Background

General somatic sensation is conveyed to the central nervous system at cranial levels by the trigeminal ganglion (TG), and at spinal levels by the dorsal root ganglia (DRG). Although these ganglia have similar functions, they have distinct embryological origins, in that both contain neurons originating from the neural crest, while only the TG includes cells derived from the placodal ectoderm.

Results

Here we use microarray analysis of E13.5 embryos to demonstrate that the developing DRG and TG have very similar overall patterns of gene expression. In mice lacking the POU-domain transcription factor Brn3a, the DRG and TG exhibit many common changes in gene expression, but a subset of Brn3a target genes show increased expression only in the TG. In the wild-type TG these Brn3a-repressed genes are silent, yet their promoter regions exhibit histone H3-acetylation levels similar to constitutively transcribed gene loci. This increased H3-acetylation is not observed in the DRG, suggesting that chromatin modifications play a role in cell-specific target gene regulation by Brn3a.

Conclusion

These results demonstrate that one developmental role of Brn3a is to repress potential differences in gene expression between sensory neurons generated at different axial levels, and to regulate a convergent program of developmental gene expression, in which functionally similar populations of neurons are generated from different embryological substrates.

Global gene expression is highly conserved in sensory neurons from different axial levels

To begin to compare the molecular program of sensory neuron development at spinal and cranial levels, we performed global analysis of gene expression in the developing DRG and TG. E13.5 was chosen for this comparison because, at this stage, most of the neurons of the DRG and TG have exited the cell cycle and have begun to express definitive markers of neurogenesis. Because sensory neurons have conserved functions at spinal and cranial levels, we hypothesized that the global pattern of gene expression in the DRG would be much more similar to the TG than to other neural tissues. Figure1 illustrates a comparison of gene expression in the DRG to a replicate analysis from the same tissue, to the TG, and to the embryonic neocortex. These results show a high degree of overall similarity in gene expression between the sensory ganglia from different axial levels, and confirm that there is much greater divergence between the patterns of gene expression in the DRG and the developing cerebral cortex.

Overall, only a very small fraction of the approximately 44,000 transcripts assayed by the array showed profound differences in expression between the DRG and TG (Table1). Significantly more transcripts were uniquely expressed in the DRG, and seven out of ten of the transcripts with the highest relative expression in the DRG encoded Hox transcription factors, an expected finding given the axial restriction of Hox expression. Several of the other differentially expressed genes revealed in the microarray analysis could be validated byin situhybridization (seeadditional file 1), although some differences appear to represent anomalous findings that may be related to factors such as high expression in peripheral glia (Ednrb, Sostdc1).

Aside from the Hox factors, the transcript with the greatest relative expression in the DRG was Etv1, an Ets-family transcription factor expressed in 1a muscle spindle afferents [13,21]. Immunofluorescence for Etv1 expression in the E13.5 DRG confirmed expression in sensory neurons of large and intermediate size, whilst expression was entirely absent in the TG at this stage (Figure2a, b). In contrast, expression of Runx3, an earlier and more general marker of proprioceptive neurons [13,14], was noted in both the DRG and TG at E13.5 (Figure2c, d).

By E16.5, Etv1 expression could be detected in some neurons of the TG, as well as the DRG. In the DRG, one population of Etv1-expressing neurons displayed large soma and nuclei consistent with 1a proprioceptors. However, in the TG nearly all of the Etv1-expressing neurons were of intermediate size, and large Etv1-positive neurons consistent with 1a proprioceptors were rare. In both the DRG and the TG, this intermediate-sized and later-developing population of Etv1-expressing sensory neurons could be further distinguished from the 1a proprioceptors by the co-expression of Islet2 (Figure2e, f).

One distinguishing feature of the TG is that a subset of neurons that are functionally part of the trigeminal system, the mesV, is located within the CNS. Examination of the mesV in late gestation revealed large sensory neurons positive for Etv1. These neurons co-expressed atauLacZtransgene integrated into the Brn3a locus, and Brn3a is a known marker of the mesencephalic trigeminal [6]. The position of these neurons in the caudal midbrain is consistent with the role of some of these neurons as muscle spindle afferents for the muscles of mastication [22]. Thus, the exclusive expression of Etv1 in the DRG but not the TG at E13.5 appears to result from a population of Etv1⁺, Islet2^-proprioceptive neurons that are present in the DRG but are largely restricted to the mesV at the cranial level.

Brn3a regulates multiple downstream targets in the DRG

To understand the developmental programs regulated by Brn3a at spinal versus cranial levels, we next analyzed global gene expression in E13.5 DRG from Brn3a mutant embryos and wild-type controls. Tables2 and3 summarize the transcripts most increased and decreased at E13.5 in the DRG of Brn3a knockout mice, and a more complete list appears inadditional file 2.In situhybridization and immunofluorescence were used to verify altered expression of several of the target genes (Figures3 and4), with an emphasis on newly identified Brn3a targets and genes for which expression has not been previously described in the sensory ganglia.

Most of the Brn3a-regulated genes of known function have specific roles in neurotransmission, axonogenesis/synaptogenesis, signal transduction, regulation of developmental pathways, or transcription. Among mediators of neurotransmission, multiple glutamate receptors (Gria3, Gria4, Grik1), a GABA transporter (Slc6a1), a serotonin receptor (Htr3a) and the neuropeptide somatostatin are increased, while a GABA receptor (Gabra2) and neuropeptides with specific sensory roles (Galanin, Adcyap1/PACAP) are decreased. Also reduced is latexin, a secreted carboxypepidase inhibitor with a role in nociception [23].

Profound changes were also observed in genes that encode known or potential mediators of sensory neurogenesis, and axon growth or guidance. Increased transcripts in this class included the cell adhesion molecule Chl1, which has a known role in the growth of cortical dendrites [24], and Nel-like 2, an epidermal growth factor (EGF)-repeat containing factor with a previously demonstrated role in sensory development [25]. Increased expression was also noted for several molecules with known or likely roles in axon guidance, including Disabled1, Sema3c, Sema3d, f-spondin, Cadherin22, Protocadherin 8, Protocadherin 17, Dcc, and NetrinG1. Decreased expression was noted for advillin, an actin binding protein that is a known mediator of sensory neurite outgrowth [26], as well as Ncam2 and Eph receptor A7. These changes suggest that the profound defects observed in the sensory axons of Brn3a null mice [17] result from the derangement of a coordinated program of expression of mediators of axon growth and guidance, rather than from a single molecular lesion.

Loss of Brn3a also resulted in significant changes in the expression of other transcription factors. Notably, deletion of Brn3a effectively resulted in a triple-knockout of the Pou4 homeodomain class, because Pou4f2 (Brn3b) expression is nearly eliminated, and Pou4f3 (Brn3c) expression is greatly reduced in Brn3a knockout ganglia. Marked decreases were also observed in specific markers of sensory neuron subclasses, including Prrxl1/DRG11, Runx1 and Runx3. In contrast, specific bHLH genes, including those encoding NeuroD1, NeuroD6, Msc, and Nhlh2, were increased, and, perhaps consistent with this, the inhibitory bHLH factor Id1, which can act to oppose the action of neurogenic bHLH genes, was decreased. The significance of some of these changes in the hierarchy of gene regulation in developing sensory neurons is discussed below.

Loss of Brn3a expression resulted in increased expression of essentially all of the anteriorHoxA,HoxBandHoxCgenes, and the changes reached statistical significance (p< 0.005 orp> 0.995, Materials and Methods) for seven genes in this group (Figure5). The posteriorHoxgenes were expressed at much lower levels than the anterior factors, as expected for brachial-level DRG, and exhibited either unchanged or decreased expression in the knockout. Members of the HoxD cluster were not significantly expressed in the DRG (data not shown), with the exception of HoxD1, which, unlike the other anteriorHoxgene products, exhibited decreased expression in Brn3a null DRG, and was also the onlyHoxgene product expressed at significant levels in the TG (seeadditional file 3) [20].

In prior studies, we have shown that Brn3a regulates its own expression via direct negative feedback, and that, in heterozygous Brn3a knockout mice, partial relief of this negative autoregulation results in increased expression of the intact allele, resulting in nearly complete gene dosage compensation. To determine whether gene dosage compensation is also present at spinal levels, we examined the levels of all increased and decreased transcripts in Brn3a^+/+, Brn3a^+/-and Brn3a^-/-ganglia (see additional files2 and4). In each case, the mean effect of the loss of oneBrn3aallele on target gene expression was 16% to 19% of the effect of the loss of both alleles, compared to the 50% change that would be expected without any compensatory mechanism, indicating significant but incomplete compensation for gene dosage.

A subset of Brn3a targets are unique to the cranial sensory ganglia

Comparison of the regulatory targets of Brn3a in the developing DRG with our prior analysis of the TG [20] suggests that there are many downstream genes in common between these ganglia. However, results from the more advanced microarray used in the present study (Affymetrix 430) cannot be compared directly to the prior TG data derived from an older array (Affymetrix U74v2). To make such a comparison, we performed an additional analysis of these E13.5 DRG samples with the U74v2 array. We also updated the interpretation of the results for the TG U74v2 dataset using annotations from Build 34 of the mouse genome. This analysis confirmed that the DRG and TG of E13.5 Brn3a knockout mice have many conserved changes in gene expression (seeadditional file 3). Transcripts increased in both the DRG and TG of Brn3a knockout mice include the bHLH transcription factors musculin and NeuroD1, the neurotransmitters/receptors somatostatin, CCK-receptor A, 5HT-receptor 3A, and GABA-transporter 1, and the growth factor FIGF/VEGF-D. Conserved decreases include the transcription factors Hmx1, Runx1 and basonuclin, the peptides galanin and PACAP, as well as advillin and latexin.

Although the majority of the downstream targets of Brn3a were conserved in the DRG and TG, a subset of genes was uniquely regulated at each axial level. The most profound differences were observed for a set of genes markedly increased in the TG of Brn3a knockout embryos, and unchanged in the DRG (Figure6). These differentially regulated genes include those encoding the transcription factors Tcfap2b (Ap2β), NeuroD4 (Math3) and Gata3, the calcium binding protein Calb2 (calretinin) and the small GTP-binding protein Rab3b. These results led us to consider possible mechanisms for cell-specific target gene regulation by Brn3a in these two closely related neural tissues.

The cell-type specific effects of transcription factors are generally attributed to distinct complements of interacting partners, or to the expression of related factors that provide redundancy in some cell types but not in others. However, with the exception of the Hox factors, the very similar gene expression profiles of the DRG and TG do not suggest many candidates for distinct Brn3a partners in these neurons. In addition, the loss of Brn3a effectively eliminates expression of all Pou4 class factors in both the DRG and TG, making selective redundancy unlikely.

One approach to understanding the cell-specific effects of the loss of Brn3a expression is suggested by recent work in which we have demonstrated a correlation between Brn3a binding to its target sitesin vivoand H3-acetylation in the vicinity of the potential binding sites [[27] in press]. Given these results, one potential mechanism for the differential regulation of target genes by Brn3a might be distinct modifications of chromatin at the target gene loci in the DRG and TG, which could modulate the effects of Brn3a or downstream regulatory factors. To examine this question for the differentially regulated loci Tcfap2b, NeuroD4 and Gata3, we used chromatin immunoprecipitation (ChIP) to profile histone H3 acetylation in wild-type E13.5 DRG and TG (Figure7). We also examined the Msc locus as an example of a gene that shows similarly increased expression in the DRG and TG of Brn3a knockout mice. ChIP assays of the promoter regions of three constitutively expressed genes,Gapdh,Mapt(tau), andEno2(neuron specific enolase), were used as positive controls, and the results were normalized to negative control assays from the promoter region of theAlb1(albumin) gene, which is not expressed in the nervous system. Because the genomic sequences that regulate these genes in the sensory ganglia have not been defined, we surveyed histone acetylation across these loci using multiple oligonucleotide pairs spaced at 500 to 1,000 base-pair (bp) intervals from approximately -10 kb to +15 kb relative to the start of transcription, using quantitative real-time PCR ('locus-ChIP') ["locus-ChIP", [27] in press].

ChIP profiling of the Msc locus revealed similar levels of H3-acetylation in the DRG and TG. However, ChIP assays of theTcfap2bandNeuroD4loci revealed significantly greater H3-acetylation in the TG relative to the DRG (p= 0.0003 andp= 0.0005, respectively), while the Gata3 locus exhibited a trend (p= 0.09) toward greater acetylation in the TG. As expected, the positive control promoters were highly acetylated, and the negative control,Alb1, was deacetylated in both the DRG and TG. Thus, the state of H3-acetylation in wild-type ganglia appears to be correlated both with basal expression and also with a state of latent potential expression that can be induced in Brn3a null ganglia.

Matings, embryos, and RNA isolation for array analysis

To generate tissue for microarray analysis, timed matings of Brn3a heterozygote animals were performed, and the embryos were harvested at E13.5. Only embryos corresponding to E13.5 ± 0.5 days based on the staging system of Theiler [37] were used for microarray analysis. TG were removed by blunt dissection and carefully freed of adherent non-neural tissue with fine forceps. DRG were isolated by stripping of the spinal cord with its adherent ganglia, followed by dissection of the ganglia with fine forceps. DRG were harvested from brachial region only, including the C5-T1 levels. Dissected ganglia were placed in RNAse inhibitor solution (RNAlater, Ambion, Austin TX), and RNA was prepared using the RNeasy system (Qiagen, Valencia, CA). Embryos were genotyped for Brn3a alleles as previously described [17] from a sample of tail or hind-limb tissue harvested at the time of ganglion dissection. Genotyped TG or DRG from five embryos were sufficient to provide approximately 5 μg of total RNA for a single microarray analysis. The generation of cDNA, production of labeled cRNA, and hybridization to GeneChip arrays were all performed according to standard protocols provided by the manufacturer (Affymetrix, Santa Clara, CA).

Analysis of expression array data

The principal microarray datasets presented here for the mouse DRG and TG were generated using the Affymetrix 430A and 430B microarrays. In addition, the same DRG samples were analyzed using Affymetrix U74A and U74B arrays for direct comparison with prior data sets for the TG [20]. The primary analysis of microarray data, including determination of the absence/presence of the assayed transcripts, transcript expression levels, and the probability of change in transcript expression between samples ('changep') was performed with Microarray Suite 5.0 (Affymetrix). Default Microarray Suite 5.0 parameters were used for increase (I) and decrease (D) calls. For the 430 array set these cutoff values werep< 0.005 andp> 0.995 for I and D, respectively, and for the U74 arrays, the values werep< 0.003 andp> 0.997. All array values were initially scaled to a mean value of 500 using global scaling. To permit more meaningful comparison of the expression levels of transcripts assayed by the 430A and 430B arrays, the initial expression values for the 430B array were rescaled based on the expression levels of 100 probe sets in common between the 430A and 430B arrays. Microarray probe sets were related to the corresponding mouse transcripts using the NetAffx database (Affymetrix), based on the NCBI Build 34 annotation of the mouse genome. The array data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (GEO) [38] and are accessible through GEO series accession numberGSE5658.

Comparisons of gene expression between wild-type DRG and TG (Table1) and between the DRG from Brn3a wild-type, heterozygous, and knockout mice (Tables2 and3) were performed in duplicate, using separate pools of isolated ganglia. To be included in the tables of changed transcripts for a given tissue or genotype, the following criteria were met: both replicates were called as 'present' in the condition of greater expression; both replicates exhibited an increased or 'I' call for the condition of higher expression; and the probe set identified an annotated and named transcript in NCBI Build 34 of the mouse genome.

Duplicate probe sets identifying the same transcript were eliminated, and the tables are annotated with the number of concordant probe sets for each transcript. Concordance was identified by the agreement of the present and increased calls for the duplicate probe set, and a minimum fold change of 2.0. In the case of multiple concordant probe sets, the probe set with the greatest fold change is listed in the tables. Results for the probe sets meeting the inclusion criteria were ranked by fold change, and the cutoff value for inclusion appears in the tables.

In situhybridization and immunofluorescence

Non-isotopicin situhybridization was performed as previously described [38]. A list of probes used and their sources appears inadditional file 5. Immunofluorescence for Brn3a was performed with rabbit polyclonal antisera as previously as previously described [39]. Other antisera used included rabbit antisera against Etv1/Er81 and Runx3 obtained from Dr Sylvia Arber [13,15], and guinea pig antisera against Islet2, obtained from Dr Sam Pfaff [28]. Immunofluorescence for other antigens was performed with commercially available antibodies, including rabbit anti-calretinin (Swant, Bellinzona, Switzerland), rabbit anti-galanin (Bachem, King of Prussia, PA), rabbit anti-somatostatin-14 (Peninsula Laboratories), and goat anti-β-galactosidase (Biogenesis (MorphoSys), Kingston, NH).

Locus-wide chromatin immunoprecipitation

Locus-ChIP assays were performed as previously described [[27] in press]. In brief, embryos for ChIP assays were generated from timed matings of ICR mice. DRG and TG were dissected from E13.5 embryos and fixed in 4% paraformaldehyde for 30 minutes, then quenched with 150 mM glycine. The fixed tissue was washed with phosphate-buffered saline and stored at -80°C until analysis.

Selection of chromatin complexes from embryonic sensory ganglia was performed by a modification of a widely used procedure [40]. For each analysis, fixed ganglia from 30 embryos (60 TG or approximately 300 DRG) were pooled and suspended in lysis buffer containing 50 mM Tris-HCl, pH 8.1, with 10 mM EDTA and 1% SDS, 1 mM 4-(2-Aminoethyl)benzenesulfonylfluoride, HCl (AEBSF) and a proprietary protease inhibitor mix (1 × Complete Mini, Roche, Indianapolis, IN; used according to instructions). Chromatin was then fragmented to an average size of 500 bp by sonication, and insoluble cellular debris was removed by centrifugation. The supernatant containing fragmented chromatin was diluted in 15 mM Tris-HCl, pH 8.1, with 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 0.01% SDS, and protease inhibitor mix. An unselected ('input') sample of 10% of the total homogenate was removed prior to antibody selection.

ChIP was performed using rabbit anti-acetylated histone H3 antibody (Upstate Biotechnology, Inc., Bellerica, MA, catalog no. 06–599), which recognizes histone H3 acetylated at lys9 and lys14. For each selection, 50 μg of anti-histone antibody was coupled to 250 μL of anti-rabbit IgG magnetic beads (Dynal M-280), in lysis buffer containing 50 mM Tris-HCl, pH 8.1, with 10 mM EDTA and 1% SDS. To reduce non-specific background, the chromatin sample was pre-cleared using the magnetic beads with the secondary antibody alone. The sample was then incubated overnight with secondary antibody-coupled beads to select the acetyl H3-containing chromatin complexes. The beads were then washed for 5 minutes at room temperature with each of the following solutions: 20 mM Tris-HCl, pH 8.1, with 150 mM NaCl, 2 mM EDTA, 0.1 % SDS, and 1% Triton-X-100; 20 mM Tris-HCl, pH 8.1, with 500 mM NaCl, 2 mM EDTA, 0.1% SDS, and 1% Triton-X-100; 10 mM Tris-HCl, pH 8.1, with 1 mM EDTA, 0.25 M LiCl, 1% NP-40, and 1% deoxycholate. Salt and detergent were removed by washing twice with 10 mM Tris-HCl, 1 mM EDTA, pH 8. DNA was extracted from the antibody-chromatin complexes and the input sample by heating in 0.1 M NaHCO₃with 200 mM NaCl and 1% SDS at 65°C for 4 hours with constant shaking. The input and selected samples were then digested with proteinase K, extracted with phenol/chloroform, and precipitated with ethanol.

Real-time locus-wide PCR analysis

Chromatin fragments recovered from the immunoprecipitated and input samples were then assayed by real-time PCR using an ABI 7300 thermocycler and SYBR Green fluorimetric detection. To screen an entire gene locus, oligonucleotide pairs were designed at 500 to 1,000 bp intervals throughout the region, and selected and unselected samples were run in parallel in a 96-well plate format. The primer pairs used for locus-ChIP assays of theNeuroD4,Msc,Tcfap2bandGata3loci appear inadditional file 5.

The enrichment of immunoprecipitated chromatin fragments was assayed by the cycle-threshold difference method [41]. For this method, real-time PCR signals are measured using the 'cycle threshold', or Ct parameter, which is the number of cycles required for the amplification product to reach an arbitrary level of fluorescence intensity (threshold), and is logarithmic to the initial abundance of the target sequence in the sample. For each PCR amplicon, a ΔCt value comparing the unselected (input) and antibody-selected DNA samples was then calculated by subtracting the Ct_selectedfrom the Ct_inputsignal:

ΔCt = Ct_input- Ct_selected

Fold enrichment values for target sequences bound by the selecting antibodies, corresponding to the y-axis of the locus-ChIP plots, were calculated using the following equation:

E = 2^{(ΔCt-ΔCtcontrol)}

Because product formation approximately doubles with each cycle in the linear range of amplification, a ΔCt of one cycle represents a two-fold difference in starting template. A significant advantage of this method is that, for each primer pair, a selected sample is compared directly to its unselected control, which differs only by the antibody selection process. Potentially confounding factors such as small differences in the PCR amplification efficiency of different primer pairs are eliminated in this comparison.

The ΔCt assays for each pool of selected material were normalized to an arbitrary baseline (one-fold enrichment) determined using two primer pairs in the promoter region of theAlb1(albumin locus), which was chosen as a negative control because it is not transcribed in the nervous system. Fold enrichment values for theAlb1locus were very similar to values for the unselected, intergenic regions of the target gene loci. Primer pairs in the promoter regions ofMapt(microtubule-associated protein tau) andEno2(neuron specific enolase), which exhibit tissue specific expression in the nervous system, andGapdh(glyceraldehyde-3-phosphate dehydrogenase), which is expressed ubiquitously, were used as positive controls. Comparisons of histone H3 acetylation at a given locus in the DRG and TG were made using three- to six-fold enrichment values using oligonucleotide pairs in a contiguous genomic region near the transcription start site of the analyzed gene loci. The statistical significance (pvalues) of the difference in H3 acetylation was determined using two-sample, unequal variancet-tests.

Acknowledgements

We would like to acknowledge Bill Wachsman and Lutfunnessa Shireen for assistance with microarray technology. Dr Sylvia Arber generously provided antisera for Runx3 and Etv1/Er81, and Islet2 antiserum was the generous gift of Dr Sam Pfaff. We would also like to thank Drs Jean-Francois Brunet, Allan Basbaum, Ryoichiro Kageyama, Nobuyuki Itoh, Eric Olson and their associates forin situhybridization probes. Microarray data used in this study are deposited in the Gene Expression Omnibus, series accession numberGSE5658. Supported in part by Department of Veterans Affairs MERIT funding and VISN 22 MIRECC (EET), and NIH awards HD33442 and MH065496. EET and NF are a NARSAD Investigators.

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