Sensory neurons residing in the dorsal root ganglia (DRGs) are essential for development of low-threshold limb mechanoreceptors such as muscle spindles, Golgi tendon organs, or Pacinian corpuscles. Formation of each of these three classes of mechanoreceptors involves a peripheral projection of functionally distinct subtype of DRG neuron contacting appropriate target cells in the limb (Zelená, 1978, 1994). Neuregulins released from afferent terminals interact with the ErbB class of receptors expressed by target cells to promote the formation and differentiation of the mechanoreceptors. In the case of muscle spindles, neuregulin-1 (NRG1) released from group Ia afferent terminals interacts specifically with ErbB2 receptors on myotubes to induce the spindle assembly (Hippenmeyer et al., 2002; Leu et al., 2003). Other members of the neuregulin family may similarly mediate the differentiation of Golgi tendon organs and Pacinian corpuscles.
Cell-specific programs of transcriptional regulation are hypothesized to be involved in the signaling process whereby distinct subpopulations of sensory neurons initiate the formation of specific classes of peripheral mechanoreceptors. Transcription factors involved in the specification of functional identity of DRG sensory neurons include members of the ETS family (Ghosh and Kolodkin, 1998; Wasylyk et al., 1998). ER81, a member of the PEA3 subfamily of ETS transcription factors, is expressed by primary sensory neurons that innervate low-threshold limb mechanoreceptors that themselves express ETS factors (Lin et al., 1998). Large-caliber TrkC- and parvalbumin (PV)-expressing DRG neurons that innervate muscle spindles and Golgi tendon organs express ER81, whereas TrkA-expressing small-caliber neurons that innervate cutaneous nociceptors do not express ER81. Deletion of both Er81 alleles in mice results in abnormal development of muscle spindles but not Golgi tendon organs (Arber et al., 2000; Kucera et al., 2002).
PEA3, another member of the ETS family of transcription factors, is also expressed by primary sensory neurons of DRGs during development. In birds, ER81 and PEA3 expression becomes segregated into largely non-overlapping subpopulations of DRG sensory neurons by the time afferents contact spinal motoneurons (Lin et al., 1998). In mice, approximately 20% of all lumbar DRG neurons express ER81 and 15% express PEA3 by E13.0 to E14.0 (Arber et al., 2000). However, unlike in the case of Er81, deletion of both Pea3 alleles alters neither the number nor morphology of muscle spindles in mice hindlimb muscles (Livet et al., 2002).
No data are available on the role of ER81 or PEA3 in the differentiation of Pacinian corpuscles, limb mechanoreceptors mediating the perception of vibration. In a preliminary study, we observed that Pacinian corpuscles were absent in postnatal Er81 mutant mice (Šedý, 2004). We undertook the present work to define and compare the extent of Pacinian corpuscle abnormalities in mice with null mutations in Er81 or Pea3 genes. We observed that the development of Pacinian corpuscles in the crus is critically dependent on the ER81 and to a lesser degree the PEA3 transcription factor.
Impact of Er81 and/or Pea3 Deletions
Clusters of Pacinian corpuscles derived from the interosseous nerve could easily be identified alongside the distal fibula in wild type neonates. In contrast, no Pacinian corpuscles were present in the crus of Er81 null mice at P0–P1 (Table 1). A 25% deficit in the number of the crural corpuscles was observed in neonates lacking PEA3 (Table 1). Deletion of one copy of the Er81 or Pea3 gene resulted in a 15–17% deficit in corpuscle numbers in both types of mutant at birth (Table 1). The deficit of Pacinian corpuscles (20%) in double heterozygots (Er81+/ETS; Pea3+/lacZ) was less than the arithmetic sum of the deficits (30%) in Er81+/ETS and Pea3+/lacZ single heterozygous mutants (Table 1). These data suggests some overlap of subpopulations of Pacinian corpuscles dependent on ER81 or PEA3 for structural integrity. Of note is that residual Pacinian corpuscles in Er81+/ETS,Pea3+/lacZ or PEA3lacZ/lacZ mutants were structurally similar to wild type corpuscles except that PEA3-deficient corpuscles were smaller than their wild type counterparts (Fig. 1A,B).
Table 1. Numbers of Crural Pacinian Corpuscles in Er81 and Pea3 Null Mutant Mice
No. of corpuscles
% of WT
aValues are means ± s.e.m. of counts of corpuscles in semi-serial plastic sections in neonatal (P0–P1) mutants and their wild type (WT) littermates. Presence (+) or absence (−) of the interosseous nerve in samples is indicated.
The early development of Pacinian corpuscles takes place in the crus over a 3-day period between E16.5 and E18.5, just prior to birth on E19, and no new corpuscles form after birth in wild type mice (Zelená, 1994; Šedý et al., 2004). In toluidine blue staining, the interosseous afferents destined to innervate crural Pacinian corpuscles can be observed to enter the crural target region in the lower leg at E15.5; nascent Pacinian corpuscles can be identified at E16.5 as enlarged terminals of the afferents that are intermingled with cells of Schwann cell origin (Šedý et al., 2004). In the current study, no Pacinian corpuscles were observed in toluidine blue-stained sections of the crus or in whole mounts stained for cholinesterase obtained from Er81ETS/ETS embryos at E16.5–E18.5. Thus, Pacinian corpuscles do not form in the absence of ER81.
Loss of Interosseous Nerve Afferents
The interosseous nerve (Fig. 1C) consists almost entirely of somatic afferent nerve fibers innervating the crural cluster of Pacinian corpuscles. Pacinian afferents and corpuscles exist in a 1:1 ratio, thus numbers of interosseous afferents provide an index of numbers of sensory neurons innervating the cluster (Šedý et al., 2004). Er81 null mutants were devoid of Pacinian corpuscles, and also lacked an interosseous nerve after birth (Fig. 1D). Thus, the deletion of Er81 impacts both Pacinian corpuscles and their afferents equally. In contrast, the interosseous nerve was present in Pea3 mutants and in Er81;Pea3 double heterozygous mutants that contained a portion of the wild type complement of Pacinian corpuscles. However, the mutant nerve appeared thinner than in wild type mice. These observations suggest that the deficits of Pacinian corpuscles parallel deficits of afferent nerve fibers (or sensory neuron bodies) in ETS-deficient mice after birth (Table 1).
In contrast to postnatal Er81 null mutants, the interosseous nerve was present in the crus of mutant embryos at E16.5, and nerve fibers originating from this sensory nerve were observed to innervate the crural region where Pacinian corpuscles normally form in wild type mice. However, nascent corpuscles were detected only in wild type embryos, but not in their mutant littermates at E16.5 (Fig. 2A–D). Thus, the absence of interosseous afferents in postnatal ER81-deficient mice is due to a failure of the afferents to survive during late embryogenesis.
ER81 and PEA3 Expression
To address whether Pacinian corpuscles express either ER81 or PEA3, we used P0–P1 neonates that had a heterozygous lacZ reporter gene substitution (Er81+/lacZ or Pea3+/lacZ) for Er81 or Pea3 (Fig. 3). Pacinian corpuscles of Er81+/lacZ newborns showed strong β-galactosidase reactivity in cell layers of the inner core, but no reactivity was detected in cells forming the external capsule (Fig. 3B). The β-galactosidase reactivity of the inner core cells decreased after birth although mild to moderate reactivity was still present in Pacinian corpuscles of adolescents at P21 (Fig. 3E). The diminution of β-galactosidase reactivity with increasing postnatal age suggests a developmental role for ER81 in the corpuscle assembly. No β-galactosidase was detected in the inner core cells of Pacinian corpuscles in Pea3+/lacZ neonates, although moderate reactivity was detected in the cells of the outer capsule (Fig. 3C). Dot-like deposits of the β-galactosidase reaction product were associated with nerve fibers of the dorsal spinal roots examined in longitudinal sections in Er81+/lacZ or Er81lacZ/lacZ mice, possibly corresponding to lacZ gene expression by the Schwann cells ensheathing the nerve fibers (Fig. 3D).
Afferents arrive at sites of Pacinian corpuscle formation at E15.5, and by E16.5–E17.5 nascent Pacinian corpuscles can be recognized as afferent terminal profiles surrounded by a few sheath cells (Šedý et al., 2004). We observed no β-galactosidase reactivity at the potential sites of Pacinian corpuscle formation at E15.5, minimal β-galactosidase reactivity at E16.5, and distinct reactivity corresponding to assembling corpuscles at E18.5 in Er81+/lacZ mice. At E16.5, the β-galactosidase reactivity appeared as small deposits of the reaction product at presumed sites of the corpuscle assembly, possibly corresponding to clusters of glial cells engaged in the formation of the inner core (Fig. 3A). Thus, the onset of ER81 expression in Pacinian corpuscles, as revealed by the lacZ reporter gene, occurs shortly after the arrival of their afferents to peripheral target sites.
Deficiency of Calretinin-Expressing Neurons
Lumbar DRGs of postnatal Er81 mutant mice show a 14% deficiency of neurons relative to counts of neurons in wild type DRGs (Kucera at al., 2002). To examine whether neurons innervating Pacinian corpuscles are among the missing sensory neurons, we reacted postnatal lumbar DRGs to an antibody specific for calretinin (CR) (Fig. 4). CR is a calcium binding protein that is normally expressed by afferents that innervate low-threshold mechanoreceptors such as Pacinian corpuscles in rats (Duc et al., 1993, 1994). We observed that the central axon of Pacinian corpuscles (Fig. 4D) and a subpopulation of the L4 DRG neurons were strongly immunoreactive to an anti-CR antibody (Fig. 4A–C) at all postnatal ages examined (P0–P21) in wild type mice, analogous to observations made in rats (Duc et al., 1993, 1994). The interosseous nerve that innervates the crural cluster of Pacinian corpuscles as well as the dorsal spinal roots and spinal nerves that contain the central and peripheral projections of DRG neurons, respectively, also contained CR-reactive axons. Thus, we assume that the subpopulation of lumbar DRGs neurons that innervate Pacinian corpuscles in mice is CR-immunoreactive.
Fewer DRG neurons expressed calretinin in Er81 null mutant than in wild type mice examined at P0–P1 or P10–P21. In random cross-sections of the L4 DRGs from three Er81−/− mutant and three wild type P0-P1 mice, neurons immunoreactive to an anti-CR antibody represented 0.2% of 9,400 neuron profiles in mutant compared to 5.6% of 700 neuron profiles in wild type mice. Thus, over 95% of CR-reactive DRG neuron profiles were absent in Er81 mutant relative to wild type mice. The deficiency of CR-expressing neurons in DRGs was associated with a deficiency of CR-reactive large caliber axons in the dorsal spinal roots of Er81 mutants as noted previously (Kucera et al., 2002). These observations, analyzed in conjunction with the loss of interosseous nerve afferents, suggested that the absence of crural Pacinian corpuscles in Er81 mutants is associated with a deficiency of DRG neurons innervating the corpuscles.
Co-Expression of Calretinin and ER81 by DRG Neurons
We next explored whether CR-reactive neurons also express the Er81nlslacZ marker, as determined from sections of the L4 DRGs that were alternatively or double reacted for CR and β-galactosidase in Er81+/lacZ mice at P0–P1 (Fig. 5). Using the β-galactosidase detection system, sites of the Er81nlslacZ transgene activation were evidenced by nuclear X-gal staining. However, some of the β-galactosidase molecules must have remained in the cytoplasm of β-galactosidase-positive DRG neurons because the X-gal reaction product also stained the cytoplasm surrounding the nucleus (Fig. 5G,H). We observed that approximately 58% (132 out of 228) of CR-reactive DRG neurons expressed CR only whereas 39% (90 out of 228) of CR-reactive neurons expressed both CR and β-galactosidase (Fig. 5A–F). Only 3% (6 out of 228) of the neurons stained for β-galactosidase but did not co-express CR in Er81+/lacZ mice. Thus, two major subpopulations of neurons coexist in DRGs, one reactive to CR only and the other expressing both CR and the Er81nlslacZ reporter.
Lumbar DRGs of Er81 null mutants were characterized by the absence of neurons reactive to CR or β-galactosidase only, and a deficiency of neurons co-expressing CR and the Er81lacZ marker. Approximately 0.2% (22 out of 9,400) of Er81−/− neurons were reactive to CR, and all of them co-labeled with β-galactosidase at P0–P1. Thus, all neurons expressing CR only or ER81 only as well as most of the neurons co-expressing CR and ER81 are among the 14% of neurons that are missing in the L4 DRGs of Er81 null mutants at birth (Kucera et al, 2002). Such missing DRG neurons likely include neurons innervating Pacinian corpuscles.
Development of two out of the three types of low-threshold limb mechanoreceptors is abnormal in Er81 null mice. The present study shows that crural Pacinian corpuscles do not form in the absence of ER81. Previous reports showed that muscle spindles form early in development but they either do not complete their differentiation or degenerate after birth in the absence of ER81 (Arber et al., 2000; Kucera et al., 2002). In contrast, Golgi tendon organs do not require ER81 to assemble and differentiate normally (Arber et al., 2000; Kucera et al., 2002).
None of three types of mechanoreceptors has an absolute requirement for PEA3. Although numbers of Pacinian corpuscles were mildly decreased in Pea3 mutants, muscle spindles and Golgi tendon organs develop normally in the absence of PEA3 (Livet et al., 2002; Kucera, unpublished data). However, whether a deletion of Pea3 or one copy of Er81 would lead to a functional defect of the residual Pacinian corpuscles is unknown. Vibration is a less effective stimulus in activating surviving spindles in Er81 mutants than in wild type mice, suggesting that ETS factors play a role in the normal function of mechanoreceptors (Arber et al., 2000).
Expression of ETS Factors by Pacinian Corpuscles
Cells comprising limb mechanoreceptors express individual members of the ETS family with distinct temporal and spatial patterns of expression. Intrafusal muscle fibers, the principal constituents of muscle spindles, express both ER81 and PEA3, though cells contributing to the Golgi tendon organs express only PEA3 (Arber et al., 2000). Using the lacZ reporter gene, we showed that Pacinian corpuscles express both ER81 and PEA3. However, the constituent cells of the corpuscles that express either of these two factors differ in their origins. The inner core cells of the corpuscles that express ER81 are derived from the Schwann cells associated with incoming afferents, whereas the outer capsular cells that expressed PEA3 differentiate locally from mesenchymal cells (Zelená, 1994). Schwann cells were reported to express ER81, but not PEA3 in a PCR screen for ETS family members (Parkinson et al., 2002). The observed pattern of distribution of the lacZ reaction product along spinal nerve fibers in ER81+/lacZ and ER81lacZ/lacZ mutants also suggests that the Schwann cells express ER81. During development, PEA3 is expressed in organs derived from different embryonic tissues, often at sites of epithelial-mesenchymal interactions, and plays a role in regulating cell proliferation, cell differentiation, and intercellular interactions (Chotteau-Lelievre et al., 1997, 2003). Using the lacZ reporter expression, we observed that PEA3 is expressed in the connective tissue capsule of Pacinian corpuscles, including the interface of the inner core and outer capsular cells. These two cell types are derived from the neural crest and local mesenchyme, respectively (Zelená, 1994), as do cells of inner core and outer capsule of Herbst corpuscle (Halata et al., 1990) that corresponds to Pacinian corpuscle in birds. Thus, the differential expression of ER81 and PEA3 in Pacinian corpuscles may reflect the Schwann cell and connective tissue origins of their inner and outer cell compartments. The differential expression of the two ETS factors suggests independent regulation of the Er81 and Pea3 genes, and may serve important functions in regulating proliferation and differentiation of the constituent cells of Pacinian corpuscles.
Absence of Pacinian Corpuscles
Crural Pacinian corpuscles form at sites of contacts between interosseous nerve afferents and specific target cells. Early in development, Schwann cells derived from sheaths of the incoming sensory axons proliferate and surround the afferent terminals to form the inner core of the corpuscles (Zelená, 1994). In the present study, no Pacinian corpuscles were observed in the crural region of Er81 embryos at E16.5–E18.5, a stage of development when Pacinian corpuscles normally form in wild type mice (Šedý et al., 2004). Thus, a failure of Pacinian corpuscles to form rather than degeneration of formed Pacinian corpuscles accounts for the absence of corpuscles in postnatal Er81 mutants.
The molecular events underlying the formation of Pacinian corpuscles are not completely understood. Development of mechanoreceptors is mediated by members of the neuregulin family of signaling proteins (Wolpowitz et al., 2000; Hippenmeyer et al., 2002). Sensory nerve fibers initiate the development of Pacinian corpuscles (Zelená, 1994) through the inductive effect of an afferent neuron-derived neuregulin (glial growth factor 2) on target cells in the periphery (Kopp et al., 1997).
However, experiments with avian chimeras suggest that peripheral factors also play a role in the formation of Pacinian corpuscles, and may regulate numbers of the corpuscles (Grim et al., 1999). These considerations, taken in conjunction with ER81 expression by the inner core cells, raise the possibility that a peripheral target tissue disturbance rather than a primary neuron dysfunction may account for the failure of Pacinian corpuscles to form in Er81 null mice.
Interactions between afferent neurons and Schwann cells, which form the inner core of Pacinian corpuscles, involve multiple reciprocal and/or bi-directional signaling systems comprising neuregulins and ErbB receptors and acting in both a paracrine and juxtacrine manner (Meintanis et al., 2001; Goodearl et al., 2001; Lyons et al., 2005). ETS factors are involved in neuregulin/ErbB signaling between afferent neurons and their target Schwann cells and mesenchymal cells, which form the inner and outer capsules of the corpuscles (Parkinson et al., 2002; Falls, 2003). Moreover, ER81 is a component of a positive regulatory feedback loop in which ErbB receptor protein activates ER81 causing the upregulation of the ErbB genes (Bosc and Janknecht, 2002). Schwann cell–derived cues that regulate Pacinian corpuscle development in response to neuregulin signaling may similarly be dependent on ER81, a factor expressed by the inner core cells of the corpuscles. In the absence of ER81, afferent-associated Schwann cells may be unable to transform themselves into cells that normally form the inner core of the corpuscles. As a result, no Pacinian corpuscles may form in Er81 null embryos.
Deficits in Sensory Neurons in Er81 Mutants
A mild (14%) deficiency of the L4 DRG neurons that provide afferent innervation to the lower leg exists in Er81 mutants (Kucera et al., 2002). The observed absence of interosseous nerve afferents is congruous with the deficiency of DRG neurons, and parallels the absence of Pacinian corpuscles in Er81 mutants. Moreover, neurons expressing calretinin, a marker of afferent neurons innervating Pacinian corpuscles (Duc et al., 1993, 1994), were deficient in the L4 DRGs of Er81 mutants. Expression of calretinin is not specific to neurons innervating Pacinian corpuscles as group Ia afferents innervating muscle spindles also express calretinin (Duc et al., 1993, 1994). However, DRG neurons expressing TrkC and parvalbumin, the two markers of spindle neurons, are not lost in Er81 mutants (Arber et al., 2000). In contrast, TrkC- and parvalbumin-expressing neurons are absent in neurotrophin-3 (NT3) null mutant mice that lack muscle spindles but contain over 50% of the wild type complement of crural Pacinian corpuscles (Ernfors et al., 1994; Klein at al., 1994; Šedý et al., 2004). Thus, the most parsimonious interpretation of the deficiency of calretinin-expressing DRG neurons and absence of the interosseous nerve afferents is that sensory neurons innervating Pacinian corpuscles are depleted in postnatal Er81 null mutants.
The deficiency of sensory neurons in Er81 mutants might be due to a lack of trophic signals that are normally provided by the inner core cells of Pacinian corpuscles. Such factors are retrogradely transported to afferent cell bodies to support neuron survival (Zelená, 1994). Sectioning of the sciatic nerve that disconnects Pacinian corpuscles from sensory neuron bodies results in degeneration of corresponding afferents in neonates (Zelená, 1980). In the absence of Pacinian corpuscles and their target-derived survival factors, sensory neurons to the corpuscles may undergo an early developmental death that results in the prenatal degeneration of interosseous nerve afferents in Er81 mutants.
Our study shows that Pacinian corpuscles, similar to muscle spindles and Golgi tendon organs (Arber et al., 2000), express ETS factors ER81 and PEA3. Unlike PEA3, ER81 is essential for the development of Pacinian corpuscles and for the survival of afferent neurons that innervate them. These data suggest that ER81 may have a direct (possibly peripheral) role in the development of low threshold limb mechanoreceptors, in addition to its known role in the specification of sensory and motor neuron subtypes and establishment of functional afferent-motor connectivity in the spinal cord (Lin et al., 1998; Arber et al., 2000).
Generation of mice carrying the mutated either Er81nlslacZ, Er81ETS, or Pea3nlslacZ alleles have been described previously (Arber et al., 2000). The frequency, pattern, and types of abnormalities observed in Pacinian corpuscles of Er81nlslacZ or Er81ETS mutant mice were similar. Therefore, data from the two types of Er81 mutants were pooled. In studies of embryos, the morning after an overnight mating of males to females was considered as embryonic (E) day 0.5. Fetuses were removed from anesthetized (sodium pentobarbital, 50 mg/100 g body weight) timed pregnant females in the morning of days E16.5–E18.5 by Cesarean section. Polymerase chain reaction of DNA extracted from tails or embryonic, newborn (P0–P1), or adolescent (P10–21) mice was used to genotype mice as null mutant (−/−), heterozygous mutant (+/−), or wild type (+/+) as detailed elsewhere (Kucera et al., 2002). Er81 null mutations were genotyped using two separate primer pairs; each pair was specific for one of the nucleotide sequences inserted into the Er81 locus (Arber et al., 2000).
Identification of Pacinian Corpuscles
Round or oval axon profiles surrounded by concentric rows of sheath cells that formed a lamellated inner core and an outer capsular envelope were the criteria used to identify Pacinian corpuscles (Zelená, 1994). At P0–P1, individual Pacinian corpuscles of the crural cluster derived from the interosseous nerve could already be identified as distinct entities scattered alongside the distal fibula. At this stage, corpuscles could be reliably counted in sections stained with toluidine blue or in whole mounts stained for cholinesterase (Šedý et al., 2004).
Counts of Pacinian Corpuscles in the Crus
Neonatal (P0–P1) and adolescent (P10–12) mutants and their wild type littermates were anesthetized with sodium pentobarbital (50 mg/kg body weight, i.p.) and were perfused with 2% paraformaldehyde-glutaraldehyde fixative in cacodylate buffer at pH 7.4. Fetuses were removed from pregnant females anesthetized with sodium pentobarbital by Cesarean section. Hindlimbs were detached and embedded in resin. Counts of crural Pacinian corpuscles were obtained from the resin-embedded specimens that were serially sectioned in the transverse plane at 1-μm thickness from the ankle to the knee. Every 10th section was stained with toluidine blue and examined by light microscopy. Pacinian corpuscles in newborn mice are considerably longer than 10 μm, so it is unlikely that any corpuscles were missed due to the sectioning protocol. No new corpuscles form after birth in wild type mice (Zelená, 1978; Nava, 1974, 1988), thus counts from newborn and neonatal mice of the same genotype could be pooled. All counts were transformed by square root prior to analysis, but were reported as untransformed values (mean ± s.e.m.). Means of variables (wild type vs. mutant) were compared by a Student's t-test using P < 0.05 as the limit for statistical significance.
Immunocytochemistry of Neurons and Pacinian Corpuscles
We used an antibody to calretinin (no. 7699/4, Swant Co.) to identify DRG neurons that innervate low-threshold limb mechanoreceptors, and to visualize their central and peripheral projections. Calretinin (CR) is a calcium-binding protein expressed by afferents innervating muscle spindles, Golgi tendon organs, and Pacinian corpuscles in birds and mammals (Duc et al., 1993, 1994). Paraformaldehyde-fixed lumbar DRGs with attached dorsal spinal roots and ipsilateral lower legs were excised, cryoprotected in 30% sucrose, frozen, serially cut into 12-μm-thick sections, reacted with the anti-CR antibody (diluted 1:10000), and processed using the Vectastain ABC commercial kit (Vector Labs, Burlingame, CA). The number of CR-reactive neurons in each DRG section was expressed as a percentage of all neurons (large nucleated cells) visible in the section. Transverse sections of the spinal roots and crus were examined for CR reactivity associated with nerve fibers and Pacinian corpuscles.
For indirect immunocytochemistry, sections were rinsed with PBS, blocked with a biotin blocking kit (no. SP 2001, Vector Labs), and incubated overnight with an anti-calretinin Ab (no. 7699/4, Swant Co.) diluted 1: 2000. After washing, the sections were incubated for 2 hr at room temperature with a secondary goat anti-rabbit biotin-conjugated Ab (no. 111-065-144, Jackson ImmunoResearch Labs, West Grove, PA) diluted 1:600. The streptavidin-TRITC Ab (no. 016-020-084, Jackson ImmunoResearch Labs) diluted 1:300 was used for visualization of the binding sites. It was followed by a 4-hr incubation with rabbit unconjugated Fab fragment (no. 111-007-003, Jackson ImmunoResearch Labs) diluted at 20 μg /1 ml, and then by application of a rabbit anti-β-galactosidase Ab (no. AB986, Chemicon, Temecula, CA) diluted 1:1.500. After a PBS wash, a secondary goat anti-rabbit FITC Ab (no. 111-095-144, Jackson ImmunoResearch labs) diluted 1:150 was applied for 90 min at room temperature. Sections were mounted with Vectashield-DAPI (H 1200, Vector Labs) and coverslipped. Images were taken with an Olympus BX51 microscope and processed with AnalySIS (Soft Imaging System) software.
Sites of lacZ reporter gene expression in Er81+/lacZ and Pea3+/lacZ mutants were visualized using β-galactosidase histochemistry. Lumbar DRGs and crura of neonatal (P0–P1) and adolescent (P10–12) mutant mice were fixed with 4% paraformaldehyde, cryoprotected in 30% sucrose, frozen, cut at 12-μm thickness in a cryostat, and stained overnight at 37°C in X-gal solution at pH 7.3 according to Fariñas et al. (1996). Deposits of a blue reaction product indicated sites of the lacZ reporter expression. Pacinian corpuscles of wild type mice were devoid of X-gal reactivity. In some DRG specimens, adjacent sections were alternately immunoreacted for CR or processed for β-galactosidase histochemistry.
We thank Drs. S. Arber and T.M. Jessell for providing Er81 and Pea3 mutant mice. Ms. E. Kluzáková, M. Pleschnerová, and A. Kautská provided excellent technical assistance. M.G. received grant support from the Ministry of Education of the Czech Republic (VZ 0021620806 and 1M0021620803). J.M.W. received funding from the National Institutes of Health, and J.K. received funding from the Veterans Administration.