The Paired Homeodomain Protein DRG11 Is Required for the Projection of Cutaneous Sensory Afferent Fibers to the Dorsal Spinal Cord

Abstract

Cutaneous sensory neurons that detect noxious stimuli project to the dorsal horn of the spinal cord, while those innervating muscle stretch receptors project to the ventral horn. DRG11, a paired homeodomain transcription factor, is expressed in both the developing dorsal horn and in sensory neurons, but not in the ventral spinal cord. Mouse embryos deficient in DRG11 display abnormalities in the spatio-temporal patterning of cutaneous sensory afferent fiber projections to the dorsal, but not the ventral spinal cord, as well as defects in dorsal horn morphogenesis. These early developmental abnormalities lead, in adults, to significantly attenuated sensitivity to noxious stimuli. In contrast, locomotion and sensori-motor functions appear normal. Drg11 is thus required for the formation of spatio-temporally appropriate projections from nociceptive sensory neurons to their central targets in the dorsal horn of the spinal cord.

Full text

Neuron, Vol. 31, 59–73, July 19, 2001, Copyright 2001 by Cell Press
The Paired Homeodomain Protein DRG11 Is
Required for the Projection of Cutaneous Sensory
Afferent Fibers to the Dorsal Spinal Cord
ceptive sensory neurons to their central targets in the
dorsal horn of the spinal cord.
Introduction
Zhou-Feng Chen,
1,7,9
Sandra Rebelo,
3,8
Fletcher White,
4,8
Annika B. Malmberg,
2,8,10
Hiroshi Baba,
5,11
Deolinda Lima,
3
Clifford J. Woolf,
5
Allan I. Basbaum,
2
and David J. Anderson
1,6
1
Division of Biology 216-76 and
Howard Hughes Medical Institute The accurate perception of external sensory information
California Institute of Technology by the brain requires appropriate synaptic connectivity
Pasadena, California 91125 between peripheral sensory neurons of a given modality,
2
Departments of Anatomy and their central targets. How such central connectivity
and Physiology and is coordinated with the specification of sensory modality
W.M. Keck Foundation Center is poorly understood, and remains a central problem in
for Integrative Neuroscience neural development. In the mammalian olfactory sys-
University of California, San Francisco tem, for example, a family of odorant receptors (Buck
San Francisco, California 94143 and Axel, 1991) both determines sensory modality and
3
Instituto de Histologia e Embriologia contributes to the specificity of topographic projections
Universidade do Porto to the olfactory bulb (Wang et al., 1998). Whether such
Porto a conservative mechanism for coordinating sensory
Portugal modality and connectivity is employed by other devel-
4
Department of Neurology and oping sensory systems is not clear, however.
PVA/EPVA Neuroscience Research Center Primary somatosensory neurons in mammalian dorsal
Yale Medical School root ganglia (DRG) are a heterogeneous population sub-
New Haven, Connecticut 06510 serving diverse sensory modalities, including pain,
and Rehabilitation Research Center touch, and body position (Scott, 1992). These different
VA Medical Center modalities are associated with topographically distinct
West Haven, Connecticut 06516 projections to the spinal cord, the first relay station in
5
Department of Anesthesia and Critical Care the central nervous system (CNS). For example, large
Massachusetts General Hospital diameter muscle stretch receptor neurons project to the
Harvard Medical School ventral spinal cord (Figure 1A, red), while cutaneous
Boston, Massachusetts 02129 afferent neurons project to superficial laminae I and II
of the dorsal horn (Willis and Coggeshall, 1991) (Figure
1A, blue and green). The latter population includes neu-
rons specialized for nociception, the sensation of nox-
Summary ious stimuli. Low-threshold mechanosensory neurons,
in turn, project to deeper layers III and IV (Brown et al.,
Cutaneous sensory neurons that detect noxious stim- 1977; Ralston et al., 1984; Shortland et al., 1989). Thus,
uli project to the dorsal horn of the spinal cord, while to a first approximation different stimulus features of
those innervating muscle stretch receptors project to primary sensory neurons are topographically mapped
the ventral horn. DRG11, a paired homeodomain tran- to different laminae of the spinal cord (Figure 1A).
scription factor, is expressed in both the developing The molecular mechanisms that govern the connectiv-
dorsal horn and in sensory neurons, but not in the ity of different subsets of primary sensory neurons with
ventral spinal cord. Mouse embryos deficient in DRG11 their central targets are only beginning to be understood.
display abnormalities in the spatio-temporal pat- Semaphorins and their receptors, the neuropilins (He
terning of cutaneous sensory afferent fiber projections and Tessier-Lavigne, 1997; Kolodkin et al., 1997), have
to the dorsal, but not the ventral spinal cord, as well been proposed to control the projections of cutaneous
as defects in dorsal horn morphogenesis. These early versus muscle sensory afferent fibers into the dorsal
developmental abnormalities lead, in adults, to signifi- versus ventral spinal cord based on in vitro assays (Mes-
cantly attenuated sensitivity to noxious stimuli. In con- sersmith et al., 1995; Puschel et al., 1996). However,
trast, locomotion and sensori-motor functions appear these molecules have not yet been shown to be essential
normal. Drg11 is thus required for the formation of for this process in vivo (Kitsukawa et al., 1997; Chen et
spatio-temporally appropriate projections from noci- al., 2000; Giger et al., 2000). Similarly, in vitro studies
have suggested that Slit molecules may control the ini-
tial branching of sensory afferent fibers into the spinal
6
Correspondence: mancusog@caltech.edu
7
To whom requests for materials should be directed.
gray matter (Wang et al., 1999), but whether they play
8
These authors contributed equally to this work.
this role in vivo is not yet clear.
9
Present address: Departments of Anesthesiology, Psychiatry, Mo-
Transcription factors that control the connectivity of
lecular Biology and Pharmacology, Washington University School
sensory neurons and their central targets are also begin-
of Medicine Pain Center, St. Louis, Missouri 63110.
ning to be identified. For example Ets-domain transcrip-
10
Present address: NeurogesX, Inc., San Carlos, California 94070.
tion factors, such as ER81 and PEA3, are coordinately
11
Present address: The Department of Anesthesia, Niigata Univer-
sity, Niigata, Japan.
expressed by subsets of muscle afferent sensory neu-

Neuron
60
DRG sensory neurons and neurons in the dorsal horn
is a paired homeodomain protein known as DRG11
(Saito et al., 1995). Expression of DRG11 occurs first in
DRG sensory neurons and approximately one day later
in the dorsal horn, around the time when cutaneous
afferent fibers first penetrate the spinal gray matter
(Ozaki and Snider, 1997). Induction of Drg11 in the dorsal
spinal cord is controlled independently of that in the
DRG (A. Greenwood, Z.-F.C. and D.J.A., unpublished
data), suggesting that its coordinated expression in
these pre- and post-synaptic neurons could contribute
causatively to their connectivity.
Here, we have generated and analyzed mice con-
taining a mutation in the Drg11 gene. Drg11
⫺/⫺
embryos
exhibit spatio-temporal abnormalities in the initial pene-
tration of cutaneous sensory afferent fibers into the lat-
eral-most part of the developing dorsal horn, while spar-
ing projections, including those of muscle afferents, to
more medial and ventral regions. Subsequent to this
initial deficiency, later defects in cellular differentiation
and survival are observed, leading in adults to a substan-
tial loss of both neurons and cutaneous afferent fibers
in the lateral-most regions of the dorsal horn. Behavioral
studies show that these anatomical defects are corre-
lated with a substantial reduction in sensitivity to various
types of noxious stimuli, while locomotion and sensori-
motor function appear normal. These data suggest that
DRG11 is required, directly or indirectly, for the initial
formation of connections between cutaneous afferent
sensory neurons and their central targets.
Results
Generation of Drg11-Deficient Mice
A mutation in Drg11 was produced using homologous
Figure 1. Organization of Primary Sensory Afferent Projections to
recombination in embryonic stem (ES) cells (Ramirez-
the Spinal Cord and Relationship to Expression of DRG11
Solis et al., 1993). The design of the targeting construct
(A) Schematic diagram illustrating the organization of sensory neu-
deletes exons 3 and 4, which encode most of the paired-
ron projections to the spinal cord. Muscle afferent sensory neurons
like homeodomain, the putative DNA binding region (Fig-
(red), which mediate proprioception (p), project to motoneurons in
ures 2A and 2B). RT-PCR experiments failed to identify
the ventral spinal cord (magenta) and to muscle spindles in the
periphery (yellow oval). Cutaneous afferents with peripheral projec-
transcripts that encoded residual homeodomain se-
tions in the skin project to different laminae in the dorsal spinal cord:
quences upstream of the deleted exons in Drg11
⫺/⫺
mice
nociceptive neurons (n, light and dark blue) to lamine I and II (ᐉI, II),
(see Experimental Procedures). Therefore, the mutation
and mechanoceptive neurons (m, green) to laminae III and IV (ᐉIII,
is likely to abolish DRG11 function. Targeted ES cell
IV). Within a given lamina, cutaneous afferents innervating proximal
clones were identified by Southern blotting (Figure 2C),
peripheral targets project to the lateral region of the dorsal horn (n,
and germline chimeras were obtained from C57BL/6J
dark blue), while those with distal peripheral targets project more
medially (n, light blue). For simplicity, only mechanoceptive afferents
host blastocysts injected with these clones (Hogan et
with distal targets are illustrated.
al., 1986). Drg11 heterozygous mice were viable, fertile,
(B) Expression of DRG11 mRNA in the DRG and spinal cord at
and apparently normal. In a mixed 129/SvJ x C57BL/6J
postnatal day 3 in the rat. Arrow indicates expression in neurons of
genetic background, Drg11
⫺/⫺
mice were born in the
the dorsal spinal cord (substantia gelatinosa), arrowhead in sensory
expected Mendelian ratio (31 ⫹/⫹:45⫹/⫺:25⫺/⫺),
neurons.
but weighed less than wild-type or heterozygous lit-
termates. By about 3 weeks of age, however, all Drg11
⫺/⫺
mice in this genetic background had died.
rons and the motoneurons to which they connect (Lin
et al., 1998). Genetic analysis has indicated that ER81
is essential for the formation of such connections (Arber Early Developmental Defects in the Dorsal Horn
of Drg11
⫺/⫺
Embryoset al., 2000). Interestingly, all Ets-domain transcription
factors thus far examined are expressed exclusively by We first looked for evidence of phenotypic abnormalities
in Drg11
⫺/⫺
embryos at stages just after the gene is firstmuscle afferent sensory neurons (Lin et al., 1998; Arber
et al., 2000). This suggests that other families of tran- expressed. In wild-type embryos, Drg11 is expressed in
the dorsal spinal cord beginning on E12-E12.5 (Figurescription factors may control the connectivity of cutane-
ous afferent sensory neurons with their central targets 2E and Saito et al., [1995]). Within this region, Drg11 is
initially expressed by newly generated neurons adjacentin the dorsal horn.
One transcription factor that is expressed by both to the ventricular zone (Figure 2E, upper small arrow),

DRG11 Controls Sensory Neuron Projections
61
Figure 2. Targeted Mutagenesis of Drg11 in Mice
(A) Amino acid sequence of the first coding exons (boxed). Black letters encode the homeodomain, and the arrows indicate the boundaries
of the deleted region.
(B) Targeting strategy for the Drg11 gene. Black box indicates the coding exons. Following homologous recombination, coding exons 3 and
4, which encode most of the homeodomain region, are replaced by the IRES-Tau-lacZ-neo cassette. B: BamH1.
(C) Southern blot of Drg11
⫹
/⫺
intercross progeny. Wild-type (6.6 kb) and mutant (12.7 kb) alleles are distinguishable by BamH1 digestion using
an 0.8kb Pst1 genomic fragment as a 3⬘external probe. Lane1, homozygote (⫺/⫺); lane 2, heterozygote (⫹/⫺); lane 3, wild-type (⫹/⫹).
(D) Phenotype of a Drg11
⫺/⫺
mouse (right, ⫺/⫺) and a wild-type littermate (left, ⫹/⫹). Note the characteristic skin lesion on the dorsal aspect
of the proximal hindlimb (arrow).
(E–G) Expression of Drg11 mRNA in wild-type mouse embryos at E12.5 (E), E13.5 (F), and E15.5 (G). Arrowheads indicate expression in the
lateral region of the dorsal horn. Upper small arrows in (E) and (F) indicate expression just outside the ventricular zone; lower small arrows
indicate expression in the DRG. Expression is also detected in the DRG at E15.5, but is not shown in (G).
as well as in scattered cells lateral to this region (Figure populated by small, darkly staining neurons (Figures 3G
and 3H, arrows). In addition, the dorsal funiculus, which2E, arrowhead). Over the next few days, expression in
this medial location is extinguished (Figure 2F, arrow), consists mainly of primary afferent fibers and second-
order projections at these embryonic stages, was shal-while it increases in the dorso-lateral region (Figures
2Fand 2G, arrowheads). lower (Figures 3E–3H, brackets, “DF”). These differ-
ences between wild-type and Drg11
⫺/⫺
dorsal horn wereNissl staining of E14.5 dorsal horns revealed no obvi-
ous difference between wild-type and Drg11
⫺/⫺
embryos more pronounced at thoraco-lumbar than at cervical
levels (cf. Figures 3E and 3F versus Figures 3G and 3H,(Figures 3A–3D). However, beginning at E15.5, several
differences could be observed. First, there appeared arrows). No defects in dorsal horn morphology were
observed in heterozygous Drg11⫹
/⫺
embryos (notto be a reduction in the intensity of Nissl staining in
the dorsal horn of Drg11
⫺/⫺
embryos (Figures 3E–3H, shown).
Despite the clear defects visible by Nissl staining, wearrows), inthe same area where Drg11 itself is expressed
at this stage in wild-type embryos (Figure 2G, arrow). were unable to identify any consistent, obvious alter-
ations in the expression of several molecular markersThis reduction in staining intensity occurs in a region

Neuron
62
Figure 3. Timing of Morphological Abnormalities and Cell Death in the Dorsal Horn of Drg11
⫺/⫺
Mice
(A–H) Nissl-stained sections of E14.5 (A–D) and E15.5 (E–H) spinal cord. No difference between mutant (⫺/⫺) and wild-type (⫹/⫹) specimens
are detectable at E14.5. At E15.5, reduced Nissl staining is visible in the dorsal horns of mutant embryos (F) and (H), arrows). The dorsal
funiculus (“DF, brackets”) is also shortened. Note that the difference between mutant and wild-type specimens is more obvious at thoracic
(G) versus (H) than at cervical (E) versus (F) levels. Heterozygous embryos were indistinguishable from wild-type (not shown).
(I–L) TUNEL labeling of apoptotic cell death. Enhanced cell death in the Drg11
⫺/⫺
spinal cord is first detected at E17.5 (L), two days after
defects are detectable by Nissl staining (F and H).
in the dorsal spinal cord of midgestational Drg11
⫺/⫺
em- (data not shown), and the axon guidance molecule
Netrin 1 (Leonardo et al., 1997) (Figures 4Gand 4H).bryos. These markers included the transcription factors
Ebf1 and Ebf2 (Garel et al., 1997; Wang et al., 1997) We next asked whether the reduction in Nissl staining
detected in the dorsal horn of Drg11
⫺/⫺
embryos was(Figures 4A–4D), Lmx1b (Chen et al., 1998a) (Figures 4E
and 4F), Math1 (Akazawa et al., 1995), LH2a (Xu et al., due to cell death. Prior to E17.5, there were few if any
TUNEL⫹cells in the spinal cord of Drg11
⫺/⫺
embryos1993; Liem et al., 1997), and Pax3 (Goulding et al., 1993)
(Figure 3J and data not shown), despite the reduced
Nissl staining evident one day earlier (Figures 3F and
3H). However, beginning on E17.5, increased cell death
was apparent in the dorsal horn (Figures 3K and 3L,
arrows), in the same region where the decrease in Nissl
staining was visible two days earlier (Figures 3G and
3H, arrows). These data suggest that the decrease in
Nissl staining intensity evident at E15.5 is unlikely to be
due to the death of small darkly staining neurons. More
likely, it reflects a defect in some aspect of their differen-
tiation. This defect is eventually followed by cell death,
but not until two days later.
A Defect in the Projection Pattern of Primary
Sensory Afferents in the Dorsal Horn
of Drg11
⫺/⫺
Embryos
We next examined the development of primary sensory
afferent projections to the embryonic dorsal horn in
Drg11
⫺/⫺
mice. In wild-type animals, the central projec-
tions of cutaneous nociceptive sensory neurons first
arrive in the dorsal root entry zone (DREZ) at E10.5, and
begin to invade the spinal gray matter at E12.5 (Ozaki
and Snider, 1997). Staining with antibody to calbindin-
28K marks a subset of cutaneous neurons and their
afferent fibers (Honda, 1995). Ingrowth of such calbin-
din⫹afferent fibers into the lateral aspect of the dorsal
horn occurred between E12.5 and E13.5, in wild-type
embryos (Figures 5C and 5G, arrow). In contrast, no
Figure 4. Expression of Molecular Markers in the Dorsal Horn of
such calbindin⫹fibers were detected in the dorsal horn
Drg11
⫺/⫺
Embryos
of Drg11
⫺/⫺
embryos at E13.5 (Figure 5H, arrow). Nissl
Sections through dorsal horn of wild-type (⫹/⫹) and Drg11
⫺/⫺
em-
staining of adjacent sections revealed no apparent mor-
bryos (⫺/⫺) at E14.5 are shown. Three transcription factor markers,
phological defects in the developing dorsal horn at this
Ebf1 (A and B), Ebf2 (C and D), and Lmx1b (E and F) are shown, along
stage (Figures 5E and 5F) or at E12.5 (Figures 5A and 5B),
with the axon guidance molecule Netrin (G and H). No differences are
consistent with the analysis of E14.5 embryos (Figures
seen except for the slight flattening of the dorsal horn in the mutant
(see also Figure 3).
3A–3D).

DRG11 Controls Sensory Neuron Projections
63
Figure 5. A Connectivity Defect Precedes
Morphological Defects in Drg11
⫺/⫺
Embryos
([A], [C]), ([B], [D]), ([E], [G]) and ([F], [H]) are
pairs of adjacent sections stained with Nissl
(A, B, E, and F) and anti-calbindin-28K anti-
body (C, D, G, and H), respectively.
(A–D) At E12.5, no difference in either Nissl
or calbindin staining is detectable between
wild-type (A) and (C) and mutant (B) and (D)
specimens. At this stage, calbindin
⫹
sensory
afferent fibers have not yet penetrated the
dorsal horn gray matter.
(E–H) At E13.5, calbindin
⫹
afferents are seen
to penetrate the gray matter of wild-type (G,
arrow) but not mutant (H, arrow) dorsal horn,
although no abnormalities are yet detectable
by Nissl staining (E and F). Heterozygous
specimens were indistinguishable from wild-
type.
To determine whether the absence of calbindin⫹fibers biased toward the medial region of the dorsal horn, in
comparison to wild-type embryos.at E13.5 reflected a complete block or rather a delay in
cutaneous afferent fiber ingrowth, we examined later Similar results were obtained using antibodies to trkA,
which is expressed by most or all cutaneous sensoryembryos. At E14.5, calbindin⫹fibers could be seen in
the DREZ of Drg11
⫺/⫺
embryos (Figure 6B, arrow), but neurons at this stage (Lewin, 1996; Snider and Silos-
Santiago, 1996) (Figures 6E–6H). At E16.5, the lateral-to-had not penetrated into the spinal gray matter to the
extent visible in wild-type embryos (Figure 6A, arrow). medial shift in the distribution of trkA⫹afferents between
wild-type and Drg11
⫺/⫺
embryos was clear, with manyIn addition, there was a marked absence of such fibers
in the lateral-most portion of the dorsal horn (Figures more fibers appearing to cross the midline in the mutant
(Figures 6G and 6H, arrows). In contrast to the abnormal6A and B; arrowheads). By E16.5, calbindin⫹fibers had
invaded the dorsal horn of Drg11
⫺/⫺
mutants (Figure 6D). cutaneous afferent projections, no differences between
wild-type and Drg11
⫺/⫺
embryos were observed in theHowever, the absence of such fibers in the lateral-most
region of the dorsal horn persisted (Figures 6C and 6D; central projections of IA muscle afferent fibers (Figures
6I and 6J, arrows) as revealed by staining with antibodiesarrowheads). At the same time, there was an increased
density of calbindin⫹fibers near the midline of Drg11
⫺/⫺
to peripherin, which marks such proprioceptive affer-
ents at these embryonic stages (Escurat et al., 1990;embryos in comparison to wild-type (Figures 6C and
6D; arrows). These data suggest that the initial absence Goldstein et al., 1991).
Because apparent differences in afferent fiber projec-of calbindin⫹fibers at E13.5 (Figure 5H) reflects a delay,
rather than a block, in afferent fiber ingrowth, but also tions revealed by antibody staining might reflect differ-
ences in the distribution of the corresponding antigens,indicate that the ingrowth that eventually does occur is

Neuron
64
Figure 6. Developmental Progression of Afferent Projection Defects in the Dorsal Horn of Drg11
⫺/⫺
Mice
(A–D) Calbindin-D28K staining. Note that at E14.5 (A and B), calbindin
⫹
fibers have already entered the spinal gray matter in wild-type embryos
(A, arrow), while in the mutant they are restricted to the most superficial layer (B, arrow) and absent from the lateral region (cf. [A] versus [B],

DRG11 Controls Sensory Neuron Projections
65
rather than of the fibers that express them, we used DiI
labeling as an independent assessment of afferent fiber
projections in Drg11
⫺/⫺
and wild-type embryos (Figures
6K–6N). These studies confirmed the abnormalities in
cutaneous afferent fiber projection to the dorsal horn
detected by antibody staining. First, there was a virtually
complete absence of afferent fiber ingrowth into the
lateral-most portion of the dorsal horn, at E14.5 (Figures
6K and 6L, arrowed bracket), even though the growth
of sensory axons to the DREZ through the dorsal root
was unaffected (Figures 6K and 6L, dr). This defect in
afferent fiber penetration to the dorso-lateral gray matter
persisted at E16.5 (Figures 6M and 6N, arrowed bracket),
although afferent fibers could be detected in the dorso-
medial region of mutant embryos at this stage (Figures
6M and 6N, arrowheads). Consistent with the results of
peripherin staining, there appeared to be no difference
in the ventro-medial projections of IA muscle afferent
fibers (Figures 6M and 6N, large arrows).
Taken together, these data indicate that in Drg11
⫺/⫺
embryos there is a defect in the spatio-temporal pat-
terning of sensory afferent fiber projections to the dorsal
horn, which selectively affects cutaneous afferents.
These abnormalities are especially prominent in the lat-
eral-most region of the dorsal horn, a site where expres-
sion of Drg11 itself is most abundant at this stage (Figure
2F, arrowhead). This is also the region where defects in
Nissl staining are eventually apparent, at E15.5 (Figures
3E–3H, arrows). However, the stage when the cutaneous
afferent fiber projection defect is first detected (E13.5;
Figures 5G and 5H) precedes the abnormalities in Nissl
staining by two days. No defects in afferent fiber projec-
tions were detectable in Drg11⫹
/⫺
heterozygous mice,
at any stage examined (data not shown).
Figure 7. Expression of Sensory Neuron Markers Appears Normal
in the DRG of Drg11
⫺/⫺
Mice
Differentiation and Survival of DRG Sensory
A series of adjacent sections through E14.5 lumbar DRG of wild-
Neurons Are Normal in Drg11
⫺/⫺
Embryos
type and Drg11 mutant embryos is shown. All photomicrographs
are nonisotopic in situ hybridizations performed with the probes
and Neonates
indicated to the left of the photomicrographs. No difference in the
DRG11 is also expressed in developing sensory neurons
expression of any of the markers is detected.
of the dorsal root ganglia (DRG), at the same time as it
is detected in the dorsal horn (Figures 2E and 2F, lower
arrows). At E14.5, no differences between wild-type and
embryos counted). Consistent with these results, there
Drg11
⫺/⫺
embryos were detectable using a battery of
was no increase in TUNEL labeling in the DRG of
molecular markers for different sensory neuron sub-
Drg11
⫺/⫺
mice, even at a stage (E17.5) when TUNEL⫹
types (Figure 7). The total number of sensory neurons
cells were apparent in the dorsal horn (Figure 3L; data
was also not significantly different in lumbar (L3-L5) DRG
not shown). To determine whether any loss of cutaneous
between wild-type (18,943 ⫾1,437; mean ⫾SEM) and
afferent sensory neurons occurred perinatally, we also
Drg11
⫺/⫺
mutant ganglia (16,334 ⫾713; p ⫽0.193; n ⫽
2 independent Drg11
⫺/⫺
mutant and wild-type littermate quantified the number of trkA⫹sensory neurons in neo-
arrowheads). At E16.5 (C and D), calbindin staining is most intense in presumptive laminae I and II at the lateral margins (C, arrowhead). In
the mutant (D), calbindin-positive fibers have penetrated the gray matter by this stage, but are more concentrated in the medial region (arrow)
and depleted from the lateral region (D, arrowhead).
(E–H) trkA antibody staining. The entry of trkA
⫹
afferent fibers into the gray matter of Drg11 mutant mice is delayed relative to wild-type (E
and F, arrows). It is also biased toward the medial region (F and H, arrows) and depleted from the lateral region (F and H, arrowheads).
(I and J) Peripherin staining reveals no difference between mutant (J) and wild-type (I) in the ingrowth of group IA muscle sensory afferents
that grow to the ventral spinal cord (arrows).
(K–N) Di I labeling of lumbar level sensory afferents in the sciatic nerve. At E14.5 (K and L), afferent ingrowth to the lateral dorsal horn is
apparent in wild-type (K, arrowed bracket), but not in Drg11
⫺/⫺
specimens (L, arrowed bracket). Nevertheless, sensory afferent fibers appear
to grow normally to the dorsal roots (dr) in the mutant. By E16.5 (M and N), afferents in the mutant have entered the gray matter and are
present in the medial region of the gray matter (N, arrowhead), but are still absent from the lateral region (N, arrowed bracket). Presumptive
IA muscle afferent projections (arrows) are unaffected, consistent with the results of anti-peripherin antibody staining (I and J).

Neuron
66
P2 (not shown). The absence of PKC␥⫹cells is not due
Table 1. Number of trkA
⫹
Neurons in Lumbar DRG
of P0 Drg11
⫺/⫺
Mice
simply to generalized neuronal death, because many
neurons in the dorsal horn were still present at this stage
Ganglion ⫹/⫹Drg11 ⫺/⫺t test
as indicated by staining for the pan-neuronal nuclear
L2 1230 ⫾156 1348 ⫾247 NS
marker NeuN (Figures 8A and 8B), as well as for other
L3 1634 ⫾54 1723 ⫾65 NS
dorsal horn markers (not shown). However, we cannot
L4 1972 ⫾58 2306 ⫾450 NS
distinguish whether the loss of this specific marker re-
L5 2344 ⫾0 2044 ⫾393 NS
flects selective cell death or, rather, defective differenti-
Numbers indicate the sum of the neurons counted in the right plus
ation.
left ganglia at each of the indicated axial levels. Data represent the
mean ⫾SEM from two separate mutant and wild-type embryos.
Numbers are not corrected. NS, not significantly different (p ⬎0.05).
Defects in Pain Sensitivity in Adult Drg11
⫺/⫺
Mice
The foregoing data suggested that the Drg11
⫺/⫺
muta-
tion selectively perturbs the development of primary
cutaneous sensory afferent projections to the dorsal horn
natal Drg11
⫺/⫺
mice (Table 1). Again, there was no sig-
(as well as subsequent development of the dorsal horn
nificant difference in the number of these neurons in
itself). As these projections mediate (among other modal-
L2-L5 DRG between wild-type and Drg11
⫺/⫺
mutants.
ities) nociception, these results implied that Drg11
⫺/⫺
mice might exhibit selective deficiencies in their behav-
Absence of PKC␥
ⴙ
Cells in Lamina IIi of Early
ioral responses to noxious stimuli. Because Drg11
⫺/⫺
Postnatal Drg11
⫺/⫺
Mice
mice die by the third postnatal week in a 129Sv x C57BL/
The abnormal Nissl staining in the dorsal horn of E15.5
6J background, we attempted to extend their lifespan by
Drg11
⫺/⫺
embryos (Figures 3E–3H) suggested that the
crossing them to CD-1 mice, which are a more vigorous
mutation might affect the development of second-order
outbred strain. In this mixed genetic background, about
neurons involved in the central processing of nocicep-
50% of Drg11
⫺/⫺
mice did survive to adulthood. Neverthe-
tive afferent input to the spinal cord. One marker of
less, analysis of Drg11
⫺/⫺
embryos in this mixed back-
such neurons is protein kinase C-␥(PCK␥), which is
ground revealed similar developmental defects as de-
specifically expressed in lamina IIi and has been func-
scribed previously for the 129Sv x C57BL/6J background
tionally implicated in nerve injury-induced (“neuro-
(data not shown).
pathic”) pain (Malmberg et al., 1997). In wild-type mice,
At the age of three or four weeks, there was no differ-
PKC␥is first expressed in the dorsal horn at postnatal
ence in normal, spontaneous behavior between the
day 2 (P2; data not shown). Strikingly, in postnatal day
Drg11
⫺/⫺
and wild-type mice. However, by about two
5 (P5) Drg11
⫺/⫺
mice, there was a virtually complete
months of age, Drg11
⫺/⫺
mice on the outbred CD-1 back-
absence of PKC␥⫹cells across the entire medio-lateral
ground could be recognized by persistent grooming of
extent of the dorsal horn (Figure 8C, arrow, versus Figure
the dorsal hindlimb, which subsequently led to fur loss
8D). The expression of this marker in the corticospinal
and skin lesions on some of the mice (Figure 2D, arrow).
tract, however, was unchanged (Figure 8D, large arrow),
Such bare patches are observed in more anterior regions
providing an internal positive control for the staining.
of mice whose cutaneous sensory neurons have been
The lack of PKC␥expression could also be observed at
destroyed by capsaicin treatment (Crowley et al., 1994;
Smeyne et al., 1994; Thomas et al., 1994). Their location
in the posterior region of adult Drg11
⫺/⫺
mice is consis-
tent with the fact that the developmental phenotype
appears more severe caudally than rostrally and is there-
fore suggestive of compromised nociception in these
mutants.
To experimentally assess nociceptive function, we
performed a battery of behavioral tests on adult Drg11
⫺/⫺
mice (Figure 9). In virtually all of these tests, there was
a clear and statistically significant reduction in the re-
sponse of Drg11
⫺/⫺
mice to a variety of noxious stimuli
applied to the hindpaw or tail, relative to both wild-
type and Drg11⫹
/⫺
heterozygous littermate controls. For
example, Drg11
⫺/⫺
mice displayed significantly higher
response latencies in the hot plate, tail-flick, and paw
withdrawal tests of thermal sensitivity (Figure 9A and
data not shown). Drg11
⫺/⫺
mice also showed reduced
Figure 8. Early Loss of PKC␥Expression in Lamina IIi of the Dorsal
Horn
sensitivity to mechanical stimulation, tested using using
von Frey filaments (Chaplan et al., 1994) (Figure 9B). In
(A and B) NeuN staining of neuronal cell bodies reveals a disorgani-
zation of the dorsal horn in the mutant at postnatal day 5 (B).
addition, they exhibited reduced responses in tests of
(D) PKC␥staining is eliminated in lamina IIi of the mutant (D), al-
chemical nociception, using either formalin or capsaicin
though it is retained in corticospinal axons in the dorsal funiculus
(Figure 9C). No significant differences were observed
(D, arrow). Similar results were also obtained at P2 (not shown).
between wild-type and Drg11⫹
/⫺
heterozygous mice in
Note that the loss of PKC␥
⫹
cells is relatively selective, in that there
any of these tests (n ⫽4Drg11⫹
/⫺
animals examined in
are still many surviving neurons in the dorsal spinal cord at this
stage (B).
each of the assays; data not shown).

DRG11 Controls Sensory Neuron Projections
67
Figure 9. Reduced Sensitivity to Nociceptive
and Mechanical Stimuli in Drg11-Deficient
Mice
(A) Tests of thermal sensitivity. In both the
hot plate and tail-flick tests, the response la-
tency of Drg11
⫺/⫺
mice is significantly longer
(**, p ⬍0.001; *, p ⬍0.01; t test) than that of
wild-type littermates.
(B) Test of sensitivity to innocuous mechani-
cal stimulation using calibrated von Frey fila-
ments. The threshold weight (in grams) nec-
essary to produce a response is significantly
higher (*, p ⬍0.001; Mann-Whitney test) for
the Drg11
⫺/⫺
mutant mice.
(C) Tests of chemical nociception. Injection
of a 5% formalin solution into the paw evokes
two phases of paw licking: phase I reflects
direct activation of primary nociceptors;
phase II reflects peripheral inflammation and
sensitization of dorsal horn neurons. Both
phases of licking are significantly reduced (*,
p⬍0.05; **, p ⬍0.01, t test) in the mutant.
Licking responses to capsaicin injection into
the hindpaw are also significantly reduced (*,
p⬍0.05, t test). In (A) through (C), n ⫽8 wild-
type and n ⫽7 mutant animals were examined. No differences were observed between wild-type and heterozygous Drg11
⫹
/⫺
animals in any
of these assays (not shown).
(D) Sensorimotor (IA afferent) function as determined by a rotarod treadmill test is not significantly different between wild-type and mutant
animals.
Taken together, these results indicate that Drg11
⫺/⫺
neurons at lumbar levels, the region where the pheno-
type was most pronounced (as was observed in em-mice exhibit significant reductions in their responses to
a broad range of noxious stimuli encompassing several bryos). These anatomical defects in the lumbar dorsal
horn may account for the localization of skin lesions tomodalities, as well as sensitivity to mechanical stimuli.
In contrast, a rotarod treadmill test revealed no differ- the posterior region of Drg11
⫺/⫺
animals (Figure 2D).
By contrast, there was no evident loss of neurons orence in motor function between Drg11
⫺/⫺
and wild-type
mice (Figure 9D), suggesting that sensorimotor func- alteration of morphology in the ventral spinal cord,
where motoneurons are located (data not shown).tions mediated by muscle afferent sensory neurons in-
nervating spindle fibers and Golgi tendon organs are To determine the molecular identity of the dorsal horn
neurons that were lost, we applied a battery of reagentsintact. Such a conclusion is consistent with the observa-
tion that the projections of IA muscle afferent sensory that mark the postsynaptic targets of cutaneous afferent
projections in laminae I and II of the dorsal horn. Thereneurons to the ventral spinal cord develop normally in
the mutant (Figures 6I–6N). Furthermore, Drg11
⫺/⫺
mice was a complete absence of PKC␥⫹cells in lamina IIi in
adult Drg11
⫺/⫺
mice (Figure 10H, arrowhead). Thus, theexhibited normal locomotion, in contrast to mice bearing
mutations in genes required for proprioceptive sensory absence of this marker at early postnatal stages (Figure
8) does not simply reflect delayed differentiation of theseneuron development or survival, in which hindlimb loco-
motion is clearly impaired (Ernfors et al., 1994; Farin
˜as cells. In addition, Drg11
⫺/⫺
mice exhibited a lack of ex-
pression of calretinin, a calcium binding protein ex-et al., 1994; Klein et al., 1994; Arber et al., 2000). Thus, the
developmental defects caused by the Drg11
⫺/⫺
mutation pressed in laminae I and II (Ren et al., 1993) (Figures
10C and 10D; arrows). Expression of PKC␤II, a proteinlead to functional deficits involving somatosensory func-
tions processed by the dorsal spinal cord. kinase C isoform (Malmberg et al., 1997), was also virtu-
ally completely eliminated at the lateral-most margins
of the dorsal horn, and was greatly reduced in more
Neuronal Loss in the Superficial Laminae
dorso-medial locations (Figures 10E and 10F; arrows).
of the Dorsal Horn of Adult Drg11
⫺/⫺
Mice
Taken together, these data reveal a pronounced loss in
To determine whether the developmental defects in
adult Drg11
⫺/⫺
mice of post-synaptic neurons in laminae
Drg11
⫺/⫺
embryos led to persistent anatomical and/or
I, IIo, and IIi, especially in the lateral-most region of the
molecular deficiencies in the adult spinal cord, which
dorsal horn.
might account for the behavioral phenotype, we first
examined the expression of NeuN (Figures 10A and 10B;
arrowheads), a general neuronal marker (Mullen et al., A Central Projection Defect in the Spinal Cord
of Adult Drg11
⫺/⫺
Mice1992). Staining with anti-NeuN antibody revealed a strik-
ing loss of neurons in the dorsal horn, particularly in the We next examined the expression of markers of presyn-
aptic cutaneous sensory afferent fibers in the dorsallateral-most region (Figures 10 A and 10B, arrowhead),
the site where the ingrowth of cutaneous afferent fibers horn. The dorsal horn is innervated in distinct topo-
graphic locations by two different classes of nociceptiveis blocked in embryos (Figure 6). Cell counts indicated
a reduction of 60%–70% of such NeuN⫹dorsal horn afferent fibers. “Peptidergic” C- and A␦fibers, con-

Neuron
68
taining the neuropeptides CGRP and/or Substance P,
project mainly to lamina I and outer lamina II (IIo) (Hunt
et al., 1992; Snider and McMahon, 1998). There was a
dramatic reduction of Substance P⫹fibers (Figures 10I
and 10J), as well as of fibers expressing CGRP (not
shown), in the adult Drg11
⫺/⫺
spinal cord. As was the
case in embryos, the lateral-most projection field of
these cutaneous afferent fibers appeared almost com-
pletely eliminated (Figures 10I and 10J, arrowheads), so
that there was an apparent dorso-medial shift in the
distribution of surviving fibers in the mutant (Figure 10J,
arrow).
The second class of nociceptive fibers are nonpeptid-
ergic afferents expressing the surface lectin IB4, the
GDNF receptor c-RET, and thiamine monophosphatase
(TMPase) (Knyihar-Csillik et al., 1986; Molliver et al.,
1997; Bennett et al., 1998). These afferents project spe-
cifically to lamina IIi (reviewed in Snider and McMahon
(1998). As was observed for the peptidergic fibers, there
was a dramatic loss of TMPase-positive fibers in the
lateral-most domain of the Drg11
⫺/⫺
dorsal horn (Figures
10K and 10L, arrowheads). Furthermore, the remaining
fibers appeared to be shifted dorso-medially, overlap-
ping the domain occupied by SubP⫹fibers in the mutant
(Figures 10J and 10L, arrows). Double-labeling with fluo-
rescent IB4 and anti-CGRP antibody confirmed an over-
lap of these peptidergic and non-peptidergic fibers in
the dorso-medial region of Drg11
⫺/⫺
mice, rather than
the well-separated staining in laminae IIi and I seen
in wild-type specimens (data not shown). Interestingly,
these nonpeptidergic fibers normally synapse onto
PKC␥-expressing cells in lamina IIi (Snider and Wright,
1996), which are absent in the mutant (Figure 10H, ar-
rowhead). Thus, the concentration of residual peptider-
gic and nonpeptidergic afferents fibers in the dorso-
medial region of the Drg11
⫺/⫺
spinal cord may reflect
the loss of their postsynaptic targets in the lateral dorsal
horn, as well as throughout lamina IIi, so that the re-
maining fibers project to the sites where surviving intrin-
sic dorsal horn neurons are located.
In preliminary experiments, we sought to determine
Figure 10. Loss of Dorsal Horn Neurons and Primary Afferent Pro-
whether there were any obvious defects in synaptic
jections in the Spinal Cord of Adult Drg11
⫺/⫺
Mice
transmission in the dorsal horn of adult Drg11
⫺/⫺
mice.
(A and B) Staining with the pan-neuronal nuclear marker NeuN re-
Whole cell patch-clamp recordings from postsynaptic
veals abnormal morphology and neuron loss in the dorsal horn of
neurons (n ⫽14) were performed in the medial third of
Drg11
⫺/⫺
(⫺/⫺, B) mice. Note that the size of the dorsal horn of the
mutant is greatly reduced, and that cell loss is particularly pro-
lamina II in slices of dorsal horn with an attached dorsal
nounced in the ventrolateral margin of the laminae I and II (arrow-
root (Baba et al., 1999). All neurons tested responded
heads). lf, lateral funiculus; df, dorsal funiculus.
to orthodromic dorsal root stimulation and exhibited
(C and D) Expression of calretinin in laminae I and II of the dorsal
either or both monosynaptic (n ⫽6) or/and polysynaptic
horn is virtually eliminated in the mutant (D).
(n ⫽11) A␦fiber-mediated EPSCs with short latencies
(E and F) Expression of PKC␤II in the dorsal horn (laminae I and II)
(2–5ms) (Yoshimura and Jessell, 1989). In eight of four-
of the mutant (F) is greatly reduced.
(G and H) Expression of PKC␥, a marker of inner lamina II (G, curved
teen lamina II neurons, C fiber-evoked long-latency (ⵑ20
arrow) is lost in the mutant (H, arrowhead), although expression in
ms) EPSCs were also observed at an appropriate stimu-
corticospinal axons is retained in the dorsal funiculus (broad arrows).
lus intensity (⬎200 ␮A, 0.5 ms) (see supplemental data
(I and J) Cutaneous nociceptive afferent fibers identified by Sub-
at http://www.neuron.org/cgi/content/full/31/1/59/DC1).
stance P immunoreactivity are reduced in number and shifted dorso-
The response properties of these lamina II neurons in
medially in the mutant (J, arrow).
(K and L) Nonpeptidergic cutaneous afferent fibers, identified by
Drg11
⫺/⫺
mice are similar to those extensively charac-
expression of TMPase, which project primarily to lamina IIi, are also
terized in prior studies of normal animals (Yoshimura
reduced in the mutant (L). Note the pronounced loss of afferent
and Jessell, 1989; Yoshimura and Nishi, 1993; Yoshi-
fibers in the lateral half of laminae I and II (I–L, arrowheads), the
mura and Nishi, 1995; Baba et al., 1999). These data
region of maximal cell loss (A and B). The dorsal shift of the TMPase-
suggest that the surviving primary nociceptive afferent
positive fibers (L) was confirmed by double-labeling for IB4 and
synapses in the dorsal horn of Drg11
⫺/⫺
mice function
CGRP (not shown) and is consistent with the loss of their normal
postsynaptic targets in lamina IIi (H).
relatively normally, although more subtle defects may

DRG11 Controls Sensory Neuron Projections
69
Table 2. Numbers of Sensory Neurons in Lumbar DRG of Wild-Type and Drg11 Mutant Mice
Genotype Specimen Nissl trkA
⫹
CGRP
⫹
trkA/Nissl (%) CGRP/Nissl (%)
⫹/⫹1 9256 4861 3086 53 33
2 12039 5448 2941 45 24
3 9338 4744 3111 51 33
4 9028 5220 3015 58 33
Avg. ⫾SD 9915 ⫾14 5068 ⫾32 3038 ⫾77 52 ⫾5.37 31 ⫾4.5
22 4
Drg11
⫺
/
⫺
1 7925 3490 1935 44 24
2 9176 3055 2141 33 23
3 5176 2631 1973 51 38
4 6017 3364 1869 56 31
Avg. ⫾SD 7073 ⫾18 3135 ⫾38 1979 ⫾11 46 ⫾9.97 29 ⫾6.97
13 2 6
t test p ⬍0.05 0.0003 1.6 ⫻10
⫺
5
NS NS
NS, not significantly different.
have been missed due to the limited number of neurons Genetic Control of Nociceptive Circuit Formation
The formation of the neural circuits that mediate painsampled.
sensation is an important subject in neural development,
yet remarkably little is known about the molecular mech-
Cell Loss in the DRG of Adult Drg11
⫺/⫺
Mice
anisms that control this process in vivo. The only pub-
Given the reduced number of cutaneous afferent fibers
lished mutations that affect the development of nocicep-
in the dorsal horn, we examined the number and pheno-
tors are those in the genes encoding NGF (Crowley et
type of sensory neurons in adult Drg11
⫺/⫺
mice as well.
al., 1994), its receptor trkA (Smeyne et al., 1994), the
There was a statistically significant (p ⬍0.05) reduction
bHLH transcription factor NGN1 (Ma et al., 1999), and
of almost 30% in the total number of Nissl-stained neu-
the POU-domain transcription factor Brn3.0 (McEvilly et
rons in L3⫹L4 DRG (Table 2, Nissl). The number of noci-
al., 1996; Xiang et al., 1996). None of these mutations,
ceptive trkA⫹or CGRP⫹neurons was also reduced in
however, affects the initial establishment of connections
Drg11
⫺/⫺
animals by about 30% (Table 2). However, the
between the DRG and the dorsal horn. NGF and trkA
proportion of DRG neurons expressing these markers
are required for the survival of sensory neurons long
was unchanged (Table 2), suggestive of generalized sen-
after they have differentiated and extended axons to
sory neuron loss. Consistent with this, the frequency
their targets, while Brn3.0 appears to control the expres-
distribution of different neuronal cell body diameters,
sion of neurotrophin receptors (Huang et al., 1999).
which are characteristic of different DRG neuronal sub-
NGN1, by contrast, controls the initial determination of
populations (Scott, 1992), did not differ significantly be-
trkA⫹sensory neuron precursors (Ma et al., 1999). Thus,
tween Drg11
⫺/⫺
mice and controls (see Supplementary
the early projection defect seen in Drg11
⫺/⫺
embryos is
Data ). These data suggest that there is a loss of sensory
distinct from other mutations affecting the development
neurons in adult Drg11
⫺/⫺
mice affecting both nocicep-
of nociceptive circuits, and may provide a useful point
tive and other classes of neurons. Nevertheless, as noci-
of entry for studies of the cell-intrinsic control of this
ceptors constitute about 70% of the adult sensory neu-
process.
ron population, most of the total neuronal loss in
Drg11
⫺/⫺
DRG reflects a diminution in this population.
Timing and Cellular Locus of the Primary Defect
in Drg11
⫺/⫺
MiceDiscussion
The earliest detectable cellular defect in Drg11
⫺/⫺
mice
is an abnormal projection of primary sensory afferentWe have analyzed the phenotypic consequences of a
mutation in Drg11, a paired homeodomain transcription fibers to the dorsal horn, at E13.5. Because Drg11 is
expressed in the sensory ganglia and spinal cord at thisfactor that is expressed from early stages of develop-
ment in both the dorsal horn and in DRG sensory neu- stage, it is not clear whether this initial projection defect
reflects an intrinsic function for the gene in the DRG,rons. Embryos deficient in DRG11 display abnormalities
in the timing and position of the initial ingrowth of sen- the dorsal horn, or both. However, the severity of the
projection defect, as detected by calbindin-28K stain-sory afferent fibers to the dorsal horn and, subsequently,
in morphogenesis of the dorsal horn itself. These early ing, appears similar at cervical and thoraco-lumbar lev-
els (data not shown), while the subsequent defects indefects perturb the development of circuits that process
nociceptive and other cutaneous sensory stimuli, as dorsal horn development are more prominent caudally.
These observations suggest that the morphological ab-confirmed by behavioral studies in adult Drg11
⫺/⫺
mice.
Drg11 is thus one of the few genes to be described normalities in the dorsal horn may develop indepen-
dently of the projection defect. Consistent with this,whose function in vivo is essential for the initial stages
of assembly of the neural pathways that detect noxious similar morphological abnormalities, including a reduc-
tion in small, darkly staining neurons and a shorteningstimuli.

Neuron
70
of the dorsal funiculus, are seen in mutants lacking mice is not more complete. However, because the re-
maining afferent fibers mediate apparently normal syn-Lmx1b, a LIM homeodomain transcription factor (Chen
et al., 1998a) expressed in the dorsal horn but not in aptic transmission with their surviving second-order tar-
gets, the incomplete loss of sensitivity to noxious stimulisensory neurons. In Lmx1b
-/-
embryos, expression of
Drg11 is lost in the spinal cord but not in the DRG (A. may simply reflect a reduced volume of synaptic infor-
mation transmitted in the dorsal horn of Drg11
⫺/⫺
mice.Kania and T.M. Jessell, personal communication). This
observation supports the idea that the dorsal horn de- What is the connection between the early develop-
mental defects observed in Drg11
⫺/⫺
embryos and thefects in Drg11
⫺/⫺
mice may reflect an intrinsic function
for the gene in the spinal cord. Whether the projection anatomical deficiencies seen in adults? The cell and
afferent fiber loss in the lateral dorsal horn of adultdefect reflects, conversely, an intrinsic role for DRG11
in sensory neurons or, rather, a requirement in the dorsal Drg11
⫺/⫺
mice are consistent with the pattern of defects
seen in embryos. The more general loss of PKC␥⫹neu-horn that is independent of axial position will require
site-specific knockouts of Drg11. rons may, however, reflect an independent, later action
of DRG11 to control the differentiation or survival of
these cells. In contrast to these embryonic and perinatal
DRG11 Is Required for the Proper Spatial
defects, the loss of sensory neurons is only observed
Patterning as Well as the Timing of Cutaneous
in adult DRG. This suggests either that this sensory
Afferent Ingrowth to the Dorsal Horn
neuron deficit is secondary to the earlier defects or that
The absence of afferent fiber ingrowth to the dorsal horn
Drg11 has a later, independent function in these periph-
in E13.5 Drg11
⫺/⫺
embryos reflects a delay and not a
eral neurons.
total arrest: by E16.5 calbindin⫹and trkA⫹fibers have
Although the adult behavioral phenotype of Drg11
⫺/⫺
penetrated the spinal gray matter. However the in-
mice is specific to modalities mediated by cutaneous
creased density of these fibers medially, and increased
afferent sensory neurons, the neuron loss in adult DRG
frequency of midline crossing, suggests that the abnor-
does not appear to be specific for this subset. While
mal trajectory reflects more than a simple deletion of
this cellular phenotype is consistent with the fact that
afferent projections to the lateral-most dorsal horn.
DRG11 is expressed in trkA⫺as well as trkA⫹sensory
Rather, both the timing and the spatial distribution of
neurons (Saito et al., 1995), it might appear inconsistent
cutaneous afferent projections into the spinal gray mat-
with the behavioral deficiencies. One possible expla-
ter are abnormal in Drg11
⫺/⫺
embryos.
nation for this paradox is that the behavioral deficits
The apparent lateral-to-medial shift in the distribution
may primarily reflect the combined effects of dorsal horn
of cutaneous afferents in Drg11
⫺/⫺
mice may reflect alter-
cell loss and afferent projection abnormalities, rather
ations in the somatotopic organization of these projec-
than the loss of DRG neurons per se. Consistent with
tions. Cutaneous afferents with distal (or ventral) periph-
this idea, defects in nociception are seen in perinatal
eral targets project to more medial regions of the dorsal
Drg11
⫺/⫺
mice (Z.F.C., S.R. and D.J.A., unpublished
horn, while those with more proximal (or dorsal) periph-
data), an age at which the dorsal horn and afferent pro-
eral targets project laterally (Figure 1A; reviewed in Wil-
jection defects are apparent, but when there is not yet
son and Kitchener [1996]). This medio-lateral somato-
any detectable loss of DRG neurons (Table 1). Accord-
topy is already evident from the earliest stages of
ingly, the relatively modest reduction in propriospinal
afferent fiber penetration to the dorsal horn (Mirnics and
sensory neurons may be insufficient to cause detectable
Koerber, 1995; Silos-Santiago et al., 1995). The fact that
abnormalities in sensorimotor function in the absence of
Drg11 is expressed more abundantly in the lateral than
a corresponding central and afferent projection defect.
in the medial dorsal horn (Figure 2F), taken together with
Nevertheless, we cannot exclude that the loss of pro-
the apparent medial bias of afferent fibers in the mutant,
priospinal neurons reflects an autonomous function for
suggests that the gene may be involved in some aspect
DRG11 in these cells that causes sensorimotor deficien-
of medio-lateral patterning that underlies such somato-
cies not detected by our behavioral assays.
topy. However, it is important to note that the loss of
PKC␥⫹neurons in Drg11
⫺/⫺
mice was observed through-
out the medio-lateral extent of lamina IIi. This may ex- The Role of Transcriptional Matching in the
plain why deficiencies in pain sensitivity were detected Control of Somatosensory Circuit Formation
distally as well as proximally in adult Drg11
⫺/⫺
mice. DRG11 is one of a relatively small number of transcrip-
tion factors that are expressed in both peripheral sen-
sory neurons and their central synaptic targets (Saito etRelationship of the Embryonic Defects in Drg11
⫺/⫺
Mice to the Adult Behavioral Phenotype al., 1995). Other genes with this property include Er81,
Phox2a/b, and Tlx1/3 (Tiveron et al., 1996; Lin et al.,Behavioral tests in adult Drg11
⫺/⫺
animals revealed a
significantly reduced sensitivity to noxious stimuli 1998; Logan et al., 1998). Such coordinated expression
is highly suggestive of a functional role for these factorsacross a broad range of modalities, including mechano-,
thermo-, and chemo-sensitivities. By contrast, locomo- in controlling connectivity. Mutations in Phox2a and
Phox2b primarily disrupt neuronal differentiation, how-tion and sensorimotor function appeared normal. Con-
sistent with this behavioral data, we observed a dramatic ever (Morin et al., 1997; Pattyn et al., 1999). In contrast,
targeted disruption of Er81 (Arber et al., 2000), like thatcell loss in the lateral regions of the dorsal horn, as well
as a reduction in afferent innervation in laminae I and II of Drg11, indeed perturbs the proper formation of con-
nections between the neurons that express these genes.which primarily represents C- and A␦fibers. Given this
neuronal and afferent fiber loss, it is somewhat surpris- Interestingly, Drg11 and Er81 are required for the forma-
tion of complementary somatosensory circuits: the for-ing that the reduction of pain sensitivity in adult Drg11
⫺/⫺

DRG11 Controls Sensory Neuron Projections
71
the magnitude of the inflammatory response by measuring paw
mer for the central projections of cutaneous afferents
diameter with a spring-loaded caliper (Mitutoyo) at 40 or 30 min,
and the latter for those of muscle afferents. Therefore, in
respectively, after the formalin or capsaicin injection.
at least some cases “transcriptional matching” between
Motor function was assessed by using an accelarating rotarod
pre- and post-synaptic neurons indeed reflects a role
treadmill (Ugo Basile, Comerio, Italy). The mice are first trained to
in neural circuit formation (Saito et al., 1995; Lin et al.,
walk on the rotating rod at a slow speed. After this, the mice undergo
three trials in which the time spent on the accelerating rotating rod
1998).
is determined. The mean of the three trials is considered representa-
Despite their requirement for proper connectivity,
tive for each animal. In all tests n ⫽7–8 animals of each genotype
however, there is no definitive evidence that ER81 and
(⫹/⫹or Drg11
⫺/⫺
) were used. Analysis of Drg11
⫹
/⫺
animals (n ⫽4)
DRG11 are actually essential in both the pre- and post-
indicated no difference from wild-type in all of the behavioral tests
synaptic neuronal populations that express them. The
performed.
known functional requirement for ER81 localizes to sen-
sory neurons (Arber et al., 2000), but the available data
Immunohistochemistry and In Situ Hybridization
do not exclude a function in motoneurons as well. In
Immunohistochemical staining and in situ hybridization were essen-
the case of DRG11, the cellular locus of the connectivity
tially done as described (Birren et al., 1993; Chen et al., 1998b; Ma
et al., 1999). Antibodies used were: Mouse anti-NeuN (Chemicon,
defect is not yet clear. Conditional knockouts of Drg11,
1:500), rabbit anti-CGRP (Chemicon, 1:500), rabbit anti-calbindin
as well as identification of its target genes, should help
D-28K (Chemicon, 1:1000), rabbit anti-peripherin (Chemicon, 1:500),
to clarify the important issue of whether its expression
rabbit anti-substance P (Peninsula, 1:500), rabbit anti-trkA (1:3000),
in both peripheral and central neurons is indeed essen-
rabbit anti-calretinin (Chemicon, 1:2000), rabbit anti-PKC␥(Santa
tial to its requirement for their proper connectivity.
Cruz, 1:500). Nissl staining was performed using 0.5% crest violet
for 15 min. TUNEL labeling to detect apoptotic cells (Gavrieli et al.,
1992) was performed using an ApopTag Peroxidase Kit (Intergen),
Experimental Procedures
according to the manufacturer’s instructions. DiI tracing was per-
formed as described (White and Behar, 2000). Cell counts were
Generation of DRG-11 Knockout Mice
determined on Nissl-stained 7 ␮m plastic sections by stereological
A mouse 129/SvEv genomic library was screened with a rat Drg11
evaluation using a systematic random sampling procedure (Gund-
cDNA probe, and one genomic clone containing the paired homeo-
ersen and Jensen, 1987).
domain region was isolated. Sequencing and restriction mapping
revealed that this clone contains multiple coding exons which span
⬎10 kb. To construct a targeting vector, an IRES-Tau-lacZ-neo cas- Acknowledgments
sette (Mombaerts et al., 1996) was fused to the third coding exon
which contains the putative DNA binding region. Electroporation, We thank Shirley Pease and the staff of TAFCIT for production and
selection, and blastocyst injection of AB-1 ES cells were essentially maintenance of mutant mice, Gaby Mosconi for laboratory manage-
as described (Ramirez-Solis et al., 1993). Germline transmission of ment, Heather Gilbert for technical help, Richard Behringer for AB-1
the mutation was confirmed by both Southern blotting and PCR. ES cells and STO cells, P. Mombaerts for the IRES-Tau-lacZ plasmid,
Subsequent genotyping was done by PCR. Primers for Neo are 5⬘Louis F. Reichardt and David Julius for antibodies, Marc Tessier-
GAT,CTC,CTG,TCA,TCT,CAC,CT 3⬘and 5⬘ATG,GGT,CAC,GAC, Lavigne for Slit and netrin probes, Haiming Xu for assistance with
GAG,ATC,CT 3⬘, for the deleted region are 5⬘TGC,AAA,GCA,AAT, RT-PCR experiments, T. Saito for Figure 2, and Artur Kania, Tom
CTG,ACC,GCT,CTG 3⬘and 5⬘GAA,CAG,AAA,CAG,CAT,GGA,GGA, Jessell, and Randy Johnson for sharing unpublished data on the
AAC 3⬘.Lmx1b knockout and for providing various probes and reagents. We
We performed a series of RT-PCR experiments to characterize are grateful to Tom Jessell for helpful comments on the manuscript.
the nature of any transcripts produced from the mutated allele, using Animal experiments were reviewed and approved by the Animal Use
as a template cDNA prepared from wild-type and Drg11
⫺/⫺
spinal and Care Committees (IACUCs) at Caltech and at UCSF. S.R. was
cord. We were unable to detect any transcripts using 5⬘primers supported in the laboratory of D.J.A. by a Gulbenkian Foundation
located in the neo
r
gene and 3⬘primers located in the Drg11 coding Fellowship. Supported by NIH grant NS38253-01 to C.J.W. and NS
region downstream of the deleted region. Nor were we able to detect 14627 to A.I.B. D.J.A. is an Investigator of the Howard Hughes Medi-
any transcripts that encoded residual homeodomain sequences up- cal Institute.
stream of the deleted coding exons, or that spliced across these
exons to downstream Drg11 coding exons. However, we did detect Received October 24, 2000; revised April 18, 2001.
a transcript using 5⬘and 3⬘primers located internal to the lacZ gene.
The fact that no Xgal reaction product was detected suggests that
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