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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC Aug 28, 2012.
Published in final edited form as:
PMCID: PMC3428801
NIHMSID: NIHMS357204

Combinatorial expression of Brn3 transcription factors in somatosensory neurons: genetic and morphologic analysis

Abstract

The three members of the Brn3 family of POU-domain transcription factors (Brn3a/Pou4f1, Brn3b/Pou4f2, and Brn3c/Pou4f3) are expressed in overlapping subsets of visual, auditory/vestibular, and somatosensory neurons. Using unmarked Brn3 null alleles and Brn3 conditional alleles in which gene loss is coupled to expression of an alkaline phosphatase reporter, together with sparse Cre-mediated recombination, we describe (1) the overlapping patterns of Brn3 gene expression in somatosensory neurons, (2) the manner in which these patterns correlate with molecular markers, peripheral afferent arbor morphologies, and dorsal horn projections, and (3) the consequences for these neurons of deleting individual Brn3 genes in the mouse. We observe broad expression of Brn3a among DRG neurons, but subtype-restricted expression of Brn3b and Brn3c. We also observe a nearly complete loss of hair follicle-associated sensory endings among Brn3a−/− neurons. Together with earlier analyses of Brn3 gene expression patterns in the retina and inner ear, these experiments suggest a deep functional similarity between primary somatosensory neurons, spiral and vestibular ganglion neurons, and retinal ganglion cells. This work also demonstrates the utility of sparse genetically-directed labeling for visualizing individual somatosensory afferent arbors and for defining cell-autonomous mutant phenotypes.

Keywords: dorsal root ganglion, mechanoreceptor, skin, spinal cord, axonal arbor

Introduction

Primary somatosensory neurons convey information from the skin and body interior about temperature, mechanical stimulation, tissue damage, and joint and muscle position (Lumpkin and Catarina, 2007; Basbaum et al., 2009; Proske and Gandevia, 2009). The diversity of somatosensation is mirrored in the diversity of peripheral nerve endings, which vary in complexity from free nociceptive endings in the skin to intricate structures such as Pacinian corpuscles and muscle spindles. Diversity is also seen in nerve conduction velocity and its anatomic counterparts, axon diameter and myelination. In the dorsal horn of the spinal cord, the central projections of the primary somatosensory neurons are organized in laminae, with distinct subtypes of somatosensory fibers projecting to only one or a few laminae (e.g., Sanderson Nydahl et al., 2004; Zylka et al., 2005; Bourane et al., 2009; Luo et al., 2009).

A growing collection of molecular markers and gene knockout mouse lines has facilitated the identification and characterization of different subtypes of somatosensory neurons. The markers include TRPV channels, Mas-related G-protein coupled receptors (Mrgprs), cytosolic proteins such as parvalbumin and neurofilament-200 (NF200), and neuropeptides such as CGRP (e.g., Fundin et al., 1997a; Tominaga et al., 1998; Zylka et al., 2005; Liu et al., 2009; Luo et al., 2009). Of special interest are genes and proteins that control the development and diversification of somatosensory neurons - in particular, the neurotrophic factor receptors RET, Trk, and GFR-alpha, and the transcription factors Sox10, Neurogenin-2, Islet-1, MafA, and the members of the Runx and Brn3 families (Marmigere and Ernfors, 2007; Eng et al., 2007; Sun et al., 2008; Bourane et al., 2009; Inoue et al., 2008; Lanier et al., 2009; Dykes et al., 2010). Sox10 controls the proliferation of neural progenitors in the DRG and TG, and Islet-1 and Brn3a control, directly or indirectly, the expression of a large number of genes in DRG and TG neurons, including Runx genes. The TrkA and TrkC neurotrophin receptors, which are controlled in part by Runx1 and Runx3, play critical roles in the differentiation and survival of nociceptors, mechanoreceptors, and proprioreceptors.

The present work focuses on the three members of the Brn3 family of transcription factors: Brn3a/Pou4f1, Brn3b/Pou4f2, and Brn3c/Pou4f3. Each Brn3 gene is expressed in distinct set(s) of neurons in each of three sensory organs – the retina, inner ear, and DRG/TG. We have recently developed Brn3a, Brn3b, and Brn3c conditional knockout/alkaline phosphatase (AP) knock-in mouse lines that permit a genetic and morphologic analysis of individual neurons (Badea et al., 2009a; present work). We report here the use of these lines to define - for both WT and Brn3 mutant mice - the overlapping patterns of Brn3 gene expression among DRG neurons, the morphologies of their afferent arbors, and their central target fields in the dorsal horn of the spinal cord. The results imply that the Brn3 proteins contribute to sensory neuron diversity by participating in a combinatorial code of transcriptional regulation and that there are deep functional similarities in transcriptional circuits across diverse sensory systems.

Materials and Methods

Mouse lines

The following lines were previously described: (a) Cre lines: Sox2Cre (Hayashi et al., 2002), Pax6αCre (Marquardt et al., 2001), ROSA26CreER (Badea et al., 2003), NFL-CreER, (Rotolo et al., 2008), R26rtTACreER (Badea et al., 2009b), (b) conventional knock-out lines: Brn3a (Xiang et al., 1996) Brn3b (Gan et al., 1996) and Brn3c (Xiang et al., 1997), and (c) conditional knock-in alleles: Brn3aCKOAP and Brn3bCKOAP (Badea et al., 2009a). The Brn3cCKOAP conditional allele was generated by homologous recombination in mouse embryonic stem cells using standard techniques. For the targeted allele, the following changes were made: a loxP site was inserted in the 5′UTR 50 bp 5′ before the initiator ATG; three repeats of the SV40 early region transcription terminator were added to the 3′UTR 600 bp 3′ of the Brn3c translation termination codon, followed by a second loxP site and the coding region of human placental alkaline phosphatase (AP). A positive selection cassette (PGK-Neo), flanked by frt sites, followed the AP coding region, and was subsequently removed by crossing to mice expressing Flp recombinase in the germline, as previously described (Badea et al 2009a).

Sparse recombination

For methods related to sparse Cre-mediated recombination, see Badea et al. (2003), Rotolo et al. (2008), Badea et al. (2009b), and Badea and Nathans, (2011). For each of the three Brn3 genes, timed matings between Brn3+/−;R26rtTACreER/+ males and Brn3CKOAP/CKOAP females were set, conception date was determined by examining the copulation plug, and pregnant females were moved to cages with food pellets containing Doxycyline (1.75 mg/gram) at gestational day 3. At gestational day 9, 200 μg of 4-hydroxytamoxifen (4HT) in sunflower seed oil vehicle was delivered by intraperitoneal (IP) injection, and the doxycycline diet was continued until gestational day 11. P1–P4 pups were used for skin AP histochemistry (Figure 57), whereas adults were used for spinal chord AP histochemistry (Figure 7). For visualizing somatosensory afferents in Brn3CKOAP/+;NFL-CreER mice (Figure 5), females were injected IP with 0–200 ug 4HT at gestational day 14–17, and mice were analyzed at P1–P3. DRG immunostaining of P5 Brn3aCKOAP/+;R26CreER DRGs was performed using mice that were not exposed to 4HT, taking advantage of the background rate of Cre-mediated recombination in the absence of 4HT.

Figure 5
Individual Brn3aAP/+, Brn3bAP/+, and Brn3cAP/+ somatosensory arbors visualized histochemically following sparse Cre-mediated recombination
Figure 7
Loss of Brn3a but not Brn3b or Brn3c causes a loss of dorsal horn projections and hair follicle-associated sensory endings in the skin

Histology

Spinal chord and brain vibratome sections (typically 200 um thickness) were fixed, AP stained, processed, and imaged as previously described (Badea et al., 2003; Badea et al., 2009a). For vibratome sections of early postnatal pups, eviscerated torsos were fixed overnight in PBS/4% paraformaldehyde at 4° C, and then decalcified for ~4 days in 50 mM EDTA at 4° C. Skin flat mounts were prepared by pinning dissected skin (external surface downward) to a Sylguard surface with insect pins and fixing overnight in PBS/4% paraformaldehyde at 4° C. Retina sections were processed and immunostained as previously described (Badea et al., 2009a). For spinal chord and DRG immunostaining, adult or early postnatal mice were perfused intracardially with PBS/4% paraformaldehyde, the vertebral column dissected, decalcified, cryoprotected in OCT, and sectioned at 14 mm thickness on a cryostat. Complete spinal cords with attached DRGs from 2–4 mice of the same genotype were cut transversely into 4 segments of equal length and these 8–16 segments were embedded together in a single block. For each immunostaining analysis cells were counted from 6–8 sections at cervical, thoracic, and lumbar levels. No significant differences in patterns of dorsal horn lamination or the frequency of Brn3a-, Brn3b-, or Brn3c- expressing DRG neurons were noted across spinal cord levels with the markers used in this study. High resolution images were captured on a Zeiss Imager.Z1 fitted with an Apotome for fluorescent imaging and Axiovision software. Skin afferents neurons were imaged with a black and white Axiocam camera using DIC/Nomarsky optics, and neuronal arbors were reconstructed using Neuromantic neuronal tracing freeware (Darren Myat, http://www.reading.ac.uk/neuromantic), and exported to the rotater visualization software using scripts written in Matlab (Mathworks).

Antibodies

Rabbit polyclonal anti-Brn3a, anti-Brn3b and anti-Brn3c antisera are described in Xiang et al. (1995). For double immunostaining with rabbit antibodies to Brn3 proteins and cytoplasmic markers, the anti-Brn3 immunostaining was performed first, the patterns of nuclear immunolabeling were captured, and then the anti-cytoplasmic marker immunostaining was performed and the labeling pattern compared to the earlier image. The sources of commercial antibodies are: sheep anti-AP (American Research Products, Belmont, MA); rabbit anti-Neurofilament 200, anti-Peripherin, and anti-TrkA, and mouse monoclonal anti-Brn3a (MAB1585; Millipore, Temecula, CA); rabbit anti-Parvalbumin (Swant, Bellinzona, Switzerland); guinea pig anti-CGRP (Bachem, San Carlos, CA); rabbit anti-PKCγ (Santa Cruz Biotech, CA). Secondary antibodies were donkey anti-sera coupled with Alexa dyes (Invitrogen/Life Technologies, Carlsbad, CA). Isolectin IB4 Conjugates were from Invitrogen/Life Technologies (Eugene, OR).

Results

Central projections of Brn3c-expressing RGCs visualized by expression of alkaline phosphatase from the Brn3c locus

To eliminate Brn3c function via Cre-mediated recombination and to simultaneously visualize individual neurons expressing the recombined Brn3c allele, one loxP site was inserted in the Brn3c 5′ untranslated region (UTR), a second loxP site was inserted 3′ of the Brn3c transcription termination signal, and an alkaline phosphatase (AP) reporter coding region was inserted distal to the 3′ loxP site (Figure 1A). Expression of the AP reporter is activated by Cre-mediated deletion of the Brn3c coding region and 3′UTR, bringing AP under the control of the Brn3c promoter. The same “conditional knockout with AP” (CKOAP) strategy was previously applied to the Brn3a and Brn3b genes (Badea et al., 2009a). When the conditional allele is placed over a WT allele (Brn3cCKOAP/+), Cre-mediated recombination generates phenotypically normal Brn3cAP/+ cells. When the conditional allele is placed over a conventional null allele (Brn3cCKOAP/−; Xiang et al. 1997), Cre-mediated recombination generates phenotypically mutant Brn3cAP/− cells. In both cases, the AP reporter permits histochemical and immunocytochemical visualization of cell bodies and arbor morphologies. Figure 1B shows the co-localization of nuclear Brn3c and plasma membrane-anchored AP in Brn3cAP/+ RGCs (upper panel), and the loss of Brn3c protein with the retention of AP expression in Brn3cAP/− RGCs (lower panel).

Figure 1
Conditional Brn3c allele with an AP reporter reveals central targets of Brn3c-expressing RGCs

In earlier work, we used Brn3aCKOAP and Brn3bCKOAP alleles to determine the central projections of RGCs expressing these two genes. A similar analysis with Brn3cCKOAP, using the retina-specific Pax6αCre transgene (Marquardt et al., 2001), shows projections to the lateral geniculate nucleus and superior colliculus (Figure 1C, panels f–j, and l). In contrast to Brn3a- and Brn3b-expressing RGCs, Brn3c-expressing RGCs do not project to the medial terminal nucleus (MTN) nor do they contribute to the lateral terminal tract, both of which are involved in eye movement control as part of the accessory optic tract (AOT; compare Figure 1C, panels a–e vs. f–j). Also in contrast to Brn3b-expressing RGCs, Brn3c-expressing RGCs bypass the suprachiasmatic nucleus (SCN; Figure 1C, panel k). An analysis of the dendritic morphologies of both Brn3cAP/+ and Brn3cAP/− RGCs is reported in Badea and Nathans (2010); loss of Brn3c appears to have little or no effect on the survival or morphology of these RGCs (see also Wang et al., 2002).

The effect of mutations in each Brn3 gene on the expression of other Brn3 family members in RGCs

A high degree of functional similarity among the members of the Brn3 family is suggested by (1) the near identity of the DNA binding domains of Brn3a, Brn3b, and Brn3c, (2) the ability of the Brn3a coding region to substitute for the Brn3b coding region to permit RGC survival (Pan et al., 2005), and (3) the partial redundancy of Brn3b and Brn3c in mediating RGC survival and intra-retinal axon guidance (Wang et al., 2002). The extensive overlap in Brn3a, Brn3b, and Brn3c expression in RGCs (Xiang et al., 1995; Badea et al., 2009a) and the evidence that Brn3a negatively regulates its own transcription in DRG neurons (Trieu et al., 2003) led us to ask whether there might be cross-regulation among Brn3 family members in those neurons in which two or more Brn3 genes are expressed. As an initial test of this idea, we have immunolocalized each Brn3 protein together with AP in RGCs of genotypes Brn3aAP/+, Brn3aAP/−, Brn3bAP/+, Brn3bAP/−, Brn3cAP/+, and Brn3cAP/−. For this analysis, the Pax6αCre transgene was used to generate retina-specific Cre-mediated recombination at ~E9.5 (Marquardt et al., 2001). For the Brn3 null mutant cells (i.e. Brn3AP/−), the AP reporter identifies those cells that were programmed to express the particular Brn3 gene but from which that gene’s coding region has been deleted. Data for Brn3a and Brn3b null RGCs are described in Badea et al (2009a); Figure 2A and B presents the complete data set for all three Brn3 genes.

Figure 2
Expression of Brn3a, Brn3b, and Brn3c in Brn3aAP/+, Brn3aAP/−, Brn3bAP/+, Brn3bAP/−, Brn3cAP/+, and Brn3cAP/− RGCs and DRG neurons determined by anti-AP and anti-Brn3 double-immunostaining

As expected, all AP+ RGCs from Brn3aAP/+, Brn3bAP/+, and Brn3cAP/+ retinas also exhibit nuclear immuno-reactivity for the corresponding Brn3 family member, and all AP+ RGCs from Brn3aAP/−, Brn3bAP/−, and Brn3cAP/− retinas lack nuclear immuno-reactivity for the corresponding Brn3 family member (Figure 2A and B). With respect to cross-regulation, loss of Brn3b has little effect on expression of Brn3a or Brn3c, and loss of Brn3c has little effect on expression of Brn3a or Brn3b. However, among Brn3aAP/− RGCs there is a statistically significant increase in the fraction of cells expressing Brn3c (P=0.0013) and a modest decrease that does not rise to statistical significance in the fraction of cells expressing Brn3b (P=0.20). These observations suggest that Brn3a may normally suppress Brn3c expression in a subset of RGCs.

Expression patterns of Brn3 family members in DRG neurons

We also determined the patterns of co-expression of Brn3 family members in DRG neurons. Figures 2C and D show the patterns of co-localization in Brn3aAP/+, Brn3bAP/+, and Brn3cAP/+ DRG neurons at P5 from Brn3aCKOAP/+;R26CreER, Brn3bCKOAP/+;R26CreER or Brn3cCKOAP/+;R26CreER mice that had undergone sparse Cre-mediated recombination. We note that at P5 as many as 10% of AP+ DRG neurons are not detectably immunostained for the transcription factor that corresponds to the AP knock-in allele (e.g. 10% of Brn3bAP/+ neurons are not detectably stained with anti-Brn3b antibodies; Figure 2D). We ascribe this apparent discrepancy to the higher sensitivity of AP compared to Brn3 immunostaining and the wide variation in Brn3 protein levels per cell. In comparing the patterns of Brn3 expression, RGCs and DRG neurons show several intriguing similarities: (1) there are far fewer Brn3c-than Brn3a- or Brn3b-expressing neurons, (2) many neurons express both Brn3a and Brn3b, and (3) among Brn3c-expressing neurons, a larger fraction co-express Brn3a than Brn3b (Figure 2A–D). The principal difference between RGC and DRG expression patterns is that, among DRG neurons, Brn3a is expressed in nearly all neurons and Brn3b and Brn3c are rarely co-expressed.

A comparison of Brn3 gene expression patterns in Brn3aAP/+ vs. Brn3aAP/−, Brn3bAP/+ vs. Brn3bAP/− and Brn3cAP/+ vs. Brn3cAP/− DRGs at P0–P1 indicates that there is little cross-regulation among Brn3 family members; that is, loss of one Brn3 family member does not lead to repression or induction of other family members (Figure 2E and F). In comparing P0/P1 vs P5 DRG neurons (Figure 2C–F), we observe a progressive increase in the fraction of Brn3aAP/+ neurons that also express Brn3b (7/81 neurons at P0/P1 vs 16/42 neurons at P5; P=0.0002), indicative of ongoing postnatal refinement within this subset of DRG neurons.

To relate the expression of Brn3 family members to functional DRG neuron subtypes during early postnatal development, we performed double labeling for a series of molecular markers and for AP in P5 Brn3aAP/+, Brn3bAP/+, and Brn3cAP/+ DRGs (Figure 3A–C). The same markers were also analyzed in adult WT DRGs using anti-Brn3a, anti-Brn3b, and anti-Brn3c antibodies (Figure 3D and data not shown). The DRG markers consist of NF-200 (mechanoreceptors and proprioreceptors with large axon diameters), parvalbumin (proprioceptors), TrkA (small and medium diameter unmyelinated nociceptors and mechanoreceptors), CGRP (peptidergic nociceptors), IB4 (nonpeptidergic nociceptors), and peripherin (nociceptors and mechanoreceptors). TrkA is expressed in and required for the development of both peptidergic nociceptors (Marmigère and Ernfors, 2007) and a variety of mechanoreceptors (Fundin et al., 1997b; Cronk et al., 2002; Sedý et al., 2004).

Figure 3
Correlation between Brn3a, Brn3b, and Brn3c expression and DRG neuron subtype as defined by molecular markers

As summarized in Figure 3C and 3D, the patterns of labeling at P5 and in the adult are distinctive for each Brn3 family member. Within the diverse set of Brn3a-expressing DRG neurons, all six markers are represented, both at P5 and in the adult. A seventh marker, tyrosine hydroxylase, which is present in 10–15% of adult mouse DRG neurons (Brumovsky et al., 2006), showed no co-labeling at P5 with Brn3bAP/+ and Brn3cAP/+ DRG neurons. Among 124 Brn3aAP/+ neurons and 50 tyrosine hydroxylase-expressing neurons, only one neuron was possibly double-positive. At P5, Brn3b-expressing DRG neurons express only NF-200, peripherin, and TrkA at appreciable frequency, and Brn3c-expressing neurons express only TrkA and CGRP at appreciable frequency.

In comparing P5 and adult DRGs, the main differences are (1) an increase among all three classes of adult Brn3-expressing DRG neurons in the proportion that express NF200, an effect that is most dramatic among Brn3c-expressing neurons, (2) a conversion of all or nearly all Brn3c-expressing neurons from peripherin-negative to peripherin-positive, (3) an increase in the percentage of Brn3c-expressing neurons that express CGRP from ~55% to ~85%, and (4) a decline in the proportion of Brn3a- and Brn3b-expressing neurons that express TrkA, with expression of TrkA persisting in a majority of Brn3c-expressing neurons. These changes between P5 and adulthood suggest that Brn3c-expressing neurons mature relatively late. Taken together, this analysis indicates that Brn3a-expressing DRG neurons encompass many, but not all, of the major classes of somatosensory neurons, Brn3b-expressing DRG neurons likely correspond to mechanoreceptors, and Brn3c-expressing DRG neurons likely correspond to peptidergic nociceptors.

Distinctive laminar targets in the dorsal horn of the spinal cord for Brn3a-, Brn3b-, and Brn3c-expressing DRG neurons

The correlations described in the preceding paragraph imply that Brn3a-expressing DRG neurons should project to many laminae in the dorsal horn of the spinal cord, whereas Brn3b- and Brn3c-expressing DRG neurons should project to only one or a few laminae. To test these predictions, the projections of Brn3aAP/+, Brn3bAP/+, and Brn3cAP/+ DRG neurons in the adult dorsal horn were visualized using AP immunostaining together with immunostaining for CGRP, IB4, or PKC-gamma, markers that define, respectively, peptidergic nociceptive laminae I and IIo, a nonpeptidergic nociceptive zone between laminae IIo and IIi, and mechanoreceptive lamina IIi (Zylka et al., 2005; Neumann et al., 2008). The central projections of Brn3a-expressing DRGs were distributed throughout the dorsal horn (Figure 4A and B, and and7A),7A), consistent with the broad expression of Brn3a in DRG neurons. In contrast, the central projections of Brn3b- and Brn3c-expressing DRG neurons were narrowly targeted to the outer half of the PKC-gamma lamina and to the CGRP lamina, respectively (Figure 4C–H, and and7A).7A). These data are consistent with an assignment of Brn3b-expressing DRG neurons as likely mechanoreceptors, based on the assignment of the PKC-gamma lamina as a target area for non-noxious stimuli (Neumann et al., 2008), and Brn3c-expressing DRG neurons as likely peptidergic nociceptors.

Figure 4
Axons of Brn3a-, Brn3b-, and Brn3c-expressing DRG neurons target distinct territories in the dorsal horn of the spinal cord

Visualizing afferent arbors of Brn3a-, Brn3b-, and Brn3c-expressing DRGs

Among somatosensory neurons, the integration of physiological, morphological, and molecular properties is most advanced in the context of pain, temperature, and itch sensation mediated by TRP channels and the Mrgprd class of GPCRs (Lumpkin and Caterina, 2007; Basbaum et al., 2009; Zylka et al., 2005; Dussor et al., 2008; Cavanaugh et al., 2009; Rau et al., 2009; Liu et al., 2009). Largely missing, thus far, has been a morphologic analysis of somatosensory afferents beyond the structures of individual sensory terminals. In particular, there is very little information regarding the size and geometry of the afferent arbors of individual somatosensory neurons. The paucity of data reflects several factors: (1) somatosensory afferents typically travel long distances from their cell bodies, thus hampering their visualization following injection of the soma with intracellular tracers; (2) immunostaining for somatosensory proteins or staining with non-specific methods such as silver impregnation generally reveals a complex meshwork of afferent processes from which individual arbors cannot be reconstructed, and (3) many of the tissues within which sensory afferents reside – skin, muscle, the viscera, and connective tissue – are relatively thick and refractile, making it difficult to immunostain and image them in whole mount preparations. This situation stands in marked contrast to the sophisticated morphologic analyses that have been conducted in the retina, where a relatively thin two-dimensional tissue offers ready access to physiologic recordings, cell filling, and immuno- or histochemical staining in both flat mount and sectioned preparations (Masland, 2001; Dacey et al., 2003; Badea and Nathans, 2004).

To our knowledge, only one published study has used a genetically-directed reporter to visualize the complete arbors of individual somatosensory afferents - that of Liu et al. (2007) in which skin afferents expressed an AP knock-in at the MrgprB4 locus. AP histochemistry lends itself to visualizing skin afferents because of the high sensitivity of the AP reporter, the efficient diffusion of the low-molecular weight AP substrate into relatively thick tissue samples, and the insolubility of the nitro-blue tetrazolium reaction product in the tissue clearing solvent benzyl benzoate:benzyl alcohol (BBBA).

Individual afferent arbors of Brn3a-, Brn3b-, and Brn3c-expressing DRG neurons were visualized following extremely sparse Cre-mediated recombination with either of two CreER lines. Most of the experiments shown in Figure 5 used an IRES-CreER cassette knocked into the 3′ untranslated region of the gene coding for the neurofilament light chain (NFL-CreER; Rotolo et al., 2008). For Figures 5U, ,6,6, and and7,7, CreER was expressed from the ROSA26 locus under the control of the reverse Tet transactivator (rtTA), thus providing pharmacologic control over both transcription and nuclear translocation of Cre recombinase (R26rtTACreER; Badea et al., 2009b). As described below, NFL-CreER appears to be expressed in many types of DRG neurons as judged by the diversity of labeled Brn3a-expressing afferents in skin and muscle and the labeling of central Brn3a-expressing projections throughout the dorsal horn of the spinal cord. This conclusion is also supported by the high degree of similarity in afferent arbor patterns observed between the NFL-CreER and R26rtTACreER data sets; the latter presumably derives from ubiquitous expression of CreER.

Figure 6
Three-dimensional reconstructions of afferent arbors from Brn3a-, Brn3b-, and Brn3c- expressing DRG neurons in P1–P3 skin

In Figure 5, Brn3aCKOAP/+;NFL-CreER, Brn3bCKOAP/+;NFL-CreER, and Brn3cCKOAP/+;NFL-CreER fetuses were exposed to 0–200 ug 4HT during late gestation and then abdominal skin from these mice at P0–P3 was processed for AP histochemistry. Neonatal rather than adult skin was analyzed because the pigment content, large size, high density of adult hair follicles interfere with imaging of labeled afferents. In the mouse, many mechanoreceptors that innervate the skin appear to be physiologically and anatomically mature by the early postnatal period (Woodbury et al., 2001; Woodbury and Koerber, 2007). Figure 5A illustrates the specificity of AP expression in a 200 um vibratome section through a P1 Brn3aCKOAP/+;NFL-CreER mouse. AP activity is restricted to DRG cell bodies, afferent fibers in the dorsal roots, projections throughout the dorsal horn of the spinal cord, and occasional multipolar neurons within the spinal cord. Figure 5B–E illustrates the diversity of non-dermal afferents labeled in Brn3aCKOAP/+;NFL-CreER mice. Labeled afferents in the P1 foot, adult esophagus, and P1 abdominal wall terminate in enlarged structures, presumably tendon organs (foot) or muscle spindles (esophagus and abdominal wall; Figure 5B, D, and E). In the adult diaphragm, the termini of the highly branched afferents are not obviously enlarged (Figure 5C).

In flat mounts of Brn3aCKOAP/+;NFL-CreER P1 skin, AP-stained arbors vary in size from single dense C-shaped endings associated with hair follicles (likely lanceolate endings; Figure 5F and L) to arbors with >100 branches and a diameter of ~1 mm (Figure 5M and N). A range of intermediate sizes is also seen (Figure 5G–K), as well as individual arbors with both lanceolate and non-lanceolate endings (e.g. Figure 5M). Similar analyses of Brn3bCKOAP/+;NF-LCreER P1 skin show a more limited range of labeled arbor types, with few very large arbors and many arbors with single lanceolate endings or with a mixture of lanceolate and non-lanceolate endings (Figure 5O–T), as well as sensory endings in the glabrous skin of the footpad (Figure 5U). Brn3b is also expressed in all or nearly all spiral ganglion neurons, as judged by the density of fibers targeting the organ of Corti in a whole mount Brn3bAP/+ P1 cochlea (Figure 5V). Brn3cCKOAP/+;NFL-CreER P1 skin flat mounts show an even further reduction in the morphologic diversity of labeled sensory arbors, with almost all labeled afferents exhibiting a coarsely branched structure devoid of follicle-associated endings (Figure 5W–Y). In Brn3cCKOAP skin, AP is expressed at low level in a semi-circle of cells – presumably Merkel cells - surrounding each guard hair (red arrows in Figure 5W–Y). These AP-expressing cells are not contiguous with labeled afferents and they are readily distinguished from the more compact semicircular follicle-associated C-shaped endings (e.g. Figures 5O–T). AP expression in Merkel cells appears to derive from low-level read-through transcription into the AP coding region at the unrecombined Brn3cCKOAP locus; a similar pattern of low-level read-through is seen for Brn3aCKOAP and Brn3bCKOAP loci in RGCs.

Three dimensional reconstruction and morphologic classification of afferent arbors in the skin

To more rigorously characterize the morphologies of individual sensory arbors, we have reconstructed representative examples from sparsely labeled Brn3aCKOAP/+;R26rtTACreER/+ and Brn3bCKOAP/+;R26rtTACreER/+ abdominal skin at P1 (Figure 6). The colored outlines in Figure 6 indicate the patterns of Brn3a, Brn3b, and/or Brn3c expression among the various morphologic classes. Arbors from DRG neurons that express Brn3c, as well as a subset that express Brn3a, cover a large area, branch sparsely, stratify either broadly within the dermis (Figure 6A) or narrowly near the dermal-epidermal boundary (Figure 6B), and do not make contacts with hair follicles. Arbors from DRG neurons that express Brn3b, as well as a subset that express Brn3a, branch more densely and, in most cases, make one or more C-shaped contacts with hair follicles (Figure 6C–H). Arbors from DRG neurons that express Brn3a exclusively are diverse, including examples that cover a large area with numerous dense branches, stratify either narrowly near the dermal-epidermal boundary (Figure 6J) or more deeply (Figure 6I and L), and make few or no contacts with hair follicles. Also in the Brn3a-only group are arbors that contact hair follicles at more than one depth (Figure 6K). The great diversity of dermal and non-dermal sensory afferents that express Brn3a is consistent with the nearly ubiquitous expression of Brn3a in DRG neurons (Figures 2 and and3)3) and the large number of dorsal horn laminae targeted by these neurons (Figures 4, ,5A,5A, and and7A7A).

Loss of Brn3a but not Brn3b or Brn3c causes a loss of DRG neuron subtypes

Previous genetic analyses of Brn3a function in the somatosensory system have been restricted to the prenatal period because Brn3a−/− mice die shortly after birth. Here we present a postnatal analysis of Brn3a function, based on the availability of the Brn3aCKOAP allele, which permits individual, histochemically marked Brn3aAP/− neurons to be produced by sparse Cre-mediated recombination at any time during development in Brn3aCKOAP/−;R26rtTACreER/+ embryos. Since the vast majority of Brn3a-expressing neurons do not undergo Cre-mediated recombination with the doxycycline and 4HT regimen used here (see Materials and Methods), the resulting mice can be studied at any postnatal age. Moreover, the phenotypes exhibited by individual Brn3aAP/− DRG neurons in the Brn3aCKOAP/−;R26rtTACreER/+ background are presumably cell autonomous since almost all of the neighboring neurons are phenotypically WT. This last point is of special relevance because the massive neuronal loss observed in Brn3a−/− DRGs would be expected to produce cell-nonautonomous effects arising from reduced competition with neighboring DRG neurons for target innervation, aberrant axonal pathfinding by neighboring mutant neurons, or a large local concentration of apoptotic cells. While cell-nonautonomous effects are of interest in their own right, experiments conducted in Brn3a−/− mice cannot distinguish autonomous from non-autonomous effects.

As an initial step in defining the cell-autonomous phenotypes associated with loss of Brn3a, Brn3b, and Brn3c, we compared the territories targeted by AP-expressing DRG neurons in the dorsal horn of adult Brn3aCKOAP/+;R26rtTACreER vs. Brn3aCKOAP/−;R26rtTACreER, Brn3bCKOAP/+;R26rtTACreER vs. Brn3bCKOAP/−;R26rtTACreER, and Brn3cCKOAP/+;R26rtTACreER, vs. Brn3cCKOAP/−;R26rtTACreER/+ spinal cords under conditions of sparse Cre-mediated recombination (Figure 7A). While no differences were seen between Brn3bAP/+ and Brn3bAP/− projections or between Brn3cAP/+ and Brn3cAP/− projections, Brn3aAP/+ and Brn3aAP/− projections differed markedly, with the latter showing a large zone of hypo-innervation (red arrowhead in Figure 7A). Similarly, in early postnatal skin, a comparison between Brn3bAP/+ and Brn3bAP/− arbors or between Brn3cAP/+ and Brn3cAP/− arbors showed no apparent differences in density or in the types of morphologies, whereas a comparison between Brn3aAP/+ and Brn3aAP/− skin showed a reduced density of AP+ arbors in the Brn3aAP/− sample (data not shown). Strikingly, the morphologies of Brn3aAP/− arbors represent only a subset of the Brn3aAP/+ arbor morphologies. In particular, the Brn3aAP/− skin is nearly devoid of AP+ C-shaped endings that contact hair follicles, a difference that is apparent even when comparing regions in which the overall density of AP+ arbors in the Brn3aCKOAP/−;R26rtTACreER skin is comparable or greater to that in the Brn3aCKOAP/+;R26rtTACreER control skin (Figure 7B and D). Many of the AP+ arbor morphologies that remain in Brn3aCKOAP/−;R26rtTACreER skins conform to types observed in control Brn3aCKOAP/+;R26rtTACreER skins, as seen, for example, in comparing Figures 6C and and7C.7C. Thus loss of Brn3a leads to a selective loss and/or fate switch of hair follicle-associated terminals.

Discussion

The experiments described here, together with those reported in Badea et al. (2009a) and Badea and Nathans (2011), use a set of three Brn3 null alleles and three AP-expressing Brn3 conditional alleles, together with sparse Cre-mediated recombination, to define the overlapping patterns of Brn3 gene expression in RGCs and somatosensory neurons, the manner in which the patterns of gene expression correlate with morphologically distinct neuronal types, and the consequences for these neurons of deleting each Brn3 gene. Among RGCs, we observe that Brn3 gene expression correlates with the patterns of dendritic arborization and lamination in the inner plexiform layer and the patterns of axonal projections to retinorecipient targets in the brain. Among somatosensory neurons we observe subtype-specific expression of Brn3b and Brn3c. We also demonstrate the general utility of sparse, genetically-directed reporter expression and histochemical labeling for visualizing individual somatosensory afferent arbors, an approach that should aid in classifying somatosensory neurons and determining the sizes and structures of their receptive fields.

Brn3 gene expression and somatosensory neuron identity

The essential role of Brn3a in the development and survival of somatosensory neurons was appreciated in the initial analyses of Brn3a−/− mice (McEvilly et al. 1996; Xiang et al., 1996). Subsequent work has refined this picture by characterizing the down-regulation of a variety of somatosensory cell-type-specific molecular markers, including Trk receptors, in the DRG and TG of Brn3a−/− mice (Huang et al., 1999; Ma et al., 2003; Ichikawa et al., 2002, 2004, 2005), by demonstrating defects in pathfinding of somatosensory afferents (Eng et al., 2001), and by showing that, either directly or indirectly, Brn3a represses genes associated with neurogenesis and induces genes associated with terminal differentiation, including Runx1 and Runx3 (Lanier et al., 2009; Dykes et al., 2010). The nearly complete loss of hair follicle-associated endings observed here among Brn3aAP/− afferents is consistent with the idea that Brn3a controls a program of Runx and Trk receptor expression that is required for mechanosensory neuron development and/or survival (Dykes et al., 2010).

Prior to the present work, the relationship between Brn3b and Brn3c expression and somatosensory neuronal subtypes was largely unexplored. Although the immunohistochemical profiles of DRG cell bodies, morphologies of afferent arbors in the skin, and locations of target laminae in the dorsal horn suggest that Brn3b is expressed principally in hair follicle-associated mechanoreceptors and that Brn3c is expressed principally in peptidergic nociceptors, a definitive assignment of sensory types must await an electrophysiologic characterization of the stimulus-response characteristics of these neurons. The persistence of each of these cell types upon elimination of the associated Brn3 protein suggests that, unlike Brn3a, Brn3b and Brn3c may play a relatively subtle role in the somatosensory system, perhaps conferring aspects of molecular identity, but not determining subtype-specific morphology, central target specificity, or survival.

Genetically-directed sparse labeling as a tool for characterizing neuronal morphology in mice

Our analyses of Brn3 expression and function have relied on the use of sparse Cre-mediated Brn3 deletion coupled to activation of an AP reporter. This strategy offers several experimental advantages over the use of conventional null alleles or conditional alleles that do not generate marked cells. First, it permits mosaic mice to survive beyond the age at which homozygous null mutants die. For Brn3a−/− mice, death occurs in the neonatal period, a severe limitation for the analysis of retinal development, much of which occurs postnatally. Second, the sparse recombination strategy limits the phenotypic analysis to cell-autonomous effects, since the vast majority of the cells surrounding the rare AP/− cell are phenotypically WT. As noted in the Results section, in Brn3a−/− embryos the large numbers of dying DRG and TG neurons and/or the reduced competition for peripheral and central targets among the surviving DRG and TG neurons could produce cell-nonautonomous effects. A similar argument applies to the Brn3b−/− retina, where ~70% of RGCs eventually die. Third, by using pharmacologic control of Cre recombinase – in the present study, a dual doxycycline and 4HT strategy (Badea et al., 2009b) – the rare recombination events that lead to loss of gene function can be precisely timed. Finally, the sparse recombination method permits single cell morphologic analysis by revealing a Golgi-like image of neuronal processes, either in AP/+ or AP/− cells, the latter being null for the protein of interest but expressing the AP reporter by virtue of continued activity of the Brn3 promoter. With respect to sparse visualization of somatosensory afferents and central projections, the combination of Brn3aCKOAP and NFL-CreER might be generally useful as a tool for surveying these structures in the context of surgical, toxicologic, genetic, or other experimental perturbations.

Combinatorial expression of the Brn3 family in the visual, somatosensory, and auditory/vestibular systems

Figure 8 summarizes the patterns of Brn3a, Brn3b, and Brn3c expression in the WT inner ear, retina, and DRG/TG. The Venn diagrams in Figure 8A illustrate both the relative numbers of neurons expressing each Brn3 gene and the extent of overlap of Brn3 gene expression among these neurons. In most cases, the Brn3 genes are expressed in the projection neurons that communicate information from the primary sensory structures to the brain or spinal cord (Figure 8B–D), the two exceptions being the expression of Brn3c in vestibular and auditory sensory hair cells (Figure 8C) and in Merkel cells (Figure 5W–Y). The general pattern of expression in projection neurons applies regardless of whether the Brn3 expressing cells are or are not primary sensory neurons or whether they are derived from surface ectoderm (spiral and vestibular ganglia), the neuroectoderm (DRG/TG), or neural tube (retina). Conceptualized in this manner, the logic of Brn3 expression suggests an evolutionary equivalence of RGCs, primary somatosensory neurons, and auditory and vestibular ganglion neurons.

Figure 8
Combinatorial expression of Brn3a, Brn3b, and Brn3c in projection neurons in the visual, auditory/vestibular, and somatosensory systems

How do the patterns of Brn3 gene expression relate to the evolution of increasingly complex sensory structures? It is reasonable to suppose that the primordial architecture of all sensory neurons initially resembled that seen in present-day nociceptors: the neuron that was the primary receiver of sensory information also projected directly to a central target. Following this logic, we imagine that the two outer layers of neurons in the tri-layered vertebrate retina represent a later addition of specialized photoreceptors and interneurons. Consistent with this view, present-day vertebrate retinas contain a minor class of intrinsically photosensitize RGCs (ipRGCs). The ipRGCs mediate non-image forming visual functions such as circadian entrainment and pupil constriction (Güler et al., 2008), and they may represent the last relics of a primordial monolayer retina. Analogous arguments apply to the auditory and vestibular systems: primordial mechanosensory neurons may have projected directly to their central targets, and only later acquired a specialized epithelial partner – hair cells in the auditory/vestibular system and a variety of specialized epithelial cells in mechanosensory end organs in the skin, muscle, tendons, and viscera – to enhance their sensitivity. In this regard, it is striking that Brn3c is not only expressed in auditory hair cells (Xiang et al., 1997) but is also expressed in Merkel cells (red arrows in Figure 5W–Y). This serendipitous finding is consistent with a growing body of evidence demonstrating that Merkel cells – specialized skin epithelial cells that synapse onto mechanosensory afferents -share molecular, morphologic, and functional similarities with auditory hair cells (Lumpkin and Caterina, 2007). As Brn3c is only required in the final stages of inner ear sensory hair cell differentiation (Xiang et al., 1998), these data suggest that Brn3c may play a general role in promoting the mechanosensory differentiation of specialized epithelial cells.

A further inference from the suggested evolutionary equivalence of RGCs, primary somatosensory neurons, and auditory and vestibular ganglion neurons is that there may be a corresponding equivalence in the logic of information processing at their initial CNS targets. Thus, the separation of somatosensory sub-modalities by targeting of DRG axons to distinct laminae in the dorsal horn of the spinal cord may be analogous to the separation of visual information streams produced by targeting of RGC axons to different retino-recipient regions in the diencephalon, or to the separation of auditory and vestibular inputs by targeting of spiral and vestibular ganglion axons to the cochlear and vestibular nuclei in the brainstem.

Functions analogous to those of mammalian Brn3 genes are seen in the single Brn3 homologues in C. elegans (Unc86) and D. melanogaster (Acj6). Unc86 is required both for the production and differentiation of primary touch sensitive neurons and the correct functioning of the chemosensory AIZ interneurons (Duggan et al., 1998; Sze and Ruvkin, 2003). Acj6 is required for specifying the identity of primary olfactory receptor neurons, at the level of olfactory receptor gene choice and at the level of axon targeting to the appropriate glomerulus (Bai et al., 2009; Komiyama et al., 2004). Intriguingly, differential splicing of the Acj6 gene generates multiple isoforms with distinct activities in olfactory receptor neuron specification (Bai and Carlson, 2010). Initial steps have been taken to identify transcriptional targets of Unc86, Acj6, and the mammalian Brn3s (Erkman et al., 2000; Eng et al., 2004, 2007; Mu et al., 2004; Lanier et al., 2009; Dykes et al., 2010). It will be of great interest to compare these targets both between species and across sensory modalities.

Acknowledgments

The authors thank Rocky Cheung, Sumit Kumar and Yanshu Wang for help with histology and immunostaining, and Xinzhong Dong, David Ginty, Qin Liu, and Ting Guo for helpful discussions and/or comments on the manuscript. Supported by the Howard Hughes Medical Institute and the National Institutes of Health.

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