributes a smallernumber of cells, which are mainly restricted to thehindgut (Le Douarin and Kalcheim, 1999). In thechick embryo, vagal NCC migrate from the neuraltube to a position posterior to the branchial arches,where they enter the foregut and then travel in a

rved.

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rostro-caudal direction to populate the gut tube.Sacral NCC initially form the nerve of Remak and,after the gut has been colonized by vagal NCC,move into the gut in a caudo-rostral direction.Among the factors that control NCC precursors,within the ENS, the ligands of the glial cell-derivedneurotrophic factor family (GFLs) seem to be themost influential (for review see Enomoto, 2005).The GFLs comprise four known members: glial cell-derived neurotrophic factor (GDNF), neurturin(NRTN), artemin (ARTN) and persephin (PSPN).They signal through multicomponent receptorsformed by a common receptor tyrosine kinase(RET) and by one of a family of glycosyl phospha-tidylinositol (GPI)-linked specific co-receptors,GRFa1–4 (Rosenthal, 1999). GDNF, the first memberof the GFLs to be discovered, was shown to play afundamental role in survival, proliferation anddifferentiation during ENS development (Fockeet al., 2003; Gianino et al., 2003; Worley et al.,2000). Moreover, GDNF is chemoattractive toenteric NCC in and along the gastrointestinal tract(Natarajan et al., 2002; Young et al., 2001). NRTNpromotes the proliferation and differentiation ofNCC in vitro (Heuckeroth et al., 1998; Taraviraset al., 1999), and it is chemoattractive to entericNCC (Yan et al., 2004). Furthermore, mice gener-ated with mutations in GDNF, RET or GFRa1 lackenteric neurons in the small and large intestines(Moore et al., 1996; Pichel et al., 1996; Sanchezet al., 1996). In contrast, deficiency of NRTN,or its corresponding co-receptor GFRa2, resultsin less severe abnormalities of the ENS, and areless severe than those obtained by gdnf and itsreceptor ablation, such as reduced myentericplexus innervation and abnormal gastrointestinalmotility (Heuckeroth et al., 1999; Rossi et al.,2003).

Regarding ARTN, it is considered a survival factorfor sensory and sympathetic neurons in culture andfor dopaminergic midbrain neurons (Baloh et al.,1998; Rosenblad et al., 2000). In the gut, ARTN wasdetected in the oesophagus and the stomach ofmouse embryos (Enomoto et al., 2001; Yan et al.,2004), but it did not appear to induce migration orneurite outgrowth in the oesophagus or anygastrointestinal region examined. There are nodata reported regarding ARTN in the gut of avianembryos, although there are some studies on RET,GFRa1 and GFRa2 (Homma et al., 2000; Robertsonand Mason, 1995; Schiltz et al., 1999; Schuchardtet al., 1995). In order to extend the knowledgeregarding GFLs in avian embryos and also tocontinue our previous studies on the developmentof ENS (Lucini et al., 1993; Vittoria et al., 1992),the aim of this study was to determine the

localization of ARTN immunoreactivity in the gutof duck embryos.

Materials and methods

Embryos and adult subjects of the domestic duck(Anas platyrhyncos platyrhyncos) were examined.The embryos were taken from fertilized eggs andincubated at 38.570.2 1C and at 8472% relativehumidity. Specimens were collected daily from d4to d9, and then on alternate days from d9 to birth.Three specimens for each developmental stage plusfive adult ducks were examined. The embryos werekilled by decapitation while the adult ducks wereanaesthetized with ether. The embryos up to d7were fixed and embedded entire and undissected.The later embryonic and adult gut was removedand divided into oesophagus, proventriculus,gizzard, antrum (gizzard–duodenum junction),duodenum, jejunum–ileum, caecum and rectum.The samples were fixed by immersion in Bouin’sfluid for 24–48 h at room temperature (RT),dehydrated, and then embedded in paraffin wax.Tranverse sections of 7-mm-thick were cut using amicrotome.

Single-immunohistochemical labeling

Immunohistochemical labeling was performedusing the peroxidase–antiperoxidase (PAP) methoddescribed by Sternberger (1986). After dewaxing,sections were rinsed in distilled water, immersed in0.01M sodium citrate buffer, pH 6.0, and heated ina microwave oven for 10min at 750W (Reynoldset al., 1994) to unmask antigens. Then, the sectionswere rinsed in distilled water and treated with 3%H2O2 for 20min to block the endogenous peroxidaseactivity. Non-specific background labeling wasprevented by incubating the sections with normalrabbit serum (S-1000; Vector, Burlingame, CA, USA)diluted 1:5, for 30min prior to incubation withprimary antibody in a humid chamber, for 24 h, at4 1C. Goat polyclonal antibody against an epitopemapping near the C-terminus of all forms ofartemin, (C-18; sc 9330 Santa Cruz Biotechnology,Santa Cruz, CA, USA), diluted 1:200, was used asthe primary antibody. Sections were then incubatedwith antiserum raised in rabbit against goat IgG(Z0228, Dako A/S Glostrup, Denmark), diluted 1:50,for 30min; then with goat PAP complex (Z0113,Dako A/S), diluted 1:100, for 30min. Immunoreac-tive sites were visualized by incubation with afreshly prepared solution of 10 mg of 3,30-diamino-benzidine tetrahydrochloride (DAB) (Sigma-Aldrich,St. Louis, MO, USA) in 15ml of 0.5M Tris buffer, at

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pH 7.6, containing 1.5ml of 0.03% H2O2. Sectionswere lightly counterstained with Mayer’s hematox-ylin in order to ascertain structural details, andobserved using a Leitz DM RA2 microscope. Allstages were performed at RT unless otherwisestated. All dilutions and thorough washes betweensteps were performed in PBS unless otherwisestated. Photomicrographs were taken with aDC300F camera, stored in IM 1000 archive, andsuccessively processed using Leica Qwin and CorelDraw 10.

Double immunolabeling

Consecutive sections were dewaxed, rehydratedand microwave treated as described previously.They were then rinsed in PBS and incubated for 48 hwith the following antisera mixtures diluted 1:5with normal donkey serum (017-000-121 Jackson,PA, USA): (a) anti-ARTN (C-18, sc9330, Santa CruzBiotechnology, Santa Cruz, CA, USA), diluted 1:20and anti-protein gene product (PGP) 9.5 (P 9102-71, US Biological, Swampscott, MA, USA) diluted1:100 and (b) anti-ARTN and S-100 (119-01, Signet,Dedham, MA, USA), both diluted 1:20. Sectionswere then incubated for 2 h with a mixturecontaining fluorescein isothiocyanate (FITC)-conju-gated donkey anti goat IgG (705-095-147, Jackson,PA, USA), diluted 1:30, and tetramethyl rhodamineisothiocyanate (TRITC)-conjugated donkey anti-rabbit IgG (711-025-152 Jackson, PA, USA), diluted1:50. Both secondary antibodies were isolated fromantisera by immunoaffinity chromatography. Final-ly, the sections were mounted with glycerin diluted1:1 with PBS. All stages were performed at RTunless otherwise stated. All dilutions and thoroughwashes between steps were performed in PBSunless otherwise stated.

Controls

The specificity of the immunoreactivity wastested by successively substituting either theprimary, the secondary antisera, or PAP complexwith PBS or normal serum, in repeated trials.Adsorption controls and dot blotting tested thecross-reactivity of the anti-artemin antibody.Adsorption controls were performed by using anti-artemin antibody pre-adsorbed with excessiveamounts of its homologous (25 mg/ml) and hetero-logous (50 mg/ml) antigens. The control peptidesused were artemin (sc-9330 P), GDNF (sc-328P),NRTN (sc-6362P) and PSPN (sc-8686P) (all fromSanta Cruz Biotechnology, Santa Cruz, CA, USA).For dot blotting analysis, strips of nitrocellulose

were cut and 2 ml drops of control peptides ofdecreasing concentration (50, 25, 12.5, 6.25, and3.125 mg) were spotted onto the strips and allowedto dry at RT. Then, the strips were fixed withBouin’s fluid for 1 h. They were then treated asfollows: (1) 25min washing in PBS and 1% Triton X(PBS-T); (2) blocking with normal rabbit serum,diluted 1:5, for 1 h; (3) washing in PBS-T for 5min;(4) incubation with anti-artemin antibody over-night at 4 1C; (5) washing in PBS-T for 30min;(6) incubation with rabbit anti-goat IgG, diluted1/100, for 30min; (7) washing in PBS-T for 30min;(8) incubation with PAP, diluted 1:200 for 30min;(9) washing in PBS-T for 30min; (10) incubationwith DAB substrate for 10–45min. All reagents aslisted previously. All steps performed at RT unlessotherwise specified.

Results

Controls

The controls obtained by substituting a bufferfor the anti-artemin antibody, the anti-rabbitIgG or the PAP complex resulted in a completeabsence of labeling. Moreover, no immunoreactiv-ity was detected in sections incubated with theantiserum adsorbed with its homologous antigen.The immunolabeling pattern was not modified byincubating sections with artemin adsorbed byheterologous antigens. Dot blot analysis showedno cross-reaction of the anti-artemin antibody withtested GDNF, PSPN and NRTN peptides (data notshown).

Immunolabeling of ARTN in the ENS duringembryonic development

ARTN immunoreactivity was first detected withinthe ENS at d7 and double immunolabeling with theneuronal marker PGP 9.5 (Figure 1A and B) or glialmarker S-100 (Figure 1C and D) allowed its specificlocalization.

Weak immunolabeling of ARTN was first observedat d7 in both the cytoplasmic region and cellularprocesses of small cell clusters that were distrib-uted peripherally in the mesenchyme of oesopha-gus, proventriculus, gizzard (Figure 2A), antrumand duodenum. At d9, the circular muscle layer waswell developed only in more cranial segments. Inthe oesophagus, proventriculus (Figure 2B), gizzardand antrum, ARTN immunoreactive glial cells andneurons were observed in the myenteric plexus.ARTN immunolabeling was also detected within

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Figure 1. Double immunofluorescent labeling. ARTN immunoreactivity in a ganglion of the submucous duodenal plexusof hatching duck (A) and ARTN immunoreactivity in a ganglion of myenteric duodenal plexus of adult duck (C) arelocalized in neurons and glial cells, respectively, as demonstrated by co-localization with the neuronal marker PGP9.5(B) and the glial cell marker S100 (D). Scale bar ¼ 10 mm.

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neurons and fibres extending towards the outerlayer of the gut wall in the subserous plexus.

At d11 the myenteric plexus of the entireintestine featured ARTN immunolabeling withinboth neurons and glial cells (Figure 2C). ARTNlabeled neurons and glial cells were also observedwithin the submucosal plexuses of the oesophagus,jejunum–ileum, caecum and rectum. This plexusnever develops in the gizzard and antrum.Rare scattered ARTN, immunoreactive fibres wereseen in the circular muscle layer of all seg-ments and in the proventriculus mucosa arounddeveloping glands. ARTN immunolabeling was alsodetected in the subserous plexus, mainly in glialcells (Figure 2C).

From d13 to d17 ARTN immunoreactivity in-creased in all gut segments examined, especiallyin glial cells of the myenteric plexus (Figure 2D).Neurons and glial cells of the submucosal plexuscontinued to label for ARTN (Figure 2D) and a smallnumber of nerve fibres extending into the circularmuscle layer were also ARTN immunoreactive(Figure 2D).

From d19 to d21 intense ARTN immunoreactivitywas mainly observed in glial cells and in someneurons of the submucosal (Figure 2E) and myen-teric (Figure 2F) plexuses. Rare ARTN immunor-eactive fibres were observed in the circular muscleplexus and in the mucosal plexus.

From d23 to hatching, ARTN immunoreactivityfurther increased in glial cells of all plexuses(Figure 3A, B), whereas only some neurons ofmyenteric and submucosal plexuses (Figure 3B)showed ARTN immunoreactivity.

Immunolabeling of ARTN in the ENSof the adult duck

In the oesophagus, intense ARTN immunoreactiv-ity was detected in numerous glial cells and in someneurons of the myenteric and submucosal plexuses.In the proventriculus ARTN, immunoreactivity wasdetected in some neurons and in glial cells ofmyenteric (Figure 3C) and submucosal plexuses.ARTN immunoreactive nerve fibres were seen also

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Figure 2. ARTN immunoreactivity in the developing enteric nervous system of the duck. PAP method. (A) Someimmunopositive neurons (arrow) in the mesenchyme of gizzard at d7, in the inset enlargement of positive neurons;(B) neurons and glial cells in ganglia (arrows) in the mesenchyme of proventriculus at d9; (C) glial cells in ganglia ofduodenal subserous plexus (asterisk) and of myenteric plexus (arrow) at d11; (D) neurons and glial cells in the myenteric(arrow) and submucous (head arrow) plexuses, and fibres in the circular muscle layer (black arrow) of ileum at d15;(E) ganglia of the submucous plexus (arrow) of caecum at d19; (F) some neurons (arrow) and glial cells (arrowhead) inmyenteric plexus ganglion of gizzard at d19. ep ¼ epithelial cells, cm ¼ circular muscle, s ¼ serosa, dw ¼ duodenalwall. Scale bar of (A–F) ¼ 10 mm; scale bar of inset ¼ 5mm.

Artemin in developing duck enteric nervous system 167

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Figure 3. ARTN immunoreactivity in the enteric nervous system of the duck at hatching (A, B) and in adults (C, D, E),PAP method. (A) Neurons (arrow) and glial cells (arrowhead) in the muscle layer of the gizzard; (B) some positiveneurons (arrows) and glial cells (arrowhead) in ganglia of the myenteric and submucous plexuses of duodenum;(C) ganglion of the myenteric plexus of proventriculus showing deeply labeled neurons; (D) ganglion of the longitudinalmuscle plexus of duodenum; (E) enlargement of (D) showing unlabeled neurons (arrows) surrounded by immunopositivefibre and glial cells (arrowheads). Ep ¼ epithelium, cm ¼ circular muscle, lm ¼ longitudinal muscle. Scale bar of(A–D) ¼ 10 mm, scale bar of (E) ¼ 5mm.

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around the composite and the simple superficialglands.

In the gizzard and antrum, the outer longitudinalmuscle primordium does not differentiate duringdevelopment, leaving the myenteric plexus lyingjust within the serosa. Moreover, no submucosalplexus ganglia are present (Bennett and Cobb,1969). ARTN immunoreactivity was observed in glialcells and in some neurons of the myenteric plexus.ARTN immunoreactive nerve fibres and glial cellsalso were seen in the myenteric, circular andmucosal plexuses.

In the small intestine and in the caecum,ARTN immunoreactivity was observed in glialcells and in rare neurons of myenteric andsubmucosal plexuses. Immunoreactive fibres andglial cells were also detected in the longi-tudinal (Figure 3D, E) and circular muscle layer.However, only a few fibres were detected inthe muscularis mucosa and in the stroma ofintestinal villa.

In the rectum, a very small number of ARTNimmunoreactive neurons and glial cells of both themyenteric and submucosal plexuses were identi-fied. Nerve fibres imunopositive for ARTN were alsodetected in the longitudinal and circular musclelayers and to a lesser degree in the muscularismucosa.

Discussion

This study reports the immunolocalization ofARTN in the ENS during development of theduck and into adult life. To our knowledge, this isthe first report of the distribution of this neuro-trophic factor in birds. Although the antiserumemployed in this study was directed againsthuman artemin, it worked well in duck tissues.Results of positive and negative controls suggestthe presence of proteins similar to mammalianARTN in duck gut.

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Artemin in developing duck enteric nervous system 169

Weak immunolabeling of ARTN was initiallydetected in neurons and glial cells of the oesophago-gastric region, and in the duodenum, then laterin the other more caudal intestinal segments. Thus,ARTN immunoreactive cells exhibit a temporalrostro-caudal pattern of distribution during earlyduck gut development which is similar to therostro-caudal wave of migration of vagal NCCdescribed in other avian species (for a review seeBurns and Le Douarin, 1998).

Furthermore, ARTN immunolabeling was initiallydetected in the outermost layers of the mesench-yme in the individual regions of the gut and thenalso within the inner layers upon completion ofdifferentiation of the circular muscle layer. Thus,ARTN immunoreactivity was detected firstlyin the myenteric plexus and then in the sub-mucous plexus. The outside-inside pattern ofdistribution has also been reported for someneurotransmitters such as vasoactive intestinalpeptide, substance P, enkephalin, neurotensin,(Lucini et al., 1993; Saffrey et al., 1982; Vittoriaet al., 1992) and for some neuronal markers(Burns and Le Douarin, 1998) in duck and chickembryos. Nevertheless, a careful comparison be-tween the localization of ARTN immunoreactivitydescribed in this study and the previously reportedlocalization of VIP and substance P in the samespecies (Lucini et al., 1993; Vittoria et al., 1992)did not suggest a co-localization. In general, ARTNimmunoreactivity was mainly detected in glialcells, whereas VIP and substance P immunor-eactivity were detected in neuronal cell bodiesand fibres.

During development of the gut in the domesticduck, the number of ARTN immunoreactive neuronsdecreased whilst the numbers of glial cells andnerve fibres labeling for ARTN immunoreacivityincreased. These results could reflect the prolif-eration rates of the different cell populationswithin the gut during its growth. Consequently,during enteric growth, fibres have to extendtowards targets. Furthermore, because the ENS isformed by multiple lineages of neural crest-derivedprecursor cells (for a review see Gershon, 1997),the majority of ARTN-positive neurons could re-present a transient subpopulation which gives riseto other differently characterized neurons duringduck development. However, we cannot excludethat the reduction of ARTN immunoreactive neu-rons could be due to cell death because of neuronalcompetition for small amounts of neurotrophicfactors produced by tissue targets (Cowan andO’ Leary, 1984).

ARTN is considered to be a survival factor forsensory and sympathetic neurons in culture and for

dopaminergic midbrain neurons (Baloh et al., 1998;Rosenblad et al., 2000). Moreover, ARTN exerts adiversity of actions on sympathetic neurons atdifferent developmental stages: promotes sympa-thetic neuroblast proliferation and neuron genera-tion, enhances neurite growth and sustainssympathetic neuron survival (Andres et al., 2001;Yan et al., 2003). ARTN mRNA is diffusely expressedin peripheral tissues in mammals (Baloh et al.,1998; Masure et al., 1999). Previous studies haveshown that ARTN is expressed in the oesophagusand stomach of mouse embryos (Yan et al., 2004),although it was unable to induce migration orneurite outgrowth (Yan et al., 2004).

The widespread and plentiful presence of ARTNin the duck gut suggests an involvement of thisprotein in duck development and different rolescould be supposed, especially in adults, as pre-viously reported for other neurotrophic factors.GDNF are widely distributed in adult rat gut (Peterset al., 1998) and in human gut (Bar et al., 1997) andhave a function in mucosal protection and regen-eration in human inflammatory bowel disease(Steinkamp et al., 2003). Neurotrophins seem toplay fundamental roles in adult human pathologies,such as inflammatory bowel disease (di Mola et al.,2000) and constipation (Coulie et al., 2000; Faceret al., 2001).

Regarding the mode of action of ARTN in duckgut, only a limited number of suggestions can bemade. According to the neurotrophic nature ofARTN (for a review, see Neet and Campenot, 2001),the presence of ARTN in neurons could be explainedby retrograde transport of ARTN from an externalsource, for instance from glial cells which wereseen to contain ARTN. However, ARTN could besynthesized and transported anterogradally alongaxons and secreted in the synaptic region, as shownfor GFLs in mammalian sensory and motorneurons(Ohta et al., 2001; Rind and von Bartheld, 2002;Russell et al., 2000; von Bartheld et al., 2001).According to the latter hypothesis, the decrease ofARTN immunoreactive neurons reported in theadult gut compared to that of embryos could bedue to a switch of the ARTN storage from the cellbody, where it is synthesized during embryonic life,to the nerve terminal.

In conclusion, our data show the immunolocali-zation of ARTN in neurons and glial cells of thedeveloping and adult duck gut. Together with ourprevious findings regarding ARTN in adult teleostfish (Lucini et al., 2003, 2005), the study suggestsa well-conserved presence of this neurotrophicfactor in the vertebrate gut and could be importantfor future understanding of its role in the ontogenyof the ENS.

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Acknowledgements

We thank Mrs. Annamaria Zollo for her skilfultechnical assistance. This study was supported by agrant from University of Naples ‘‘Federico II’’, Polodelle Scienze e Tecnologie per la Vita, Napoli, Italy.

References

Andres R, Forgie A, Wyatt S, Chen Q, de Sauvage FJ,Davies AM. Multiple effects of artemin on sympatheticneurone generation, survival and growth. Develop-ment 2001;128:3685–95.

Baloh RH, Tansey MG, Lampe PA, et al. Artemin a novelmember of the GDNF ligand family, supports periph-eral and central neurons and signals through theGFRalpha 3-RET receptor complex. Neuron 1998;21:1291–302.

Bar KJ, Facer P, Williams NS, Tam PK, Anand P. Glial-derived neurotrophic factor in human adult and fetalintestine and in Hirschsprung’s disease. Gastroenter-ology 1997;12:1381–5.

Bennett T, Cobb JLS. Studies on the avian gizzard:the development of the gizzard and its innervation.Z Zellforsch Mikrosk Anat 1969;98:599–621.

Burns JA, Le Douarin NM. The sacral neural crestcontributes neurons and glia to the postumbilicalgut: spatiotemporal analysis of the development ofthe enteric nervous system. Development 1998;125:4335–47.

Coulie B, Szarka LA, Camilleri M. Recombinant humanneurotrophic factors accelerate colonic transit andrelieve constipation in humans. Gastroenterology2000;119:41–50.

Cowan WM, O’ Leary DDM. Cell death and processelimination: the role of regressive phenomena in thedevelopment of the vertebrate nervous system. In:Isselbacker KJ, editor. Medicine, science and society.New York: Wiley; 1984.

di Mola FF, Friess H, Zhu ZW, et al. Nerve growth factorand Trk high affinity receptor (TrkA) gene expression ininflammatory bowel disease. Gut 2000;46:670–9.

Enomoto H. Regulation of neural development by glialcell line-derived neurotrophic factor family ligands.Anat Sci Int 2005;80:42–52.

Enomoto H, Crawford PA, Gorodinsky A, Heuckeroth RO,Johnson Jr EM, Milbrandt J. RET signaling is essentialfor migration, axonal growth and axon guidance ofdeveloping sympathetic neurons. Development 2001;128:3963–74.

Facer P, Knowles CH, Thomas PK, Tam PK, Williams NS,Anand P. Decreased tyrosine kinase C expression mayreflect developmental abnormalities in Hirschsprung’sdisease and idiophatic slow-transit constipation. Br JSurg 2001;88:545–52.

Focke PJ, Swetlik AR, Schilz JL, Epstein ML. GDNF andinsulin cooperate to enhance the proliferation anddifferentiation of enteric crest-derived cells. J Neu-robiol 2003;55:151–64.

Gershon MD. Genes and lineages in the formation of theenteric nervous system. Curr Opin Neurobiol 1997;7:101–9.

Gianino S, Grider JR, Cresswell J, Enomoto H, HeuckerothRO. GDNF availability determines enteric neuronnumber by controlling precursor proliferation. Devel-opment 2003;130:2187–98.

Heuckeroth RO, Lampe PA, Johnson EM, Milbrandt J.Neurturin and GDNF promote proliferation and survi-val of enteric neuron and glial progenitors in vitro.Dev Biol 1998;200:116–29.

Heuckeroth RO, Enomoto H, Grider JR, et al. Genetargeting reveals a critical role for neurturin in thedevelopment and maintenance of enteric, sensory,and parasympathetic neurons. Neuron 1999;22:253–63.

Homma S, Oppenheim RW, Yaginuma H, Kimura S.Expression pattern of GDNF, c-ret and GFRalphassuggests novel roles for GDNF ligands during earlyorganogenesis in the chick embryo. Dev Biol 2000;217:121–37.

Le Douarin NM, Kalcheim C. The neural crest. Cambridge:Cambridge University Press; 1999.

Lucini C, Castaldo L, Cocca T, La Mura E, Vittoria A.Distribution of substance P-like immunoreactive ner-vous structures in the duck gut during development.Eur J Histochem 1993;37:173–82.

Lucini C, Maruccio L, de Girolamo P, Castaldo L. Brain-derived neurotrophic factor in higher vertebratepancreas: immunolocalization in glucagon cells. AnatEmbryol (Berl) 2003;206:311–8.

Lucini C, Maruccio L, Tafuri S, Bevaqua M, Staiano N,Castaldo L. GDNF family ligand immunoreactivity inthe gut of teleostean fish. Anat Embryol (Berl) 2005;210:265–74.

Masure S, Geerts H, Cik M, et al. Enovin a member of theglial cell-line-derived neurotrophic factor (GDNF)family with growth promoting activity on neuronalcells. Existence and tissue-specific expression ofdifferent splice variants. Eur J Biochem 1999;266:892–902.

Moore MW, Klein RD, Farinas I, et al. Renal and neuronalabnormalities in mice lacking GDNF. Nature 1996;382:76–9.

Natarajan D, Marcos-Gutierrez C, Pachnis V, de Graaff E.Requirement of signalling by receptor tyrosine kinaseRET for the directed migration of enteric nervoussystem progenitor cells during mammalian embryo-genesis. Development 2002;129:5151–60.

Neet KE, Campenot RB. Receptor binding, internaliza-tion, and retrograde transport of neurotrophic fac-tors. Cell Mol Life Sci 2001;58:1021–35.

Ohta K, Inokuchi T, Gen E, Chang J. Ultrastructural studyof anterograde transport of glial cell line-derivedneurotrophic factor from dorsal root ganglion neuronsof rats towards the nerve terminal. Cells TissuesOrgans 2001;169:410–21.

Peters RJ, Osinski MA, Hongo JA, et al. GDNF is abu-ndant in the adult rat. J Auton Nerv Syst 1998;70:115–22.

ARTICLE IN PRESS

Artemin in developing duck enteric nervous system 171

Pichel JG, Shen L, Sheng HZ, et al. Defects in entericinnervation and kidney development in mice lackingGDNF. Nature 1996;382:73–6.

Reynolds GM, Young FI, Young JA, Williams A, RowlandsDC. Microwave oven antigen retrieval applied to theimmunostaining of cytopathology specimens. Cyto-pathology 1994;5:345–58.

Rind HB, von Bartheld CS. Anterograde axonal transportof internalized GDNF in sensory and motor neurons.Neuroreport 2002;13:659–64.

Robertson K, Mason I. Expression of ret in the chickenembryo suggests roles in regionalisation of the vagalneural tube and somites and in development ofmultiple neural crest and placodal lineages. MechDev 1995;53:329–44.

Rosenblad C, Gronborg M, Hansen C, Blom N, Meyer M,Johansen J, et al. In vivo protection of nigraldopamine neurons by lentiviral gene transfer of thenovel GDNF-family member neublastin/artemin. MolCell Neurosci 2000;15:199–214.

Rosenthal A. The GDNF protein family: gene ablationstudies reveal what they really do and how. Neuron1999;22:201–3.

Rossi J, Herzig KH, Voikar V, Hiltunen PH, Segerstrale M,Airaksinen MS. Alimentary tract innervation deficitsand dysfunction in mice lacking GDNF family receptoralpha2. J Clin Invest 2003;112:707–16.

Russell FD, Koishi K, Jiang Y, McLennan IS. Anterogradeaxonal transport of glial cell line-derived neurotrophicfactor and its receptors in rat hypoglossal nerve.Neuroscience 2000;97:575–80.

Saffrey MS, Polak JM, Burnstock G. Distribution ofvasoactive intestinal polypeptide-, substance P-,enkephalin and neurotensin-like immunoreactivenerves in the cicken gut during development. Neu-roscience 1982;7:279–93.

Sanchez MP, Silos-Santiago I, Frisen J, He B, Lira SA,Barbacid M. Renal agenesis and the absence of entericneurons in mice lacking GDNF. Nature 1996;382:70–3.

Schiltz CA, Benjamin J, Epstein ML. Expression of theGDNF receptors ret and GFRalpha1 in the developingavian enteric nervous system. J Comp Neurol 1999;414:193–211.

Schuchardt A, Srinivas S, Pachnis V, Costantini F. Isolationand characterization of a chicken homolog of the c-retproto-oncogene. Oncogene 1995;10:641–9.

Steinkamp M, Geerling I, Seufferlein T, von Boyen G,Egger B, Grossmann J, et al. Glial-derived neuro-trophic factor regulates apoptosis in colonic epithelialcells. Gastroenterology 2003;124:1748–57.

Sternberger LA. Immunohistochemistry, 3rd ed. New York:Wiley; 1986.

Taraviras S, Marcos-Gutierrez CV, Durbec P, et al.Signalling by the RET receptor tyrosine kinase and itsrole in the development of the mammalian entericnervous system. Development 1999;126:2785–97.

Vittoria A, Castaldo L, la Mura E, Lucini C, Cecio A. Vip-immunoreactive nerve structures of the gastrointest-inal tract in the developing and adult domestic duck.Arch Histol Cytol 1992;55:361–74.

von Bartheld CS, Wang X, Butowt R. Anterograde axonaltransport, transcytosis, and recycling of neurotrophicfactors: the concept of trophic currencies in neuralnetworks. Mol Neurobiol 2001;24:1–28.

Worley DS, Pisano JM, Choi ED, et al. Developmentalregulation of GDNF response and receptor expressionin the enteric nervous system. Development 2000;127:4383–93.

Yan H, Newgreen DF, Young HM. Developmental changesin neurite outgrowth responses of dorsal root andsympathetic ganglia to GDNF, neurturin, and artemin.Dev Dyn 2003;227:395–401.

Yan H, Bergner AJ, Enomoto H, Milbrandt J, Newgreen DF,Young HM. Neural cells in the esophagus respond to glialcell line-derived neurotrophic factor and neurturin,and are RET-dependent. Dev Biol 2004;272:118–33.

Young HM, Hearn CJ, Farlie PG, Canty AJ, Thomas PQ,Newgreen DF. GDNF is a chemoattractant for entericneural cells. Dev Biol 2001;229:503–16.