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Expression of the RET Proto-Oncogene in Human Embryos Tania Attie ´ -Bitach, 1 Marc Abitbol, 2 Marion Ge ´ rard, 2 Anne-Lise Delezoide, 1 Joelle Auge ´, 1 Anna Pelet, 1 Jeanne Amiel, 1 Vassilis Pachnis, 3 Arnold Munnich, 1 Stanislas Lyonnet, 1 and Michel Vekemans 1 * 1 De ´partment de Ge ´ne ´tique et Unite ´ de Recherches sur les Handicaps Ge ´ne ´tiques de l’Enfant, Hôpital Necker-Enfants Malades, Paris, France 2 Centre d’Etude et de Recherche The ´rapeutique en Ophtalmologie, Faculte ´ de Me ´decine Necker-Enfants Malades, Paris, France 3 MRC, The Ridgeway, Mill Hill, London, United Kingdom The patterns of RET proto-oncogene expres- sion in mouse, rat, and chicken and the anomalies observed in targeted RET mu- tants suggest that RET plays a major role in development of mouse enteric nervous sys- tem and in kidney organogenesis. Here, we report on in situ hybridization studies describing the pattern of RET proto- oncogene expression during early develop- ment of human embryos between 23 and 42 days. We show that the RET gene is ex- pressed in the developing kidney (nephric duct, mesonephric tubules, and ureteric bud), the presumptive enteric neuroblasts of the developing enteric nervous system, cranial ganglia (VII+VIII, IX, and X) and in the presumptive motor neurons of the spi- nal cord. Yet, despite the high level of RET gene expression in the kidney and in the mo- tor neurons of the developing central ner- vous system in human embryos, only rare cases with renal agenesis have been re- ported in Hirschsprung disease patients, and no clinical evidence of spinal cord in- volvement has been shown in patients car- rying RET germline mutations (i.e., multiple endocrine neoplasia syndromes and Hirsch- sprung disease). Am. J. Med. Genet. 80:481– 486, 1998. © 1998 Wiley-Liss, Inc. KEY WORDS: RET proto-oncogene; gene expression; human embryos; in situ hybridization; Hirsch- sprung disease INTRODUCTION The RET proto-oncogene encodes a tyrosine-kinase receptor of unknown function that requires ligands for dimerization (Glial cell-derived neurotrophic factor, GDNF [Durbec et al., 1996; Trupp et al., 1996], or neu- turin, NTN [Baloh et al., 1997; Klein et al., 1997; Kotzbauer et al., 1996]). RET germline mutations cause Hirschsprung disease [Edery et al., 1994; Romeo et al., 1994] and familial predispositions to cancer, namely multiple endocrine neoplasia type 2A (MEN 2A [Donis-Keller et al., 1993; Mulligan et al., 1993]), 2B (MEN 2B [Carlson et al., 1994; Eng et al., 1994; Hofstra et al., 1994]), and familial medullary thyroid carcinoma (FMTC [Donis-Keller et al., 1993; Mulligan et al, 1994]). The functional role of c-Ret during development was clarified by targeted mutagenesis experiments. Homozygous ret-/- mice have malformations of the urinary excretory system with lack of enteric neurons and die soon after birth [Schuchardt et al., 1994]. More- over, expression studies in mouse [Pachnis et al., 1993], rat [Tsuzuki et al., 1995], and chicken [Schu- chardt et al., 1995] have further confirmed the role of c-Ret in kidney and enteric nervous system develop- ment. However, nothing is known regarding the pattern of RET gene expression during human embryonic devel- opment. Here, we study the pattern of RET gene ex- pression in developing kidney and central and enteric nervous system of human embryos. Considering the high level of RET gene expression in motor neurons of the developing central nervous system and kidney, it is surprising that patients carrying RET germline muta- tions show no clinical evidence of spinal cord involve- ment and rarely show renal involvement [Calabrese et al., 1994]. Contract grant sponsor: The Association Française contre les Myopathies (AFM); Contract grant sponsor: The Association pour la Recherche contre le Cancer (ARC); Contract grant sponsor: The Caisse Nationale des Assurances Maladies (CANAM); Contract grant sponsor: IMAGE; Contract grant sponsor: The Projet Hos- pitalier de Recherche Clinique; Contract grant number: PHRC AOA 94060. *Correspondence to: Michel Vekemans, Département de Géné- tique et Unité de Recherches sur les Handicaps Génétiques de l’Enfant, INSERM U-393, Hôpital Necker-Enfants Malades, 149, rue de Se `vres, 75743 Paris Cedex 15, France. E-mail: michel. [email protected] Received 14 April 1998; Accepted 3 August 1998 American Journal of Medical Genetics 80:481–486 (1998) © 1998 Wiley-Liss, Inc.

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Page 1: Expression of theRET proto-oncogene in human Embryos

Expression of the RET Proto-Oncogene inHuman Embryos

Tania Attie-Bitach,1 Marc Abitbol,2 Marion Gerard,2 Anne-Lise Delezoide,1 Joelle Auge,1Anna Pelet,1 Jeanne Amiel,1 Vassilis Pachnis,3 Arnold Munnich,1 Stanislas Lyonnet,1 andMichel Vekemans1*1Department de Genetique et Unite de Recherches sur les Handicaps Genetiques de l’Enfant, Hôpital Necker-EnfantsMalades, Paris, France

2Centre d’Etude et de Recherche Therapeutique en Ophtalmologie, Faculte de Medecine Necker-Enfants Malades,Paris, France

3MRC, The Ridgeway, Mill Hill, London, United Kingdom

The patterns of RET proto-oncogene expres-sion in mouse, rat, and chicken and theanomalies observed in targeted RET mu-tants suggest that RET plays a major role indevelopment of mouse enteric nervous sys-tem and in kidney organogenesis. Here,we report on in situ hybridization studiesdescribing the pattern of RET proto-oncogene expression during early develop-ment of human embryos between 23 and 42days. We show that the RET gene is ex-pressed in the developing kidney (nephricduct, mesonephric tubules, and uretericbud), the presumptive enteric neuroblastsof the developing enteric nervous system,cranial ganglia (VII+VIII, IX, and X) and inthe presumptive motor neurons of the spi-nal cord. Yet, despite the high level of RETgene expression in the kidney and in the mo-tor neurons of the developing central ner-vous system in human embryos, only rarecases with renal agenesis have been re-ported in Hirschsprung disease patients,and no clinical evidence of spinal cord in-volvement has been shown in patients car-rying RET germline mutations (i.e., multipleendocrine neoplasia syndromes and Hirsch-sprung disease). Am. J. Med. Genet. 80:481–486, 1998. © 1998 Wiley-Liss, Inc.

KEY WORDS: RET proto-oncogene; geneexpression; human embryos;in situ hybridization; Hirsch-sprung disease

INTRODUCTION

The RET proto-oncogene encodes a tyrosine-kinasereceptor of unknown function that requires ligands fordimerization (Glial cell-derived neurotrophic factor,GDNF [Durbec et al., 1996; Trupp et al., 1996], or neu-turin, NTN [Baloh et al., 1997; Klein et al., 1997;Kotzbauer et al., 1996]). RET germline mutationscause Hirschsprung disease [Edery et al., 1994; Romeoet al., 1994] and familial predispositions to cancer,namely multiple endocrine neoplasia type 2A (MEN 2A[Donis-Keller et al., 1993; Mulligan et al., 1993]), 2B(MEN 2B [Carlson et al., 1994; Eng et al., 1994; Hofstraet al., 1994]), and familial medullary thyroid carcinoma(FMTC [Donis-Keller et al., 1993; Mulligan et al,1994]). The functional role of c-Ret during developmentwas clarified by targeted mutagenesis experiments.Homozygous ret−/− mice have malformations of theurinary excretory system with lack of enteric neuronsand die soon after birth [Schuchardt et al., 1994]. More-over, expression studies in mouse [Pachnis et al.,1993], rat [Tsuzuki et al., 1995], and chicken [Schu-chardt et al., 1995] have further confirmed the role ofc-Ret in kidney and enteric nervous system develop-ment.

However, nothing is known regarding the pattern ofRET gene expression during human embryonic devel-opment. Here, we study the pattern of RET gene ex-pression in developing kidney and central and entericnervous system of human embryos. Considering thehigh level of RET gene expression in motor neurons ofthe developing central nervous system and kidney, it issurprising that patients carrying RET germline muta-tions show no clinical evidence of spinal cord involve-ment and rarely show renal involvement [Calabrese etal., 1994].

Contract grant sponsor: The Association Française contre lesMyopathies (AFM); Contract grant sponsor: The Association pourla Recherche contre le Cancer (ARC); Contract grant sponsor: TheCaisse Nationale des Assurances Maladies (CANAM); Contractgrant sponsor: IMAGE; Contract grant sponsor: The Projet Hos-pitalier de Recherche Clinique; Contract grant number: PHRCAOA 94060.

*Correspondence to: Michel Vekemans, Département de Géné-tique et Unité de Recherches sur les Handicaps Génétiques del’Enfant, INSERM U-393, Hôpital Necker-Enfants Malades, 149,rue de Sevres, 75743 Paris Cedex 15, France. E-mail: [email protected]

Received 14 April 1998; Accepted 3 August 1998

American Journal of Medical Genetics 80:481–486 (1998)

© 1998 Wiley-Liss, Inc.

Page 2: Expression of theRET proto-oncogene in human Embryos

MATERIALS AND METHODSSections

The tissue sections were taken from whole humanembryos, ranging from Carnegie stages 11 to 16, ob-tained after induced abortions performed according tothe French Legislation and after allowance of the Ethi-cal Committee. Two embryos were studied at each ofthe following stages (Table I): Carnegie 11 (24 to 26days), 12 (26 to 28 days), 13 (28 to 32 days), and 16 (37to 41 days). Tissues were frozen using powdered dry iceand stored at −80°C. Cryostat sections (15 mm) weremounted on slides, fixed for 20 min with 2% paraform-aldehyde, rinsed briefly in water, dehydrated in agraded ethanol series (70, 95, and 100%), air-dried, andstored at −80°C. This procedure was devised to protectembryonic mRNAs from rapid degradation.

Hybridization ProbesHybridization probes were 45- and 60-mer oligonu-

cleotides for the short and long RET mRNA isoforms,respectively (Genset, Paris, France). The 45-mer oligo-nucleotide specific for the short RET9 isoform encom-passed the 38 end of exon 19 and the 58 end of intron 19(reverse, antisense: 58-AGCATCACAGAGAGGAAG-GATAGTGCAGAGGGGACAGCGGTGCTA-38; for-ward, sense: 58-TAGCACCGCTGTCCCCTCTGCAC-TATCCTTCCTCTCTGTGATGCT-38). The 60-mer oli-gonucleotide was in exon 20 (reverse antisense:5 8 - A A C C C A G T G T T A G T G C C A T C A G C T C T C -GTGAGTGGTACAGGACTCTCTCCAGGCCAGTTC-38; forward sense: 58-GAACTGGCCTGGAGAGA-GTCCTGTACCACTCACGAGAGCTGATGGCACTA-ACACTGGGTT-38) and thus could hybridize someRET9 transcripts also [Tahira et al., 1990]. Probeswere 38 end-labelled with a(35S)dATP (Dupont ofNemours, Mechelen, Belgium) using deoxyribonucleo-tidyl transferase (Gibco-BRL Life Technologies, CergyPontoise, France) at a specific activity of 7.108 cpm/mg,purified on Biospin Columns (Bio-Rad, Ivry sur Jeine,France) and stored at −20°C.

In Situ HybridizationThe hybridization mixture contained 50% formam-

ide, 4× SSC, 1× Denhardt’s solution, 0.25 mg/ml tRNA,0.25 mg/ml denatured herring sperm DNA, 0.25 mg/mlpoly(A), 10% dextran sulphate, 100 mmol/L DTT, anda(35S)dATP-labeled probes (6.106 cpm/ml). 100 ml of

the hybridization solution was deposited on each sec-tion. The sections were then incubated in a humidifiedchamber at 43°C for 20 hr. After hybridization, the sec-tions were washed twice in decreasing SSC solutions.After dehydration, the sections were exposed to Amer-sham Betamax X-ray films for 10 days and then toKodak NTB-2 photographic emulsion for 10 weeks at+4°C. After revelation and fixation, the sections werecounterstained, coverslipped with Eukitt, and ana-lyzed using a microscope Leitz with dark field andbright field illumination.

RESULTS

At 23 to 26 days (Carnegie 11), the RET mRNAs areonly detected in the differentiating mesonephric duct(Fig. 1). At 26 to 30 days, the RET gene is also ex-pressed in the mesonephric tubules and in the Wolffianduct merging with the cloaca (Carnegie 12, Fig. 1). At37 to 42 days, RET is expressed in the ureteric bud butnot in the surrounding metanephric blastema. Thegene expression in the developing excretory system isintense and homogeneous throughout all stages.

At 26 to 30 days, RET mRNAs are also detected inthe neural crest-derived part of the facioacoustic gan-glion complex (VII+VIII; Fig. 2). A weak RET signal isalso observed in developing neural crest-derived gan-glia of the Xth and XIth cranial nerves (data notshown). In each of these ganglia, the RET mRNAs arestill detected at 37 to 42 days (Carnegie 16), whereasthey were never detected in the Vth ganglion in ourstudy.

In the enteric nervous system, RET expression startsin the foregut at 26 to 30 days (Carnegie 12) and issubsequently detected in midgut and hindgut until 37to 42 days (Carnegie 16; Fig. 3). In the mesenchyme ofthe gut wall, the RET signal appears punctate, sug-gesting an expression in coalescent groups of cells.

In neural tube, the RET mRNAs are detected at 28 to32 days (Carnegie 13; Fig. 4). The signal extends alongthe anteroposterior axis in the ventral half of the de-veloping spinal cord in which the presumptive motorneurons are differentiating. This signal persists until37 to 42 days (Carnegie 16).

In order to study the differential expression of theshort and long RET mRNAs isoforms (RET9 andRET51 (19–21)), two different probes were used: a 45-nt-long probe specific of the short isoform (ret9) and a60-nt-long probe that could hybridize with both species(ret51). Both probes give similar signals suggestingthat the long isoform is not specifically associated withearly development in human (data not shown). Finally,no hybridization signal is detected with the senseprobes, confirming that the hybridization patterns ob-served with antisense probes are specific.

DISCUSSION

The pattern of RET gene expression during embry-onic development has been previously studied in vari-ous species including mouse, rat, and chicken [Pachniset al., 1993; Schuchardt et al., 1995; Tsuzuki et al.,1995]. However, nothing is known regarding the pat-

TABLE I. Summary of RET Expression DuringHuman Embryogenesis

Carnegie stage(age in days) Kidney

Cranialganglia

(VII+VIII,IX, X)

Entericnervoussystem

Ventralneuraltube

11(23–26 days)

Wolffian duct − − −

12(26–30 days)

Wolffian ductmesonephrictubules

+ + −

13(28–32 days)

Wolffian ductmesonephrictubules

+ + +

16(37–42 days)

Ureteric bud + + +

482 Attie-Bitach et al.

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tern of RET gene expression during human embryonicdevelopment. Here, we describe the sequential expres-sion of the RET gene in the developing excretory sys-tem, the VII+VIII cranial ganglia, presumptive entericneuroblasts, and motor neurons of the spinal cord(Table I).

In human embryos, RET mRNAs are detected in the

Wolffian duct prior to ureteric bud formation. HumanRET gene expression is also observed in the mesoneph-ric tubules. In the developing mouse, RET transcriptshave been detected in the nephric duct (day 8.5 to 10.5),the ureteric bud epithelium (day 10 to 11.5), and thegrowing tips of the renal collecting ducts (day 13.5 to17.5). Additional investigations at later stage human

Fig. 1. RET expression in the Wolffian duct. a,c,e: Photomicrographs of sections under bright-field illumination; b,d,f: photomicrographs of the sameseries of sections under dark-field illumination. a,b: Transverse section through the dorsal region of a Carnegie stage 11 embryo; c,d: same section at ahigher magnification view showing a signal restricted to the Wolffian duct (w). e,f: Transverse section through the caudal region of a Carnegie stage 12embryo; the signal is observed in the Wolffian duct merging in the cloaca (cl). n, neural tube; c, intraembryonic coelom; pi, primitive intestine. Bars,200 mm.

RET Proto-Oncogene in Human Embryos 483

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embryos are being performed to determine whetherRET is also expressed in the derivatives of the uretericbud in the metanephros. Because kidney organogenesisis based on mesenchyme-epithelium reciprocal induc-tion [Saxen, 1987], it has been postulated that RET

was the receptor for a signal from the mesenchyme thatwould induce the growth and/or branching of the ure-teric bud. Our results in human embryos along withthe recent identification of GDNF as RET ligand[Durbere et al., 1996; Trupp et al., 1996] and its ex-

Fig. 3. RET expression in the enteric nervous system. a,b: Bright-field and dark-field photomicrographs of a transverse section through the midgut(mg) of a Carnegie stage 13 embryo at a high magnification view. c,d: Bright-field and dark-field photomicrographs of a parasagittal section through themidgut of a Carnegie stage 16 embryo at high magnification view. The signal is observed in the mesenchyme of the gut wall in groups of cells that startto coalesce to form the enteric ganglia. Bars, 200 mm.

Fig. 2. RET expression in the facioacoustic ganglion (VII+VIII). Bright-field (a) and dark-field (b) photomicrographs of a transverse section throughthe otic vesicle region of a Carnegie stage 12 embryo. A strong signal is observed in the facioacoustic ganglion (VII+VIII) localized immediately anteriorto the otic vesicle (ov). Bars, 200 mm.

484 Attie-Bitach et al.

Page 5: Expression of theRET proto-oncogene in human Embryos

pression in the metanephric mesenchyme [Durbec etal., 1996; Hellmich et al., 1996; Suvanto et al., 1996]suggest that the RET signaling pathway plays a majorrole in kidney organogenesis. In keeping with this, it isworth remembering that both c-ret−/− and gdnf−/−mouse knock-out mutants have kidney agenesis [Mooreet al., 1996; Pichel et al., 1996; Sanchez et al.,. 1996;Schuchardt et al., 1994].

The human RET gene is also expressed early in neu-ral crest cells migrating rostro-caudally into the gas-trointestinal tract. Both the location and the punctatedaspect of the RET signal are suggestive of RET expres-sion in groups of cells that might coalesce to form thepresumptive enteric ganglia. Consistently, ret gene ex-pression in mouse developing gut has been detected inpresumptive neuroblasts of the vagal crest (day 9.5 to11.5) and in myenteric ganglia (day 13.5 to 14.5 [Pach-nis et al., 1993]).

In humans, RET mRNAs were detected in the neuralcrest-derived part of the facioacoustic ganglion complexand, to a lesser extent, in that of the Xth and XIth cra-nial nerve complex ganglia. The RET gene was neverexpressed in the Vth ganglion in our study. By contrast,mouse ret transcripts were first detected in neuralcrest cells migrating from rhombomere 4 (day 8.5 to9.5), then in the facioacoustic ganglion complex (day9.5), the inferior ganglia of the IXth and Xth cranialnerve ganglion complexes (day 10.5), and finally in allganglia, including the IXth and Xth superior gangliaand the trigeminal ganglion (day 13.5 to 14.5 [Pachniset al., 1993]). These results differ therefore from thatobserved in early human embryos in which RET geneexpression is limited to the neural crest-derived part ofthe cranial ganglia.

In human neural tube, RET gene expression is firstdetected in the motor neurons at day 28 to 32 and isstill present at day 37 to 42. No RET expression wasdetected in neuroepithelial cells of the neural tube. Inmouse, however, ret transcripts were detected early inthe neuroepithelial cells of the ventral half of the neu-ral tube (day 8.75) prior to expression in the motorneurons of the spinal cord (day 10.5 to 14.5) and somemotor neurons of the hindbrain [Pachnis et al., 1993].

As far as pathological findings in patients with RETgermline mutations are concerned, our study supportsthe role of RET in the development of the enteric ner-vous system and fits well with the absence of ganglia inthe distal colon of HSCR patients. Consistently, entericnervous system ganglia are lacking throughout thegastrointestinal tract of c-ret−/− mice [Schuchardt etal., 1994]. By contrast, as renal abnormalities are rarein HSCR patients (only 3 in 200 in our series), bothc-ret−/− and gdnf−/− mutant mice display kidney agen-esis or dysgenesis {Moore et al., 1996; Pichel et al.,1996; Sanchez et al., 1996; Schuchardt et al., 1994].The discrepancy between human and mouse could berelated to either gene dosage effect or to redundancy inhuman developing kidney. Interestingly, as most pa-tients carrying heterozygous RET mutations do notdisplay cranial nerve anomalies, it is worth remember-ing that the congenital central hypoventilation syn-drome (CCHS, Ondine’s curse) was observed occasion-ally in HSCR patients [Nakahara et al., 1995; Verloeset al., 1993]. Most of these patients failed to have RETor GDNF mutations [Amiel et al., 1998; Bolk et al.,1996a]. These data suggest a low susceptibility of neu-ral crest-derived cells of the central nervous system toRET or GDNF mutations [Amiel et al., 1998] and thepossible involvement of other genes in CCHS patients[Bolk et al., 1996b]. Finally, as RET gene expression inthe motor neurons is particularly high in the develop-ing central nervous system, the question of why RETmutations have apparently no clinical expression inspinal cord both in mouse and human remains unan-swered.

ACKNOWLEDGMENTS

We thank the medical staff of Orthogeny Center(Hôpital Broussais) and of Gynecology Department(Hopital Boucicaut) and J.L. Dufier (Ophtalmologie,Hôpital Necker) for their cooperation. We also thank A.Beauvais, C. Esculpavit, S. Fahy, and P. Brice for tech-nical assistance. This study was supported by the As-sociation Française contre les Myopathies (AFM), the

Fig. 4. RET expression in the ventral neural tube. Bright-field (a) and dark-field (b) photomicrographs of a transverse section through the neural tubeof a Carnegie stage 13 embryo at high magnification view. A strong signal is detected in the motor neuron columns (mn) of the ventral part of the neuraltube (n). Bars, 200 mm.

RET Proto-Oncogene in Human Embryos 485

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Association pour la Recherche contre le Cancer (ARC),the Caisse Nationale des Assurances Maladies(CANAM), the association IMAGE, and the Projet Hos-pitalier de Recherche Clinique (PHRC AOA 94060).

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