Download Axonal projections of mechanoreceptive dorsal root ganglion

RESEARCH ARTICLE 2319
Development 137, 2319-2328 (2010) doi:10.1242/dev.046995
© 2010. Published by The Company of Biologists Ltd
Axonal projections of mechanoreceptive dorsal root
ganglion neurons depend on Ret
Yutaka Honma1,*, Masako Kawano1, Shinichi Kohsaka2 and Masaharu Ogawa1
SUMMARY
Establishment of connectivity between peripheral and central organs is essential for sensory processing by dorsal root ganglion
(DRG) neurons. Using Ret as a marker for mechanoreceptive DRG neurons, we show that both central and peripheral projections
of mechanoreceptive neurons are severely impaired in the absence of Ret. Death of DRG neurons in Ret-deficient mice can be
rescued by eliminating Bax, although their projections remain disrupted. Furthermore, ectopic expression of the Ret ligand
neurturin, but not Gdnf, in the spinal cord induces aberrant projection of mechanoreceptive afferents. Our results demonstrate
that Ret expression in DRG neurons is crucial for the neurturin-mediated formation of precise axonal projections in the central
nervous system.
INTRODUCTION
Establishing precise neuronal circuits during development is
essential for the proper execution of various neural activities.
Dorsal root ganglion (DRG) neurons form a circuit that relays
signals from peripheral sensory organs to the central nervous
system. DRG neurons that transmit different sensory modalities
project their axons to distinct laminae in the spinal cord (Mirnics
and Koerber, 1995b; Sanes and Yamagata, 1999), and the
development of these projections is a complex process (Altman and
Bayer, 2001). First, DRG proximal afferents travel to the spinal
cord, where they bifurcate and extend ascending and descending
branches within the prospective dorsal funiculus. Then, collaterals
invade the gray matter of the spinal cord and terminate in the target
lamina. Finally, the ascending fibers of DRG neurons within the
dorsal funiculus proceed to the dorsal column nuclei of the
medulla. Peripheral projections of sensory afferents have been
examined in detail in rat embryos (Mirnics and Koerber, 1995a).
First, afferent fibers exit the lumbosacral DRG at E12, and by E14
they are present in the epidermis of the proximal hindlimb. Fibers
originating from L3 to L5 reach the paw by E14.5-15, and the
epidermis of the most distal toes is innervated by E16-16.5. Given
that the development of afferent projections in the spinal cord has
been shown in several studies to be delayed relative to the
innervation of the hindlimb, it has been proposed that peripheral
innervation may stimulate central axon growth (Smith and Frank,
1988). However, recent experiments using carbocyanine dyes argue
against this hypothesis (Mirnics and Koerber, 1995a).
DRG neurons can be divided into three groups based on sensory
modality: nociceptive, mechanoreceptive and proprioceptive
(Marmigere and Ernfors, 2007). Nociceptive afferent neurons
penetrate into the dorsal horn of the spinal cord and terminate in
laminae I and II. Mechanoreceptive neuron afferents invade the
1
Ogawa Research Unit, Brain Science Institute, RIKEN, Saitama 351-0198, Japan.
Department of Neurochemistry, National Institute of Neuroscience, Kodaira, Tokyo
187-8502, Japan.
2
*Author for correspondence ([email protected])
Accepted 10 May 2010
medial gray matter of the spinal cord, then turn and enter the dorsal
horn ventrally to terminate in laminae III and IV of the dorsal horn.
Proprioceptive afferents pass through the medial part of the dorsal
horn without branching and reach the ventral spinal cord, where
they finally synapse to motoneurons (Caspary and Anderson,
2003). Whereas the axon terminals of nociceptive neurons are
strictly confined to the dorsal horn and do not enter the ventral
spinal cord, proprioceptive central afferents do not branch in the
dorsal horn and are able to invade the ventral spinal cord (Eide and
Glover, 1997). Thus, it has been suggested that repulsive and
attractive cues residing in the dorsal horn and/or the ventral spinal
cord guide each afferent to the proper region (Perrin et al., 2001;
Sharma and Frank, 1998). However, despite some progress (Frank,
2006), the mechanisms governing central afferent projections to the
spinal cord remain unclear.
Neurotrophins [Ngf, Bdnf, NT-3 (Ntf3) and NT-4 (Ntf5)] play a
crucial role in the differentiation, innervation pattern and survival
of sensory neurons (Lewin and Barde, 1996; Markus et al., 2002).
The sensory modality of a DRG neuron is tightly correlated with
Trk subtype expression (Moqrich et al., 2004; Sun et al., 2008).
Nociceptive neurons express TrkA (Ntrk1) and proprioceptive
neurons express TrkC (Ntrk3). However, only a subset of
mechanoreceptive neurons expresses TrkB (Ntrk2) (GonzalezMartinez et al., 2004), and no histochemical marker for all
mechanoreceptive neurons has yet been identified (Snider and
Wright, 1996).
Ret is a receptor tyrosine kinase that binds to the glial cell linederived neurotrophic factor (GDNF) family ligands (GFLs) Gdnf,
neurturin (Nrtn), artemin and persephin (Airaksinen and Saarma,
2002). Recent studies of genetically modified mice have
demonstrated that GFL/Ret signaling is important for cell migration,
axonal outgrowth and cell survival during peripheral nervous system
development (Fundin et al., 1999; Kramer et al., 2006a). In the
sensory nervous system, Ret expression first starts in TrkA+ smalldiameter neurons during embryonic development, but these Ret+
neurons downregulate TrkA expression and eventually become
TrkA–, nonpeptidergic, isolectin-B4 (IB4)+ nociceptive DRG neurons
after birth, while Ret– TrkA+ neurons become peptidergic nociceptive
neurons. Ret is responsible for the acquisition of several properties
DEVELOPMENT
KEY WORDS: Neuroscience, Axon guidance, Mechanoreceptive neuron, Sensory neuron, Mouse
of nonpeptidergic sensory neurons (Luo et al., 2007; Molliver et al.,
1997). Since Ret-deficient mice die perinatally due to renal agenesis,
conditional deletion of Ret in sensory neurons has been performed
to analyze the role of Ret in nonpeptidergic nociceptive neuron
development (Luo et al., 2007). Loss of Ret affects only the
peripheral, and not the central, projections of nonpeptidergic
nociceptive neurons. In addition, the number of cells expressing
Gfr1 and Gfr2 is significantly reduced in conditional Ret-null
mutants, whereas nonpeptidergic neuronal loss is not observed. Thus,
Ret signaling regulates the expression of these Gfr co-receptors
during nonpeptidergic nociceptive neuron development, without
affecting cell survival in these neuronal populations.
Furthermore, the expression of Ret in small TrkA+ nociceptive
neurons is regulated by the neurotrophic factor Ngf, which instructs
a subset of TrkA+ sensory neurons to adopt a nonpeptidergic
neuronal fate by suppressing TrkA expression (Luo et al., 2007).
Several experiments have provided evidence that Ret is also
expressed in large-diameter DRG neurons from early in DRG
development (Kramer et al., 2006b; Luo et al., 2007), and Ret+
fibers have been observed in the deep dorsal horn. However, the
physiological role of Ret in large-diameter DRG neurons is not
well understood (Ernsberger, 2008). In order to determine whether
Ret signaling plays a role in large-diameter neurons, we examined
Ret mutant mice in which tau-GFP was knocked into the Ret locus.
We found that Ret is required for the projections of
mechanoreceptive neurons during development.
MATERIALS AND METHODS
Animals
The generation of RetGFP/GFP and Gfra3–/– mice was described previously
(Enomoto et al., 2001; Honma et al., 2002). Bax-deficient mice were
purchased from Jackson Laboratory (Knudson et al., 1995). Nrtn-GFP
transgenic mice were obtained from Mutant Mouse Regional Resource
Centers (Gong et al., 2003). This study was carried out in accordance with
the Guide for the Care and Use of Laboratory Animals from the Society
for Neuroscience and was authorized by the Animal Care and Use
Committee of RIKEN.
In utero electroporation
The introduction of DNA into E12.5 spinal cord was performed by in utero
electroporation. The expression vector (1-5 g/l) was mixed with 0.05%
Fast Green as a tracer and injected into the central canal of the embryo
through the uterine wall. After injection, electrodes (CUY 650-5, Nepa
Gene) were placed on both sides of the embryo and electroporation was
performed using a square-pulse electroporator CUY21 EDIT (Nepa Gene).
Antibodies
The following antibodies were used: mouse anti-Isl1 (DSHB); rabbit anticleaved caspase 3 and rabbit anti-Aml1 (Runx1) (Cell Signaling); rabbit
anti-Runx3 (Osaki et al., 2004); goat anti-Gfr1, goat anti-Gfr2, goat
anti-Ret, goat anti-TrkB, goat anti-TrkC and goat anti-neurturin (R&D);
rabbit anti-Ret (IBL); rabbit anti-TrkA, rabbit anti-NF-200, rabbit antiperipherin and guinea-pig anti-vGlut1 (Millipore); mouse anti-cytokeratin
20 and rabbit anti-S100 (DAKO); rabbit anti-PGP9.5 (Uchl1) (Lab Vision);
rabbit anti-GFP (MBL and Invitrogen); rat anti-GFP (Nacalai); and chick
anti-GFP (Aves Labs).
Development 137 (14)
To determine the percentage of neurons expressing Isl1, GFP, Gfra1 and
Gfra2 per ganglion, we prepared five adjacent sets of 12-m sections from
L4 and L5 DRG from three E14.5 embryos per group, and then stained each
separately with an antibody or riboprobe. To compare the number of neurons
labeled with each marker, Isl1 staining was used to determine the total
number of L4 and L5 DRG neurons at E14.5. To quantitate the number of
neurons undergoing apoptosis, we prepared five adjacent sets of 12-m
sections, and the number of cells labeled with GFP and caspase 3 antibodies
was counted. The number of neurons expressing each marker in controls was
set to 100%. To investigate the cell size distribution, L3-L5 DRG of E18.5
embryos were collected and dissociated with trypsin (n3). After plating and
staining with GFP antibody, the cell size distribution of the mean soma area
for GFP+ neurons was examined. To quantitate central and peripheral
projections, we prepared five adjacent sets of 16-m sections from the entire
series of axial spinal cord (corresponding to C5-C6 DRG levels) or of the
forelimb. The positive fibers were framed and quantitated from ImageJ
(version 1.41, NIH). We set the fluorescence intensity in controls to 100%.
n3 for each genotype. To evaluate the attractive effect of ectopic Nrtn
quantitatively, the lower edge of the dorsal horn to the upper edge of the
central canal of spinal cord was divided horizontally into three bins, as
indicated in Fig. 7. The average pixel intensity of fibers in each bin was
determined using ImageJ. Twenty sections from five embryos electroporated
with Nrtn were used for this analysis.
Data are presented as mean ± s.e.m. and were compared using Student’s
t-test.
RESULTS
Two distinct types of Ret+ DRG neurons during
development
To characterize Ret expression in the sensory nervous system, we
used mice in which tau-GFP was knocked into the Ret locus
(RetGFP/GFP) to map the projections of Ret+ DRG neurons in vivo
(Enomoto et al., 2001). Consistent with previous data, the GFP
reporter accurately reflected the Ret expression pattern. To assess
which population of early embryonic DRG neurons expresses Ret
and to examine the central and peripheral axonal projection
patterns of Ret+ neurons, we examined GFP expression in an
extensive array of sensory neuron targets.
DRG neurons began expressing GFP at ~E11.5 (see Fig. S1A,B
in the supplementary material). Most of these GFP+ neurons had
larger somata than TrkA+ small-diameter neurons. Few, if any,
small-diameter DRG neurons, which express TrkA, were GFP+
even at E14.5 (see Fig. S1C in the supplementary material).
However, by E18.5, the number of GFP+ small-diameter neurons
had increased, even though the percentage of GFP+ large-diameter
DRG neurons did not change during embryonic development
(E13.5-18.5), remaining at ~7-10% of Isl1+ neurons. This suggests
that there are two distinct populations of Ret+ DRG neurons
(Kramer et al., 2006b). One is a small population of large-diameter
neurons that begin expressing Ret at E11.5, the percentage of
which, relative to the total number of DRG neurons, is stable
throughout embryonic development. The other population
comprises small-diameter neurons that emerge at ~E13.5, express
TrkA (see Fig. S1D in the supplementary material), and gradually
become the major population of Ret+ DRG neurons at later stages
of embryonic development.
Histological and statistical analyses
To compare the phenotype of central afferents between Ret+/GFP and
RetGFP/GFP mice, lower cervical level and upper thoracic level spinal cords
were prepared. To examine the Ret+ central and peripheral projection, 1015 mice for each genotype at E12.5-18.5 were used, except where noted
otherwise in the figure legends and experimental procedures.
To perform whole-mount immunostaining, we cut the skin of the left
upper forelimb in half; the radial side was stained with NF-200 antibody,
while the ulnar side was stained with peripherin antibody.
Development of Ret+ central afferents in the
spinal cord
We carefully examined GFP+ axonal projections in the spinal cord
during early embryonic stages (E12.5-16.5) in order to understand
the behavior of central afferent projections of Ret+ large-diameter
neurons. The axon collaterals of GFP+ DRG neurons began to
penetrate the spinal cord gray matter at E12.5 (Fig. 1C). At E12.5,
DEVELOPMENT
2320 RESEARCH ARTICLE
Ret function in mechanoreceptive neurons
RESEARCH ARTICLE 2321
the oval bundle of His, which is the primordium of the dorsal
funiculus formed by GFP+ fibers, was distinct from both TrkA+ and
TrkC+ oval bundles, suggesting that Ret+ afferents form a unique
path within the dorsal funiculus (Fig. 1A,B). Central afferents
projected laterally underneath the deep dorsal horn at E14.5 (Fig.
1H), and finally terminated in the deep dorsal horn with upward
endings at E16.5 (Fig. 1J). GFP+ terminal plexuses were also
observed in the medial part of the gray matter (Fig. 1J). Even at
postnatal day (P) 6, GFP-labeled Ret+ axon collaterals were found
in the deep dorsal horn that were labeled with vesicular glutamate
transporter 1 (vGlut1; Slc17a7 – Mouse Genome Informatics),
which marks the central afferents that transmit low-threshold
mechanoreceptive sensory inputs to the deep dorsal horn of the
spinal cord (Todd et al., 2003; Yoshida et al., 2006) (Fig. 1L,M).
We also observed weak GFP expression in the superficial layer of
the dorsal horn (lamina II), which probably reflects labeling of the
terminals of IB4+ Ret+ neurons. This signal grew more intense after
P7 (see Fig. S1E in the supplementary material).
Mechanoreceptive afferents terminating in the
deep dorsal horn are severely disturbed in
Ret-deficient mice
To determine whether loss of Ret affects the development of the
mechanoreceptive sensory neurons that normally express Ret
during embryonic development, we examined the phenotype of
GFP+ fibers in RetGFP/GFP mice. GFP+ DRG neurons were detected
in RetGFP/GFP and Ret+/GFP mice at E11.5 (see Fig. S1A,B in the
supplementary material). At E12.5, the first GFP+ axonal
projections were observed, but no GFP+ axon collaterals were
observed in the spinal cord gray matter in RetGFP/GFP mice (Fig.
1D). At E13.5, GFP+ axon collaterals were found in the gray matter
of RetGFP/GFP mice (Fig. 1G), but were few in number compared
with those in Ret+/GFP mice (Fig. 1E-G). Although the number of
axon collaterals invading the spinal cord increased from E13.5-14.5
in RetGFP/GFP mice (Fig. 1G,I), the fibers proceeding to the medulla
within the dorsal funiculus were sparse at E14.5 (Fig. 1I). These
results suggest that there are fewer distal DRG central axons in
RetGFP/GFP mice than in control mice. Furthermore, the axon
collaterals of RetGFP/GFP mice appeared to lose their direction
within the spinal cord (Fig. 1I), whereas those of Ret+/GFP mice
extended laterally along the developing dorsal horn with proper
directionality (Fig. 1H). Finally, GFP+ axon collaterals were almost
entirely excluded from the deep dorsal horn in RetGFP/GFP mice at
E16.5 (Fig. 1K), suggesting that Ret-ablated neurons might die due
to their inability to project axons to the proper targets. Collectively,
these results indicate that the loss of Ret in large-diameter DRG
neurons delays their central afferent projections and impairs
directional axonal extension within the spinal cord.
We next examined whether all mechanoreceptive afferents
terminating in the deep dorsal horn are affected in RetGFP/GFP mice.
We first performed DiI tracing to label all centrally projecting DRG
afferents. DiI labeling at E16.5 revealed severe fiber loss in the
deep dorsal horn, despite a lack of obvious abnormalities in
proprioceptive axonal projections to the ventral spinal cord (Fig.
DEVELOPMENT
Fig. 1. Characterization of Ret+ central afferents in the spinal cord, which are aberrant in Ret mutants. (A,B)The oval bundle of Ret+ fibers
(green in A) encircles the TrkC+ oval bundle (red in A) and differs from TrkA+ fibers (blue in B). (C,D)Central projections of Ret+ neurons are impaired
in RetGFP/GFP mice. GFP+ axon collaterals invade the developing dorsal horn normally at E12.5 in RetGFP/GFP mice, but fail to penetrate into the spinal
cord (D). Furthermore, GFP+ fibers are sparse within the oval bundle in RetGFP/GFP mice (D), as compared with those in Ret+/GFP mice (C). (F-K)Central
afferents invading the spinal cord of RetGFP/GFP mice are increased at E13.5 (G), but still sparse compared with those in Ret+/GFP mice (F). Axon
collaterals in Ret+/GFP mice project laterally (H), but those of RetGFP/GFP mice appear to lose their direction at E14.5 (I). Collaterals finally terminate in
the deep dorsal horn with upward endings at E16.5 in Ret+/GFP mice (J), but these fibers are severely impaired in RetGFP/GFP mice (K). Vertically
running fibers projecting to the medulla within the dorsal funiculus in Ret+/GFP mice (arrow in H) are severely impaired in RetGFP/GFP mice at E14.5
(arrow in I). (L,M)Central afferents projecting into the deep dorsal horn are apparent even at P6 (L), and these afferents are also labeled with
vGlut1, a marker for the deep dorsal horn (M). (E)Central afferent projections are significantly reduced in RetGFP/GFP mice at E12.5 and E13.5. n3
for each group. *P<0.05; **P<0.01 (Student’s t-test). Scale bars: 50m.
2322 RESEARCH ARTICLE
Development 137 (14)
Fig. 2. The selective loss of central afferents of
mechanoreceptive neurons in the deep dorsal horn. (A-D)DiI
labeling and vGlut1 immunostaining in E16.5 mice revealed that
mechanoreceptive central afferents projecting into the deep dorsal horn
are exclusively eliminated in RetGFP/GFP mice (arrowheads).
(E-H)Parvalbumin+ proprioceptive afferents and TrkA+ nociceptive
afferents are identical in RetGFP/GFP and Ret+/GFP mice. (I-N)Double
immunostaining for GFP and TrkA, vGlut1 and parvalbumin was
performed at E18.5. GFP+ fibers are impaired (J,L,N) and double
staining of GFP with vGlut1 is severely reduced in RetGFP/GFP mice
(arrowheads in I,J). Punctate GFP staining reflects Ret+ cells in the deep
dorsal horn (J,L,N). Scale bars: 50m.
2A,B). We then investigated the expression of vGlut1 because it
marks the central afferents that transmit low-threshold
mechanoreceptive sensory inputs to the deep dorsal horn of the
spinal cord. We found that vGlut1 expression was exclusively
eliminated from laminae III-IV, but was retained in the intermediate
and ventral gray matter of spinal cord at E16.5 in Ret-deficient
mice (Fig. 2C,D). Thus, all mechanoreceptive sensory afferents are
lost from the deep dorsal horn in the absence of Ret. Consistent
with the preservation of vGlut1 expression in the intermediate and
ventral spinal cord in RetGFP/GFP mice, parvalbumin+
proprioceptive afferents were not affected (Fig. 2E,F). In addition,
TrkA+ cutaneous nociceptive afferents in laminae I-II were
comparable in RetGFP/GFP and Ret+/GFP mice (Fig. 2G,H). We also
found similar abnormalities at E18.5 in the deep dorsal horn of
RetGFP/GFP mice (Fig. 2I-N). These results indicate that Ret is
exclusively required for mechanoreceptive neuron terminals in the
deep dorsal horn, and that loss of these terminals does not result in
invasion of proprioceptive and nociceptive afferents into the deep
dorsal horn.
Ret+ fibers innervate mechanoreceptors in the
periphery and are severely impaired in
Ret-deficient mice
Next, we examined GFP-labeled peripheral projections in Ret+/GFP
mice. Because we still observed GFP immunostaining in the deep
dorsal horn, as described above (Fig. 1L,M), it seemed likely that
GFP staining marks early large-diameter Ret+ DRG sensory neuron
fibers during early postnatal stages. Thus, we examined GFP+ axon
terminals in the forelimb skin at P3-P6. GFP+ fibers with lanceolate
or Ruffini endings were found around hair follicles (Fig. 3A).
Furthermore, we found GFP+ fibers innervating several types of
mechanoreceptors, including Meissner corpuscles (Fig. 3D),
Merkel cells (Fig. 3E) and Pacinian corpuscles (Fig. 3F) in the skin
and the crural interosseous membrane. Thus, Ret+ DRG neurons
project into the deep dorsal horn (laminae III-IV) and innervate
mechanoreceptors in the skin, suggesting that large-diameter Ret+
DRG neurons are mechanoreceptive neurons.
To explore whether Ret deficiency also influences peripheral
projections, we examined GFP+ fibers in the body wall skin at
E12.5 and in the forelimb skin at E18.5 in RetGFP/GFP mice. GFP+
fibers were observed in the body wall at E12.5 (data not shown)
and were abundant around hair follicles in forelimb skin at E18.5
(Fig. 3G) in Ret+/GFP mice. These fibers were severely reduced in
RetGFP/GFP mice (Fig. 3G), suggesting that Ret is required for both
the central and peripheral projections of these neurons.
Since Ret+ peripheral afferents are defined as mechanoreceptive
fibers, which are usually myelinated, we next explored whether
loss of Ret affects NF-200+ fibers innervating the skin. As
expected, NF-200 (Nefh – Mouse Genome Informatics)
immunostaining, which marks prospective myelinated axons
(Zylka et al., 2005), was significantly reduced in the skin of E18.5
RetGFP/GFP as compared with Ret+/GFP mice (Fig. 3H). However,
these axons were not completely lost, consistent with the
observation of a few GFP+ fibers in RetGFP/GFP skin (Fig. 3G). By
contrast, fibers that are positive for peripherin, a marker for
prospective unmyelinated axons (Kosaras et al., 2009; Lariviere et
al., 2002), were unaffected in RetGFP/GFP mice (Fig. 3I), consistent
with the idea that Ret+ neurons are myelinated mechanoreceptive
neurons, although the staining specificity of NF-200 and peripherin
is somewhat controversial (Jackman and Fitzgerald, 2000). To
examine the cutaneous branch of the sensory nerve in Ret-deficient
mice, we performed whole-mount NF-200 and peripherin
immunostaining of the dorsal skin of the left upper forelimb at
E14.5. NF-200+ fibers in RetGFP/GFP mice were thinner than those
in Ret+/GFP mice, but their branching appeared to be intact (Fig.
3J,K). By contrast, peripherin+ fibers were not impaired in
RetGFP/GFP mice (Fig. 3L,M). Thus, our data show that the
peripheral projections of NF-200+ sensory fibers are severely
reduced in RetGFP/GFP mice.
DEVELOPMENT
Since laminar structure could influence the lamina-specific
termination of DRG neurons, we examined the laminar structure of
the dorsal horn using lamina-specific molecular markers, including
Ebf1, Drg11 (Prrxl1 – Mouse Genome Informatics), Brn3a
(Pou4f1) and c-Maf (Li et al., 2006). At E14.5, expression of these
molecules in dorsal horn was normal in RetGFP/GFP mice,
suggesting that the dorsal horn structure is not impaired by the lack
of Ret (see Fig. S2A-H in the supplementary material), despite
expression of Ret in the developing dorsal horn (see Fig. S2I in the
supplementary material). Thus, it is unlikely that the defects in
mechanoreceptive central afferent projections are due to aberrant
dorsal horn formation.
Fig. 3. Characterization of Ret+ peripheral afferents, which are
impaired in Ret-deficient mice. (A)Ret+ fibers with lanceolate and
Ruffini endings innervate hair follicles in hairy skin at P3. (B,C)Most
Ret+ fibers are positive for neurofilament 200 (NF-200), a myelinated
axon marker (B), but not for peripherin, an unmyelinated axon marker
(C). (D-F)Ret+ peripheral fibers also innervate Meissner corpuscles
(positive for PGP9.5) (D), Merkel cells [positive for cytokeratin 20
(Krt20)] (E) and Pacinian corpuscles (S100+) (F) in P6 peripheral tissues.
(G-I)At E18.5, GFP+ and NF-200+ axons in the skin are severely
impaired in RetGFP/GFP mice (G,H), but peripherin+ axons are spared (I).
Quantitative analysis revealed significant reduction of GFP+ and NF200+ peripheral projections in Ret-deficient mice at E18.5 (G,H),
whereas peripherin+ fibers are intact (I). (J-M)Whole-mount
immunohistochemistry for NF-200 and peripherin in the upper forelimb
skin at E14.5 revealed that NF-200+ fibers are dense in Ret+/GFP mice
(arrowheads in J), but severely impaired in RetGFP/GFP mice (arrowhead in
K). By contrast, peripherin+ fibers are unaffected even in RetGFP/GFP mice
(L,M). n3 for each group. **P<0.01 (Student’s t-test). Scale bars:
50m.
The role of Nrtn-Gfr2 in Ret+ mechanoreceptive
neurons
GFL signaling requires a receptor complex comprising Ret and a
Gfr co-receptor. Since the effects of Ret deficiency on the
expression of Gfr co-receptors have only been examined during
the later stages of embryonic development (Luo et al., 2007), we
examined co-receptor expression in DRG neurons during early
embryogenesis in Ret mutant mice. Because we did not observe
any abnormalities in central and peripheral Ret+ DRG afferents in
RESEARCH ARTICLE 2323
Ret+/GFP; Gfra3–/– mice (see Fig. S3A-D in the supplementary
material), we focused on Gfr1 and Gfr2. Antibodies against
either Gfr1 or Gfr2 and GFP were used to double label DRG
neurons. At E13.5, Gfr2+ cells (93±1%) were GFP+ largediameter neurons (Fig. 4D-F), whereas Gfr1+ cells (30±2.8%)
were GFP+ small-diameter neurons (Fig. 4A-C). Thus, during
embryonic development, Gfr2 is the best candidate for a Ret coreceptor in large-diameter DRG neurons.
Because Gfr2 is a known co-receptor for Nrtn (Heuckeroth et
al., 1999; Rossi et al., 1999), we next examined Nrtn expression
during embryonic development. Since Nrtn expression is scarcely
detectable by in situ hybridization, we used transgenic mice
carrying GFP under the control of the Nrtn promoter in order to
follow Nrtn expression during development. We found Nrtn
expression in the DRG and in peripherally projecting axons at
E12.5 (Fig. 4J). At E13.5, Nrtn was expressed in the dorsal root
entry zone and in the path of the central afferents (Fig. 4K). We
also detected signal in the prospective dorsal funiculus and deep
dorsal horn at E13.5 (Fig. 4M). Although Nrtn expression in the
proximal afferents and DRG became weak at E14.5 (data not
shown), expression in the deep dorsal horn was clearly evident at
E14.5 (Fig. 4O). The signal in the deep dorsal horn was scattered
at E13.5 (Fig. 4M), but was concentrated on the lateral side of the
dorsal horn at E14.5 (Fig. 4O). The Nrtn signal appeared to precede
the GFP+ fiber projections in the deep dorsal horn of Ret+/GFP mice
(Fig. 4L-O). Finally, the Nrtn expression level in the dorsal horn
was decreased by E16.5 (data not shown). At E15.5, Nrtn was also
expressed in the periosteum and ligaments of the developing
forelimb at E12.5 (data not shown) and around the hair follicles, a
potential target of mechanoreceptive neurons (Fig. 4P).
Intriguingly, we observed that Ret+ central afferents terminated in
the deep dorsal horn in chick embryos (Fig. 4Q) and that Nrtn
expression was detected in the region where those fibers project
(Fig. 4R), as in the mouse (Fig. 4O). Moreover, chick Nrtn
expression was also seen around the hair follicles, as in the mouse
(Fig. 4P,S), suggesting that the mechanism by which
mechanoreceptive neuron projections find their path might be
evolutionary conserved. These observations support the idea that
Nrtn is involved in the development of the peripheral and central
projections of mechanoreceptive neurons via Ret and Gfr2.
Characterization of DRG neuron abnormalities in
Ret-deficient mice
Several lines of evidence indicate that functionally distinct DRG
neurons express specific neurotrophic factor receptors, and that this
diversification underlies the segregation of DRG neurons according
to sensory modality (Woolf and Ma, 2007). To assess whether the
loss of Ret influences the segregation of neurotrophic factor
receptor expression in DRG neurons, we performed doublelabeling experiments at E13.5 with an anti-GFP antibody and
antibodies against different neurotrophic factor receptors. There
was little overlap between GFP expression and neurotrophic factor
receptor expression in the DRG neurons of E13.5 Ret+/GFP mice
(Fig. 5C,E,G), in agreement with a previous report (Kramer et al.,
2006b). The GFP and Trk receptor expression patterns in
RetGFP/GFP mice were almost identical to those in Ret+/GFP mice
(Fig. 5C-H), suggesting that Ret signaling is not involved in the
segregation of Trk receptor expression. Furthermore, we found that
there was little overlap between GFP expression and that of Runx1
(a marker for nociceptive neuron) or Runx3 and parvalbumin
(markers for proprioceptive neurons) in Ret+/GFP mice (Patel et al.,
2003; Sun et al., 2008; Yoshikawa et al., 2007), and the expression
DEVELOPMENT
Ret function in mechanoreceptive neurons
2324 RESEARCH ARTICLE
Development 137 (14)
Fig. 4. Role of Gfr2-Nrtn in Ret+ mechanoreceptive neuron development. (A-F)Double immunolabeling for GFP (B,E) and Gfr1 (A) or
Gfr2 (D) in E13.5 DRG sections. Most GFP and Gfr1 double-positive neurons have small somata (C), whereas almost all GFP and Gfr2 doublepositive neurons have large somata (F). Notably, most Gfr2+ cells coexpress GFP (F), whereas many Gfr1+ cells do not (C). (G-I)GFP+ cells were
stained for Isl1. (J-M)At E12.5, Nrtn is detected on the path of peripherally projecting sensory afferents (arrowheads in J). At E13.5, Nrtn is also
expressed in proximal axons (arrow in K) and the dorsal root entry zone of DRG neurons (arrowhead in K), as well as in the primordium of the
dorsal funiculus (arrowhead in L,M) and deep dorsal horn (arrow in M). (N-P)At E14.5, Nrtn is expressed in the deep dorsal horn of the spinal cord
(arrowhead in O), where Ret+ axon collaterals project (arrowhead in N). In the peripheral tissue, Nrtn is expressed in the hair follicles, which are
target tissues of Ret+ mechanoreceptive neurons at E15.5 (P). (Q-S)Similar to mouse Nrtn, chick Nrtn (cNRTN) expression is also found in the deep
dorsal horn (arrowhead in R), where Ret+ fibers innervate (arrowhead in Q), as well as in hair follicles (S). Scale bars: 50m.
in number, which might reflect the observation that most
peripherin+ neurons are hypotrophic in Ret-deficient mice, as
previously described (Luo et al., 2007). Collectively, these results
indicate that large-diameter sensory neurons are severely reduced
in the DRG of Ret-deficient mice.
Bax deficiency rescues loss of GFP+ and
Gfra2-expressing neurons, but fails to rescue
mechanoreceptive neuron projection defects
In order to determine whether the reduction in the number of
neurons expressing GFP or Gfra2 was caused by cell loss or by
transcriptional downregulation, we used an antibody against
cleaved caspase 3 to measure cell death. The number of labeled
neurons in DRG of Ret+/GFP and RetGFP/GFP mice was counted at
E13.5, before the stage at which we observed the massive reduction
in GFP+ and Gfra2-expressing neurons (see above). There were
more cleaved caspase 3+ cells in RetGFP/GFP than in Ret+/GFP mice
(121% of control) (Fig. 6F), suggesting that cell death is
responsible for the reduction in Ret+ DRG neurons at E14.5.
The axonal defects we observed might simply reflect DRG
neuron loss in the absence of Ret. To eliminate the influence of cell
death, we generated mice that were double null for Ret and Bax. In
Bax knockout mice, cell death is virtually eliminated in peripheral
ganglia, including the DRG (White et al., 1998). The number of
GFP+ and Gfra2-expressing neurons in the DRG was normal in
RetGFP/GFP; Bax–/– mice (Fig. 6G), suggesting that Bax-mediated
programmed cell death is involved in neuronal loss during
DEVELOPMENT
pattern in RetGFP/GFP mice was comparable to that in Ret+/GFP mice
(Fig. 5I-N). These observations indicate that Ret is not involved in
the segregation of DRG neurons with respect to sensory modality.
Because GFP+ afferent projections were impaired during early
embryogenesis in Ret mutants, we examined whether Ret functions
in mechanoreceptive neurons during early embryonic development.
We first tested whether impaired central and peripheral DRG
projections in Ret-deficient early embryos affect sensory neuron
development. In situ hybridization revealed loss of Gfra2
expression in the DRG of E14.5 RetGFP/GFP mice (Fig. 6C,D),
whereas Gfra1 expression was identical to that of controls (Fig.
6A,B). We then quantitatively examined the number of Isl1+,
GFP+, Gfra1-expressing and Gfra2-expressing neurons (Fig. 6E).
Isl1 labeling was used to evaluate the total number of DRG
neurons. We detected a 37% reduction in the number of GFP+
neurons in Ret-deficient embryos at E14.5, even though the
number of Isl+ neurons was not significantly reduced. The small
subset of GFP+ DRG neurons at E14.5 (7% of Isl1+ neurons) might
explain this discrepancy. The number of Gfra2-expressing neurons
was also markedly reduced in RetGFP/GFP embryos (by 44%,
compared with Ret+/GFP embryos), whereas the number of Gfra1expressing neurons was unchanged. We also observed a GFP+
neuron reduction in RetGFP/GFP mice at E18.5 (by 23%, compared
with Ret+/GFP embryos). In addition, when we examined the mean
soma area of GFP+ neurons, we found that the number of largediameter GFP+ neurons in RetGFP/GFP mice at E18.5 was reduced
(Fig. 5A,B). Cells with small somata also appeared to be increased
Ret function in mechanoreceptive neurons
RESEARCH ARTICLE 2325
Fig. 5. DRG neuron abnormalities in Ret-deficient mice. (A,B)The number of GFP+ large-diameter neurons is dramatically reduced in RetGFP/GFP
mice at E18.5. Cell size distribution analysis using dissociated DRG neurons also revealed severe reduction of large-diameter neurons in RetGFP/GFP
mice. (C-N)Double labeling for GFP (green) and the proteins indicated (red) in E14.5 DRG sections from control Ret+/GFP (C,E,G,I,K,M) and RetGFP/GFP
(D,F,H,J,L,N) mice. A small number of GFP+ neurons from control Ret+/GFP and RetGFP/GFP mice express one of the Trk proteins. Notably, the number of
GFP+ DRG neurons was reduced in RetGFP/GFP mice (C-H). Staining with other molecular markers for nociceptive (Runx1) and proprioceptive (Runx3
and parvalbumin) neurons also revealed no DRG neuron segregation abnormalities in RetGFP/GFP mice (I-N). Scale bar: 50m.
Ectopic Nrtn attracts Ret+ DRG central afferents to
an aberrant region of the spinal cord
Given that Ret-ablated mechanoreceptive neurons in RetGFP/GFP
mice fail to extend axons in the proper direction and to project
into the deep dorsal horn, we hypothesized that the Ret signal
might be involved in the attraction of mechanoreceptive afferents
to the deep dorsal horn of the spinal cord. To test this hypothesis
directly, we introduced an expression vector for the Gfr2 ligand
Nrtn into Ret+/GFP mouse spinal cords by in utero electroporation
at E12.5, and harvested the embryos 3 days later to determine
whether Ret+ fibers were attracted to exogenously expressed
Nrtn. Consistent with our hypothesis, GFP+ fibers were attracted
to the region of ectopic Nrtn expression (Fig. 7A-D). By contrast,
neither Gdnf nor DsRed attracted GFP+ afferents (Fig. 7H; data
not shown). TrkA+ nociceptive afferents and parvalbumin+
proprioceptive afferents in spinal cord did not respond to ectopic
Nrtn expression (Fig. 7F,G), indicating that mechanoreceptive
central fibers are the only afferents that are able to respond to
Nrtn. Moreover, Nrtn had no attractive effect on central afferents
in Ret-deficient mice (Fig. 7I), indicating that the attractive
effect of Nrtn is Ret dependent.
DISCUSSION
Ret expression has been observed in large-diameter DRG neurons,
but the sensory modality of these Ret+ large-diameter neurons was
unclear (Ernsberger, 2008). Here, we show that Ret+ DRG neurons
send peripheral axons to mechanoreceptors in the skin and central
axon collaterals to the deep dorsal horn in the spinal cord. These
results indicate that Ret+ large-diameter neurons are
mechanoreceptive neurons. Moreover, we found that Ret is
required for proper laminar termination of mechanoreceptive
neuron central projections in the spinal cord. The peripheral
projections of mechanoreceptive neurons are also severely
disturbed in the absence of Ret. Finally, we show that Nrtn, a Ret
ligand, is expressed in the region where Ret+ neurons send their
axons, and that ectopic Nrtn attracts Ret+ fibers to an aberrant
region of the spinal cord. These results demonstrate that Ret
signaling is required for mechanoreceptive neurons to establish the
circuit between peripheral and central organs, and also suggest that
central afferent growth within the spinal cord is independently
regulated from the signal for peripheral projections.
Neurotrophic factors for mechanoreceptive
neurons
Neurotrophins (i.e. Bdnf, NT-3 and NT-4) play a developmental
role in the survival, function and axonal projections of
mechanoreceptive DRG neurons (Airaksinen et al., 1996; Cronk et
al., 2002; Stucky et al., 1998). However, neurotrophins have only
been implicated in the postnatal development of these neurons
(Carroll et al., 1998), and no trophic factors involved in their
development during embryonic stages have thus far been described.
Our data show that Ret is essential for the development of
mechanoreceptive
neuron
axonal
projections
during
DEVELOPMENT
embryonic development of Ret-deficient DRG neurons.
Remarkably, although the number of neurons was restored by
knocking out Bax in RetGFP/GFP mice (Fig. 6G), the central and
peripheral axonal projections of mechanoreceptive neurons were
still impaired (69% and 89% reduction, respectively, as compared
with Ret+/GFP; Bax–/– mice) (Fig. 6H-M). Taken together, these data
suggest that cell death is not the direct cause of the axonal defects.
Instead, the axonal projections themselves are dependent on Ret in
mechanoreceptive neurons.
Fig. 6. Cell death of large-diameter neurons in Ret-deficient mice.
(A-D)DRG neurons from E14.5 control Ret+/GFP and RetGFP/GFP mice
were hybridized with Gfra1 and Gfra2 in situ probes. Representative
sections show a reduction in Gfra2, but not Gfra1, expression in
RetGFP/GFP mice compared with control mice. (E)The percentage of Isl1+,
GFP+, Gfra1-expressing and Gfra2-expressing DRG neurons was
quantitatively analyzed at E14.5. The number of positive cells for each
marker in control Ret+/GFP mice was set to 100%. n3 for each group.
*P<0.05; **P<0.01 (Student’s t-test). (F)Quantitative analysis of the
percentage of cleaved caspase 3+ cells in E13.5 Ret+/GFP and RetGFP/GFP
mice shows an increase in apoptotic cells in the homozygous mutant.
The number of positive cells for each marker in Ret+/GFP mice was set to
100%. n4 for each group. **P<0.01 (Student’s t-test).
(G)Quantitative analysis of the percentage of Isl1+, GFP+, Gfra1expressing and Gfra2-expressing DRG neurons in E14.5 Ret+/GFP; Bax–/–
and RetGFP/GFP; Bax+/– and RetGFP/GFP; Bax–/– mice. Bax deficiency rescued
the number of DRG neurons expressing GFP or Gfra2. n3 for each
group. **P<0.01 (Student’s t-test). (H-K)Ret+ fibers in the deep dorsal
horn and skin are severely impaired in E16.5 RetGFP/GFP; Bax+/– mice
compared with control Ret+/GFP; Bax–/– mice. (L,M)Bax deficiency failed
to rescue the axonal defect in the deep dorsal horn and skin of mice
lacking Ret. Scale bars: 50m.
embryogenesis. Previous work showed that D-hair
mechanoreceptors switch their neurotrophin dependence from NT3 to NT-4 during postnatal development, even after DRG neurons
are terminally differentiated (Stucky et al., 2002). It therefore
appears that DRG neurons change their dependence on trophic
factors several times before maturation.
Development 137 (14)
Fig. 7. Attractive effect of Nrtn on Ret+ central afferents in the
spinal cord. (A-C)Ectopic Nrtn-induced aberrant extension of Ret+
afferents (arrowheads in A). Although a slight attraction of Ret+ fibers
on the control side was observed (Bin 2), ectopic Nrtn significantly
induced Ret+ fiber projection compared with the non-electroporated
control side (Bin 3). Horizontal black dashed lines in B indicate the lines
used to divide bins 1-3, as quantified in C. n5. *P<0.01 (Student’s ttest). (D,E)Higher magnification views also reveal the attractive effect
of ectopic Nrtn on GFP+ fibers in the spinal cord (arrowheads). Double
staining for GFP with DsRed , as superimposed in E, shows the GFP+
fibers winding around DsRed+ cells expressing Nrtn. (F,G)TrkA+ and
parvalbumin+ central afferents do not respond to ectopic Nrtn. (H)Gdnf
does not have any attractive effect on Ret+ afferents. (I)GFP+ fibers are
not attracted to ectopic Nrtn in Ret-deficient mice. EP, electroporated.
Scale bars: 50m.
It has been suggested that a subset of mechanoreceptive neurons
express TrkB (Kramer et al., 2006b). Is there a correlation between
Ret and TrkB in mechanoreceptive neuron development before
birth? In TrkB–/– mice, central afferents terminating in the
intermediate zone of the spinal cord, where myelinated
mechanoreceptors innervate, are severely reduced, although the
central afferents in the deep dorsal horn are intact (Silos-Santiago
et al., 1997). By contrast, central afferents terminating in the
intermediate zone are not impaired in RetGFP/GFP mice. Together
with the fact that TrkB+ neuron number is not reduced in
RetGFP/GFP mice, this suggests that TrkB-dependent
mechanoreceptive neurons are distinct from Ret-dependent
mechanoreceptive neurons. It is possible that mechanoreceptive
neurons that project to the intermediate gray matter of the spinal
cord depend on TrkB, but are not Ret-dependent for their
innervation, at least before birth.
DEVELOPMENT
2326 RESEARCH ARTICLE
The role of Nrtn in the survival of
mechanoreceptive neurons
Ret signaling is dispensable for the viability of nonpeptidergic
DRG neurons in vivo (Luo et al., 2007). By contrast,
mechanoreceptive neurons undergo cell death in Ret-deficient mice
at E13.5. In mechanoreceptive neurons, Ret may function in the
survival of Ret+ and Gfr2+ neurons, as Bax deficiency rescued the
number of cells expressing GFP and Gfr2 co-receptors in Retdeficient mice. However, we could not conclude whether or not
Nrtn is the survival factor for mechanoreceptive neurons because
a small number of GFP+ Gfra2-expressing neurons survive in
RetGFP/GFP mice. Moreover, a few GFP+ fibers are already found in
the peripheral tissues and spinal cord of RetGFP/GFP mice at E12.5.
These observations support the idea that once GFP+ neurons reach
the skin, a target-derived trophic factor other than Nrtn promotes
their survival. Indeed, GFLs fail to support embryonic DRG
sensory neuron survival in the absence of Ngf in vitro, despite the
expression of Ret and its co-receptors in DRG neurons (Baudet et
al., 2000). It is likely that Ret signaling is required only for axonal
projections, but not for the survival of mechanoreceptive neurons.
However, it is still possible that other molecules, such as
neurotrophins, induce mechanoreceptive neuron axonal growth
even before birth, similar to what occurs in postnatal development.
Thus, Nrtn might be involved in both mechanoreceptive neuron
survival and axonal projections.
Nrtn is required for the axonal projections of Ret+
mechanoreceptive neurons
DRG neurons that convey different sensory modalities send axons
into distinct laminae within the spinal cord, but the molecular
mechanisms underlying lamina-specific projection patterns are
largely unknown. Proprioceptive central afferents are disrupted in
Nt3-null mice (Ernfors et al., 1994), and NT-3 attracts axons in
DRG explant cultures in vitro (Genc et al., 2004). We found that
mechanoreceptive neurons require Ret to establish their terminal
projections in the deep dorsal horn. Ectopic Nrtn induces aberrant
axonal extension of mechanoreceptive neurons via Ret in vivo. Nrtn
is expressed in the dorsal horn, suggesting that Nrtn acts as a
diffusible guidance cue for mechanoreceptive afferent neurons.
Thus, we propose that a diffusible guidance cue exists for each class
of DRG neuron that attracts afferents in the spinal cord. However,
when ectopic Nrtn is introduced into the superficial dorsal horn by
electroporation, Ret+ afferents fail to invade the region (our
preliminary observation). This raises the possibility that repulsive
cues in the superficial lamina of the dorsal horn prevent Ret+ fibers
from invading the region. Sema3a repels Ngf-responsive axons, but
has little effect on NT-3-responsive axons (Messersmith et al.,
1995), suggesting that a repulsive cue might also exist for each class
of DRG neuron. Mechanoreceptive afferents take a unique path into
the spinal cord, which might indicate that they use their own
repulsion molecules to terminate in the deep dorsal horn. It will be
interesting to explore which repulsive cues act in concert with the
attractive Nrtn cue to guide mechanoreceptive afferent projections
and to reveal how precise neural circuits are established during
DRG development. Repulsive cues for mechanoreceptive afferents
remain to be identified, but histochemical Ret labeling should allow
mechanoreceptive afferent behavior within the spinal cord to be
traced. In the periphery, we have observed Nrtn expression along
the peripheral sensory afferents and in their target tissues, but not
along the ventral motor efferents. These findings suggest that Nrtn
is required for the peripheral afferents to find their target organs, as
is the case for the central projections. The generation of mice that
RESEARCH ARTICLE 2327
ectopically express Nrtn in the spinal cord and/or the peripheral
tissues might allow us to better understand the mechanism by which
Nrtn regulates the projections of mechanoreceptive neurons.
Ret signaling in pathological conditions
Previous studies have shown that Gfr2 is the main co-receptor of
Ret in nonpeptidergic nociceptive neurons (Luo et al., 2007). Thus,
Gfr2 appears to play a major role in both nonpeptidergic
nociceptive and mechanoreceptive Ret+ sensory neurons. In some
pathological conditions, non-nociceptive stimuli evoke pain; for
example, in allodynia, tactile stimuli cause pain (Sandkuhler, 2009).
It is hypothesized that mechanoreceptive neurons undergo an
abnormal phenotypic switch to nociceptive neurons in allodynia, and
that non-nociceptive stimuli might evoke pain sensation through
mechanoreceptive neurons. Although ligand-receptor binding may
normally activate different downstream targets in different cell types
and therefore elicit distinct actions, under pathological conditions it
is possible that this distinction is lost. Thus, one intriguing hypothesis
is that Ret-Gfr2 signaling is partly responsible for this phenotypic
switch of mechanoreceptive neurons to nociceptive neurons in
allodynia.
Acknowledgements
We thank Dr Jeffrey Milbrandt for providing Ret and Gfra3 knockout mice; Dr
Mitsuhiko Osaki for providing Runx3 antibody; Drs Masato Hoshi and Kazuyo
Kamitori for their technical support; Dr Takayoshi Inoue for providing the pCA
expression vector for in utero electroporation; Drs Tomomi Shimogori and
Toshiyuki Araki for their comments on the manuscript; and Dr Toshio Ohshima
and members of the M.O. laboratory for their helpful discussions.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.046995/-/DC1
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