DRG11 immunohistochemical expression during embryonic development in the mouse

Abstract

DRG11 is a paired domain transcription factor that is necessary for the assembly of the nociceptive circuitry in the spinal cord dorsal horn. It is expressed in small dorsal root ganglion (DRG) neurons and in their projection area in the spinal cord. Drg11 knockout mice exhibit structural and neurochemical defects both at the DRG and spinal superficial dorsal horn and present reduced nociceptive responses. In this study, a polyclonal antibody against DRG11 was generated and used for a detailed systematic spatio-temporal analysis of DRG11 expression during development. DRG11 is first detected at E10.5 in the spinal dorsal horn, DRG and trigeminal ganglion, where it persists until P14-21. At the cranial level, DRG11 expression is observed from E10.5 up to the same early post-natal ages in several cranial sensory ganglia and brain nuclei. These results suggest that DRG11 is required for the establishment of the first neuronal sensory relay along development. Developmental Dynamics 236:2653–2660, 2007. © 2007 Wiley-Liss, Inc.

INTRODUCTION

Spinal cord dorsal horn neurons are responsible for modulating and relaying various types of sensory information to higher brain centers using a complex, still poorly understood circuitry that is established during embryonic development (for review, see Gillespie and Walker,2001; Julius and Basbaum,2001; Goulding et al.,2002). In the brain, somatic sensory information is relayed through the trigeminal nuclei, while visceral sensory information is relayed through the solitary nucleus (Saper,2000). The mechanisms of differentiation of the various sensory relay neurons in the hindbrain and spinal cord began to be understood only recently mainly due to the lack of specific molecular markers. At the spinal cord level, DRG11, a paired-like homeodomain protein, is detected in primary sensory neurons and in their projection area in the superficial dorsal horn (Saito et al.,1995). Because superficial dorsal horn laminae are involved in nociception, Drg11 was proposed to be required for the development of the nociceptive system. This hypothesis was later supported by the finding that Drg11 knockout mice (Drg11^−/−) exhibit reduced reflex responses to painful stimuli together with abnormalities in superficial dorsal horn structure and neurochemistry (Chen et al.,2001). Failure to establish normal vibrisal somatosensory maps in the thalamus and SI cortex was also demonstrated (Ding et al.,2003). A recent study addressing the expression of various primary afferent neuronal markers along development pointed to a role for DRG11 in early postnatal survival of apparently normally differentiated small primary afferent neurons innervating various kinds of peripheral tissues (Rebelo et al.,2006).

In the present study, a polyclonal antibody was generated against the C-terminal part of mouse DRG11 and used to carry out a detailed systematic spatiotemporal analysis of DRG11 expression along embryonic development. The results indicate that DRG11 is expressed early in the embryo both in central nervous system (CNS) and peripheral nervous system (PNS) neurons subserving somatosensory and viscerosensory functions. The protein is first immunodetected at embryonic day 10.5 (E10.5) and is maintained throughout development. It is expressed in the dorsal root ganglia (DRG), trigeminal, facial, vestibulocochlear, glossopharyngeal, and vagus cranial ganglia, spinal superficial dorsal horn and sensory hindbrain nuclei. Postnatally, DRG11 expression progressively decreases, being undetectable by P21. These findings suggest that DRG11 plays a crucial role in the development of primary afferent neurons and second-order sensory neurons.

RESULTS AND DISCUSSION

Generation and Characterization of the Antibody Directed Against DRG11

Drg11 expression has first been detected by in situ hybridization in the DRG and the spinal cord dorsal horn (Saito et al.,1995; Chen et al.,2001). To gain further insight on DRG11 function, we generated a rabbit anti-DRG11 antibody and tested its specificity by immunostaining the DRG and spinal cord of wild-type and Drg11^−/− E18.5 embryos. The antibody was raised against the C-terminal region, spanning amino acids 103 to 263, which excludes the homeodomain (Fig. 1A). Immunohistochemical analysis showed that the protein expression pattern correlates entirely with the previously reported mRNA expression in the nervous system (Saito et al.,1995; Chen et al.,2001), and no expression was detected in any other tissues (Fig. 1B). DRG11 immunostaining was observed in the DRG and in the spinal cord superficial dorsal horn of wild-type animals, but not in the Drg11^−/− mice (Fig. 1B). Moreover, immunoblotting analysis of protein extracts from several tissues of wild-type mice revealed that the expression of DRG11 is present in DRG and spinal cord (Fig. 1C). As expected, no signal was observed in the spinal cord of Drg11^−/− mice (Fig. 1C). Although DRG11 has a predicted molecular mass of 28 kDa, it was detected by immunoblotting around 36 kDa (Fig. 2). Such an altered migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is occasionally observed in homeodomain proteins (Jackson et al.,2002). Nonetheless, the signal must correspond to DRG11, because it is located in the same areas as Drg11 mRNA and in Drg11^−/− littermates no immunostaining or immunoblotting bands were detected. All together, these findings indicate that the antibody specifically recognizes the DRG11 protein.

Figure 1. Characterization of the antibody raised against DRG11, a paired-like homeodomain protein. A: Schematic representation of Drg11 structure, which includes a DNA-binding homeodomain and an OAR domain. The region of amino acids 103–263 was used for antibody generation. B: Expression of DRG11 protein in wild-type (+/+) and Drg11^−/− mice (−/−) at embryonic day (E) 18.5. Arrows indicate expression of DRG11 in the dorsal horn of the spinal cord (DHsc) and the dorsal root ganglia (DRG). C: Western blotting analysis of DRG11 expression pattern. Several tissue extracts were immunoblotted for DRG11, but bands were obtained only from the DRG and spinal cord (sc) of wild-type mice. Beta-actin was blotted on the same membrane and used as a sample loading control.

Figure 2. Subcellular expression of DRG11, a paired-like homeodomain protein. Cells from spinal cords of embryonic day (E) 18.5 wild-type mice were fractionated by differential centrifugation into cytoplasmic (cyt) and nuclear (nu) fractions. Fifty micrograms of each fraction was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of DRG11 was detected by immunoblotting. Lamin A/C and Cu, Zn SOD were used as, respectively, nucleus and cytoplasmic marker proteins to validate the fractionation. Values represent the mean ± SEM of three replicate experiments.

To further characterize the anti-DRG11 antibody, we addressed the subcellular localization of DRG11 in the spinal cord of embryonic day (E) 18.5 wild-type embryos. Immunoblotting revealed a pronounced DRG11 signal in the nucleus and weaker staining in the cytosol. Nuclear and cytosolic fractions were isolated to quantitatively address this difference. Each fraction was resolved in SDS-PAGE and immunoblotted with the anti-DRG11 antibody. DRG11 expression amounted to approximately two-thirds (64.9%) in the nucleus and one-third (35.1%) in the cytosol (Fig. 2).

DRG11 Expression Pattern During Nervous System Development

Previous in situ hybridization data from mice (Saito et al.,1995; Chen et al.,2001; Qian et al.,2002; Ding et al.,2003) pointed to the restricted expression of Drg11 in the DRG, spinal cord dorsal horn, and trigeminal brainstem complex. However, to date, no comprehensive overview of DRG11 expression in the entire embryo and how it evolves along the development of the nervous system was attained. We sought to carefully analyze the DRG11 expression in the entire neuroaxis at various time points of embryonic development and early postnatal life by taking advantage of our novel generated antibody. The study was carried out in mice from the time of neural tube closure (E10.5) up to adulthood. DRG11 immunostaining was observed in the DRG, spinal cord, cranial sensory ganglia, and brainstem sensory nuclei (results are summarized in Table 1).

Table 1. Expression Analysis of DRG11 in the Peripheral and Central Mouse Nervous System at Various Time Points Along Embryonic Development and Postnatal Lifea

	Developmental age
a Indicated are the relative amounts of DRG11, a paired-like homeodomain protein, present in each region. Symbols: +, strong expression; (+), weak expression; −, no expression; na, not analyzed.
Nervous system	10.5	12.5	15.5	18.5	P0	P7	P14	P21
Peripheral
Dorsal root ganglia	+	+	+	+	+	+	(+)	−
Trigeminal (V) ganglia	+	+	+	+	+	+	(+)	−
Facial (VII) ganglia	+	+	+	+	+	na	na	na
Vestibulocochlear (VIII) ganglia	+	+	+	+	+	na	na	na
Glossopharyngeal (IX) ganglia	+	+	+	+	+	na	na	na
Vagus (X)	+	+	+	+	+	na	na	na
Central
Spinal cord	+	+	+	+	+	+	(+)	−
Spinal trigeminal nucleus	−	+	+	+	+	+	(+)	−
Principal trigeminal nucleus	−	+	+	+	+	+	(+)	−
Nucleus tractus solitarus	−	+	+	+	+	+	(+)	−
Prepositus nucleus	−	+	+	+	+	+	(+)	−

DRG11 Expression in Sensory Ganglia

DRG11 was immunodetected in the DRG and the trigeminal (V) ganglia (both the ophthalmic and maxillomandibular lobes) throughout development, beginning at age E10.5 (Table 1; Fig. 3A,C). DRG11 expression was also observed in other cranial sensory ganglia, namely the facial (VII), vestibulocochlear (VIII), glossopharyngeal (IX), and vagus (X; Table 1; Fig. 3A–C,E). Immunostaining persisted in all areas with no apparent changes until perinatal age (P0). Postnatally, due to difficulties in dissecting the cranial ganglia, DRG11 expression was only sought in the DRG and trigeminal ganglia. In both structures, DRG11 expression was still present, although it was lower at P14 and nil at P21 (Table 1).

Figure 3. Expression of DRG11, a paired-like homeodomain protein, in the mouse cranial ganglia. A: Trigeminal (V), facial (VII), vestibulocochlear (VIII), and glossopharygeal (IX) ganglia at embryonic day (E) 18.5. B: Higher magnification of the boxed region in A. C–F: Sagittal adjacent sections of E11.5 facial (C,D), glossopharygeal (C,D), and vagus (X; E,F) visceral sensory ganglia showing DRG11 expression (C,E) and Phox2b expression (D,F). Scale bars = 100 μm.

DRG11 expression in the DRG and the trigeminal ganglia had been previously reported (Ding et al.,2003; Rebelo et al.,2006). The trigeminal ganglion is analogous to the DRG, containing the cell bodies of somatic sensory fibers from the head and oral cavity. As in the DRG, trigeminal primary sensory neurons are distributed through large size and small size populations. DRG11 was exclusively expressed in small neurons in both the trigeminal ganglion and the DRG. Coimmunostaining for DRG11 with either trkA, CGRP, or IB4 showed colocalization with the three markers of small DRG neurons, which supports a role for these neurons in nociceptive and thermal sensations (Fig. 4A–C).

Figure 4. Expression of DRG11, a paired-like homeodomain protein, in the mouse dorsal root ganglion. A–C: DRG11 coexpresses with trkA (A) and CGRP (B) in small peptidergic DRG neurons and with IB4 (C) in nonpeptidergic neurons. Scale bars = 50 μm.

The facial, glossopharyngeal, and vagus ganglia contain visceral primary afferent neurons and convey nociceptive input to the spinal trigeminal nucleus. To confirm that DRG11 is expressed in visceral ganglia, we immunostained adjacent sections with Phox2b, a molecular visceral sensory marker (Fig. 3D–F). DRG11 expression was present in all Phox2b-positive visceral ganglia. Because the available DRG11 and Phox2b antibodies are raised in the same species, colocalization of the two markers at the cellular level was not addressed. In agreement with what was reported in the zebrafish (McCormick et al.,2007), expression of DRG11 seemed to be weaker in these visceral sensory ganglia compared with the somatic trigeminal ganglion. No DRG11 expression was detected in cranial ganglia exclusively subserving motor function.

DRG11 Expression in the Brain

In the brain, expression of DRG11 was first observed in the principal trigeminal nucleus (Pr5) at E12.5 (Table 1; Fig. 5A,B,G,H), followed by the spinal trigeminal nucleus (subnucleus caudalis and subnucleus oralis; Table 1; Fig. 5C,G,H). DRG11 was also detected in the nucleus of the solitary tract (NTS) and nucleus prepositus (NP; Table 1; Fig. 5D–F,I). From P7 on, DRG11 expression decreased, being virtually absent by age P21 (Table 1).

Figure 5. Expression of DRG11, a paired-like homeodomain protein, in the mouse brain. A,B: Principal trigeminal nucleus (Pr5). B: Magnification of the boxed region in A. C: Spinal trigeminal nucleus (Sp5). D–F: Nucleus of the solitary tract (NTS; D,F) and nucleus prepositus (NP; D,E). A–F: Taken from embryonic day (E) 18.5 embryos. G,H: Expression of DRG11and Lmx1b (G) and Drg11 and Tlx3 (H) in the prospective Pr5 (pons) and medulla at E13.5 (horizontal sections). I,J: Expression of DRG11 (I) and Phox2b (J) on adjacent sections at E13.5 (coronal sections). Scale bars = 100 μm in A–E; 50 μm in G–J.

All the brainstem nuclei expressing DRG11 function as first-relay stations for sensory input arriving through cranial nerves. The trigeminal system is made up of various components processing different kinds of somatic peripheral input. The subnucleus caudalis of the spinal trigeminal nucleus processes mainly nociceptive and thermal information, and the principal trigeminal nucleus processes tactile information. Among trigeminal areas that did not express DRG11 is the subnucleus interpolaris, which is less well characterized but thought to be involved in tactile-evoked reflex activity.

Expression of DRG11 in the spinal trigeminal nucleus (Sp5) is in accordance with previous findings by Ding et al. (2003) and supports the assumption that DRG11 plays an important role in the development of the nociceptive neuronal circuit at its first relay (Chen et al.,2001; Rebelo et al.,2006). However, DRG11 is also expressed very early in a trigeminal area devoted to tactile sensation, the principal nucleus (Qian et al.,2002; Ding et al.,2003; present results), raising the possibility that DRG11 may be also required for the establishment of the sensory innocuous processing circuit. Unfortunately, the capability of experimentally testing tactile functioning is very limited.

Immunohistochemical colocalization of DRG11 with either Lmx1b or Tlx3, markers expressed in both trigeminal nuclei (Qian et al.,2002; Ding et al.,2003), was assessed. The majority of DRG11 neurons in the principal nucleus colocalized with Lmx1b (Fig. 5G), while only a small portion colocalized with Tlx3 (Fig. 5H). In the medulla, three immunoreactive columns were depicted. A lateral column, which is likely to originate the Sp5 was densely populated by neurons double-stained for DRG11 and either Lmx1b or Tlx3 (Fig. 5G–H, arrowhead). At an intermediate position laid a second column with neurons that only stained for DRG11 (Fig. 5G,H). More medially neurons also colocalized DRG11 and Lmx1b/Tlx3, although the proportion of double-stained neurons was not as abundant as laterally (Fig. 5G,H, arrow). According to the localization of Lmx1b and Tlx3 at the age of E13.5–E14.5 (Qian et al.,2001; Dauger et al.,2003), the two medial columns may be at the origin of the NTS.

The NTS is targeted by visceral afferent fibers supporting a role for DRG11 in the development of visceral sensory systems. Its formation during development is dependent on the expression of Tlx3, which is upstream of Drg11 (Qian et al.,2001). DRG11 coimmunostaining with Lmx1b or Tlx3 indicates that, earlier in development, there is a population in the NTS that is DRG11-specific and a another that colocalizes extensively either with Lmx1b or Tlx3 (Fig. 5G,H). Phox2b has been used as a molecular marker for visceral neurons during their development and migration (Dauger et al.,2003). Immunostaining for both transcription factors on adjacent sections showed a partial overlap, indicating that, although most NTS neurons are DRG11-positive, there is a fraction that is not (Fig. 5I,J). As to the nucleus prepositus, it is thought to play a role in the regulation of eye movements. Interestingly, Drg11^−/− mice exhibit defects in eye movement control shortly after birth. The data here collected on the brain expression of DRG11 clearly indicate that Drg11 is preferentially involved in the development of sensory processing areas from early differentiation stages until shortly after birth.

DRG11 Expression in the Spinal Cord

In the spinal cord, DRG11 expression was first detected at E10.5 in dI3 and dI5 neurons (Fig. 6A). This stage corresponds to the first wave of neurogenesis, which specifies the dorsal neural tube into six classes of early-born neurons (dI1-6) according to their dorsoventral position and expression profile of some homeodomain proteins (Liem et al.,1997; Lee and Jessell,1999; Caspary and Anderson,2003; Helms and Johnson,2003). Periventricular staining decreased at E11.5 (Fig. 6B) to increase again at E12.5 (Fig. 6C). At this time point, staining was massive of the dorsal periventricular area and extended to the dorsolateral spinal cord region (Fig. 6C). Most of these neurons are likely to represent the late born-populations, which will populate the spinal cord superficial laminae (Gross et al.,2002; Matisse,2002; Müller et al.,2002). At E11.5, a few neurons could be observed migrating ventrally (Fig. 6B, arrows). Ventrally located neurons were detected until E15.5 (Fig. 6B–F, arrows). Recently, it was suggested that early-born neurons (born between E10.5 and E11.5) migrate ventrally and do not contribute to the formation of the superficial dorsal horn (Caspary and Anderson,2003). At E13.5–E14.5, DRG11-positive neurons were packed in the lateral most portion of the superficial spinal cord, immunostaining in the periventricular zone being still detected, but much less intensely (Fig. 6D,E). From E15.5 on, DRG11 neurons occupied the most superficial region of the spinal cord (Fig. 6F), so that at E18.5, when spinal cord lamination is distinguishable, DRG11 expression extended from lamina I to III (Fig. 6G). This expression pattern was maintained postnatally (Fig. 6H–K), although decreasing in intensity along time (Fig. 6H,I). Even though Lmx1b and Tlx3 expression spanned the entire dorsal horn of the spinal cord, DRG11 expression was restricted to the superficial spinal cord laminae (Fig. 6L,M). In addition, almost all DRG11-expressing neurons were Lmx1b-positive (Fig. 6L), while colocalization with Tlx3 was observed in a lesser extent (Fig. 6M).

Figure 6. Time course analysis of the expression of DRG11, a paired-like homeodomain protein, in the developing spinal cord. A: DRG11 is first expressed in dI3 and dI5 interneurons at embryonic day (E) 10.5. Lim1/2 was used as a dI 2, 4, 6 marker. B,C: At E11.5 and E12.5, DRG11 is detected in the periventricular zone and neuronal migration is initiated. D,E: DRG11 expression starts to be concentrated in laminae I–III neurons. Periventricular staining is still detected. F,G: The migration of DRG11-expressing neurons is settled. H,I: Postnatally, DRG11-positive neurons are localized predominantly in laminae I–III, but DRG11 expression progressively decreases. J: Higher magnification of the boxed region shown in I. K: Laminae I–IV were depicted by Nissl (blue) staining at P14 to show the location at laminae I–III of DRG11-positive neurons (brown). L: DRG11-positive neurons are mostly Lmx1b-positive and are restricted to the superficial laminae I–III. M: Neurons expressing both Drg11 and Tlx3 are preferentially located in lamina II of the superficial spinal dorsal horn. Scale bars = 50 μm.

The superficial dorsal horn, in particular laminae I and II, has for long been claimed to process nociceptive information. DRG11-positive spinal neurons heavily populate this spinal area and are, therefore, likely to constitute an important component of the spinal nociceptive circuitry. In this light, it should be recalled that, in Drg11^−/− mice, small peptidergic and nonpeptidergic primary afferent neurons appear to develop normally until birth but undergo apoptosis immediately after, decreasing their numbers to approximately half their normal amount (Rebelo et al.,2006). Because primary afferent fibers establish connections with superficial spinal neurons at late stages of embryonic development (Reynolds et al.,1991; Fariñas et al.,1996; White et al.,1996), it is possible that this apoptotic fate is due to the absence of their neuronal spinal targets. It remains to be clarified, however, whether ablation of spinal second-order neurons is the primary trigger of nociceptive disruption in these mice.

Also relevant is the here found expression of DRG11 in neurons of lamina III. Lamina III is the site of termination of Aδ D-hair follicle primary afferents (Light and Perl,1979; Willis et al.,2004), which probably suggests that processing of innocuous input may also be, at least partly, dependent on DRG11. In this respect, it is worth mentioning that brain areas devoted to tactile processing, such as the principal trigeminal nucleus, heavily express DRG11 at early stages of embryonic development.

Semiquantitative Characterization of DRG11 Along Development

To access the relative expression of DRG11 along development, a time course immunoblotting analysis was performed using spinal cord protein extracts from mice at E15.5, E18.5, P0, P7, P14, and P21 (Fig. 7). Developmental ages before E15.5 were not considered due to the difficulty in micro-dissecting correctly the neural tube of these embryos. At E15.5 and E18.5, expression of DRG11 was very high. It was reduced to approximately half at birth and kept decreasing until P14, where the levels of DRG11 were almost nil (Fig. 7). In the adult spinal cord (P21), no DRG11 was detected by immunoblotting. The DRG11 signal was normalized with beta-actin, used as a loading control.

Figure 7. Western-blotting analysis of DRG11, a paired-like homeodomain protein, expression along development. A: Time-course analysis of DRG11 expression was performed using mouse spinal cord extracts from embryonic day (E) 15.5, E18.5 and postnatal ages P0, P7, P14, and P21. Knockout mice were used as negative control. B: Relative quantification of DRG11 expression at the various time points was compared taking E15.5 expression as 100%.

In this study, we used an anti-DRG11 antibody generated in our laboratory to address the immunohistochemical expression pattern of the homeodomain transcription factor DRG11 during development in the mouse. We show that DRG11 expression is restricted to the nervous system and occurs in peripheral ganglia as well as in brain and spinal cord areas related to the processing of somatosensory and viscerosensory information. In the spinal cord, DRG11 expression coincides with the two waves of neurogenesis, defining two subpopulations of early-born (dI3 and dI5) as well as late-born neurons. As development proceeds, DRG11-positive neurons settle in layers I–III of the spinal cord dorsal horn.

EXPERIMENTAL PROCEDURES

Genotyping and Maintenance of Animals

Drg11 mutant mice were generated by intercross between heterozygous mice. Wild-type mice and Drg11^−/− littermates were genotyped as previously described (Chen et al.,2001). Animals used in this study were bred and maintained at the IBMC animal facility. The day when the vaginal plug was formed was considered to be the E0.5. The ethical guidelines for investigation of experimental pain in animals (Zimmermann,1983) and the European Community Council Directive of 24 November 1986 (86/609/EEC) were followed.

Generation of Anti-Drg11 Antisera

A 480 bp cDNA fragment from the murine Drg11, corresponding to amino acids 103 to 263 (excluding the homeobox domain), was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from total RNA of newborn spinal cord using the one-shot RT-PCR kit (Roche) following the manufacturer's instructions. The following primers were used: 5′-aaggaacccatggcagag-3′ and 5′-tcatacactcttctctccctcgc-3′. The amplified PCR fragment was cloned using the TA cloning kit (Invitrogen) into the pCR2.1 plasmid, and digested with the XbaI and BamHI restriction enzymes to subclone the Drg11 cDNA fragment into the bacterial expression vectors pGEX-4T3 (Amersham Biosciences) and pRSET-A (Invitrogen) in frame with glutathione-S-transferase (GST) and (His)6-tag sequences, respectively. The fusion proteins (His)6-Drg11(C-terminal) and GST-DRG11(C-terminal) were propagated in BL21(DE3)pLysS Escherichia coli cells and affinity purified on nickel or GST resins (Sigma), respectively, according to the manufacturer's recommendations. The purified (His)6-Drg11(C-terminal) recombinant protein was injected into rabbits. The antiserum was affinity purified using a CNBr-activated resin (Amersham Biosciences) conjugated with the GST-DRG11 (C-terminal) recombinant protein according to the manufacturer's protocol.

Tissue Preparation

Embryos were removed by cesarian surgery of pregnant females under anesthesia (sodium pentobarbital 50 mg/kg intraperitoneally), fixed in 4% paraformaldehyde for at least 4 hr, cryoprotected in 30% sucrose overnight, and sectioned on a cryostat at 12 μm. Mice at postnatal ages were also anesthetized before perfusion through the ascending aorta with phosphate-buffered saline 0.1 M (PBS) followed by 4% paraformaldehyde. The spinal cord, DRG, and brain were dissected, post-fixed for 2 hr, cryoprotected in 30% sucrose overnight and embedded in Jung Tissue Freezing Medium before being sectioned into 12-μm sections in a cryostat.

Subcellular Fractionation and Western Blotting

Nuclear and cytosolic fractions were obtained as previously described (Manitt et al.,2001). Briefly, in a typical experiment, 500 μg of spinal cord protein extract were homogenized in SEI buffer containing 10 mM imidazole, pH 7.2, 5 mM ethylenediaminetetraacetic acid, 0.32 M sucrose and protease inhibitor cocktail (Sigma), and centrifuged at 1,000 × g for 10 min at 4°C. Nuclear pellet was washed three times in SEI buffer, and the supernatant was centrifuged at 16,000 × g for 10 min at 4°C, to obtain the cytosolic fraction. Of the total protein, 51% was recovered in the cytosolic fraction, whereas 26% was found in the nuclear fraction. These values were considered for the estimation of the amount of DRG11 in each fraction.

For Western blotting analysis, protein samples were resolved in a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (Bio-Rad). Immunoblot analysis was performed using classic protocols and signal was detected with the Immun-Star Chemiluminescent kit (Bio-Rad). Signal intensities were determined using the 1D Image Analysis Software (Kodak), and normalized with beta-actin. The values were presented as a mean of three independent experiments (n = 3).

Immunohistochemistry

For immunohistochemical staining, tissue endogenous peroxidase was quenched in PBS containing 10% methanol and 3% hydrogen peroxide. Sections were immersed in PBS containing 0.4% Triton X-100 and 10% normal serum from the host species of the secondary antibody to be used (see below), followed by immersion overnight in rabbit anti-DRG11 (1:500) or rabbit anti-Phox2b (gift from Qiufu Ma, 1:200). Sections were washed and incubated for 1 hr, at room temperature, in biotinylated swine anti-rabbit antiserum (Dakopatts, Dako A75, Copenhagen, Denmark, 1:200). The antigen signal was visualized with the Vectastain ABC kit (Vector Labs). For immunofluorescent detection, primary antibodies were rabbit anti-Drg11 (1:500), mouse anti-Lim1/2 (Developmental Studies Hybridoma Bank, University of Iowa, 1:20), guinea-pig anti-Lmx1b and Tlx3 (gift from Thomas Müller and Carmen Birchmeier, 1:1,000), rabbit anti-trkA (Chemicon, 1:1,000), rabbit anti-CGRP (Chemicon, 1:1,000), and IB4 (Sigma, 1:1,000). The antigen signal was detected by Alexa-conjugated secondary antibodies (Molecular Probes). Fluorescent samples were captured on a confocal microscope (Bio-Rad 1024). Omitting the primary antibodies resulted in a complete absence of staining in neuronal profiles. Nissl staining was performed using 0.5% crest violet for 15 min, rinsed in tap water, dehydrated, and mounted in Eukitt.

Acknowledgements

The authors thank Thomas Müller, Carmen Birchmeier, and Qiufu Ma for gifts of antibodies.