BACKGROUND
the primary receptor cells of the somatosensory system are the neurons of the dorsal root ganglia .
their role is to detect environmental stimuli such as noxious stimuli, temperature, mechanical pressure, proprioception and to conduct these signals to the central nervous system.
noxious stimuli are sensed by nociceptors that innervate the skin, muscle and internal organs.
touch and proprioceptive stimuli are detected by low-threshold mechanoreceptors that innervate the skin and muscle, respectively.
there exist up to  <dig> different sub-types of sensory neurons in the drg  <cit> .
different sub-types can be distinguished by a variety of criteria: cell body diameter; conduction velocity; neurotrophic factor dependence; sensitivity to specific stimuli; expression of neuropeptides, ion channels, calcium-binding proteins and transcription factors  <cit> .
however, for many sub-types no specific molecular markers have been found.
for example, slowly adapting mechanoreceptors  that interact with merkel cells in the skin and whose phenotypic development depends on bdnf/trkb signaling  <cit>  can only be identified using physiological criteria.
thus identification of specific molecular markers is an essential step in understanding the development and the function of this system.
a large body of evidence shows that nociceptors depend for their survival during development on ngf signaling through trka receptors specifically expressed on these cells  <cit> .
trka mutant mice are insensitive to painful stimuli and mutations in trka are associated with the syndrome "congenital insensitivity to pain" in humans  <cit> .
around the time of birth, nociceptors divide into  <dig> main populations, one of which maintains trka expression and secretes neuropeptides such as cgrp and substance p. the other population down-regulates trka, expresses the ret tyrosine kinase receptor and requires gdnf for its survival  <cit> .
this non-peptidergic population is further characterized by the capacity of binding the lectin ib <dig> and it has recently been shown that the transcription factor runx <dig> is necessary for the phenotypic development of this cell population  <cit> .
in the adult mouse, peptidergic  and non-peptidergic  nociceptors project to the different laminae in the dorsal horn, and may be responsible for different pain modalities .
skin mechanoreceptors and muscle proprioceptors depend for their survival on nt- <dig>  bdnf and nt- <dig> and project to deeper laminae in the spinal cord, reviewed in  <cit> .
to study the physiology of somatosensory neurons and the molecular changes in functionally-identified drg neuron sub-types during development and after peripheral trauma, we have created several sage  libraries from drg tissues  <cit> .
sage generates global gene expression data from thousands of transcripts in a given tissue or cell-type  <cit> .
since nociceptors constitute up to 80% of all neurons in the drg, transcripts representing this cell type should be enriched in wild-type tissue.
trka mutant mice lose all nociceptive neurons during development due to inactivation of the ngf survival signaling pathway  <cit> , leaving only trkb and trkc mechanoreceptor neurons, thus the trka mutant drg is enriched for transcripts representing low-threshold myelinated mechanoreceptors.
in the study presented here, we compared the transcription profiles of wild-type and trka mutant drg from neonatal mice in order to identify markers of sub-populations.
double labeling analysis of a selection of these genes with known markers of drg neuron sub-types revealed expression in sub-populations of drg neurons in the adult mouse from birth to adulthood.
RESULTS
general results from sage libraries analysis
we used sage technology to generate global gene expression profiles from wild-type and trka mutant drgs.
this methodology consists in isolating, from a given cdna preparation, short 14-bp tags which are small nucleotides sequences representative of a particular transcript  <cit> .
sequencing and counting of tags generates information about the presence and frequency of thousands of transcripts in the original tissue.
one thousand plasmid inserts were sequenced for each library, resulting in  <dig>  and  <dig>  tags for the wild-type and the mutant drg libraries, respectively.
the overall results of the bioinformatics analysis are shown in figure  <dig>
differential analysis of p <dig> wt versus p <dig> trka mutant mice libraries
tag frequencies were compared between the wt and trka mutant sage libraries in order to identify genes preferentially expressed in one or the other tissues.
this analysis revealed that  <dig> genes are differentially expressed  between the two banks including  <dig> tags more highly expressed in wild type library and  <dig> tags more represented in mutant mice library, and might represent new markers of subpopulations of drg neurons.
a preliminary analysis of  <dig> genes, chosen on the basis of differential tag numbers, was carried out by whole-mount in situ hybridisation on p <dig> dorsal root ganglia .
subsequently we analyzed, by in situ hybridisation on cryostat sections, three genes chosen on the basis of restricted expression pattern and published information about their potential function.
crip <dig> .
these quantitative rt-pcr results confirmed the data obtained from the sage tag analysis.
when expressed as "fold-change in expression" the sage results demonstrate a down regulation of grik <dig>  dock <dig> and crip <dig> expression between p <dig> wt and p <dig> trka-/- drg of about  <dig>   <dig> and  <dig> fold respectively, while qrt-pcr provided an expression ratio between p <dig> wt and p <dig> trka-/- drg of  <dig>   <dig> and  <dig> fold respectively.
in situ hybridization patterns of selected genes
in order to assess the types of cells in which the candidate genes were expressed and to gain idea about the potential expression in functional sub-types, we carried out in situ hybridization on sections of wild-type or mutant ganglia using dig-labelled probes generated from pcr products amplified using primers specific for each gene.
in all cases the pcr products were sequenced to confirm the identity of the probe.
figure  <dig> shows the in situ hybridization profile for these transcripts on cryostat sections from wild-type p <dig>  trka mutant and adult drgs.
as controls we used riboprobes generated against trka, the neuropeptide cgrp and the sodium channel scn10a, all known markers of nociceptive neurons.
as shown in figure  <dig> , trka, cgrp and scn10a all label sub-populations of neurons in p <dig> wild-type and adult drgs.
in accordance with the loss of nociceptive neurons in trka mutant drgs, no labelling for these transcripts was observed in trka mutant drgs .
transcripts representing the adaptor protein dok <dig> were observed in broad range of neuron-like cells in wild-type p <dig> and adult drg , with negative cells scattered throughout the tissue .
labelled cells were also observed in trka mutant drg sections from new-born mice .
crip <dig> was expressed in a large proportion  of cells of neuronal morphology in the p <dig> wild-type and adult drg .
however, unlabeled cells were observed.
in p <dig> trka mutant drg, a number of crip <dig> labelled cells were observed , suggesting that the expression of this gene is not exclusive to the nociceptor population.
crip <dig> expression persisted in adult drg, and negative cells of large cell diameter were observed .
the kainate receptor grik1/glur <dig> was expressed in a sub-population of cells of neuronal morphology in wild-type drg and was completely absent in trka mutant drg, strongly suggesting nociceptor specific expression .
grik1/glur <dig> expression persisted in a sub-population of cells  in the adult .
sub-population specific expression pattern revealed by double labeling
to further characterise the sub-population in which the genes of interest were expressed, we carried out double labelling using known markers of neuronal drg sub-populations .
grik1/glur <dig> expression was absent from each of the trka, trkb and trkc expressing populations .
however, grik1/glur <dig> was expressed in a sub-population  of neurons of small cell body diameter labelled by c-ret .
these cells correspond to the isolectin b <dig> positive nociceptor population as shown by double labelling where there was  <dig> % +/-  <dig>  co-localisation of grik1/glur <dig> and isolectin b <dig> .
virtually all grik1/glur5-expressing neurons were ib4-positive.
dok <dig> was expressed in a wide range  of neurons of different cell diameters in the adult drg.
in co-localisation studies we observed that most neurons expressing members of the trk receptor family also expressed dok <dig>  in addition, most c-ret and isolectin b4+ neurons also expressed dok <dig> .
however, we consistently observed that about 5% of all cells with neuron-like morphology were negative for dok <dig>  in co-labeling experiments with trks, c-ret and ib <dig> lectin, it was observed that these dok <dig> negative cells were never labelled by any of the  <dig> markers used.
most trka-expressing neurons were also crip2-positive, as were trkb and c-ret expressing neurons .
however, all trkc expressing neurons were negative for crip <dig> , and as expected, crip <dig> was never co-localised with parvalbumin, a definitive marker of muscle proprioceptors .
discussion
in this report we describe the use of a combination of sage analysis and in situ hybridization to identify markers of sensory neuron sub-types in the dorsal root ganglion of the mouse.
we took advantage of the fact that in the trka mutant mouse all neurons of the nociceptive sub-type die during development.
by creating sage banks from wild-type  and trka mutant  we could compare global expression profiles of the  <dig> major classes of neurons in the drg i.e.
mainly non-myelinated or thinly-myelinated nociceptors and myelinated low-threshold mechanoreceptors of skin and muscle.
the choice of the developmental time-point p <dig> was determined by the fact that trka mutant mice die in the days following birth, and that we are interested in identifying molecules involved in the functioning of drg neurons in the maturing somatosensory system.
although the starting material, whole drg, necessarily contains non-neuronal cells including satellite glia and immature schwann cells, methods based on isolating purified neurons in culture were considered to be overly artificial, since the cultured neurons undergo axotomy during dissection.
we carried out more detailed analysis of a limited sample of these genes, whose exact sub-population specificity had not previously been determined, by qrt-pcr and double labeling using known markers of sensory neuron sub-populations.
in each case, the sage results were confirmed by quantitative rt-pcr.
qualitatively, we observed three types of in situ hybridization expression patterns.
for dok <dig> there is a quantitative difference in expression, while the double in situ hybridization pattern does not reveal remarkable sub-population specificity of expression.
we can suggest that while dok <dig> is expressed in most neurons, expression is generally higher in the nociceptive population.
however, it cannot be ruled out that the basal expression level of this gene is changed in the surviving neurons of the trka mutant drg.
downstream of tyrosine kinase/docking proteins  comprise a family of intracellular adaptors that modulate signaling pathways mediated by receptor and non-receptor tyrosine kinases.
for example dok <dig> regulates neuromuscular junction formation by interaction with musk  <cit> .
in biochemical studies, it was shown that dok <dig> could interact specifically with trkb and trkc receptors, but not trka, and was involved in neurotrophin induced mapk activation  <cit> .
dok <dig> was shown to regulate gdnf/ret dependent neurite outgrowth in neuroblastoma cells  <cit> .
expression of dok <dig> has been described in the drg during embryonic development, and an interaction with c-ret was demonstrated in a heterologous cell expression system  <cit> .
our results show that, in adult drg, dok <dig> is expressed in ret-expressing neurons, but the broad expression of this molecule suggests a potential role in association with other tyrosine kinase receptors.
interestingly, about 5% of adult drg neurons were dok-negative and these cells did not express any of the  <dig> typical drg neuron tyrosine kinase receptors , suggesting that there might be an as yet unidentified sub-population that uses a ligand receptor signaling system other than trks or ret.
this observation is in line with the study of kashiba et al.,  <cit>  who showed, using a cocktail of in situ hybridization probes for the trks and ret receptors, that a small proportion  of neurons remained unlabeled.
our results show for the first time that crip <dig> is expressed in drg neurons.
intriguingly, this molecule is very specifically excluded from the trkc-expressing population that includes the parvalbumin expressing proprioceptors  <cit>  and certain low-threshold mechanoreceptors  <cit> .
thus it is possible that this gene is negatively regulated by trkc signaling.
on the other hand, it has been shown that the transcription factor runx <dig> plays an essential role in the establishment of the proprioceptive neuron phenotype  <cit>  and the targeting of proprioceptive  afferents to the ventral region of the spinal cord  <cit> , raising the possibility that crip <dig> expression is under the control of this transcriptional program.
the cysteine-rich lim-only protein crip <dig> is a member of a family of related proteins, crip <dig>  crip <dig> and tlp/crip <dig>  <cit> .
these proteins contain  <dig> or  <dig> lim domains and are thought to act as intracellular adaptors, although the signaling pathway involved have not been identified.
the expression of crip <dig> has been described in developing and adult mesenchymal tissue and in particular in the developing and adult heart  <cit> .
an interaction between crip <dig> and the mouse protein tyrosine phosphatase ptp-bl  <cit>  was demonstrated by 2-hybrid screen, and it was proposed that crip2/ptp-bl interactions might play a role in the dynamics of the actin cytoskeleton.
the functional significance of crip <dig> expression in drg neurons remains to be explored, concerning grik1/glur <dig>  our results show unequivocally that, in the adult mouse drg, this kainate receptor is expressed in the great majority of isolectin b <dig> binding neurons and is excluded from other neuronal sub-types.
a body of evidence shows that grik1/glur <dig> receptors are expressed in sensory neurons of the drg and are transported to the spinal cord where they are an essential component of presynaptic kainate receptors thought to play a role in the modulation of pain sensation  <cit> .
grik1/glur <dig> mutant mice display altered behavioral responses to noxious stimuli  <cit> .
several studies have indirectly pointed to a localization of grik1/glur <dig> in the isolectin b <dig> binding drg neuron sub-population.
by pharmacological experiments on cultured neonatal rat drg neurons, grik1/glur <dig> was identified in isolectin b <dig> binding cells  <cit>  and was shown by double immunhistochemistry to be highly-represented in the p2x <dig> positive nociceptor population, that are mostly isolectin b <dig> binding  <cit> .
in previous studies, using in situ hybridization, the expression of grik1/glur <dig> was detected in mouse drg from e <dig> onward, using a rat probe on mouse tissue  <cit> , and by rt-pcr in e <dig> rat drg tissue  <cit> .
in our experiments on mouse tissue, by both qrt-pcr and in situ hybridization, we could not detect this mrna at  <dig> time-points prior to birth, e <dig> and e <dig> .
this discrepancy may be due to the use of cross-species probes in the earlier study, or the fact that earlier in situ studies used more sensitive radioactive in situ technique.
nevertheless, our in situ hybridization and qrt-pcr results suggest that high grik1/glur <dig> expression appears at the late embryonic phase.
at p <dig>  grik1/glur <dig> was largely co-localized with c-ret .
in the adult, grik1/glur <dig> never co-localized with trka and was restricted to small cell-diameter c-ret+/isolectin b4+ neurons.
thus grik1/glur <dig> expression seems to coincide with the appearance of the population of nociceptors that down-regulate trka and express c-ret and bind isolectin b <dig>  <cit> .
this switch in expression also parallels that of runx <dig>  a transcription factor that is broadly co-localized with trka at p <dig>  and whose expression becomes restricted to the isolectin b4+ nociceptor population during early postnatal life  <cit> .
indeed, runx <dig> appears to regulate the molecular phenotype of this cell-type as demonstrated by the analysis of runx <dig> mutant mice, in which was observed an expansion of the trka/peptidergic nociceptor phenotype and a lack of expression of a range of genes normally expressed in the non-peptidergic/ib4+ nociceptor population  <cit> .
runx <dig> mutant mice display loss of sensitivity to acute thermal, but not mechanical, pain stimuli, as well as reduced responses to inflammatory stimuli  <cit>  a phenotype partially mirrored by that of the grik1/glur <dig> mutant  <cit> .
our results suggest that grik1/glur <dig> could be a target gene of runx <dig> and should play a role in the specific functions of these neurons.
CONCLUSIONS
in conclusion, we have identified and characterized the detailed expression patterns of three genes in the developing drg, placing them in the context of the known major neuronal sub-types as defined by several molecular markers.
further analysis of differentially expressed genes in this tissue promises to extend our knowledge of the molecular diversity of different cell types and forms the basis for understanding their particular functional specificities.

 the primary receptor cells of the somatosensory system are the neurons of the dorsal root ganglia. their role is to detect environmental stimuli such as noxious stimuli, temperature, mechanical pressure, proprioception and to conduct these signals to the central nervous system. noxious stimuli are sensed by nociceptors that innervate the skin, muscle and internal organs. touch and proprioceptive stimuli are detected by low-threshold mechanoreceptors that innervate the skin and muscle, respectively. there exist up to different sub-types of sensory neurons in the drg. different sub-types can be distinguished by a variety of criteria: cell body diameter; conduction velocity; neurotrophic factor dependence; sensitivity to specific stimuli; expression of neuropeptides, ion channels, calcium-binding proteins and transcription factors. however, for many sub-types no specific molecular markers have been found. for example, slowly adapting mechanoreceptors that interact with merkel cells in the skin and whose phenotypic development depends on bdnf/trkb signaling can only be identified using physiological criteria. thus identification of specific molecular markers is an essential step in understanding the development and the function of this system. a large body of evidence shows that nociceptors depend for their survival during development on ngf signaling through trka receptors specifically expressed on these cells. trka mutant mice are insensitive to painful stimuli and mutations in trka are associated with the syndrome "congenital insensitivity to pain" in humans. around the time of birth, nociceptors divide into main populations, one of which maintains trka expression and secretes neuropeptides such as cgrp and substance p. the other population down-regulates trka, expresses the ret tyrosine kinase receptor and requires gdnf for its survival. this non-peptidergic population is further characterized by the capacity of binding the lectin ib and it has recently been shown that the transcription factor runx is necessary for the phenotypic development of this cell population. in the adult mouse, peptidergic and non-peptidergic nociceptors project to the different laminae in the dorsal horn, and may be responsible for different pain modalities. skin mechanoreceptors and muscle proprioceptors depend for their survival on nt- bdnf and nt- and project to deeper laminae in the spinal cord, reviewed in. to study the physiology of somatosensory neurons and the molecular changes in functionally-identified drg neuron sub-types during development and after peripheral trauma, we have created several sage libraries from drg tissues. sage generates global gene expression data from thousands of transcripts in a given tissue or cell-type. since nociceptors constitute up to 80% of all neurons in the drg, transcripts representing this cell type should be enriched in wild-type tissue. trka mutant mice lose all nociceptive neurons during development due to inactivation of the ngf survival signaling pathway, leaving only trkb and trkc mechanoreceptor neurons, thus the trka mutant drg is enriched for transcripts representing low-threshold myelinated mechanoreceptors. in the study presented here, we compared the transcription profiles of wild-type and trka mutant drg from neonatal mice in order to identify markers of sub-populations. double labeling analysis of a selection of these genes with known markers of drg neuron sub-types revealed expression in sub-populations of drg neurons in the adult mouse from birth to adulthood.  general results from sage libraries analysis we used sage technology to generate global gene expression profiles from wild-type and trka mutant drgs. this methodology consists in isolating, from a given cdna preparation, short 14-bp tags which are small nucleotides sequences representative of a particular transcript. sequencing and counting of tags generates information about the presence and frequency of thousands of transcripts in the original tissue. one thousand plasmid inserts were sequenced for each library, resulting in and tags for the wild-type and the mutant drg libraries, respectively. the overall results of the bioinformatics analysis are shown in figure differential analysis of p wt versus p trka mutant mice libraries tag frequencies were compared between the wt and trka mutant sage libraries in order to identify genes preferentially expressed in one or the other tissues. this analysis revealed that genes are differentially expressed between the two banks including tags more highly expressed in wild type library and tags more represented in mutant mice library, and might represent new markers of subpopulations of drg neurons. a preliminary analysis of genes, chosen on the basis of differential tag numbers, was carried out by whole-mount in situ hybridisation on p dorsal root ganglia. subsequently we analyzed, by in situ hybridisation on cryostat sections, three genes chosen on the basis of restricted expression pattern and published information about their potential function. crip. these quantitative rt-pcr results confirmed the data obtained from the sage tag analysis. when expressed as "fold-change in expression" the sage results demonstrate a down regulation of grik dock and crip expression between p wt and p trka-/- drg of about and fold respectively, while qrt-pcr provided an expression ratio between p wt and p trka-/- drg of and fold respectively. in situ hybridization patterns of selected genes in order to assess the types of cells in which the candidate genes were expressed and to gain idea about the potential expression in functional sub-types, we carried out in situ hybridization on sections of wild-type or mutant ganglia using dig-labelled probes generated from pcr products amplified using primers specific for each gene. in all cases the pcr products were sequenced to confirm the identity of the probe. figure shows the in situ hybridization profile for these transcripts on cryostat sections from wild-type p trka mutant and adult drgs. as controls we used riboprobes generated against trka, the neuropeptide cgrp and the sodium channel scn10a, all known markers of nociceptive neurons. as shown in figure, trka, cgrp and scn10a all label sub-populations of neurons in p wild-type and adult drgs. in accordance with the loss of nociceptive neurons in trka mutant drgs, no labelling for these transcripts was observed in trka mutant drgs. transcripts representing the adaptor protein dok were observed in broad range of neuron-like cells in wild-type p and adult drg, with negative cells scattered throughout the tissue. labelled cells were also observed in trka mutant drg sections from new-born mice. crip was expressed in a large proportion of cells of neuronal morphology in the p wild-type and adult drg. however, unlabeled cells were observed. in p trka mutant drg, a number of crip labelled cells were observed, suggesting that the expression of this gene is not exclusive to the nociceptor population. crip expression persisted in adult drg, and negative cells of large cell diameter were observed. the kainate receptor grik1/glur was expressed in a sub-population of cells of neuronal morphology in wild-type drg and was completely absent in trka mutant drg, strongly suggesting nociceptor specific expression. grik1/glur expression persisted in a sub-population of cells in the adult. sub-population specific expression pattern revealed by double labeling to further characterise the sub-population in which the genes of interest were expressed, we carried out double labelling using known markers of neuronal drg sub-populations. grik1/glur expression was absent from each of the trka, trkb and trkc expressing populations. however, grik1/glur was expressed in a sub-population of neurons of small cell body diameter labelled by c-ret. these cells correspond to the isolectin b positive nociceptor population as shown by double labelling where there was % +/- co-localisation of grik1/glur and isolectin b. virtually all grik1/glur5-expressing neurons were ib4-positive. dok was expressed in a wide range of neurons of different cell diameters in the adult drg. in co-localisation studies we observed that most neurons expressing members of the trk receptor family also expressed dok in addition, most c-ret and isolectin b4+ neurons also expressed dok. however, we consistently observed that about 5% of all cells with neuron-like morphology were negative for dok in co-labeling experiments with trks, c-ret and ib lectin, it was observed that these dok negative cells were never labelled by any of the markers used. most trka-expressing neurons were also crip2-positive, as were trkb and c-ret expressing neurons. however, all trkc expressing neurons were negative for crip, and as expected, crip was never co-localised with parvalbumin, a definitive marker of muscle proprioceptors. discussion in this report we describe the use of a combination of sage analysis and in situ hybridization to identify markers of sensory neuron sub-types in the dorsal root ganglion of the mouse. we took advantage of the fact that in the trka mutant mouse all neurons of the nociceptive sub-type die during development. by creating sage banks from wild-type and trka mutant we could compare global expression profiles of the major classes of neurons in the drg i.e. mainly non-myelinated or thinly-myelinated nociceptors and myelinated low-threshold mechanoreceptors of skin and muscle. the choice of the developmental time-point p was determined by the fact that trka mutant mice die in the days following birth, and that we are interested in identifying molecules involved in the functioning of drg neurons in the maturing somatosensory system. although the starting material, whole drg, necessarily contains non-neuronal cells including satellite glia and immature schwann cells, methods based on isolating purified neurons in culture were considered to be overly artificial, since the cultured neurons undergo axotomy during dissection. we carried out more detailed analysis of a limited sample of these genes, whose exact sub-population specificity had not previously been determined, by qrt-pcr and double labeling using known markers of sensory neuron sub-populations. in each case, the sage results were confirmed by quantitative rt-pcr. qualitatively, we observed three types of in situ hybridization expression patterns. for dok there is a quantitative difference in expression, while the double in situ hybridization pattern does not reveal remarkable sub-population specificity of expression. we can suggest that while dok is expressed in most neurons, expression is generally higher in the nociceptive population. however, it cannot be ruled out that the basal expression level of this gene is changed in the surviving neurons of the trka mutant drg. downstream of tyrosine kinase/docking proteins comprise a family of intracellular adaptors that modulate signaling pathways mediated by receptor and non-receptor tyrosine kinases. for example dok regulates neuromuscular junction formation by interaction with musk. in biochemical studies, it was shown that dok could interact specifically with trkb and trkc receptors, but not trka, and was involved in neurotrophin induced mapk activation. dok was shown to regulate gdnf/ret dependent neurite outgrowth in neuroblastoma cells. expression of dok has been described in the drg during embryonic development, and an interaction with c-ret was demonstrated in a heterologous cell expression system. our results show that, in adult drg, dok is expressed in ret-expressing neurons, but the broad expression of this molecule suggests a potential role in association with other tyrosine kinase receptors. interestingly, about 5% of adult drg neurons were dok-negative and these cells did not express any of the typical drg neuron tyrosine kinase receptors, suggesting that there might be an as yet unidentified sub-population that uses a ligand receptor signaling system other than trks or ret. this observation is in line with the study of kashiba et al., who showed, using a cocktail of in situ hybridization probes for the trks and ret receptors, that a small proportion of neurons remained unlabeled. our results show for the first time that crip is expressed in drg neurons. intriguingly, this molecule is very specifically excluded from the trkc-expressing population that includes the parvalbumin expressing proprioceptors and certain low-threshold mechanoreceptors. thus it is possible that this gene is negatively regulated by trkc signaling. on the other hand, it has been shown that the transcription factor runx plays an essential role in the establishment of the proprioceptive neuron phenotype and the targeting of proprioceptive afferents to the ventral region of the spinal cord, raising the possibility that crip expression is under the control of this transcriptional program. the cysteine-rich lim-only protein crip is a member of a family of related proteins, crip crip and tlp/crip. these proteins contain or lim domains and are thought to act as intracellular adaptors, although the signaling pathway involved have not been identified. the expression of crip has been described in developing and adult mesenchymal tissue and in particular in the developing and adult heart. an interaction between crip and the mouse protein tyrosine phosphatase ptp-bl was demonstrated by 2-hybrid screen, and it was proposed that crip2/ptp-bl interactions might play a role in the dynamics of the actin cytoskeleton. the functional significance of crip expression in drg neurons remains to be explored, concerning grik1/glur our results show unequivocally that, in the adult mouse drg, this kainate receptor is expressed in the great majority of isolectin b binding neurons and is excluded from other neuronal sub-types. a body of evidence shows that grik1/glur receptors are expressed in sensory neurons of the drg and are transported to the spinal cord where they are an essential component of presynaptic kainate receptors thought to play a role in the modulation of pain sensation. grik1/glur mutant mice display altered behavioral responses to noxious stimuli. several studies have indirectly pointed to a localization of grik1/glur in the isolectin b binding drg neuron sub-population. by pharmacological experiments on cultured neonatal rat drg neurons, grik1/glur was identified in isolectin b binding cells and was shown by double immunhistochemistry to be highly-represented in the p2x positive nociceptor population, that are mostly isolectin b binding. in previous studies, using in situ hybridization, the expression of grik1/glur was detected in mouse drg from e onward, using a rat probe on mouse tissue, and by rt-pcr in e rat drg tissue. in our experiments on mouse tissue, by both qrt-pcr and in situ hybridization, we could not detect this mrna at time-points prior to birth, e and e. this discrepancy may be due to the use of cross-species probes in the earlier study, or the fact that earlier in situ studies used more sensitive radioactive in situ technique. nevertheless, our in situ hybridization and qrt-pcr results suggest that high grik1/glur expression appears at the late embryonic phase. at p grik1/glur was largely co-localized with c-ret. in the adult, grik1/glur never co-localized with trka and was restricted to small cell-diameter c-ret+/isolectin b4+ neurons. thus grik1/glur expression seems to coincide with the appearance of the population of nociceptors that down-regulate trka and express c-ret and bind isolectin b. this switch in expression also parallels that of runx a transcription factor that is broadly co-localized with trka at p and whose expression becomes restricted to the isolectin b4+ nociceptor population during early postnatal life. indeed, runx appears to regulate the molecular phenotype of this cell-type as demonstrated by the analysis of runx mutant mice, in which was observed an expansion of the trka/peptidergic nociceptor phenotype and a lack of expression of a range of genes normally expressed in the non-peptidergic/ib4+ nociceptor population. runx mutant mice display loss of sensitivity to acute thermal, but not mechanical, pain stimuli, as well as reduced responses to inflammatory stimuli a phenotype partially mirrored by that of the grik1/glur mutant. our results suggest that grik1/glur could be a target gene of runx and should play a role in the specific functions of these neurons.  in conclusion, we have identified and characterized the detailed expression patterns of three genes in the developing drg, placing them in the context of the known major neuronal sub-types as defined by several molecular markers. further analysis of differentially expressed genes in this tissue promises to extend our knowledge of the molecular diversity of different cell types and forms the basis for understanding their particular functional specificities. 
we have identified and characterized the detailed expression patterns of three genes in the developing drg, placing them in the context of the known major neuronal sub-types defined by molecular markers. further analysis of differentially expressed genes in this tissue promises to extend our knowledge of the molecular diversity of different cell types and forms the basis for understanding their particular functional specificities. the different sensory modalities temperature, pain, touch and muscle proprioception are carried by somatosensory neurons of the dorsal root ganglia. in order to detect genes expressed in sub-populations of somatosensory neurons, gene profiling was carried out on wild-type and trka mutant neonatal dorsal root ganglia using sage methodology. several potential markers of sub-populations, dok crip and grik1/glur were further analyzed by quantitative rt-pcr and double labeling with trka,-b,-c, c-ret, parvalbumin and isolectin b known markers of drg neuron sub-types. in trka mutant drgs, the nociceptor sub-class of sensory neurons is lost due to absence of nerve growth factor survival signaling through its receptor trka. study of this system is hampered by the lack of molecular markers for many of these neuronal sub-types. some of these genes, cgrp, scn10a are known markers of sensory neuron sub-types.
 the different sensory modalities temperature, pain, touch and muscle proprioception are carried by somatosensory neurons of the dorsal root ganglia. study of this system is hampered by the lack of molecular markers for many of these neuronal sub-types. in order to detect genes expressed in sub-populations of somatosensory neurons, gene profiling was carried out on wild-type and trka mutant neonatal dorsal root ganglia using sage methodology. thermo-nociceptors constitute up to % of the neurons in the drg. in trka mutant drgs, the nociceptor sub-class of sensory neurons is lost due to absence of nerve growth factor survival signaling through its receptor trka. thus, comparison of wild-type and trka mutants allows the identification of transcripts preferentially expressed in the nociceptor or mechano-proprioceptor subclasses, respectively.  our comparison revealed genes differentially expressed between the two tissues. some of these genes, cgrp, scn10a are known markers of sensory neuron sub-types. several potential markers of sub-populations, dok crip and grik1/glur were further analyzed by quantitative rt-pcr and double labeling with trka,-b,-c, c-ret, parvalbumin and isolectin b known markers of drg neuron sub-types. expression of grik1/glur was restricted to the isolectin b4+ nociceptive population, while dok and crip had broader expression profiles. crip expression was however excluded from the proprioceptor sub-population.  we have identified and characterized the detailed expression patterns of three genes in the developing drg, placing them in the context of the known major neuronal sub-types defined by molecular markers. further analysis of differentially expressed genes in this tissue promises to extend our knowledge of the molecular diversity of different cell types and forms the basis for understanding their particular functional specificities. 