Expression of the C-Ret Proto-Oncogene During Mouse Embryogenesis

Development 119, 1005-1017 (1993) 1005 Printed in Great Britain © The Company of Biologists Limited 1993

Expression of the c-ret proto-oncogene during mouse embryogenesis

Vassilis Pachnis1,2,*, Baljinder Mankoo2 and Frank Costantini1 1Department of Genetics and Development, Columbia University, 701 West 168th Street, New York, NY, 10032, USA 2National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK *Author for correspondence (present address: NIMR)

SUMMARY

The c-ret proto-oncogene encodes a receptor tyrosine vagal crest (day 9.0-11.5), and in the myenteric ganglia kinase whose normal function has yet to be determined. of the gut (day 13.5-14.5). c-ret mRNA was observed in To begin to investigate the potential role of this gene in several regions of the central nervous system, including vertebrate development, we have isolated cDNA clones the undifferentiated neuroepithelial cells of the ventral representing the murine c-ret gene, and have analyzed neural tube (8.5 days), the motor neurons in the spinal the pattern of expression during mouse embryogenesis, cord and the hindbrain (10.5-14.5 days), the embryonic using northern blotting, in situ hybridization to histo- neuroretina (day 13.5) and the layers of the postnatal logical sections and whole-mount hybridization histo- retina containing ganglion, amacrine and horizontal chemistry. c-ret transcripts were detected beginning at cells. Outside the nervous system, c-ret was expressed in day 8.5 of embryogenesis, and were observed in a the nephric (Wolffian) duct at day 8.5-10.5, the ureteric number of cell lineages in the developing peripheral and bud epithelium (but not the surrounding metanephric central nervous systems, as well as in the excretory mesenchyme) at day 11.0-11.5, and the growing tips of system. In the cranial region at day 8.5-9.5, c-ret mRNA the renal collecting ducts (but not the previously formed, was restricted to a population of neural crest cells subcortical portions of the collecting ducts, or the mes- migrating from rhombomere 4 and forming the anlage enchyme-derived renal vesicles) at day 13.5-17.5. Our of the facioacoustic ganglion, as well as to a closely asso- results suggest that the c-ret gene may encode the ciated domain of surface ectoderm and pharyngeal receptor for a factor involved in the proliferation, endoderm. At later stages (10.5-14.5 days), c-ret mRNA migration, differentiation or survival of a variety of was observed in all cranial ganglia. In the peripheral neuronal cell lineages, as well as in inductive interactions nervous system of the trunk, c-ret was expressed in the during organogenesis of the kidney. autonomic ganglia and in subsets of cells in the dorsal root ganglia. In the enteric nervous system, c-ret was Key words: c-ret, tyrosine kinase receptor, mouse embryogenesis, expressed in the presumptive enteric neuroblasts of the nervous system, excretory system

INTRODUCTION transformation has been studied extensively (Cantley et al., 1991), but their function during embryogenesis of multicel- Normal development of the vertebrate embryo depends on lular organisms is less well understood. The best evidence the proper communication between diverse cell types. that RTKs play a critical role in diverse developmental Several membrane-bound or diffusible molecules have been processes is derived from genetic studies in both inverte- identified as components of such an intercellular communi- brates and vertebrates (Hafen and Basler, 1990; Pawson and cation system. These molecules mediate their effects by Bernstein, 1990). In Drosophila melanogaster (D. binding to specific cell surface receptors, a class of which melanogaster), several well-characterized developmental has intrinsic and ligand-dependent tyrosine kinase activity. mutations have been shown to reside in genes encoding All receptor tyrosine kinases (RTKs) have a similar RTKs. One of the best studied examples is the sevenless topology: they possess an extracellular ligand-binding locus, which encodes a tyrosine kinase receptor that domain, a transmembrane domain, and a cytoplasmic interacts with a cell surface molecule encoded at the boss segment containing the catalytic tyrosine kinase domain locus. Mutations at either of these loci prevent the differen- (Yarden and Ullrich, 1988). Binding of the ligand leads to tiation of R7 photoreceptors and lead to abnormal pattern- dimerization of the receptor and activation of the kinase ing of the D. melanogaster eye. In mice, mutations in the domain, manifested as autophosphorylation of the receptor Dominant White Spotting (W) and Steel (Sl) loci, which and phosphorylation of specific substrates that mediate the encode the kit RTK and its ligand respectively, cause similar intracellular signaling (Ullrich and Schlessinger, 1990). defects in the haematopoietic, germ cell and melanocytic The role of RTKs in cellular proliferation and oncogenic lineages (Green, 1989). Finally, RTKs of the trk subfamily 1006 V. Pachnis, B. Mankoo and F. Costantini have recently been shown to function as the high affinity In situ hybridization analysis on sections and whole-mount receptors for the nerve growth factor (NGF)-related neu- hybridization histochemistry were performed essentially as rotrophins (Kaplan et al., 1991a,b; Klein et al., 1991a,b; described (Wilkinson and Green, 1990, Wilkinson, 1992). Lamballe et al., 1991; Soppet et al., 1991). Although the phenotypic effects of mutations in members of the trk gene family have yet to be described, the established importance RESULTS of NGF as a trophic factor for sympathetic neurons and neural-crest-derived sensory neurons of the peripheral Isolation of cDNA clones from the murine c-ret nervous system (PNS), and certain cholinergic neurons in locus the central nervous system (CNS) (Levi-Montalcini, 1987; To isolate cDNA clones of the murine homologue of the c- Martinez et al., 1985), strongly suggests a critical develop- ret proto-oncogene, a cDNA library was constructed with mental role for the trk RTKs. poly(A)+ RNA from the murine neuroblastoma cell line The c-ret proto-oncogene is a member of the RTK gene Nb/2a (which, as shown below, expresses high levels of c- superfamily. It was originally identified as a transforming ret mRNA), and screened under stringent hybridization con- gene by transfection of T-cell lymphoma DNA into NIH3T3 ditions with the insert of recombinant plasmid pret2.2, cells (Takahashi et al., 1985). In vivo mutations of c-ret have which contains human c-ret mRNA sequences (Takahashi also been implicated in tumorigenesis. The PTC (papillary and Cooper, 1987). Several positive clones were identified thyroid carcinoma) oncogenes, detected frequently in and a partial restriction enzyme map of two of them, thyroid papillary carcinomas (25%), result from somatic pmcret7 and pmcret10, is shown in Fig. 1A. Partial sequenc- rearrangements that juxtapose the transmembrane and kinase domains of c-ret to unrelated 5′ sequences (Grieco et al., 1990; Jhiang et al., 1992; Bongarzone et al., 1993; Fabien et al., 1992). Recently, missense germ-line mutations in the extracellular domain of ret have been detected in a high proportion of cases of Multiple Endocrine Neoplasia type 2A (MEN2A; Mulligan et al., 1993), a dominantly inherited cancer syndrome consisting of thyroid medullary carcinoma, pheochromocytoma and parathyroid hyperplasia. These findings strongly suggest that mutations at the c- ret locus are the cause of dominantly inherited human neoplasia such as the MEN2A syndrome. Despite the established role of c-ret in human tumorigenesis, the function of the normal locus is currently unknown. c-ret mRNA and/or protein have been found in tumours of neuroectodermal origin (neuroblastomas, pheochromocytomas and medullary thyroid carcinomas) and in human neuroblastoma cell lines (Tahira et al., 1991; Ikeda et al., 1990; Santoro et al., 1990; Takahashi et al., 1991). Reports on the expression of c-ret during mammalian embryogenesis are, however, inconclusive and conflicting (Tahira et al., 1988; Szentirmay et al., 1990). We report here the identification of cDNA clones of the murine homologue of the c-ret proto- oncogene and studies on the expression of c-ret mRNA during mouse embryogenesis. Our results demonstrate that c-ret is expressed predominantly in the developing nervous Fig. 1. (A) Partial restriction enzyme map of the mouse c-ret and excretory systems, and lead us to propose that the ret cDNAs identified and used in this study. Shown at the top is a RTK is normally involved in several aspects of neurogene- diagrammatic representation of the various domains of the human ret receptor as deduced from its nucleotide sequence. The longest of sis and kidney organogenesis. the two mouse c-ret cDNA clones, pmcret7, contains sequences encoding a small part of the extracellular ligand-binding domain and the entire intracellular portion of the ret receptor, as well as 3′ MATERIALS AND METHODS untranslated sequences. pmcret10 corresponds to most of the intracellular part of the ret receptor plus 3′ untranslated sequences. Embryos were derived from crosses between FVB/N inbred mice, Abbreviations: TK, tyrosine kinase domain; TM, transmembrane or between (FVB/N×C57BL/6)F1, or (C57BL10×CBA)F1 hybrid domain. Restriction enzymes: B, BamHI; E, EcoRI; H, HindIII. mice. The morning of vaginal plug was considered 0.5 days post (B) Comparison of the amino acid sequence of the kinase domains coitum (dpc). The 8.5 dpc embryos were staged under the dissect- of the mouse and human ret receptors. Vertical lines indicate ing microscope by counting somites. identical amino acids, two dots indicate conservative substitutions RNA was isolated from tissue culture cells or embryos according and one dot indicates semiconservative substitutions. The two to published procedures (Auffray and Rougeon, 1980). The cDNA sequences are 95% identical and all the differences are either library was generated from poly(A)+ RNA isolated from Nb/2a conservative or semiconservative substitutions. The numbers cells using the ZAP-cDNATM synthesis kit (Stratagene) according correspond to the first and the last amino acid of the kinase domain to the manufacturer's recommendations. of the human receptor. Expression of c-ret proto-oncogene in mouse 1007 ing of pmcret7 revealed a potential product with high similarity to the published human ret receptor sequence (Fig. 1B) (Takahashi and Cooper, 1987; Takahashi et al., 1988), and identity (with the exception of two conservative amino acid substitutions which are likely to represent polymorphisms) to the sequence of the murine c-ret proto-oncogene reported during preparation of this manuscript (Iwamoto et al., 1993). Furthermore, hybridization of radiolabelled pret2.2 and the corresponding sequences of pmcret7 on Southern blots of mouse genomic DNA, under conditions of low and high stringency, identified an identical set of restriction enzyme fragments (data not shown). These data demonstrate that pmcret7 is derived from the mouse homologue of the c-ret proto-oncogene and that there are no additional genes in the mouse genome closely related to c-ret. Northern blot analysis of c-ret expression The tissue distribution of c-ret mRNA in adult animals was examined by northern blot analysis of poly(A)+ RNA extracted from various organs. As shown in Fig. 2A, c-ret expression is tissue specific. Among the tissues analyzed, salivary gland and brain contained the highest levels of c- ret mRNA, while heart and spleen contained lower but detectable levels. No expression was detected in kidney, liver, ovary, thymus, lung or testis. To determine whether c-ret is expressed during embryogenesis, poly(A)+ RNA was isolated from mouse embryos at different stages of development (8.5-16.5 dpc), and subjected to northern blot analysis. c-ret mRNA was detected in the embryo proper throughout the period examined (Fig. 2B), as well as in the extraembryonic Fig. 2. Northern blot analysis of c-ret expression. (A) Samples (5 µg) of poly(A)+ RNA isolated from the designated tissues of 8- to membranes (Fig. 2B, lane Em). We also detected c-ret 12-week-old mice were hybridized with antisense pmcret7 mRNA in the murine neuroblastoma cell line Nb/2a (Fig. riboprobe. The sizes of the four c-ret transcripts are indicated. The 2C) and, at low levels, in the rat pheochromocytoma cell line apparent signal in the kidney lane was not observed in other PC12 (not shown), a finding consistent with previous reports experiments. (B) Similar analysis of poly(A)+ RNA samples (5 of c-ret expression in other tumours and cell lines of neu- µg), isolated from embryos at the designated stages of roectodermal origin (Tahira et al., 1991; Ikeda et al., 1990; development (8.5-16.5 dpc) and from extraembryonic membranes Santoro et al., 1990; Takahashi et al., 1991). of 12.5 dpc embryos (lane Em). (C) 1 µg of poly(A)+ RNA from In all RNA samples analyzed so far, representing three the murine neuroblastoma cell line Nb/2a hybridized with the vertebrate species, i.e. human (Tahira et al., 1990), rat pmcret7 riboprobe. The set of c-ret transcripts observed in the (Tahira et al., 1988) and mouse (this report), a similar set of embryo, the extraembryonic membranes and the Nb/2a cells are identical to those observed in RNA preparations from the adult c-ret transcripts has been identified, migrating at 7.0, 6.0, tissues. 4.5 and 3.9 kb, with the 4.5 kb transcript being the most abundant. Analysis of human (Tahira et al., 1990), mouse (C. Marcos, M. Sukumaran and V. P.; unpublished data) and genesis, raised the possibility that the gene plays a role in the chicken (A. Schuchardt, V. P. and F. C., unpublished data) development of the vertebrate nervous system. Therefore, we c-ret cDNA clones has demonstrated that the various tran- sought to determine the spatial distribution of c-ret transcripts are generated from alternative splicing and/or scripts during mammalian embryogenesis. In situ hybridiza- polyadenylation of the primary c-ret transcript. Such events tion analysis was performed on mouse embryos between 7.5 are likely to be under tissue-specific regulation, as suggested and 14.5 days of gestation, a period during which the most by the reversed relative abundance of the 6.0 and 7.0 kb tran- critical events of organogenesis are taking place. For these scripts in mouse brain and salivary gland mRNA prepara- experiments, we used a riboprobe derived from pmcret7 tions (Fig. 2A). which contains sequences for a small part of the extracellular domain, the transmembrane and intracellular domains as In situ hybridization analysis of c-ret expression well as the entire 3′ untranslated sequence of the 4.5 kb tran- during mouse embryogenesis script (Fig. 1A and C. Marcos, M. Sukumaran and V. P., Expression in cranial neural crest cells and their unpublished observations). At 7.5 days c-ret mRNA was neurogenic derivatives absent from the embryo proper, but was detected in the tro- The expression of c-ret in tissues (e.g. brain) and cell lines phoblast cells of the extraembryonic membranes (not of neuroectodermal origin (neuroblastomas and pheochro- shown). 24 hours later (8.5-9.0 dpc), as predicted by the mocytomas), and its expression in embryos during organo- northern blot analysis, c-ret transcripts were detected in 1008 V. Pachnis, B. Mankoo and F. Costantini

Fig. 3. Expression of c-ret in the head of 9.0-9.5 day mouse embryos. (A,C,E,G) Photomicrographs of sections under bright-field illumination; (B,D,F,H) photomicrographs of the same series of sections under dark-field illumination. (A,B) Frontal section through the hindbrain of a 9.25 dpc embryo. Signal is detected in the group of neural crest cells emerging from rhombomere 4 (r4), and in a domain of surface ectoderm extending posterior to the c-ret-positive neural crest cells. (C,D) Parasagital section through the head of a 9.25 dpc embryo. Expression of c-ret is restricted to a group of neural crest cells immediately anterior to the otic vesicle (ov), and extending from the dorsal edge of the embryo into the forming second branchial arch. (E,F) Frontal section through the head of a 9.5 dpc embryo. This section is oblique such that dorsal structures such as cranial ganglia (VII) are on the left while ventral structures such as branchial arches (ba 1,2,3,4) are on the right. c-ret-specific signal is localized in the anlage of the facioacoustic ganglion (VII) and over the posterior branchial arches. In the c-ret-positive branchial arch (ba4), expression is observed in the pharyngeal endoderm, the surface ectoderm and the mesenchymal cells lying in between. (G,H) Transverse section through the head of a 9.5 dpc embryo at the posterior edge of the otic vesicle (ov). Signal is observed in the pharyngeal (p) endoderm and the overlying ectoderm. sp e c i fi c groups of cells within the embryo. In the head, strong mount preparations of 8.75-9.0 day embryos, a stage of signal was localized to a cohesive group of cells immediately embryogenesis at which the migration of all groups of cranial anterior to the otic vesicle and extending from the dorsal edge neural crest is well under way. In agreement with the data of ventrally towards the forming second branchial arch (Fig. Fig. 3A-D, strong c- r e t -s p e c i fi c signal was restricted to a 3A-D). The position of these cells suggests that they are single group of cranial neural crest cells emerging from the neural crest cells emigrating from rhombomere 4 (r4) of the neuroepithelium of the hindbrain at the level of r4 (Fig. 4A). hindbrain. Similar groups of cells emigrating from the neu- Two components of the hindbrain neural crest have been roepithelium anterior or posterior to r4 were negative for c- described: the early migrating cells, which populate the re t transcripts (Fig. 3A-D). This apparently segment-specific ventrally located branchial arches and adopt a mesenchymal expression of c- r e t in the neural crest of the hindbrain was fate, and the late emerging cells, which remain closer to the further examined by hybridization histochemistry on whole- neural tube and adopt a neural fate (Nichols, 1981, 1986). The Expression of c-ret proto-oncogene in mouse 1009 detection of c- r e t mRNA in both the dorsal and ventral neural neural crest. In sections from 9.5 day embryos, a diffuse and crest of r4 (Fig. 3C,D) suggests that during this stage of relatively weak (but reproducible) signal was present embryogenesis, both the neurogenic and the mesenchymal dorsally between the neural tube and the surface ectoderm components of the r4 crest express c- r e t mRNA at compara- and ventrally in the sclerotome, areas of the somites in ble levels. Interestingly, during the embryonic period under which the trunk neural crest cells first migrate and subse- consideration, the brain neuroepithelium including r4, was quently condense to form the dorsal root ganglia (DRG; not devoid of hybridization signal (Fig. 3A,B and data not shown). shown). In the DRG of later stage embryos (11.5-13.5 days), In addition to the migrating neural crest cells, c-ret is the hybridization pattern was modified. Intense punctate expressed in the 8.5-9.0 day embryo in a well-defined signal was observed in the ganglion itself, but was absent domain of cranial surface ectoderm and pharyngeal from the emerging peripheral spinal nerve (Fig. 5C). This endoderm associated with the posterior branchial arches suggests that expression of c-ret in the DRG is restricted to (Fig. 3E-H). The c-ret-positive surface ectoderm includes subsets of neurons and is absent from the glial cells of the the epibranchial placodes, which give rise to the sensory ganglion or the spinal nerve. neurons of the inferior ganglia of the IXth and Xth cranial nerve ganglion complexes (Altman and Bayer, 1982). No Expression in the developing enteric nervous system signal was detected in the more anteriorly located epi- The expression of c-ret in the sensory ganglia raised the pos- branchial placodes. sibility that the gene is also expressed in the other major Given the positionally restricted expression of c- r e t in the branches of the PNS, i.e. the enteric and the autonomic r4 neural crest and the epibranchial placodes, it was of interest nervous system. The enteric nervous system (ENS) is to examine its pattern of expression in cranial structures from composed of the myenteric and the submucosal ganglion later stage embryos. In the head of 9.5 day embryos, and con- complexes of the gut (Furness and Costa, 1987). The sistent with the restricted expression of ret in r4 neural crest majority of the cells in the ganglia of the ENS are derived of earlier stage embryos, signal was localized in the con- from the vagal crest originating from the postotic hindbrain densing facioacoustic ganglion complex (VII-VIIIth ) but not (corresponding to somites 1-7) (Yntema and Hammond, in the anlage of other cranial ganglia present anteriorly (Vth ) 1954; Le Douarin and Teillet, 1973). The presumptive or posteriorly (IXth , Xth ) (Fig. 4B). Despite the expression of enteric ganglioblasts of the vagal crest migrate first ventrally c- r e t in the anlage of the facioacoustic ganglion, no signal was through the posterior branchial arches into the foregut mes- detected in the second branchial arch (Fig. 4B, ba2), which is enchyme, and then rostrocaudally inside the gut wall mes- populated to a large extent by the mesenchymal component enchyme, where they eventually coalesce and form the of r4 crest. This indicates that by the time the r4 crest has enteric ganglia along the entire length of the gut (Tucker et completed its migration into the second branchial arch, the al., 1986; Baetge and Gershon, 1989; Gershon et al., 1993; expression of c- r e t is down-regulated. In 10.5 day embryos, Kapur et al., 1992). c-ret-positive cells have been identified in addition to the facioacoustic ganglion, the inferior ganglia in the migratory pathway of the presumptive enteric neu- of the IXth and Xth cranial nerve ganglion complexes are roblasts of the vagal crest and in the myenteric ganglia of positive for c- r e t transcripts, while the trigeminal ganglion the gut. In the 9.0-9.5 day mouse embryo, strong expression (V) is still negative (Fig. 4C). Finally, in 13.5-14.5 day was detected in mesenchymal cells of the third and fourth embryos, all cranial ganglia, including the trigeminal ganglion branchial arches, between the c-ret-positive pharyngeal (Fig. 5A,B) and the superior ganglia of the IXth and Xth endoderm and surface ectoderm (Fig. 3E,F). 12 hours later, cranial nerve ganglion complexes (not shown) are positive for a stream of c-ret-positive cells could be seen emerging from c- r e t mR N A . the posterior branchial arch mesenchyme migrating towards Overall, our data demonstrate that, in the head of the the foregut (Fig. 6A). At subsequent stages (10.5-11.5 days) postimplantation mouse embryo, c-ret expression is associ- c-ret-positive cells were found at increasingly more caudal ated with the development of cranial ganglia. Two distinct levels of the embryonic gut (Fig. 6B,C). Finally, in the 13.5- phases of c-ret expression can be discerned: an early, 14.5 day embryo, in which the rostrocaudal migration of the segment-specific, phase (8.5-9.5 day embryo), during which ENS precursors has been completed, strong c-ret expression c-ret mRNA is restricted to neural crest cells derived from was detected in the myenteric plexus along the entire axis r4 and the anlage of the facioacoustic ganglion, and a later of the gut (Fig. 6D). Although no independent molecular phase (10.5-14.5 days) during which signal appears markers were used in these experiments, the overall spatial gradually in all cranial ganglia irrespective of the origin of and temporal pattern of expression of c-ret in the develop- the contributing neural crest cells along the anteroposterior ing gut is consistent with the gene being expressed in the axis of the brain neuroepithelium. The positionally restricted majority of the migrating precursors and postmigratory cells localization of c-ret mRNA in the cranial neural crest and of the ENS. the surface ectoderm and pharyngeal endoderm of the c-ret mRNA is also expressed in the autonomic ganglia branchial arches suggests that expression of c-ret is related of the PNS. At embryonic day 9.5, c-ret transcripts were to the intrinsic mechanisms of segmentation of the ver- detected in groups of cells by the dorsal aorta, where deriv- tebrate head. atives of the trunk neural crest coalesce to form the sympathetic ganglia anlage (Fig. 6A). Expression is maintained at Expression in the developing sensory and autonomic later stages, e.g. 14.5 days, when sympathetic gangliogene- ganglia of the trunk sis has been essentially completed (Fig. 6D). At this stage, c-ret mRNA was also present in the migrating neural crest no expression of c-ret was observed in the endocrine deriv- cells of the trunk, albeit at lower levels compared to the r4 atives of the sympathoadrenal lineage, the adrenal chromaf- 1010 V. Pachnis, B. Mankoo and F. Costantini

Fig. 4. Expression of c- r e t in neural crest and cranial ganglia of 8.5-10.5 dpc embryos detected by whole-mount hybridization histochemistry. (A) In the head of the 8.5 dpc embryo, staining is restricted to the neural crest (nc) cells emerging from rhombomere 4 (r4) of the hindbrain and to a well-deﬁned domain of surface ectoderm (se) located ventrocaudally to the c-ret-positive r4 neural crest. (B) In the 9.5 dpc embryo, staining is observed in the anlage of the facioacoustic ganglion (VII) present immediately anterior to the otic vesicle (ov), and in cells of the pharyngeal clefts corresponding to the posterior branchial arches (open triangle). (C) In the 10.5 dpc embryo, c- ret-speciﬁc signal is observed in the facioacoustic ganglion (VII) and the inferior complex of the IXth and Xth cranial ganglia. No signal is present at this stage in the trigeminal ganglion, present anteriorly to the VIIth ganglion, and in the superior complex of the IXth and Xth ganglia.

Fig. 5. Expression of c-ret mRNA in the trigeminal and the dorsal root ganglia of the 13.5-14.5 dpc embryo. (A,B) Bright-field and dark-field photomicrograph of a frontal section through the head of a 14.5 dpc embryo. The two ovoid symmetrical structures expressing high levels of c-ret mRNA are the trigeminal ganglia (tg). (C) Bright-field photomicrograph of a section through thoracic dorsal root ganglia (drg) of a 13.5 dpc embryo. Signal (dark grains) is observed over a subset of cells of the dorsal root ganglia and is absent from the emerging spinal nerve (sn). Expression of c-ret proto-oncogene in mouse 1011

Fig. 6. Expression of c-ret in the vagal neural crest and the derivative myenteric plexus of embryonic gut. (A) Frontal section through the branchial arch region of a 9.75 dpc mouse embryo (p, pharynx). Strong signal is observed in single mesenchymal cells migrating from the posterior branchial arches towards the foregut, as well as in the sympathetic ganglia (sg) forming on either side of the dorsal aorta (ao). (B) At a slightly later stage (10.0 dpc embryo), c-ret-positive cells are migrating in a rostrocaudal direction within the mesenchyme of the midgut (mg) wall. (C) Section through the gut of 11.5 dpc embryo. c-ret-positive cells have migrated into the distal portion of the developing small intestine (i), and are starting to coalesce to form the enteric ganglia. (D) Section through the gut of a 14.5 dpc embryo. At this stage only the outer myenteric plexus has formed. Signal is observed in a ring-like fashion in the myenteric ganglia of the stomach (s), duodenum (d) and small intestine (i). 1012 V. Pachnis, B. Mankoo and F. Costantini

Fig. 7. Expression of c-ret in the developing spinal cord. (A,B) Bright- field and dark-field photomicrographs of a transverse section through the caudal part of an 8.75 dpc embryo. c- ret-specific signal is localised in the ventral part of the neural tube (nt) and laterally to the somites (s), in the nephrotome (n). (C,D) Bright-field and dark-field photomicrographs of a transverse section through the spinal cord of a 13.5 dpc embryo. Signal is observed in the motor neuron columns at the ventral part of the spinal cord (closed triangles) and in individual cells of the dorsal root ganglia (drg). atives of the sympathoadrenal lineage, the adrenal chromaf- in the layers that contain the bulk of the bipolar cells and fin cells (data not shown). the photoreceptors. Expression in the developing central nervous system Expression in the developing excretory system During the period of embryogenesis that we examined, c-ret Expression of c-ret in the nephrotome of 8.5 day embryos is also expressed in the developing CNS. More specifically, prompted us to examine in more detail its expression in the expression was observed in undifferentiated neuroepithelial developing excretory system of the mouse. In vertebrate cells of the neural tube and their postmitotic derivatives. embryos, the excretory system can be subdivided into three Shown in Fig. 7A,B is a transverse section through the trunk sequentially appearing organs, the pronephros, mesonephros of a 8.75 dpc embryo. Hybridization signal is localized in and metanephros (Saxen, 1987). The pronephros and the neuroepithelial cells of the ventral half of the neural tube. mesonephros are transient embryonic structures, which Strong signal was also detected laterally to the epithelial consist of a series of segmentally arranged tubules somites, in the nephrotome. At 9.5 days, hybridization was connected to the nephric (Wolffian) duct. In situ hybridiz- further restricted to the ventrolateral compartment of the ation on sections and hybridization histochemistry on spinal cord, where the motor neurons are differentiating (not whole-mount preparations of 9.0-10.5 day embryos revealed shown). An identical labelling pattern was maintained that c-ret is expressed in the nephric duct of the pronephros during the subsequent periods of embryogenesis that we (not shown) and mesonephros (Fig 9A). analyzed (10.5-14.5 dpc, Fig. 7C,D). The ventrally localized The permanent, or metanephric kidney, is formed through domain of c-ret expression extends along the entire antero- a series of reciprocal inductive interactions between the posterior axis of the developing spinal cord, and extends ureteric bud, which emerges from the caudal part of the anteriorly into groups of motor neurons in the hindbrain nephric duct, and the surrounding metanephric mesenchyme (data not shown). (Saxen, 1987). Upon invasion of the metanephric mes- We also detected c-ret mRNA in the neuroretina of the enchyme (day 11.5) the ureteric bud begins to branch embryonic and postnatal mouse eye. Shown in Fig. 8A,B is dichotomously. In embryonic kidneys at this stage, c-ret in situ hybridization to a section through the eye of a 13.5 transcripts were detected only in the epithelial cells of the day mouse embryo. Hybridization is restricted to the branching ureteric bud, but not in the surrounding mes- innermost layers of the neuroretina, where the first postmi- enchymal cells (Fig. 9B). Subsequently, the branching tips totic neurons are born. To determine whether all groups of of the ureteric epithelium induce the surrounding undiffer- postmitotic cells in the mammalian retina express c-ret, we entiated mesenchyme to condense and differentiate into the analyzed sections from eyes of postnatal day 7 mice, a stage epithelial renal tubules and glomeruli, while the mes- by which all classes of neurons and glial cells have been enchyme induces the ureteric bud to grow and branch, generated (Turner and Cepko, 1987). As shown in Fig. forming the collecting ducts. At more advanced stages of 8C,D, c-ret mRNA is localized to certain layers of the organogenesis, these inductive interactions take place only mature mammalian retina, those containing ganglion, in a narrow region around the outer edge of the kidney, the amacrine and horizontal cells. No expression was detected nephrogenic zone (Saxen, 1987). In sections of embryonic Expression of c-ret proto-oncogene in mouse 1013

ret is expressed in a complex and dynamic pattern, predominantly in subsets of cells of the PNS, the CNS and the excretory system. These findings suggest that c-ret, in addition to its role in cellular transformation and tumour formation, plays an important role in normal mammalian embryogenesis. In the developing nervous system, c- r e t is expressed in undifferentiated neuroectodermal precursors (subsets of migrating neural crest cells and neuroepithelial cells of the ventral neural tube) as well as in their differentiated postmitotic derivatives. The expression of c- r e t in cranial neural crest and its derivatives is particularly intriguing. During the migratory phase of the neural crest and the early stages of cranial gangliogenesis (8.5-9.5 days), c- r e t mRNA is restricted to the r4 crest and the anlage of the facioacoustic ganglion complex. Subsequently, over a period of three days (day 10.5-13.5), the apparent segment-specific expression of c- r e t dissipates and its mRNA is detected in groups of cells in all cranial ganglia irrespective of the origin of the contributing neural crest. In the posterior head, neural crest emerges from the hindbrain as distinct streams of cells (Serbedzija et al., 1992; Lumsden et al., 1991). It has been suggested that the patterning information for the mesenchymal and neuronal components of each branchial arch is contained within the neural crest that populates that arch and that this information has been imprinted on the neural crest by its origin along the anteroposterior axis of the brain neuroepithelium (Noden, 1983, 1988). It has been postulated that the combinatorial expression of members of the Ho x ge n e clusters provides at least part of the patterning information for the specification of individual rhombomeres of the hindbrain and the neural crest cells derived from them (Hunt et al., 1991). How might such a ‘Hox code’ in the cranial Fig. 8. c-ret expression in the mouse eye. (A,B) Bright-field and neural crest translate into different neuronal and mesenchy- dark-field photomicrograph of a section through the eye of a 13.5 mal structures in the head of the adult animal? The differen- dpc embryo. Signal is observed in the innermost layer of the tial activation of downstream target genes could provide the neuroretina (r), where the first postmitotic neurons are born (pe, means of translating such positional information into diverse pigmented epithelium). (C,D) Bright-field and dark-field developmental programs. The restricted expression of the c- photomicrograph of a section through the retina of an albino postnatal day 7 mouse. Signal is observed in the ganglion (g), the re t gene in the r4 neural crest of the 8.5-9.5 day embryo amacrine (a) and the horizontal (h) cell layers. No signal is provides additional evidence that the various groups of observed over the bipolar (b) or the photoreceptor (ph) cell layers. cranial neural crest cells are molecularly distinct. Moreover, it raises the possibility that the ret receptor is part of the molecular cascade that leads to the establishment of the kidneys at these stages (13.5-17.5 dpc), c-ret mRNA was unique properties of the r4 crest derivatives. detected only in the nephrogenic zone (Fig. 9C). At high In the PNS, c - r e t is expressed in the developing magnification, it could be seen that the hybridization signal autonomic nervous system, the ENS and the sensory was restricted to the growing tips of the collecting ducts, but ganglia of the head and the trunk, suggesting that the ret was absent from all of the more centrally located structures, signal transduction pathway is involved in the organogen- including the condensing renal vesicles and nephrons (of esis of all major branches of the PNS. Phenotypic analysis mesenchymal origin), and the subcortical segments of the of the sympathoadrenal progenitors and the progenitors of collecting ducts (Fig. 9D). the ENS in rodents has established that these two lineages share a number of independent molecular markers (Baetge et al., 1990; Baetge and Gershon, 1989; Carnahan et al., DISCUSSION 1991; Johnson et al., 1990; Lo et al., 1991). This led to the suggestion that sympathoadrenal and enteric lineages are We have cloned the murine homologue of the c-ret proto- derived from progenitors that are either identical or closely oncogene and studied its spatial and temporal pattern of related (Carnahan et al., 1991), and that the particular phe- expression during mouse embryogenesis, using northern notypes eventually acquired by these progenitors are deter- blot analysis, in situ hybridization to sections and hybridiz- mined by the specific microenvironment at their final des- ation histochemistry on whole-mount preparations. Our tination. The concomitant expression of c - r e t in the anlage studies demonstrate that, in the postimplantation embryo, c- of the sympathetic ganglia and the precursors of the ENS 1014 V. Pachnis, B. Mankoo and F. Costantini

Fig. 9. c-ret expression in the excretory system of the mouse embryo. (A) Whole-mount hybridization histochemistry of the posterior trunk region of a 9.5 dpc embryo. c-ret mRNA is expressed throughout the nephric duct (nd), with highest levels observed in its caudal part (open triangles). (B) In situ hybridization of a section through the metanephric region of an 11.5dpc embryo. High levels of c-ret mRNA is detected in the ureteric bud and its branches (u), while no signal is detected in the surrounding mesenchyme (m). (C) A section through the kidney of a 17.5 dpc embryo. Signal is restricted in the outer zone of the kidney where induction of new nephrons is still taking place. (D) Bright-ﬁeld photograph of a high magniﬁcation view of part of kidney shown in C. The c-ret-positive collecting ducts (arrows) are derivatives of the ureteric bud.

in the mouse embryo further supports their close relation- embryonic stem cells, fail to develop enteric ganglia ship. Moreover, it raises the possibility that c - r e t plays a (Schuchardt et al., unpublished data). role in the proliferation, migration, differentiation or The apparent localization of c-ret mRNA in subsets of survival of the cells of these lineages. The expression of c - neurons of the sensory ganglia (cranial nerve ganglia and r e t mRNA in the migratory ENS progenitors is particularly DRG) is consistent with previous studies indicating that interesting, since, despite the established importance of distinct gene expression programmes exist in the various signals encountered along the migratory pathway of the functional classes of the sensory neurons. Using monoclonal vagal crest and in the microenvironment of the embryonic antibodies, Dodd and colleagues were able to identify bowel for the differentiation of the enteric neurons subsets of primary sensory neurons in rat DRG expressing (Gershon et al., 1993), very little is known about the unique carbohydrate differentiation antigens (Dodd et al., molecular nature of these signals. Our data suggest that the 1984; Dodd and Jessell, 1985). More recently, in situ hybrid- ret receptor might play a critical role in the cellular and ization analysis of the pattern of expression of members of molecular processes that lead to the organogenesis of the the trk RTK subfamily has revealed that, similarly to the ret ENS, i.e. migration of the vagal crest, commitment to the receptor, individual members of the trk subfamily are neuroblast or glioblast fate, cell proliferation and survival expressed in subclasses of DRG cells (Carroll et al., 1992). of postmitotic cells in the enteric ganglia. This hypothesis Furthermore, immune deprivation of NGF in utero resulted is strongly supported by our recent finding that mouse in selective ablation of trk-expressing cells of the DRG embryos homozygous for a loss-of-function mutation of the (Carroll et al., 1992; Ruit et al., 1992). Our data suggest that, c - r e t gene, generated by homologous recombination in as is the case for the trk subfamily of RTKs, the ret receptor Expression of c-ret proto-oncogene in mouse 1015 plays a role in the differentiation and survival of a specific lineage expresses c - r e t mRNA from the early embryonic class of primary sensory neurons. The identity of this class stages. Despite lack of expression in the adrenal medulla, and the particular sensory modality that it conveys are it is likely that the chromaffin cell precursors are also currently unknown. expressing c - r e t mRNA since they originate from the ret- In the CNS, expression has been detected predominantly positive cells of the sympathetic ganglia condensations in the motor neuron lineages of the spinal cord and the (Fig. 6A; (Pankratz, 1931)). Finally, the parathyroid cell hindbrain, and in subsets of cells of the neuroretina. Lineage precursors are derived from the c - r e t-positive endoderm of analysis of the cells of the vertebrate retina has shown that the posterior branchial arches (Fig. 3E,F). Overall, the all cell types of the mature retina are derived from an early common expression of c - r e t reveals a developmental link common progenitor (Turner and Cepko, 1987; Turner et al., between the cell types affected in the MEN2A syndrome 1990; Wetts et al., 1989), and strongly suggests that cellular and provides further support for the hypothesis that germ- interactions in the developing vertebrate retina play a critical line mutations in c - r e t are responsible for all the manifes- role in the establishment of the various cellular phenotypes. tations of the MEN2A syndrome. Although certain growth factors have been identified as In addition to its postulated role in the developing nervous playing an important role in the differentiation of retinal system, the expression pattern of the c-ret gene suggests that cells in vitro (Lillien and Cepko, 1992; Guillemot and the ret receptor could function during the development of Cepko, 1992), the molecules that mediate such cellular inter- the excretory system. c-ret mRNA was first detected at day actions in vivo have yet to be identified. Our expression data, 8.5 in the nephrotome, a region of lateral mesoderm from combined with the nature of the ret protein, suggest that it which the embryonic (pronephric and mesonephric) and may play a role in the cell-cell interactions that determine permanent (metanephric) kidneys are formed and continued the cellular phenotypes of particular classes of neurons in to be detected in the nephric (Wolffian) ducts during the the mammalian retina. development of the pronephric and mesonephric kidneys The molecular mechanisms underlying the potential role (day 9.5-10.5). The expression pattern of c-ret at this stage of c-ret in the development of the mammalian nervous suggests that the ret receptor might play a role in the system are currently unknown. However, the structural sim- formation of the pronephros and mesonephros. ilarity of ret to other members of the RTK family and its The development of the mammalian metanephric kidney pattern of expression in the developing PNS and CNS, has been extensively studied as a model system for the role suggest that it functions as the receptor to a (potentially of inductive tissue interactions during organogenesis novel) neurotrophic factor(s) required for the migration, (Saxen, 1987). Formation of the kidney is initiated on day commitment, differentiation, or survival of certain cell 11 of mouse embryogenesis, when the ureteric bud emerges lineages of the mammalian nervous system. near the caudal end of the nephric duct and grows dorsally, Molecular characterization of c - r e t cDNAs from patients eventually contacting the metanephric blastema, a specific with MEN2A syndrome suggests strongly that gain-of- condensate of mesenchymal cells. At this point, the ureteric function mutations in the c - r e t proto-oncogene are respon- bud begins to grow and branch repeatedly, dependent upon sible for this inherited cancer syndrome (Mulligan et al., s p e c i fic induction by the metanephric mesenchymal cells 1993). Although the documented expression of c - r e t in cell (Grobstein, 1953, 1955; Erickson, 1968), and eventually lines of neural crest origin is consistent with the neural crest giving rise to the entire renal collecting system (calyces, origin of medullary thyroid carcinoma (C-cells of the papillae and collecting ducts). At the same time, the tips of thyroid) and pheochromocytoma (sympathoadrenal prog- the branching ureteric bud induce the surrounding mes- enitor), the occurrence of parathyroid hyperplasia in enchymal cells to condense into epithelial renal vesicles, patients with MEN2A syndrome has been puzzling, as which eventually differentiate to form the various segments parathyroid cells are derived embryologically from the of the nephron, including the proximal and distal tubules endoderm of the fourth pharyngeal pouch (Balinsky, 1970). and glomeruli (Grobstein, 1955, 1956; Saxen, 1987). At a This led to the suggestion that the tumours of the MEN2A stage when the ureteric bud has first branched within the syndrome may result from defects in closely linked genes metanephric mesenchyme, we observed that c - r e t w a s that independently control neural crest or endocrine tissue strongly expressed in the ureteric bud epithelium, but was development (Lairmore et al., 1991). Our expression not expressed at detectable levels in the surrounding undif- studies further support the suggestion that the entire ferentiated mesenchyme. At later stages of renal organo- MEN2A syndrome is caused by mutations in the c - r e t genesis, growth and branching of the ureteric derivatives, proto-oncogene by providing evidence that all lineages as well as the induction of new nephrons, is limited to a affected in this syndrome are likely to express the c - r e t narrow ‘nephrogenic’ zone at the perimeter of the kidney proto-oncogene. The precursors of the C-cells, originating (Saxen, 1987). At these stages (day 13.5-17.5), c - r e t m R N A from the posterior hindbrain, are transiently localized, was observed only in the tips of the growing collecting along with the precursors to the ENS, in the mesenchyme ducts in the outer nephrogenic zone, and not in the previ- of the posterior branchial arches (Le Douarin, 1982), an ously formed and more centrally located collecting ducts. area where c - r e t is strongly expressed (Fig. 3E-H). This, Like the undifferentiated metanephric mesenchyme, both along with the high levels of c - r e t expression in C-cell- the newly condensed renal vesicles and the more mature derived medullary thyroid carcinomas (Santoro et al., 1990) mesenchymal derivatives continued to be negative for c - r e t and our preliminary experiments, which show low levels of e x p r e s s i o n . expression in the C-cells of adult mouse thyroid (P. Durbec, Based on this restricted pattern of expression, it appears B. M. and V. P., unpublished data), suggests that the C-cell likely that the ret receptor could play a role in the growth 1016 V. Pachnis, B. Mankoo and F. Costantini and development of the ureteric bud and its derivative Carnahan, J. F., Anderson, D. J. and Patterson, P. H. (1991). Evidence structures in the kidney. More specifically, ret might serve that enteric neurons may derive from the sympathoadrenal lineage. Dev. as the receptor for an inductive factor, produced by the Biol. 148, 552-561. Carroll, S. L., Silos-Santiago, I., Frese, S. E., Ruit, K. G., Milbrandt, J. metanephric mesenchymal cells, which stimulates the and Snider, W. D. (1992). Dorsal root ganglion neurons expressing trk growth and/or branching of the ureteric bud epithelium. are selectively sensitive to NGF deprivation in utero. Neuron 9, 779-788. Support for the hypothesis that the c - r e t gene serves an Dodd, J., Solter, D. and Jessell, T. M. (1984). 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