1 Departments of Anesthesiology, Psychiatry, Molecular Biology and Pharmacology,
Washington University School of Medicine Pain Center, St. Louis, MO 63110,
USA
2 Department of Biochemistry and Molecular Biophysics, Columbia University, New
York, NY 10027, USA
3 Department of Biochemistry and Molecular Biology, University of Texas M.D.
Anderson Cancer Center, Houston, TX 70030, USA
Author for correspondence (e-mail:
chenz{at}morpheus.wustl.edu)
Accepted 4 May 2004
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SUMMARY |
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Key words: Lmx1b, Dorsal horn, Migration, Differentiation, Cutaneous afferents, Mouse
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Introduction |
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During spinal cord development, the specification of dorsal interneurons is
mediated by both extrinsic and intrinsic factors
(Caspary and Anderson, 2003;
Helms and Johnson, 2003
;
Lee and Jessell, 1999
;
Liem et al., 1997
).
Specifically, the intrinsic factors that control the specification and
development of several classes of early-born neurons (dI1-6) have been
analyzed in greater detail. In mice lacking transcription factors
Lbx1 (Lbx1h - Mouse Genome Informatics) or Rnx
(Tlx3 - Mouse Genome Informatics), the specification of dI5 neurons
is affected, as indicated by the loss or downregulation of Lmx1b (dI5
marker) (Gross et al., 2002
;
Muller et al., 2002
;
Qian et al., 2002
). These two
genes are also involved in the development of late-born neurons that make up
the dorsal horn of the spinal cord
(Caspary and Anderson, 2003
).
In addition, the transcription factors Drg11 (Prxxl1 - Mouse
Genome Informatics), Ebf1, Ebf3 and Zic1 have also been
shown to be important for the early specification of the dorsal spinal cord
neurons (Aruga et al., 1998
;
Aruga et al., 2002b
;
Chen et al., 2001
;
Ebert et al., 2003
;
Garcia-Dominguez et al., 2003
;
Garel et al., 1997
;
Wang et al., 1997
). Among
them, Zic1 has been shown to be a negative regulator of the
differentiation of the dorsal horn neurons in mice
(Aruga et al., 2002a
;
Aruga et al., 1998
;
Aruga et al., 1996a
;
Aruga et al., 2002b
;
Aruga et al., 1994
;
Aruga et al., 1996b
). Despite
these studies, our knowledge about the molecular mechanisms that govern the
development of early-born neurons and the assembly of the dorsal horn circuits
is still rather fragmentary.
Lmx1b is an LIM homeobox-containing gene, and was originally
isolated as a mouse ortholog of the chicken Lmx1
(Chen et al., 1998a;
Riddle et al., 1995
;
Vogel et al., 1995
). In the
chicken, Lmx1 is involved in the specification of the dorsal cell
fate in the limb, and the differentiation and morphogenesis of the isthmic
organizer (Adams et al., 2000
;
Matsunaga et al., 2002
;
Riddle et al., 1995
;
Vogel et al., 1995
). Mice
lacking Lmx1b exhibit abnormal limbs and kidneys
(Chen et al., 1998a
). Mutation
of Lmx1b results in nail patella syndrome in humans, an autosomal
dominant disease characterized by abnormal skeletal patterning and renal
dysplasia (Dreyer et al.,
1998
). In the central nervous system, Lmx1b is involved
in multiple developmental processes, including the formation of eye,
dopaminergic neurons, serotonergic neurons and the trajectory of the motor
axons in the limb (Cheng et al.,
2003
; Ding et al.,
2003
; Kania and Jessell,
2003
; Kania et al.,
2000
; Pressman et al.,
2000
; Smidt et al.,
2000
). In the nematode Caenorhabditis elegans, the
Lmx1b ortholog, Lim6, has been shown to be important for the
differentiation of GABAergic neurons
(Hobert et al., 1999
).
In the developing dorsal spinal cord, Lmx1b is expressed in dI5
neurons and late-born neurons destined to populate the superficial layer of
the dorsal spinal cord (Chen et al.,
2001; Gross et al.,
2002
; Muller et al.,
2002
). In this study, we performed detailed studies of the dorsal
spinal cord development in Lmx1b mutants. Our study reveals that
Lmx1b is important for the development of dI5 neurons, late-born
dorsal horn neurons and the projection of cutaneous afferents in the dorsal
spinal cord. Our study uncovers a central role for Lmx1b in
transcriptional cascades that govern the dorsal horn development.
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Materials and methods |
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BrdU labeling and detection by immunocytochemistry
Pregnant female mice derived from timed matings between Lmx1b or
Rnx or Drg11 heterozygous mice were given a single
intraperitoneal injection of BrdU (5 mg/ml solution in PBS and 60 µg/g of
body weight) at 11.5 days postcoitum (dpc) and 12.5 dpc. After time periods of
2 hours, 1 day, 2 days or 3 days, embryos were removed, genotyped and
sectioned as described (Chen et al.,
1998a; Chen et al.,
2001
; Roberts et al.,
1994
). The sections were processed and stained sequentially with a
mouse anti-BrdU antibody (Dako), biotinylated donkey anti-mouse IgG (Jackson
Immunoresearch) and ABC Elite reagents (Vector). For double staining with
anti-LMX1B antibody (Kania et al.,
2000
), the slides were first incubated with a guinea pig
anti-LMX1B antibody detected enzymatically through production of a brown
precipitate. For anti-BrdU antibody detection, a nickel intensification
technique was used, producing a black precipitate. To quantify the
distribution of BrdU-labeled neurons, we used the Photoshop (Adobe) program
after dividing the dorsal horn into two parts: medial one-third and lateral
two-thirds (from the midline to the lateral edge of the dorsal horn,
Fig. 2). BrdU-labeled neurons
in ten sections each from wild-type (n=6) and mutant (n=6)
embryos were counted at the thoracic segmental level, and a comparison was
performed using Student's t-test.
|
DiI labeling
For study of the projection from the DRG to the spinal cord, a small amount
of 1,1''-dioctadecyl-3,3,3'',3''-tetramethylindocarbocyanine
perchlorate (DiI; Molecular Probes) crystals was placed in the DRG
unilaterally. The samples were kept in the fixative at 37°C for 2-4 days,
and then were sectioned transversely with a vibratome at a thickness of 50-100
µm. Labeling was observed with epifluorescent or laser confocal
microscopy.
In utero electroporation
For in utero electroporation, previously detailed procedures were followed
(Saito and Nakatsuji, 2001).
Pregnant mice at 12 dpc were anesthetized with sodium pentobarbital (40
mg/kg), followed by the exposure of the uterus and cutting of the uterine wall
on both horns along the antiplacental side. pCAGGS-Lmx1b:EGFP or
pCAGGS-EGFP (Niwa et al.,
1991
) (1-3 µl; 0.5-1.5 µg/µl) was injected into the
central canal of the spinal cord using an orally controlled pipette system.
After injection, square electrical pulses were delivered by the use of an
Electro Square Porator (ECM830) at a rate of one pulse per second (voltage
35V, five pulses, 50 ms) to embryos by holding the utero with forceps-type
electrodes. The embryos were repositioned into the abdominal cavity without
sewing the uterine wall. The abdomen was filled with warmed saline and the
abdominal wall and skin were sutured. Embryos were allowed to survive for 2
days. Genotyping and analysis of gene expression in the spinal cord of
electroporated embryos was performed as described above.
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Results |
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A migratory defect in the dorsal spinal cord of Lmx1b mutants
To examine the effect of Lmx1b mutation on the dorsal horns of
Lmx1b-/- embryos, we performed Nissl staining. At E12.5,
the dorsal horns of Lmx1b-/- embryos and wild-type
controls were indistinguishable (Fig.
2A,B). At E15.5, in wild-type embryos, numerous laminae I-II
neurons, distinguishable from laminae III-IV neurons by their smaller size,
aggregate to form a separate layer, whereas laminae III-IV neurons also become
distinct in being more loosely distributed
(Fig. 2C). In Lmx1b
mutants, these laminar boundaries were not recognizable
(Fig. 2D) and the dorsal
funiculus was smaller relative to wild-type controls
(Fig. 2E,F).
We next used BrdU to determine the migratory behavior of the dorsal horn
neurons in Lmx1b mutants. To do this, we labeled dorsal horn neurons
of wild-type and Lmx1b mutant embryos with BrdU at E11.5 and E12.5,
and examined their settling position at E14.5. Interestingly, neurons labeled
with BrdU at E11.5 in the wild-type embryo did not migrate to the most
superficial region of the dorsal horn (Fig.
2G). By contrast, neurons labeled at E12.5 migrated through
earlier-born neurons and occupied the most superficial layer of the dorsal
horn, consistent with previous studies
(Nornes and Carry, 1978;
Nornes and Das, 1974
).
However, unlike previous studies in which [3H]thymidine
autoradiography was used, our BrdU labeling clearly revealed an inside-out
migration pattern for laminae I-II neurons. We next examined the migration of
BrdU-labeled neurons in Lmx1b mutants. First, we did not find any
significant change in the total number of BrdU-labeled neurons between
wild-type and Lmx1b mutants (data not shown). However, some neurons
labeled with BrdU at E11.5 were present in the most superficial region of the
dorsal horn of E14.5 Lmx1b-/- embryos
(Fig. 2H), while a
significantly higher number of BrdU+ neurons were accumulated near
the midline region as compared with the control
(Fig. 2I-K). In line with this
observation, fewer neurons labeled with BrdU at E11.5 were located in the
lateral region of the dorsal horn in Lmx1b-/- embryos
(Fig. 2J). This altered
distribution of dorsal neurons was not due to abnormal neuronal death, as the
TUNEL staining pattern was similar in both Lmx1b-/- and
wild-type embryos between E11.5 and E15.5 (data not shown). Together, these
results indicate that there is a major defect in the migration of neurons in
the dorsal horn of Lmx1b-/- embryos.
Lmx1b controls Drg11, Rnx and Ebf1, and Ebf3 expression in the dorsal horn
To further characterize the dorsal horn defects in Lmx1b mutants,
we examined the expression profile of dorsal horn neuron molecular markers
Drg11, Ebf1, Ebf2, Ebf3 and Rnx. In
Lmx1b-/- embryos, Drg11 expression was completely
abolished from the normal onset of its expression
(Fig. 3A,B; see Fig. S1 at
http://dev.biologists.org/supplemental).
At E11.5, Ebf1, Ebf2, Ebf3 and Rnx appeared to be normally
expressed in Lmx1b-/- embryos (see Fig. S1 at
http://dev.biologists.org/supplemental).
However, by E12.5, Ebf3 and Rnx expression levels were lower
in Lmx1b mutants (see Fig. S1) and by E15.5, Ebf3 expression
was completely absent in the Lmx1b mutants
(Fig. 3C,D). At this stage, in
wild-type embryos, Ebf1 was most strongly expressed in laminae III
neurons and weakly in laminae III-IV neurons. By contrast, Ebf1
expression was markedly reduced in Lmx1b mutant embryos
(Fig. 3E,F). Similarly,
Rnx was dramatically downregulated in the Lmx1b mutants
(Fig. 3G,H).
|
Lmx1b-independent dorsal horn-specific transcription factors
The expression of Lbx1, a lamina IIi-III marker which acts
upstream of Lmx1b was examined in wild-type and Lmx1b mutant
embryos (Fig. 4G,H). Although a
few LBX1+ neurons were found in the most superficial layer of the
Lmx1b-/- dorsal horn, no significant difference in the
number of LBX1+ neurons was detected between the Lmx1b
mutants and wild-type controls (Fig.
4I). The expression of Brn3a, a laminae III-V marker
(Fig. 4J), was examined in
Lmx1b mutants (Fig.
4K). Although a seemingly higher number of BRN3A+
neurons were located more medially in Lmx1b mutants
(Fig. 4K), neuronal counts
revealed no difference in the number of BRN3A+ neurons between
wild-type and Lmx1b mutants (Fig.
4L).
|
Hox6 and Hox8 are homeobox genes that are expressed in
laminae I-II (Graham et al.,
1991). In Lmx1b mutants, Hoxb6 and
Hoxb8 expression domains appeared to have expanded
(Fig. 4A,B,D,E). Although the
expansion of the Hoxb6 and Hoxb8 domains could be attributed
to an abnormal location of laminae I-II neurons in the deep dorsal horn, it is
also possible that the expression of Hoxb6 and Hoxb8 is
upregulated in laminae III-V neurons in the absence of Lmx1b
function. If it were the latter case, the total number of Hoxb6 and
Hoxb8 neurons would increase in the mutants. However, no significant
difference in the number of Hoxb6 and Hoxb8 neurons was
evident between wild-type and mutant animals
(Fig. 4C,F). Our data thus
suggest that abnormal distribution of Hox6/8 neurons is due to
migration defects.
Together, these results suggest that expression of Hox6, Hox8, Lbx1, Brn3a and Pax2 is Lmx1b independent; however, the distribution pattern of neurons expressing these markers appears changed in Lmx1b mutants.
Lmx1b, Rnx and Drg11 repress Zic1 and Zic4 genes
We next asked whether there is a causal relationship between the expression
of Zic genes and Lmx1b as Zic1 appears to be required for
maintaining the progenitor state of undifferentiated dorsal neurons by
repressing the differentiation of spinal cord progenitors
(Aruga et al., 2002b;
Ebert et al., 2003
).
In wild-type embryos, three Zic genes (Zic1, Zic2 and Zic4) are expressed in the developing dorsal spinal cord (Fig. 5). Their expression is mainly concentrated around the midline region, suggesting that the Zic genes may play a role in modulating the differentiation of the dorsal horn neurons. Strikingly, in Lmx1b-/- mutants, while Zic2 expression remained unaltered (Fig. 5E,F), expression of Zic1 and Zic4 was significantly upregulated (Fig. 5A-D). This was observed not only around the midline region, but also in the superficial layer of the dorsal horn where numerous laminae I-II neurons were present but failed to differentiate further at a later stage (Fig. 5B,D). An upregulation of Zic1 and Zic4 was also detected in Rnx mutants (Fig. 5H,J).
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Stereotyped expression of attractive or repulsive axonal guidance molecules
is crucial to guiding the precise axonal growth of neurons in the developing
nervous system (Tessier-Lavigne and
Goodman, 1996). To determine whether there was a change of
expression of axonal guidance cues/molecules in the dorsal horn of
Lmx1b mutants, we examined their expression by in situ hybridization.
Sema3a is a secreted cell-surface protein that functions as a
chemorepellent in the projection of cutaneous afferents in the spinal cord of
the chick and the mouse (Luo et al.,
1993
; Messersmith et al.,
1995
). Its expression, however, was normal in Lmx1b
mutants (data not shown). We also found that Sema3c
(Feiner et al., 2001
) was
completely lost in the dorsal horn of Lmx1b mutants
(Fig. 8G,H). By contrast,
expression of the receptors for Sema3a, neuropilin 1 (Nrp1)
and plexin A2 (He and Tessier-Lavigne,
1997
; Kolodkin et al.,
1997
; Takahashi et al.,
1999
), were markedly reduced in the mutants
(Fig. 8I-L). We also examined
the expression of the members of the Slit family, which have been implicated
in axonal guidance and neuronal migration
(Brose and Tessier-Lavigne,
2000
; Wong et al.,
2002
). Slit1 and Robo2 were most strongly
expressed in laminae I-II (Fig.
8M,O). In the mutants, Slit1 and Robo2
expression was dramatically reduced (Fig.
8N,P). In addition, netrin 1 expression was largely lost in the
medial region of the deep dorsal horn of Lmx1b mutants
(Serafini et al., 1994
)
(Fig. 8Q,R). Thus, the altered
expression of multiple axonal guidance molecules could account for the failure
of cutaneous sensory axon ingrowth in Lmx1b mutants.
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Discussion |
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Lmx1b controls the ingrowth of cutaneous afferents into the dorsal horn
In Lmx1b mutants, the selective blocking of cutaneous afferent
ingrowth raises the possibility that Lmx1b may coordinate the
projection of TrkA+ afferents into the dorsal horn by regulating
local axonal guidance cue(s). Altered ingrowth of TrkA+ afferents
is also found in Lbx1 mutants and Rnx/Tlx1 double mutants
(Gross et al., 2002;
Muller et al., 2002
;
Qian et al., 2002
).
Nevertheless, unlike Lbx1 and Rnx, Lmx1b is not expressed in
DRG neurons, therefore, the TrkA guidance defect most probably resides in the
dorsal horn.
The specific block of the entry of TrkA+ afferents suggests that certain attractants may be missing in the mutants. Alternatively, the expression level of some repulsive molecules may be increased to repel the cutaneous afferents. Although these possibilities exist, no evidence for increased expression of any repellants examined in Lmx1b mutants was found. Given that multiple axonal guidance cues are expressed in the dorsal horn and their expression is lost in Lmx1b mutants, it is possible that multiple axonal guidance molecules could work synergistically to coordinate the projections of cutaneous afferents. Future analysis of mice lacking multiple axonal guidance cues may be required to reveal the identity of the cues responsible for the ingrowth of TrkA+ afferents in the dorsal spinal cord.
Lmx1b guides the differentiation and migration of the dorsal horn neurons
Our study suggests that Zic1, Zic4, Ebf1 and Ebf3
function downstream of Lmx1b and Rnx. Forced expression of
Zic1 in chicks represses the differentiation of
Math1-expressing neurons in the dorsal neural tube
(Ebert et al., 2003).
Transgenic mice overexpressing Zic1 exhibit inhibited neuronal
differentiation with extension of the progenitor state in the dorsal spinal
cord (Aruga et al., 2002b
).
Loss of Zic1 in mice also leads to premature expression of the
ßIII tubulin in the dorsal spinal cord
(Aruga et al., 2002b
). In the
context of these findings, the Zic gene expression upregulation is
interesting, and consistent with the migration and differentiation defects in
Lmx1b mutants. Zic1 could be an important component of the
dorsal spinal cord neuron differentiation pathway.
The role of Zic4, Ebf1 and Ebf3 in the development of the dorsal horn is unknown. Whether dysregulation of these genes plays a causal role in aberrant development of the dorsal horn also remains to be determined. Together with previous studies, we hypothesize that the Lbx1/Lmx1b/Rnx/Drg11 pathway represents a major pathway to control the differentiation and migration of laminae I-II neurons and subsequent projection of cutaneous afferents (Fig. 9). The effects of Lmx1b and Rnx may be mediated in part through the Drg11, Ebf and Zic genes and in part via other unidentified transcription factors to control the assembly of the dorsal horn circuits.
|
The finding that an inside-out migration pattern and a normal
differentiation program are disrupted in the dorsal horn of Lmx1b
mutants raises the question of whether aberrant migratory behavior could be
attributed to aberrant differentiation of the dorsal horn neurons or vice
versa. Although we are not able to determine when the migration deficit first
occurs in Lmx1b mutants, studies of the possible downstream targets
of Lmx1b shed light onto this issue. For example, an alteration of
axonal guidance cues such as netrin 1 in Lmx1b mutants might have
contributed to aberrant neuronal migration
(Brose and Tessier-Lavigne,
2000). Moreover, some of the transcription factors downstream of
Lmx1b have also been shown to be required for neuronal migration. In
mice, Ebf1 is important for the migration of facial branchiomotor
neurons (Garel et al., 2000
).
In chicks, Ebf1 and Ebf3 appear to control migration and
differentiation of the dorsal neurons independently
(Garcia-Dominguez et al.,
2003
; Garel et al.,
2000
). Thus, Lmx1b may have a unique role in neuronal
migration in the developing spinal cord. However, this does not exclude the
possibility that aberrant migration reflects some aspects of impaired
differentiation of the dorsal horn neurons in the absence of Lmx1b.
In fact, in addition to the dorsal horn cells, Lmx1b has also been
implicated in neuronal differentiation of dopaminergic neurons and
serotonergic neurons in the developing brain
(Cheng et al., 2003
;
Ding et al., 2003
;
Pressman et al., 2000
). Thus,
it is likely that Lmx1b could play an important role in both cellular
events.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Laboratory of Neural Development, Institute of
Neuroscience, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai,
20031, PR China
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