|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 2 April 2008
doi: 10.1242/dev.015248
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1
Tennodai, Tsukuba, Ibaraki 305-8577, Japan.
2 Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore
138673, Singapore.
Author for correspondence (e-mail:
tshiga{at}md.tsukuba.ac.jp)
Accepted 3 March 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Runx, Transcription factor, Dorsal root ganglion, Proprioceptive neuron, TrkC (Ntrk3), TrkA (Ntrk1), Parvalbumin, Cell fate specification, Axonal projection
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). These distinct
sensory neuron subpopulations have different molecular characteristics, and
terminate in different laminar targets within the spinal cord and peripheral
targets. Trk receptors play crucial roles in neuronal survival,
differentiation and axon projection of DRG neurons
(Bibel and Barde, 2000;
Huang and Reichardt, 2001;
Markus et al., 2002;
Patel et al., 2003;
Snider, 1994). A recent study
suggested that the selective expression and signaling of Trk receptors in DRG
neurons are involved in axonal target selection in the spinal cord
(Moqrich et al., 2004).
However, the molecular mechanisms that govern sensory neuron differentiation
and the axonal projections remain unclear.
In mammals, the Runt-related (Runx) transcription factor family consists of
three members: Runx1, 2 and 3. These Runx transcription factors interact with
a common co-factor, polyomavirus enhancer-binding protein 2 [Pebp2; also known
as core binding factor β (Cbfβ)] with highly conserved Runt
DNA-binding domain that belongs to the Runx transcription factors, and play
important roles in developmental processes of various cell types including
hematopoietic cells, osteoblasts and gastric epithelial cells (for reviews,
see Coffman, 2003;
Ito, 2004). Runx1 and Runx3
are also expressed in subtypes of neurons in the peripheral and central
nervous systems (Simeone et al.,
1995; Theriault et al.,
2004; Theriault et al.,
2005). In the DRG, reciprocal expression during early stages of
development has been reported: Runx1 is initially expressed in
TrkA+ presumptive cutaneous neurons, while Runx3 is expressed in
TrkC+ presumptive proprioceptive neurons
(Chen et al., 2006a;
Chen et al., 2006b;
Kramer et al., 2006;
Levanon et al., 2001;
Marmigère et al.,
2006). These Runx transcription factors play crucial roles in the
cell fate specification and axonal projections of DRG neurons
(Chen et al., 2006a;
Chen et al., 2006b;
Inoue et al., 2002;
Kramer et al., 2006;
Levanon et al., 2001;
Levanon et al., 2002;
Marmigère et al., 2006;
Yoshikawa et al., 2007) (for a
review, see Marmigère and Ernfors,
2007). However, there seem to be some discrepancies concerning the
role of Runx3. Inoue et al. showed that TrkC+ DRG neurons are
differentiated and maintained, but the projection of proprioceptive axons to
both central and peripheral targets is severely impaired in
Runx3-/- embryos and newborns
(Inoue et al., 2002). By
contrast, initial appearance and subsequent loss of proprioceptive phenotypes
including TrkC expression was reported in DRG neurons of
Runx3-/- embryos, suggesting the possibility of cell death
of proprioceptive DRG neurons (Levanon et
al., 2002). More recently, it has been shown that TrkC+
DRG neuron numbers are decreased, with a concomitant increase in TrkB
(Ntrk2)-expressing (TrkB+) DRG neurons, in
Runx3-/- embryos by embryonic day (E) 12.5
(Kramer et al., 2006;
Inoue et al., 2007). Runx3
promotes DRG neuron differentiation to a solitary TrkC+ phenotype
by repressing TrkB expression in hybrid TrkB+/TrkC+ DRG
neurons, and by maintaining the expression of TrkC within prospective
proprioceptive DRG neurons (Kramer et al.,
2006; Inoue et al.,
2007). These findings raise the question as to the role of Runx3
in the control of cell fate specification of proprioceptive DRG neurons,
including its role in the expression of TrkC.
In this study, to gain further insight into the function of Runx3 in cell
type specification and axonal projections of DRG neurons, we performed a cell
fate analysis of Runx3-/- dorsal root ganglia (DRGs)
during embryonic and neonatal stages. We show that Runx3 is required for the
cell fate specification and the axonal projection of proprioceptive DRG
neurons. However, a subset of TrkC+ DRG neurons appears
independently of Runx3 that may subserve cutaneous sensation. Therefore,
TrkC+ DRG neurons are divided into two subpopulations on the basis
of Runx3 dependency: Runx3-dependent early-appearing proprioceptive neurons;
and Runx3-independent late-appearing cutaneous neurons. Moreover, we show that
Runx3 positively regulates the expression of TrkA and calcitonin gene-related
peptide (CGRP; Calca) in addition to TrkC and parvalbumin (PV; Pvalb),
suggesting that Runx3 might be involved in the development of cutaneous, as
well as of proprioceptive, DRG neurons. Considering our previous study
reporting that Runx1 negatively regulates the expression of TrkA, CGRP and
TrkC (Yoshikawa et al., 2007),
it can be suggested that Runx1 and Runx3 have antagonistic roles in the
development of DRG neuron subpopulations.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Li et al.,
2002). Bax-deficient mice
(Knudson et al., 1995) were
purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were bred in a
clean room in the Laboratory Animal Resource Center at the University of
Tsukuba. Runx3+/- and Runx3+/+ mice
were obtained from mating of Runx3+/- mice.
Runx3+/-; Bax+/- double-knockout mice and
Runx3+/+; Bax+/- control mice were
obtained from the mating of Runx3+/-; Bax+/-
double heterozygotes. Timed embryos were obtained by overnight mating, and the
morning when the vaginal plug was observed was considered E0.5. For genotyping
of Runx3 deficiency, PCRs were performed using primer pairs for
wild-type (NA, 5'-GACTGTGCATGCACCTTTCACCAA-3' and CB,
5'-TAGGGCTCAGTAGCACTTACGTCG-3') or mutant (NA and C2,
5'-ATGAAACGCCGAGTTAACGCCATCA-3') allele detection. For genotyping
of Bax deficiency, PCRs were performed using a set of three primers:
Bax exon 5 forward primer (5'-TGATCAGAACCATCATG-3'),
Bax intron 5 reverse primer (5'-GTTGACCAGAGTGGCGTAGG-3')
and Neo reverse primer (5'-CCGCTTCCATTGCTCAGCGG-3'). All
experiments followed the Guide for the Care and Use of Laboratory Animals
described by the National Institutes of Health (USA), and were approved by the
Animal Experimentation Committee of the University of Tsukuba.
Immunohistochemistry
For cryostat sections, E11.5 and E13.5 whole mouse embryos were immersed
overnight at 4°C in a fixative containing 4% paraformaldehyde (PFA) in 0.1
M phosphate buffer (PB) (pH 7.4). Embryos at E14.5 and older were perfused
transcardially with the same fixative and immersed overnight at 4°C. The
trunk at thoracic segments (Th) and lower limbs including the gastrocnemius
muscle were dissected and immersed in 30% sucrose in 0.1 M PB and frozen in
Tissue-Tek OCT compound (Sakura Finetek, Japan). Ten or 12 µm transverse
sections of Th or axial sections of legs were cut and collected onto
MAS-coated glass slides (Matsunami Glass, Japan) and air-dried for 1 hour. If
needed, sections were subjected to heat-induced epitope retrieval by heating
to 105°C for 5 minutes in REAL Target Retrieval Solution (Dako). After
treatment for 30 minutes at room temperature (RT) with 0.3%
H2O2 in methanol, the sections were incubated for 1 hour
at RT in a blocking solution containing 1% BSA and 0.1-0.5% Triton X-100 in
PBS.
For immunohistochemical analysis, the following antibodies were used:
rabbit anti-Runx1 (Sigma; 1:1000 dilution), mouse anti-Runx3 (Abnova; 1:2000),
rabbit anti-PV (Swant; 1:2000), goat anti-TrkC (R&D Systems; 1:2500),
rabbit anti-calretinin (CR) (Swant; 1:2000), rabbit anti-vesicular glutamate
transporter 1 (Vglut1) (a gift from Dr M. Watanabe, Hokkaido University,
Sapporo, Japan; 1:1000), mouse anti-Islet1 (Developmental Studies Hybridoma
Bank; 1:200), rabbit anti-CGRP (Chemicon; 1:4000), rabbit anti-TrkA (a gift
from Dr F. Reichardt, University of California, San Francisco, CA; 1:4000),
rabbit anti-TrkB (Upstate; 1:2000), rabbit anti-somatostatin (SOM) (Protos
Biotech; 1:2000), rabbit anti-c-Ret (IBL; 1:100) and rabbit anti-calbindin-28K
(CB) (Swant; 1:1000). The specificity of antibodies against Runx1 and Runx3
was verified by the absence of immunoreactivity in DRGs of
Runx1-/- and Runx3-/- mice,
respectively (Yoshikawa et al.,
2007) (data not shown). The sections were incubated for 24-48
hours at 4°C with each of the primary antibodies in the blocking solution.
For single staining, the sections were incubated with a biotinylated secondary
antibody for 1 hour at RT, followed with the peroxidase-conjugated
avidin-biotin complex (Vector Laboratories; 1:100) for 30 minutes at RT. The
positive reactions were visualized with 3,3'-diaminobenzidine (DAB)
using the ImmunoPure Metal Enhanced DAB Substrate Kit (Pierce). For caspase 3
immunostaining, cryostat sections were incubated with anti-active caspase 3
antibodies (Promega; 1:1000), followed by incubation with Alexa Fluor
488-labeled donkey anti-rabbit IgG (Invitrogen; 1:1000). For double or triple
staining, cryostat sections were incubated with anti-Runx3 antibodies,
followed by incubation with biotinylated horse anti-mouse IgG (Vector
Laboratories; 1:500) and Pacific Blue-conjugated streptavidin (Invitrogen;
1:500). The sections were then incubated with antibody against PV, CR, TrkC,
TrkA, TrkB, CGRP or Runx1, followed by Alexa Fluor 488-labeled donkey
anti-rabbit or anti-goat IgG, or Alexa Fluor 594-labeled donkey anti-rabbit or
anti-goat IgG (Invitrogen; 1:1000). Samples from Runx3-/-
and Runx3+/+ littermates were processed simultaneously for
immunohistochemistry.
DiI labeling
Newborn mice were deeply anesthetized with diethylether and perfused
transcardially with 4% PFA in 0.1 M PB. In order to retrogradely label DRG
axons, crystals of a lipophilic fluorescent dye (DiI) were placed in several
segments of the thoracic nerves after cutting off the ventral roots. The dyes
were allowed to transport for 3 days at 37°C and the preparations were
then cut in 50 µm sections for microscopy. Images of DiI-labeled spinal
cord were collected on an Axioimager microscope (Carl Zeiss).
Cell counting
For counts of Islet1+ DRG neurons, DRGs in the tenth thoracic
segment (Th10) at E13.5, E16.5 and postnatal day (P) 0, and for counts of
caspase 3+ DRG neurons, Th4 DRGs at E17.5 and P0, were serially
sectioned at 10 µm. For counts of TrkC+, CR+ and
PV+ DRG neurons, Th10, Th11 and Th12 DRGs from E13.5 to P0, and for
counts of Vglut1+, TrkA+, TrkB+,
c-Ret+, CB+, SOM+ and CGRP+ DRG
neurons, Th8, Th12, Th6, Th7, Th11, Th12 and Th9 DRGs at P0, were serially
sectioned at 10 µm. The number of immunoreactive neurons was determined by
counting neurons that contained a nucleus and showed a signal intensity in the
cytoplasm >2.5-fold above the noise level in the same sections. The total
number of immunoreactive neurons was counted from the level-matched DRGs of
each genotype (5-6 DRGs from three animals at each stage were examined).
Measurement of DRG volume
Using serial sections (10 µm) containing whole DRGs (Th10) at E13.5,
E16.5 and P0, the areas of the DRG in each section were measured and the DRG
volume calculated using AxioVision imaging software (Carl Zeiss).
Measurement of signal intensity of the immunoreactivity of axonal projections in the spinal cord
For measurements of TrkC, PV and Vglut1 immunoreactivity within the spinal
cord, Th10, Th11 and Th8 spinal cord, respectively, at P0 were sectioned by
cryostat at 10 µm. After immunostaining, images of TrkC, PV and Vglut1
immunoreactive axons in the spinal cord were collected on a LSM510META
confocal microscope (Carl Zeiss) or an Axioplan2 imaging microscope (Carl
Zeiss). The spinal cord was divided into ten equal parts along the
dorsoventral axis and the signal intensity of each part measured using ImageJ
(version 1.34s, NIH). The signal intensity of each part in
Runx3-/- and wild-type spinal cord was quantified by
comparison to the total signal intensity of the whole spinal cord of the wild
type. Spinal cord sections from wild-type and Runx3-/-
littermates were processed simultaneously during the immunohistochemical
process, and six spinal cord sections from two to three mice of each genotype
were analyzed quantitatively.
|
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
|
;
Honda, 1995;
Kucera et al., 2002;
Mu et al., 1993). In the
wild-type mice, PV was expressed at a low level in the DRG (Th12) at E13.5
(data not shown), and subsequently PV expression increased. The number of
PV+ DRG neurons increased by about 3-fold from E14.5 to E15.5, and
thereafter decreased slightly until P0
(Fig. 2A-C,G). By contrast,
PV+ DRG neurons were virtually absent from E13.5 to P0 in the
Runx3-/- mice (Fig.
2D-F,G). TrkC was expressed in DRGs (Th10) of both the wild-type and Runx3-/- mice at E11.5 (Fig. 3A,F). In the wild-type mice, TrkC expression was maintained at E13.5 and the number of TrkC+ neurons increased by about 3-fold from E13.5 to E15.5 (Fig. 3B,C,K). By contrast, TrkC+ neurons were hardly detected in Runx3-/- mice at E13.5 (Fig. 3G,K). TrkC expression reappeared by E14.5 in Runx3-/- mice, but the number of TrkC+ neurons was significantly smaller than in the wild type (Fig. 3C-E,H-K). At P0, TrkC+ neurons were decreased to about 65% in Runx3-/- mice (213.2±8.1, n=6, P<0.01) as compared with the wild-type mice (330.0±12.7, n=6) (Fig. 3K). Next, to evaluate the contribution of apoptosis to the disappearance of TrkC+ DRG neurons in Runx3-/- mice at E13.5, we examined the expression of TrkC in DRGs of Runx3-/-; Bax-/- double-knockout and Runx3+/+; Bax-/- control mice. TrkC expression was maintained in Runx3+/+; Bax-/- but not in Runx3-/-; Bax-/- DRGs at E13.5 (Fig. 1L-O), suggesting that the disappearance of TrkC+ DRG neurons is not due to apoptosis.
CR was expressed in a limited number of DRG neurons (see Fig. S1 in the supplementary material). In the wild-type mice, CR was expressed at a low level in DRGs (Th11) at E13.5 and the expression increased subsequently (see Fig. S1A-C in the supplementary material). In Runx3-/- mice, CR was also expressed, but the number of CR+ neurons was reduced to 51.4%, 63.1%, 54.3% and 77.7% at E14.5, E15.5, E18.5 and P0, respectively, as compared with that in wild type (see Fig. S1D-G in the supplementary material).
|
Projection of proprioceptive DRG afferents in Runx3-/- mice
Because the expression of PV and TrkC was lost or decreased in
Runx3-/- mice, we next analyzed the axonal projection of
these neurons. We also analyzed Vglut1 (Slc17a7) expression, as a marker for a
subpopulation of proprioceptive DRG neurons
(Alvarez et al., 2004;
Landry et al., 2004).
TrkC+ afferents projected to the dorsal horn, the intermediate zone and the ventral horn of the spinal cord in P0 wild-type mice. In Runx3-/- mice, however, the projection to the dorsal horn was observed, but not to the intermediate zone and the ventral horn (Fig. 4A). PV+ afferents projected to the ventral horn through the dorsal horn in the wild-type mice at P0. In Runx3-/- mice, no projection to the spinal cord was observed throughout developmental stages from E14.5 to P0 (Fig. 4B, data not shown). Vglut1+ afferents projected to the deep layer of the dorsal horn, the intermediate zone and the ventral horn in the wild-type mice, whereas the projection to the ventral horn was not observed in Runx3-/- mice (Fig. 4C).
To investigate whether the loss of projections to the ventral horn in Runx3-/- mice results from the apoptosis of proprioceptive DRG neurons, we examined the projections of DRG afferents using DiI labeling in Runx3-/-; Bax-/- mice and Runx3+/+; Bax-/- mice at P0. DiI-labeled afferents projected to the ventral horn of the spinal cord in Runx3+/+; Bax-/- mice, but not in Runx3-/-; Bax-/- mice (see Fig. S2 in the supplementary material), suggesting that the disappearance of proprioceptive DRG afferents is not due to apoptosis of the DRG neurons.
Next, we identified the TrkC+ neurons by examining the co expression of cutaneous markers in the wild-type and Runx3-/- mice at P0. In the skin of the lower limb, the co expression of TrkC and TrkA (Fig. 4D,E), and of TrkC and CGRP (Fig. 4F,G), were observed in some fibbers of both the wild-type and Runx3-/- mice. In the DRG, some TrkC+ neurons coexpressed CGRP or TrkB, both in the wild-type and Runx3-/- mice (see Fig. S3 in the supplementary material). In the muscle, however, TrkC+ and PV+ afferents were observed in wild-type but not in Runx3-/- mice (Fig. 4H-J, data not shown).
In summary, these results suggest that Runx3 is required for the proper projection of proprioceptive afferents. In addition, TrkC+ neurons observed in Runx3-/- mice may subserve cutaneous sensation.
Expression of various markers of DRG neuron subtypes
As mentioned above, the number of Islet1+ neurons was reduced by
as much as 1300 in the Runx3-/- mice at P0, but only a
limited decrease of TrkC+, PV+ and CR+
neurons was detected. To clarify the participation of other types of neurons
in this decrease, we examined the number of Vglut1+ neurons as the
subpopulation of proprioceptive DRG neurons, and TrkA+,
TrkB+, c-Ret+, calbindin-28K (CB)+,
somatostatin (SOM)+ and CGRP+ neurons as cutaneous DRG
neurons (Fig. 5).
The number of Vglut1+ neurons was decreased to 69% in Runx3-/- mice as compared with the wild-type mice (788.5±39.5 versus 546.2±18.7, n=6 for both Runx3+/+ and Runx3-/-, P<0.001) (Fig. 5A). Moreover, the number of TrkA+ neurons was decreased to 69% in Runx3-/- mice (4767.2±59.7 versus 3268.5±148.8, n=6, P<0.001) (Fig. 5B), SOM+ neurons to 57% (471.8±39.4 versus 268.0±13.9, n=6, P<0.01) (Fig. 5F) and CGRP+ neurons to 82% (804.2±31.6 versus 659.0±29.7, n=6, P<0.01) (Fig. 5G). TrkB+ neurons were decreased, but not significantly (1484.2±133.0 versus 1207.7±134.3, n=6, P=0.067) (Fig. 5C). By contrast, the number of CB+ neurons increased to 128% (510.8±17.5 versus 651.3±22.0, n=6, P<0.01) (Fig. 5E), whereas the number of c-Ret+ neurons was unchanged in Runx3-/- mice (1186.0±129.0 versus 1246.5±53.4, n=6, P=0.35) (Fig. 5D). These results indicate that Runx3 deficiency affected the expression of cutaneous marker molecules differentially in DRG neurons at P0.
Co expression of Runx3 with DRG neuron subtype markers and Runx1
To clarify whether the changes in the expression of TrkC, PV, CR, TrkA and
TrkB in Runx3-/- mice were regulated cell-autonomously by
Runx3, the co expression of Runx3 with these marker molecules was examined in
wild-type mice.
The majority of TrkC+ DRG neurons (83.0±1.3%, n=69) were Runx3-positive at E13.5 (Fig. 6A,C), but only one-third of TrkC+ neurons coexpressed Runx3 at P0 (30.1±3.0%, n=17, P<0.001) (Fig. 6B,C). By contrast, almost all PV+ neurons coexpressed Runx3 and TrkC (Fig. 6D), whereas CR+ neurons coexpressed TrkC but not Runx3 at P0 (see Fig. S4 in the supplementary material). Moreover, the subpopulation of TrkA+ and TrkB+ neurons coexpressed Runx3 at P0 (Fig. 6E,F). These findings suggest that Runx3 might regulate the expression of TrkC, PV, TrkA and TrkB cell-autonomously.
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
), it
can be suggested that Runx1 and Runx3 have antagonistic roles in the
development of DRG neuron subsets.
Proprioceptive DRG neurons are lost from the onset of development under Runx3 deficiency
Several lines of evidence have suggested that Runx3 plays a crucial role in
the cell fate specification of proprioceptive DRG neurons
(Inoue et al., 2002;
Levanon et al., 2002;
Kramer et al., 2006). The
expression of PV, a specific marker for proprioceptive DRG neurons
(Copray et al., 1994;
Honda, 1995), was not detected
in DRGs of Runx3-/- newborn mice
(Inoue et al., 2002;
Levanon et al., 2002). The
present study confirmed the loss of PV+ DRG neurons in
Runx3-/- mice at P0 and further showed that PV+
DRG neurons were virtually absent from Runx3-/- mice from
E13.5 to P0, whereas PV+ DRG neurons appeared by E13.5 in the
wild-type mice (Fig. 2).
Therefore, the present study suggests that Runx3 is required for the induction
of PV expression, and it is likely that proprioceptive DRG neurons fail to
develop from the onset under Runx3 deficiency.
The loss of proprioceptive DRG neurons in Runx3-/- mice
was also supported by the absence of axonal projections to the central and
peripheral targets (Inoue et al.,
2002; Levanon et al.,
2002) (the present study). In the present study, PV+,
TrkC+ and Vglut1+ afferents did not extend to the
ventral horn in Runx3-/- mice, whereas these afferents
projected to the ventral horn in the wild-type mice
(Fig. 4A-C). In accordance with
the central projection, no TrkC+ afferents projected to the muscle
in Runx3-/- mice (Fig.
4H-J). The loss of proprioceptive DRG neurons and their axonal
projections in Runx3-/- mice may not be caused by
apoptosis, as no proprioceptive neuron-specific markers and axonal projections
were observed in Runx3-/-; Bax-/-
double-knockout mice, in which DRG neurons were rescued from apoptosis during
embryonic development (White et al.,
1998). A recent study has shown that Runx3 regulates the axon
guidance of proprioceptive DRG neurons
(Chen et al., 2006a). Gain- and
loss-of-function studies in chick embryos revealed that Runx3 activity
determines the axonal projections to the ventral horn, the intermediate zone
and the dorsal horn in the spinal cord.
|
). By
contrast, Inoue et al. demonstrated that Runx3-/- DRG
neurons maintained both TrkC immunoreactivity at E14.0 and TrkC mRNA
expression at P0 (Inoue et al.,
2002). The present study examined in detail TrkC expression in
DRGs from E11.5 to P0. TrkC was expressed in DRGs of both wild-type and
Runx3-/- mice at E11.5, as shown in the previous study
(Levanon et al., 2002). In
contrast to the continuous TrkC expression in the wild-type DRG, we found that
TrkC+ DRG neurons disappeared completely by E13.5 in
Runx3-/- mice, and then TrkC expression resumed by E14.5
to be maintained through P0. Therefore, the present study revealed that
Levanon et al. and Inoue et al. observed different aspects of the dynamic TrkC
expression in Runx3-/- DRGs: the initial appearance of
TrkC expression, its transient loss and recovery. We also showed that the
disappearance of TrkC+ DRG neurons in E13.5
Runx3-/- mice is not due to apoptosis, as TrkC expression
was maintained in Runx3+/+; Bax-/- but
not in Runx3-/-; Bax-/- DRGs at E13.5
(Fig. 3L-O). Therefore, the
present study showed that Runx3 is required for the maintenance of TrkC
expression until E13.5.
|
35%) in the number of TrkC+ DRG neurons in
Runx3-/- mice at P0. Taken together, it is likely that
Runx3 is required for the maintenance of the initial TrkC expression and the
proper development of proprioceptive neurons.
In Runx3-/- mice, a subset of DRG neurons recovers TrkC
expression independently of Runx3 from E14.5
(Fig. 3). Because these
later-appearing TrkC+ DRG neurons projected to the dorsal spinal
cord and the skin and coexpressed markers of cutaneous DRG neurons, such as
TrkA and CGRP, these neurons seem to be cutaneous in nature. Indeed, previous
studies have shown that a subset of TrkC+ DRG neurons send axons to
the skin (Bronzetti et al.,
1995; McMahon et al.,
1994; Oakley and Karpinski,
2002; Oakley et al.,
2000). The expression of TrkC in non-proprioceptive DRG neurons
has also been reported (Genç et
al., 2004). Erzurumlu's group examined neurotrophin 3
(Ntf3) knockout mice and found that no DRG neurons express TrkC at
E15, but TrkC+ DRG neurons appear at P0, although these do not
express PV (Genç et al.,
2004). Considering that Ntf3 knockout mice lack
proprioceptive DRG neurons (Ernfors et al.,
1994; Fariñas et al.,
1994), these TrkC+/PV- DRG neurons should be
other than proprioceptive neurons. Taken together, it seems likely that TrkC
expression around E13.5 is regulated by Runx3, and that during the critical
period TrkC plays pivotal roles in the proper cell fate specification of
proprioceptive neurons.
Runx3 regulates the expression of cutaneous DRG neuron-specific markers
We demonstrated that the total number of DRG neurons was greatly reduced
(by as much as 1300) in Runx3-/- mice at P0
(Fig. 1C). The reduction of DRG
neurons at P0 was not affected by caspase 3-dependent apoptosis
(Fig. 1E-G). Because the
magnitude of this reduction cannot be explained by the loss of PV+
(nearly 150), TrkC+ (nearly 150) and/or Vglut1+ (nearly
250) proprioceptive DRG neurons, we examined the number of TrkA+,
TrkB+, CB+, CGRP+, SOM+,
c-Ret+ cutaneous DRG neurons. We found that DRG neurons that
express TrkA, CGRP and SOM were decreased by nearly 1500, 150 and 200 in
Runx3-/- mice, respectively
(Fig. 5). TrkB+ DRG
neurons seemed to be decreased, but no significant difference was detected. By
contrast, the number of CB+ DRG neurons was increased, whereas that
of c-Ret+ neurons was unchanged in Runx3-/-
mice. The expression of c-Ret may be regulated by Runx1, because the number of
c-Ret + DRG neurons was increased in Runx1-/-
mice (Chen et al., 2006b;
Yoshikawa et al., 2007).
|
;
Inoue et al., 2007). In
contrast to this increase of TrkB+ DRG neurons in
Runx3-/- mice during early embryonic stages, the present
study showed that these neurons tended to decrease in
Runx3-/- mice at birth. The tendency at P0 might result
from cell death due to the mismatch of neurotrophic factors between
overproduced TrkB+ DRG neurons and their axonal targets
(Oppenheim, 1991).
Runx3 and Runx1 have antagonistic effects on the development of subsets of DRG neurons
The present study showed that the total number of DRG neurons was decreased
in P0 Runx3-/- mice. It is interesting to see that the
total number of DRG neurons increases in Runx1-/- mice
(Chen et al., 2006b;
Yoshikawa et al., 2007).
Furthermore, the present study showed that the number of TrkA+,
CGRP+, SOM+ and TrkC+ neurons was decreased
in Runx3-/- mice, suggesting that Runx3 positively
regulates the expression of TrkA, CGRP, SOM and TrkC. In striking contrast, we
have previously shown that DRG neurons that express TrkA, CGRP, SOM and TrkC
were increased in Runx1-/- mice
(Yoshikawa et al., 2007).
These results suggest that Runx1 and Runx3 might be involved antagonistically
in the development of subsets of DRG neurons. Further studies that include
their downstream targets will be needed to understand the roles of Runx1 and
Runx3.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/9/1703/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Alvarez, F. J., Villalba, R. M., Zerda, R. and Schneider, S.
P. (2004). Vesicular glutamate transporters in the spinal
cord, with special reference to sensory primary afferent synapses.
J. Comp. Neurol. 472,257
-280.[CrossRef][Medline]
Bibel, M. and Barde, Y. A. (2000).
Neurotrophins: key regulators of cell fate and cell shape in the vertebrate
nervous system. Genes Dev.
14,2919
-2937.
Bronzetti, E., Ciriaco, E., Germana, G. and Vega, J. A.
(1995). Immunohistochemical localization of neurotrophin receptor
proteins in human skin. Ital. J. Anat. Embryol.
100,565
-571.[Medline]
Chen, A. I., de Nooij, J. C. and Jessell, T. M.
(2006a). Graded activity of transcription factor Runx3 specifies
the laminar termination pattern of sensory axons in the developing spinal
cord. Neuron 49,395
-408.[CrossRef][Medline]
Chen, C. L., Broom, D. C., Liu, Y., de Nooij, J. C., Li, Z.,
Cen, C., Samad, O. A., Jessell, T. M., Woolf, C. J. and Ma, Q.
(2006b). Runx1 determines nociceptive sensory neuron phenotype
and is required for thermal and neuropathic pain.
Neuron 49,365
-377.[CrossRef][Medline]
Coffman, J. A. (2003). Runx transcription
factors and the developmental balance between cell proliferation and
differentiation. Cell Biol. Int.
27,315
-324.[CrossRef][Medline]
Copray, J. C., Mantingh-Otter, I. J. and Brouwer, N.
(1994). Expression of calcium-binding proteins in the
neurotrophin-3-dependent subpopulation of rat embryonic dorsal root ganglion
cells in culture. Brain Res. Dev. Brain Res.
81, 57-65.[CrossRef][Medline]
Ernfors, P., Lee, K.-E., Kucera, J. and Jaenish, R.
(1994). Lack of neurotrophin-3 leads to deficiencies in the
peripheral nervous system and loss of limb proprioceptive afferents.
Cell 77,503
-512.[CrossRef][Medline]
Fariñas, I., Jones, K. R., Backus, C., Wang, X. Y. and
Reichardt, L. F. (1994). Severe sensory and sympathetic
deficits in mice lacking neurotrophin-3. Nature
369,658
-661.[CrossRef][Medline]
Genç, B., Özdinler, P. H., Mendoza, A. E. and
Erzurumlu, R. S. (2004). A chemoattractant role for NT-3 in
proprioceptive axon guidance. PLoS Biol.
2,2112
-2121.
Honda, C. N. (1995). Differential distribution
of calbindin-D28k and parvalbumin in somatic and visceral sensory neurons.
Neuroscience 68,883
-892.[CrossRef][Medline]
Huang, E. J. and Reichardt, L. F. (2001).
Neurotrophins: roles in neuronal development and function. Annu.
Rev. Neurosci. 24,677
-736.[CrossRef][Medline]
Inoue, K., Ozaki, S., Shiga, T., Ito, K., Masuda, T., Okado, N.,
Iseda, T., Kawaguchi, S., Ogawa, M., Bae, S. C. et al.
(2002). Runx3 controls the axonal projection of proprioceptive
dorsal root ganglion neurons. Nat. Neurosci.
5, 946-954.[CrossRef][Medline]
Inoue, K., Ito, K., Osato, M., Lee, B., Bae, S. C. and Ito,
Y. (2007). The transcription factor Runx3 represses the
neurotrophin receptor TrkB during lineage commitment of dorsal root ganglion
neurons. J. Biol. Chem.
282,24175
-24184.
Ito, Y. (2004). Oncogenic potential of the RUNX
gene family: overview. Oncogene
23,4198
-4208.[CrossRef][Medline]
Knudson, C. M., Tung, K. S., Tourtellotte, W. G., Brown, G. A.
and Korsmeyer, S. J. (1995). Bax-deficient mice with lymphoid
hyperplasia and male germ cell death. Science
270, 96-99.
Kramer, I., Sigrist, M., de Nooij, J. C., Taniuchi, I., Jessell,
T. M. and Arber, S. (2006). A role for Runx transcription
factor signaling in dorsal root ganglion sensory neuron diversification.
Neuron 49,379
-393.[CrossRef][Medline]
Kucera, J., Cooney, W., Que, A., Szeder, V., Stancz-Szeder, H.
and Walro, J. (2002). Formation of supernumerary muscle
spindles at the expense of Golgi tendon organs in ER81-deficient mice.
Dev. Dyn. 223,389
-401.[CrossRef][Medline]
Landry, M., Bouali-Benazzouz, R., El Mestikawy, S., Ravassard,
P. and Nagy, F. (2004). Expression of vesicular glutamate
transporters in rat lumbar spinal cord, with a note on dorsal root ganglia.
J. Comp. Neurol. 468,380
-394.[CrossRef][Medline]
Levanon, D., Brenner, O., Negreanu, V., Bettoun, D., Woolf, E.,
Eilam, R., Lotem, J., Gat, U., Otto, F., Speck, N. et al.
(2001). Spatial and temporal expression pattern of Runx3 (Aml2)
and Runx1 (Aml1) indicates non-redundant functions during mice embryogenesis.
Mech. Dev. 109,413
-417.[CrossRef][Medline]
Levanon, D., Bettoun, D., Harris-Cerruti, C., Woolf, E.,
Negreanu, V., Eilam, R., Bernstein, Y., Goldenberg, D., Xiao, C., Fliegauf, M.
et al. (2002). The Runx3 transcription factor regulates
development and survival of TrkC dorsal root ganglia neurons. EMBO
J. 21,3454
-3463.[CrossRef][Medline]
Li, Q. L., Ito, K., Sakakura, C., Fukamachi, H., Inoue, K., Chi,
X. Z., Lee, K. Y., Nomura, S., Lee, C. W., Han, S. B. et al.
(2002). Causal relationship between the loss of RUNX3 expression
and gastric cancer. Cell
109,113
-124.[CrossRef][Medline]
Markus, A., Patel, T. D. and Snider, W. D.
(2002). Neurotrophic factors and axonal growth, Curr.
Opin. Neurobiol. 12,523
-531.[CrossRef][Medline]
Marmigère, F. and Ernfors, P. (2007).
Specification and connectivity of neuronal subtypes in the sensory lineage.
Nat. Rev. Neurosci. 8,114
-127.[CrossRef][Medline]
Marmigère, F., Montelius, A., Wegner, M., Groner, Y.,
Reichardt, L. F. and Ernfors, P. (2006). The Runx1/AML1
transcription factor selectively regulates development and survival of TrkA
nociceptive sensory neurons. Nat. Neurosci.
9, 180-187.[CrossRef][Medline]
McMahon, S. B., Armanini, M. P., Ling, L. H. and Phillips, H.
S. (1994). Expression and co expression of Trk receptors in
subpopulations of adult primary sensory neurons projecting to identified
peripheral targets. Neuron
12,1161
-1171.[CrossRef][Medline]
Moqrich, A., Earley, T. J., Watson, J., Andahazy, M., Backus,
C., Martin-Zanca, D., Wright, D. E., Reichardt, L. F. and Patapoutian, A.
(2004). Expressing TrkC from the TrkA locus causes a subset of
dorsal root ganglia neurons to switch fate. Nat.
Neurosci. 7,812
-818.[CrossRef][Medline]
Mu, X., Silos-Santiago, I., Carroll, S. L. and Snider, W. D.
(1993). Neurotrophin receptor genes are expressed in distinct
patterns in developing dorsal root ganglia. J.
Neurosci. 13,4029
-4041.[Abstract]
Oakley, R. A. and Karpinski, B. A. (2002).
Target-independent specification of proprioceptive sensory neurons.
Dev. Biol. 249,255
-269.[CrossRef][Medline]
Oakley, R. A., Lefcort, F. B., Plouffe, P., Ritter, A. and
Frank, E. (2000). Neurotrophin-3 promotes the survival of a
limited subpopulation of cutaneous sensory neurons. Dev.
Biol. 224,415
-427.[CrossRef][Medline]
Oppenheim, R. W. (1991). Cell death during
development of the nervous system. Annu. Rev.
Neurosci. 14,453
-501.[CrossRef][Medline]
Patel, T. D., Kramer, I., Kucera, J., Niederkofler, V., Jessell,
T. M., Arber, S. and Snider, W. D. (2003). Peripheral NT3
signaling is required for ETS protein expression and central patterning of
proprioceptive sensory afferents. Neuron
38,403
-416.[CrossRef][Medline]
Simeone, A., Daga, A. and Calabi, F. (1995).
Expression of runt in the mice embryo. Dev. Dyn.
203, 61-70.[Medline]
Snider, W. D. (1994). Functions of the
neurotrophins during nervous system development: what the knockouts are
teaching us. Cell 77,627
-638.[CrossRef][Medline]
Theriault, F. M., Roy, P. and Stifani, S.
(2004). AML1/Runx1 is important for the development of hindbrain
cholinergic branchiovisceral motor neurons and selected cranial sensory
neurons. Proc. Natl. Acad. Sci. USA
101,10343
-10348.
Theriault, F. M., Nuthall, H. N., Dong, Z., Lo, R.,
Barnabe-Heider, F., Miller, F. D. and Stifani, S. (2005).
Role for Runx1 in the proliferation and neuronal differentiation of selected
progenitor cells in the mammalian nervous system. J.
Neurosci. 25,2050
-2061.
White, F. A., Keller-Peck, C. R., Knudson, C. M., Korsmeyer, S.
J. and Snider, W. D. (1998). Widespread elimination of
naturally occurring neuronal death in Bax deficient mice. J.
Neurosci. 18,1428
-1439.
Yoshikawa, M., Senzaki, K., Yokomizo, T., Takahashi, S., Ozaki,
S. and Shiga, T. (2007). Runx1 selectively regulates cell
fate specification and axonal projections of dorsal root ganglion neurons.
Dev. Biol. 303,663
-674.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Kitago, S. R. Martinez, T. Nakamura, M.-S. Sim, and D. S.B. Hoon Regulation of RUNX3 Tumor Suppressor Gene Expression in Cutaneous Melanoma Clin. Cancer Res., May 1, 2009; 15(9): 2988 - 2994. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
