1005
Development 119, 1005-1017 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
Expression of the c-ret proto-oncogene during mouse embryogenesis
Vassilis Pachnis 1,2,*, Baljinder Mankoo2 and Frank Costantini1
1Department of Genetics and Development, Columbia University, 701 West 168th Street,
2National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
New York, NY, 10032, USA
*Author for correspondence (present address: NIMR)
SUMMARY
The c-ret proto-oncogene encodes a receptor tyrosine
kinase whose normal function has yet to be determined.
To begin to investigate the potential role of this gene in
vertebrate development, we have isolated cDNA clones
representing the murine c-ret gene, and have analyzed
the pattern of expression during mouse embryogenesis,
using northern blotting, in situ hybridization to histological sections and whole-mount hybridization histochemistry. c-ret transcripts were detected beginning at
day 8.5 of embryogenesis, and were observed in a
number of cell lineages in the developing peripheral and
central nervous systems, as well as in the excretory
system. In the cranial region at day 8.5-9.5, c-ret mRNA
was restricted to a population of neural crest cells
migrating from rhombomere 4 and forming the anlage
of the facioacoustic ganglion, as well as to a closely associated domain of surface ectoderm and pharyngeal
endoderm. At later stages (10.5-14.5 days), c-ret mRNA
was observed in all cranial ganglia. In the peripheral
nervous system of the trunk, c-ret was expressed in the
autonomic ganglia and in subsets of cells in the dorsal
root ganglia. In the enteric nervous system, c-ret was
expressed in the presumptive enteric neuroblasts of the
vagal crest (day 9.0-11.5), and in the myenteric ganglia
of the gut (day 13.5-14.5). c-ret mRNA was observed in
several regions of the central nervous system, including
the undifferentiated neuroepithelial cells of the ventral
neural tube (8.5 days), the motor neurons in the spinal
cord and the hindbrain (10.5-14.5 days), the embryonic
neuroretina (day 13.5) and the layers of the postnatal
retina containing ganglion, amacrine and horizontal
cells. Outside the nervous system, c-ret was expressed in
the nephric (Wolffian) duct at day 8.5-10.5, the ureteric
bud epithelium (but not the surrounding metanephric
mesenchyme) at day 11.0-11.5, and the growing tips of
the renal collecting ducts (but not the previously formed,
subcortical portions of the collecting ducts, or the mesenchyme-derived renal vesicles) at day 13.5-17.5. Our
results suggest that the c-ret gene may encode the
receptor for a factor involved in the proliferation,
migration, differentiation or survival of a variety of
neuronal cell lineages, as well as in inductive interactions
during organogenesis of the kidney.
INTRODUCTION
transformation has been studied extensively (Cantley et al.,
1991), but their function during embryogenesis of multicellular organisms is less well understood. The best evidence
that RTKs play a critical role in diverse developmental
processes is derived from genetic studies in both invertebrates and vertebrates (Hafen and Basler, 1990; Pawson and
Bernstein, 1990). In Drosophila melanogaster (D.
melanogaster), several well-characterized developmental
mutations have been shown to reside in genes encoding
RTKs. One of the best studied examples is the sevenless
locus, which encodes a tyrosine kinase receptor that
interacts with a cell surface molecule encoded at the boss
locus. Mutations at either of these loci prevent the differentiation of R7 photoreceptors and lead to abnormal patterning of the D. melanogaster eye. In mice, mutations in the
Dominant White Spotting (W) and Steel (Sl) loci, which
encode the kit RTK and its ligand respectively, cause similar
defects in the haematopoietic, germ cell and melanocytic
lineages (Green, 1989). Finally, RTKs of the trk subfamily
Normal development of the vertebrate embryo depends on
the proper communication between diverse cell types.
Several membrane-bound or diffusible molecules have been
identified as components of such an intercellular communication system. These molecules mediate their effects by
binding to specific cell surface receptors, a class of which
has intrinsic and ligand-dependent tyrosine kinase activity.
All receptor tyrosine kinases (RTKs) have a similar
topology: they possess an extracellular ligand-binding
domain, a transmembrane domain, and a cytoplasmic
segment containing the catalytic tyrosine kinase domain
(Yarden and Ullrich, 1988). Binding of the ligand leads to
dimerization of the receptor and activation of the kinase
domain, manifested as autophosphorylation of the receptor
and phosphorylation of specific substrates that mediate the
intracellular signaling (Ullrich and Schlessinger, 1990).
The role of RTKs in cellular proliferation and oncogenic
Key words: c-ret, tyrosine kinase receptor, mouse embryogenesis,
nervous system, excretory system
1006 V. Pachnis, B. Mankoo and F. Costantini
have recently been shown to function as the high affinity
receptors for the nerve growth factor (NGF)-related neurotrophins (Kaplan et al., 1991a,b; Klein et al., 1991a,b;
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
of NGF as a trophic factor for sympathetic neurons and
neural-crest-derived sensory neurons of the peripheral
nervous system (PNS), and certain cholinergic neurons in
the central nervous system (CNS) (Levi-Montalcini, 1987;
Martinez et al., 1985), strongly suggests a critical developmental role for the trk RTKs.
The c-ret proto-oncogene is a member of the RTK gene
superfamily. It was originally identified as a transforming
gene by transfection of T-cell lymphoma DNA into NIH3T3
cells (Takahashi et al., 1985). In vivo mutations of c-ret have
also been implicated in tumorigenesis. The PTC (papillary
thyroid carcinoma) oncogenes, detected frequently in
thyroid papillary carcinomas (25%), result from somatic
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 cret 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 protooncogene 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
and excretory systems, and lead us to propose that the ret
RTK is normally involved in several aspects of neurogenesis and kidney organogenesis.
MATERIALS AND METHODS
Embryos were derived from crosses between FVB/N inbred mice,
or between (FVB/N×C57BL/6)F1, or (C57BL10×CBA)F1 hybrid
mice. The morning of vaginal plug was considered 0.5 days post
coitum (dpc). The 8.5 dpc embryos were staged under the dissecting microscope by counting somites.
RNA was isolated from tissue culture cells or embryos according
to published procedures (Auffray and Rougeon, 1980). The cDNA
library was generated from poly(A)+ RNA isolated from Nb/2a
cells using the ZAP-cDNATM synthesis kit (Stratagene) according
to the manufacturer's recommendations.
In situ hybridization analysis on sections and whole-mount
hybridization histochemistry were performed essentially as
described (Wilkinson and Green, 1990, Wilkinson, 1992).
RESULTS
Isolation of cDNA clones from the murine c-ret
locus
To isolate cDNA clones of the murine homologue of the cret proto-oncogene, a cDNA library was constructed with
poly(A)+ RNA from the murine neuroblastoma cell line
Nb/2a (which, as shown below, expresses high levels of cret mRNA), and screened under stringent hybridization conditions with the insert of recombinant plasmid pret2.2,
which contains human c-ret mRNA sequences (Takahashi
and Cooper, 1987). Several positive clones were identified
and a partial restriction enzyme map of two of them,
pmcret7 and pmcret10, is shown in Fig. 1A. Partial sequenc-
Fig. 1. (A) Partial restriction enzyme map of the mouse c-ret
cDNAs identified and used in this study. Shown at the top is a
diagrammatic representation of the various domains of the human
ret receptor as deduced from its nucleotide sequence. The longest of
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′
untranslated sequences. pmcret10 corresponds to most of the
intracellular part of the ret receptor plus 3′ untranslated sequences.
Abbreviations: TK, tyrosine kinase domain; TM, transmembrane
domain. Restriction enzymes: B, BamHI; E, EcoRI; H, HindIII.
(B) Comparison of the amino acid sequence of the kinase domains
of the mouse and human ret receptors. Vertical lines indicate
identical amino acids, two dots indicate conservative substitutions
and one dot indicates semiconservative substitutions. The two
sequences are 95% identical and all the differences are either
conservative or semiconservative substitutions. The numbers
correspond to the first and the last amino acid of the kinase domain
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 cret 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
membranes (Fig. 2B, lane Em). We also detected c-ret
mRNA in the murine neuroblastoma cell line Nb/2a (Fig.
2C) and, at low levels, in the rat pheochromocytoma cell line
PC12 (not shown), a finding consistent with previous reports
of c-ret expression in other tumours and cell lines of neuroectodermal origin (Tahira et al., 1991; Ikeda et al., 1990;
Santoro et al., 1990; Takahashi et al., 1991).
In all RNA samples analyzed so far, representing three
vertebrate species, i.e. human (Tahira et al., 1990), rat
(Tahira et al., 1988) and mouse (this report), a similar set of
c-ret transcripts has been identified, migrating at 7.0, 6.0,
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
chicken (A. Schuchardt, V. P. and F. C., unpublished data)
c-ret cDNA clones has demonstrated that the various transcripts are generated from alternative splicing and/or
polyadenylation of the primary c-ret transcript. Such events
are likely to be under tissue-specific regulation, as suggested
by the reversed relative abundance of the 6.0 and 7.0 kb transcripts in mouse brain and salivary gland mRNA preparations (Fig. 2A).
In situ hybridization analysis of c-ret expression
during mouse embryogenesis
Expression in cranial neural crest cells and their
neurogenic derivatives
The expression of c-ret in tissues (e.g. brain) and cell lines
of neuroectodermal origin (neuroblastomas and pheochromocytomas), and its expression in embryos during organo-
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
12-week-old mice were hybridized with antisense pmcret7
riboprobe. The sizes of the four c-ret transcripts are indicated. The
apparent signal in the kidney lane was not observed in other
experiments. (B) Similar analysis of poly(A)+ RNA samples (5
µg), isolated from embryos at the designated stages of
development (8.5-16.5 dpc) and from extraembryonic membranes
of 12.5 dpc embryos (lane Em). (C) 1 µg of poly(A)+ RNA from
the murine neuroblastoma cell line Nb/2a hybridized with the
pmcret7 riboprobe. The set of c-ret transcripts observed in the
embryo, the extraembryonic membranes and the Nb/2a cells are
identical to those observed in RNA preparations from the adult
tissues.
genesis, raised the possibility that the gene plays a role in the
development of the vertebrate nervous system. Therefore, we
sought to determine the spatial distribution of c-ret transcripts during mammalian embryogenesis. In situ hybridization analysis was performed on mouse embryos between 7.5
and 14.5 days of gestation, a period during which the most
critical events of organogenesis are taking place. For these
experiments, we used a riboprobe derived from pmcret7
which contains sequences for a small part of the extracellular domain, the transmembrane and intracellular domains as
well as the entire 3′ untranslated sequence of the 4.5 kb transcript (Fig. 1A and C. Marcos, M. Sukumaran and V. P.,
unpublished observations). At 7.5 days c-ret mRNA was
absent from the embryo proper, but was detected in the trophoblast cells of the extraembryonic membranes (not
shown). 24 hours later (8.5-9.0 dpc), as predicted by the
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.
specific groups of cells within the embryo. In the head, strong
signal was localized to a cohesive group of cells immediately
anterior to the otic vesicle and extending from the dorsal edge
ventrally towards the forming second branchial arch (Fig.
3A-D). The position of these cells suggests that they are
neural crest cells emigrating from rhombomere 4 (r4) of the
hindbrain. Similar groups of cells emigrating from the neuroepithelium anterior or posterior to r4 were negative for cret transcripts (Fig. 3A-D). This apparently segment-specific
expression of c-ret in the neural crest of the hindbrain was
further examined by hybridization histochemistry on whole-
mount preparations of 8.75-9.0 day embryos, a stage of
embryogenesis at which the migration of all groups of cranial
neural crest is well under way. In agreement with the data of
Fig. 3A-D, strong c-ret-specific signal was restricted to a
single group of cranial neural crest cells emerging from the
neuroepithelium of the hindbrain at the level of r4 (Fig. 4A).
Two components of the hindbrain neural crest have been
described: the early migrating cells, which populate the
ventrally located branchial arches and adopt a mesenchymal
fate, and the late emerging cells, which remain closer to the
neural tube and adopt a neural fate (Nichols, 1981, 1986). The
Expression of c-ret proto-oncogene in mouse 1009
detection of c-ret mRNA in both the dorsal and ventral neural
crest of r4 (Fig. 3C,D) suggests that during this stage of
embryogenesis, both the neurogenic and the mesenchymal
components of the r4 crest express c-ret mRNA at comparable levels. Interestingly, during the embryonic period under
consideration, the brain neuroepithelium including r4, was
devoid of hybridization signal (Fig. 3A,B and data not shown).
In addition to the migrating neural crest cells, c-ret is
expressed in the 8.5-9.0 day embryo in a well-defined
domain of cranial surface ectoderm and pharyngeal
endoderm associated with the posterior branchial arches
(Fig. 3E-H). The c-ret-positive surface ectoderm includes
the epibranchial placodes, which give rise to the sensory
neurons of the inferior ganglia of the IXth and Xth cranial
nerve ganglion complexes (Altman and Bayer, 1982). No
signal was detected in the more anteriorly located epibranchial placodes.
Given the positionally restricted expression of c-ret in the
r4 neural crest and the epibranchial placodes, it was of interest
to examine its pattern of expression in cranial structures from
later stage embryos. In the head of 9.5 day embryos, and consistent with the restricted expression of ret in r4 neural crest
of earlier stage embryos, signal was localized in the condensing facioacoustic ganglion complex (VII-VIIIth) but not
in the anlage of other cranial ganglia present anteriorly (Vth)
or posteriorly (IXth, Xth) (Fig. 4B). Despite the expression of
c-ret in the anlage of the facioacoustic ganglion, no signal was
detected in the second branchial arch (Fig. 4B, ba2), which is
populated to a large extent by the mesenchymal component
of r4 crest. This indicates that by the time the r4 crest has
completed its migration into the second branchial arch, the
expression of c-ret is down-regulated. In 10.5 day embryos,
in addition to the facioacoustic ganglion, the inferior ganglia
of the IXth and Xth cranial nerve ganglion complexes are
positive for c-ret transcripts, while the trigeminal ganglion
(V) is still negative (Fig. 4C). Finally, in 13.5-14.5 day
embryos, all cranial ganglia, including the trigeminal ganglion
(Fig. 5A,B) and the superior ganglia of the IXth and Xth
cranial nerve ganglion complexes (not shown) are positive for
c-ret mRNA.
Overall, our data demonstrate that, in the head of the
postimplantation mouse embryo, c-ret expression is associated with the development of cranial ganglia. Two distinct
phases of c-ret expression can be discerned: an early,
segment-specific, phase (8.5-9.5 day embryo), during which
c-ret mRNA is restricted to neural crest cells derived from
r4 and the anlage of the facioacoustic ganglion, and a later
phase (10.5-14.5 days) during which signal appears
gradually in all cranial ganglia irrespective of the origin of
the contributing neural crest cells along the anteroposterior
axis of the brain neuroepithelium. The positionally restricted
localization of c-ret mRNA in the cranial neural crest and
the surface ectoderm and pharyngeal endoderm of the
branchial arches suggests that expression of c-ret is related
to the intrinsic mechanisms of segmentation of the vertebrate head.
Expression in the developing sensory and autonomic
ganglia of the trunk
c-ret mRNA was also present in the migrating neural crest
cells of the trunk, albeit at lower levels compared to the r4
neural crest. In sections from 9.5 day embryos, a diffuse and
relatively weak (but reproducible) signal was present
dorsally between the neural tube and the surface ectoderm
and ventrally in the sclerotome, areas of the somites in
which the trunk neural crest cells first migrate and subsequently condense to form the dorsal root ganglia (DRG; not
shown). In the DRG of later stage embryos (11.5-13.5 days),
the hybridization pattern was modified. Intense punctate
signal was observed in the ganglion itself, but was absent
from the emerging peripheral spinal nerve (Fig. 5C). This
suggests that expression of c-ret in the DRG is restricted to
subsets of neurons and is absent from the glial cells of the
ganglion or the spinal nerve.
Expression in the developing enteric nervous system
The expression of c-ret in the sensory ganglia raised the possibility that the gene is also expressed in the other major
branches of the PNS, i.e. the enteric and the autonomic
nervous system. The enteric nervous system (ENS) is
composed of the myenteric and the submucosal ganglion
complexes of the gut (Furness and Costa, 1987). The
majority of the cells in the ganglia of the ENS are derived
from the vagal crest originating from the postotic hindbrain
(corresponding to somites 1-7) (Yntema and Hammond,
1954; Le Douarin and Teillet, 1973). The presumptive
enteric ganglioblasts of the vagal crest migrate first ventrally
through the posterior branchial arches into the foregut mesenchyme, and then rostrocaudally inside the gut wall mesenchyme, where they eventually coalesce and form the
enteric ganglia along the entire length of the gut (Tucker et
al., 1986; Baetge and Gershon, 1989; Gershon et al., 1993;
Kapur et al., 1992). c-ret-positive cells have been identified
in the migratory pathway of the presumptive enteric neuroblasts of the vagal crest and in the myenteric ganglia of
the gut. In the 9.0-9.5 day mouse embryo, strong expression
was detected in mesenchymal cells of the third and fourth
branchial arches, between the c-ret-positive pharyngeal
endoderm and surface ectoderm (Fig. 3E,F). 12 hours later,
a stream of c-ret-positive cells could be seen emerging from
the posterior branchial arch mesenchyme migrating towards
the foregut (Fig. 6A). At subsequent stages (10.5-11.5 days)
c-ret-positive cells were found at increasingly more caudal
levels of the embryonic gut (Fig. 6B,C). Finally, in the 13.514.5 day embryo, in which the rostrocaudal migration of the
ENS precursors has been completed, strong c-ret expression
was detected in the myenteric plexus along the entire axis
of the gut (Fig. 6D). Although no independent molecular
markers were used in these experiments, the overall spatial
and temporal pattern of expression of c-ret in the developing gut is consistent with the gene being expressed in the
majority of the migrating precursors and postmigratory cells
of the ENS.
c-ret mRNA is also expressed in the autonomic ganglia
of the PNS. At embryonic day 9.5, c-ret transcripts were
detected in groups of cells by the dorsal aorta, where derivatives of the trunk neural crest coalesce to form the sympathetic ganglia anlage (Fig. 6A). Expression is maintained at
later stages, e.g. 14.5 days, when sympathetic gangliogenesis has been essentially completed (Fig. 6D). At this stage,
no expression of c-ret was observed in the endocrine derivatives of the sympathoadrenal lineage, the adrenal chromaf-
1010 V. Pachnis, B. Mankoo and F. Costantini
Fig. 4. Expression of c-ret 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, cret-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) Brightfield and dark-field photomicrographs
of a transverse section through the
caudal part of an 8.75 dpc embryo. cret-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 chromaffin cells (data not shown).
in the layers that contain the bulk of the bipolar cells and
the photoreceptors.
Expression in the developing central nervous system
During the period of embryogenesis that we examined, c-ret
is also expressed in the developing CNS. More specifically,
expression was observed in undifferentiated neuroepithelial
cells of the neural tube and their postmitotic derivatives.
Shown in Fig. 7A,B is a transverse section through the trunk
of a 8.75 dpc embryo. Hybridization signal is localized in
the neuroepithelial cells of the ventral half of the neural tube.
Strong signal was also detected laterally to the epithelial
somites, in the nephrotome. At 9.5 days, hybridization was
further restricted to the ventrolateral compartment of the
spinal cord, where the motor neurons are differentiating (not
shown). An identical labelling pattern was maintained
during the subsequent periods of embryogenesis that we
analyzed (10.5-14.5 dpc, Fig. 7C,D). The ventrally localized
domain of c-ret expression extends along the entire anteroposterior axis of the developing spinal cord, and extends
anteriorly into groups of motor neurons in the hindbrain
(data not shown).
We also detected c-ret mRNA in the neuroretina of the
embryonic and postnatal mouse eye. Shown in Fig. 8A,B is
in situ hybridization to a section through the eye of a 13.5
day mouse embryo. Hybridization is restricted to the
innermost layers of the neuroretina, where the first postmitotic neurons are born. To determine whether all groups of
postmitotic cells in the mammalian retina express c-ret, we
analyzed sections from eyes of postnatal day 7 mice, a stage
by which all classes of neurons and glial cells have been
generated (Turner and Cepko, 1987). As shown in Fig.
8C,D, c-ret mRNA is localized to certain layers of the
mature mammalian retina, those containing ganglion,
amacrine and horizontal cells. No expression was detected
Expression in the developing excretory system
Expression of c-ret in the nephrotome of 8.5 day embryos
prompted us to examine in more detail its expression in the
developing excretory system of the mouse. In vertebrate
embryos, the excretory system can be subdivided into three
sequentially appearing organs, the pronephros, mesonephros
and metanephros (Saxen, 1987). The pronephros and
mesonephros are transient embryonic structures, which
consist of a series of segmentally arranged tubules
connected to the nephric (Wolffian) duct. In situ hybridization on sections and hybridization histochemistry on
whole-mount preparations of 9.0-10.5 day embryos revealed
that c-ret is expressed in the nephric duct of the pronephros
(not shown) and mesonephros (Fig 9A).
The permanent, or metanephric kidney, is formed through
a series of reciprocal inductive interactions between the
ureteric bud, which emerges from the caudal part of the
nephric duct, and the surrounding metanephric mesenchyme
(Saxen, 1987). Upon invasion of the metanephric mesenchyme (day 11.5) the ureteric bud begins to branch
dichotomously. In embryonic kidneys at this stage, c-ret
transcripts were detected only in the epithelial cells of the
branching ureteric bud, but not in the surrounding mesenchymal cells (Fig. 9B). Subsequently, the branching tips
of the ureteric epithelium induce the surrounding undifferentiated mesenchyme to condense and differentiate into the
epithelial renal tubules and glomeruli, while the mesenchyme induces the ureteric bud to grow and branch,
forming the collecting ducts. At more advanced stages of
organogenesis, these inductive interactions take place only
in a narrow region around the outer edge of the kidney, the
nephrogenic zone (Saxen, 1987). In sections of embryonic
Expression of c-ret proto-oncogene in mouse 1013
Fig. 8. c-ret expression in the mouse eye. (A,B) Bright-field and
dark-field photomicrograph of a section through the eye of a 13.5
dpc embryo. Signal is observed in the innermost layer of the
neuroretina (r), where the first postmitotic neurons are born (pe,
pigmented epithelium). (C,D) Bright-field and dark-field
photomicrograph of a section through the retina of an albino
postnatal day 7 mouse. Signal is observed in the ganglion (g), the
amacrine (a) and the horizontal (h) cell layers. No signal is
observed over the bipolar (b) or the photoreceptor (ph) cell layers.
kidneys at these stages (13.5-17.5 dpc), c-ret mRNA was
detected only in the nephrogenic zone (Fig. 9C). At high
magnification, it could be seen that the hybridization signal
was restricted to the growing tips of the collecting ducts, but
was absent from all of the more centrally located structures,
including the condensing renal vesicles and nephrons (of
mesenchymal origin), and the subcortical segments of the
collecting ducts (Fig. 9D).
DISCUSSION
We have cloned the murine homologue of the c-ret protooncogene and studied its spatial and temporal pattern of
expression during mouse embryogenesis, using northern
blot analysis, in situ hybridization to sections and hybridization histochemistry on whole-mount preparations. Our
studies demonstrate that, in the postimplantation embryo, c-
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-ret 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-ret 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-ret 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-ret 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 Hox gene
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
neural crest translate into different neuronal and mesenchymal structures in the head of the adult animal? The differential activation of downstream target genes could provide the
means of translating such positional information into diverse
developmental programs. The restricted expression of the cret gene in the r4 neural crest of the 8.5-9.5 day embryo
provides additional evidence that the various groups of
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
unique properties of the r4 crest derivatives.
In the PNS, c-ret is expressed in the developing
autonomic nervous system, the ENS and the sensory
ganglia of the head and the trunk, suggesting that the ret
signal transduction pathway is involved in the organogenesis of all major branches of the PNS. Phenotypic analysis
of the sympathoadrenal progenitors and the progenitors of
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.,
1991; Johnson et al., 1990; Lo et al., 1991). This led to the
suggestion that sympathoadrenal and enteric lineages are
derived from progenitors that are either identical or closely
related (Carnahan et al., 1991), and that the particular phenotypes eventually acquired by these progenitors are determined by the specific microenvironment at their final destination. The concomitant expression of c-ret in the anlage
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 relationship. Moreover, it raises the possibility that c-ret plays a
role in the proliferation, migration, differentiation or
survival of the cells of these lineages. The expression of cret mRNA in the migratory ENS progenitors is particularly
interesting, since, despite the established importance of
signals encountered along the migratory pathway of the
vagal crest and in the microenvironment of the embryonic
bowel for the differentiation of the enteric neurons
(Gershon et al., 1993), very little is known about the
molecular nature of these signals. Our data suggest that the
ret receptor might play a critical role in the cellular and
molecular processes that lead to the organogenesis of the
ENS, i.e. migration of the vagal crest, commitment to the
neuroblast or glioblast fate, cell proliferation and survival
of postmitotic cells in the enteric ganglia. This hypothesis
is strongly supported by our recent finding that mouse
embryos homozygous for a loss-of-function mutation of the
c-ret gene, generated by homologous recombination in
embryonic stem cells, fail to develop enteric ganglia
(Schuchardt et al., unpublished data).
The apparent localization of c-ret mRNA in subsets of
neurons of the sensory ganglia (cranial nerve ganglia and
DRG) is consistent with previous studies indicating that
distinct gene expression programmes exist in the various
functional classes of the sensory neurons. Using monoclonal
antibodies, Dodd and colleagues were able to identify
subsets of primary sensory neurons in rat DRG expressing
unique carbohydrate differentiation antigens (Dodd et al.,
1984; Dodd and Jessell, 1985). More recently, in situ hybridization analysis of the pattern of expression of members of
the trk RTK subfamily has revealed that, similarly to the ret
receptor, individual members of the trk subfamily are
expressed in subclasses of DRG cells (Carroll et al., 1992).
Furthermore, immune deprivation of NGF in utero resulted
in selective ablation of trk-expressing cells of the DRG
(Carroll et al., 1992; Ruit et al., 1992). Our data suggest that,
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
class of primary sensory neurons. The identity of this class
and the particular sensory modality that it conveys are
currently unknown.
In the CNS, expression has been detected predominantly
in the motor neuron lineages of the spinal cord and the
hindbrain, and in subsets of cells of the neuroretina. Lineage
analysis of the cells of the vertebrate retina has shown that
all cell types of the mature retina are derived from an early
common progenitor (Turner and Cepko, 1987; Turner et al.,
1990; Wetts et al., 1989), and strongly suggests that cellular
interactions in the developing vertebrate retina play a critical
role in the establishment of the various cellular phenotypes.
Although certain growth factors have been identified as
playing an important role in the differentiation of retinal
cells in vitro (Lillien and Cepko, 1992; Guillemot and
Cepko, 1992), the molecules that mediate such cellular interactions in vivo have yet to be identified. Our expression data,
combined with the nature of the ret protein, suggest that it
may play a role in the cell-cell interactions that determine
the cellular phenotypes of particular classes of neurons in
the mammalian retina.
The molecular mechanisms underlying the potential role
of c-ret in the development of the mammalian nervous
system are currently unknown. However, the structural similarity of ret to other members of the RTK family and its
pattern of expression in the developing PNS and CNS,
suggest that it functions as the receptor to a (potentially
novel) neurotrophic factor(s) required for the migration,
commitment, differentiation, or survival of certain cell
lineages of the mammalian nervous system.
Molecular characterization of c-ret cDNAs from patients
with MEN2A syndrome suggests strongly that gain-offunction mutations in the c-ret proto-oncogene are responsible for this inherited cancer syndrome (Mulligan et al.,
1993). Although the documented expression of c-ret in cell
lines of neural crest origin is consistent with the neural crest
origin of medullary thyroid carcinoma (C-cells of the
thyroid) and pheochromocytoma (sympathoadrenal progenitor), the occurrence of parathyroid hyperplasia in
patients with MEN2A syndrome has been puzzling, as
parathyroid cells are derived embryologically from the
endoderm of the fourth pharyngeal pouch (Balinsky, 1970).
This led to the suggestion that the tumours of the MEN2A
syndrome may result from defects in closely linked genes
that independently control neural crest or endocrine tissue
development (Lairmore et al., 1991). Our expression
studies further support the suggestion that the entire
MEN2A syndrome is caused by mutations in the c-ret
proto-oncogene by providing evidence that all lineages
affected in this syndrome are likely to express the c-ret
proto-oncogene. The precursors of the C-cells, originating
from the posterior hindbrain, are transiently localized,
along with the precursors to the ENS, in the mesenchyme
of the posterior branchial arches (Le Douarin, 1982), an
area where c-ret is strongly expressed (Fig. 3E-H). This,
along with the high levels of c-ret expression in C-cellderived medullary thyroid carcinomas (Santoro et al., 1990)
and our preliminary experiments, which show low levels of
expression in the C-cells of adult mouse thyroid (P. Durbec,
B. M. and V. P., unpublished data), suggests that the C-cell
lineage expresses c-ret mRNA from the early embryonic
stages. Despite lack of expression in the adrenal medulla,
it is likely that the chromaffin cell precursors are also
expressing c-ret mRNA since they originate from the retpositive cells of the sympathetic ganglia condensations
(Fig. 6A; (Pankratz, 1931)). Finally, the parathyroid cell
precursors are derived from the c-ret-positive endoderm of
the posterior branchial arches (Fig. 3E,F). Overall, the
common expression of c-ret reveals a developmental link
between the cell types affected in the MEN2A syndrome
and provides further support for the hypothesis that germline mutations in c-ret are responsible for all the manifestations of the MEN2A syndrome.
In addition to its postulated role in the developing nervous
system, the expression pattern of the c-ret gene suggests that
the ret receptor could function during the development of
the excretory system. c-ret mRNA was first detected at day
8.5 in the nephrotome, a region of lateral mesoderm from
which the embryonic (pronephric and mesonephric) and
permanent (metanephric) kidneys are formed and continued
to be detected in the nephric (Wolffian) ducts during the
development of the pronephric and mesonephric kidneys
(day 9.5-10.5). The expression pattern of c-ret at this stage
suggests that the ret receptor might play a role in the
formation of the pronephros and mesonephros.
The development of the mammalian metanephric kidney
has been extensively studied as a model system for the role
of inductive tissue interactions during organogenesis
(Saxen, 1987). Formation of the kidney is initiated on day
11 of mouse embryogenesis, when the ureteric bud emerges
near the caudal end of the nephric duct and grows dorsally,
eventually contacting the metanephric blastema, a specific
condensate of mesenchymal cells. At this point, the ureteric
bud begins to grow and branch repeatedly, dependent upon
specific induction by the metanephric mesenchymal cells
(Grobstein, 1953, 1955; Erickson, 1968), and eventually
giving rise to the entire renal collecting system (calyces,
papillae and collecting ducts). At the same time, the tips of
the branching ureteric bud induce the surrounding mesenchymal cells to condense into epithelial renal vesicles,
which eventually differentiate to form the various segments
of the nephron, including the proximal and distal tubules
and glomeruli (Grobstein, 1955, 1956; Saxen, 1987). At a
stage when the ureteric bud has first branched within the
metanephric mesenchyme, we observed that c-ret was
strongly expressed in the ureteric bud epithelium, but was
not expressed at detectable levels in the surrounding undifferentiated mesenchyme. At later stages of renal organogenesis, growth and branching of the ureteric derivatives,
as well as the induction of new nephrons, is limited to a
narrow ‘nephrogenic’ zone at the perimeter of the kidney
(Saxen, 1987). At these stages (day 13.5-17.5), c-ret mRNA
was observed only in the tips of the growing collecting
ducts in the outer nephrogenic zone, and not in the previously formed and more centrally located collecting ducts.
Like the undifferentiated metanephric mesenchyme, both
the newly condensed renal vesicles and the more mature
mesenchymal derivatives continued to be negative for c-ret
expression.
Based on this restricted pattern of expression, it appears
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
structures in the kidney. More specifically, ret might serve
as the receptor for an inductive factor, produced by the
metanephric mesenchymal cells, which stimulates the
growth and/or branching of the ureteric bud epithelium.
Support for the hypothesis that the c-ret gene serves an
important function in renal organogenesis has recently
been obtained by the production of mice carrying a
targeted, loss-of-function mutation of the c-ret gene
(Schuchardt et al., unpublished data). The homozygous
mutant mice, in addition to the ENS defects noted above,
never develop normal kidneys, and display a spectrum of
excretory development ranging from tiny, hypodysplastic
kidney rudiments, to blind-ending ureters with no renal
tissue, to the complete absence of ureters and kidneys.
Analysis of cDNA clones derived from the human
(Tahira et al., 1990), the murine (C. Marcos, M.
Sukumaran and V. P., unpublished observations) and the
chicken (A. Schuchardt, V. P. and F. C.) c-ret locus
indicates that its transcripts are capable of encoding at least
two distinct receptors differing at their intracellular
carboxy-terminal tails. Since the probe that we used for our
in situ hybridization analysis includes cRNA sequences
common to all transcripts, we are currently unable to distinguish the two potential murine ret isoforms. However,
it is possible that activation of the ret receptor by its
cognate ligand could lead to distinct cellular responses
depending on the predominant ret isoform present on the
cell surface. It would therefore be of interest to examine
the differential distribution of the various c-ret transcripts
and their protein products during mouse embryogenesis,
using transcript-specific probes and isoform-specific antibodies.
We thank Geoffrey Cooper for allowing us to use the pret2.2
plasmid. We also thank David Wilkinson for critical reading of the
manuscript. This work was supported by a fellowship by the
Leukemia Society of America to V. P., by NIH grant HD25335 to
F. C. and by the Medical Research Council (MRC, UK).
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(Accepted 1 September 1993)