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The POU-domain transcription factor Brn3a is expressed in developing sensory neurons at all levels of the neural axis, including the trigeminal ganglion, hindbrain sensory ganglia, and dorsal root ganglia. Changes in global gene expression in the trigeminal ganglion from E11.5 to E13.5 reflect the repression of early neurogenic genes, exit from the cell cycle, and initiation of the expression of definitive markers of sensory function. A majority of these developmental changes are perturbed in the trigeminal ganglia of Brn3a knockout mice. At E13.5, Brn3a−/− trigeminal neurons fail to repress a battery of developmental regulators which are highly expressed at E11.5 and are normally down-regulated as development progresses, and also fail to appropriately activate a set of definitive sensory genes. Remarkably, developing Brn3a−/− trigeminal neurons also ectopically express multiple regulatory genes associated with cardiac and/or cranial mesoderm development, although definitive myogenic programs are not activated. The majority of these genes are not ectopically expressed in the dorsal root ganglia of Brn3a null mice, perhaps due to redundant mechanisms of repression at spinal levels. These results underscore the importance of gene repression in regulating neuronal development, and the need for unbiased screens in the determination of developmental gene regulatory programs.
Information about the external world is conveyed to the vertebrate brain by special sense organs for vision, olfaction and hearing, and by the peripheral sensory nervous system mediating general somatic sensation. Somatic sensory neurons innervate the skin and muscles throughout the body, and produce specialized structures and molecular receptors for sensory modalities including pain, touch, temperature and position. At trunk levels, somatic sensory neurons reside in the dorsal root ganglia (DRG) associated with each spinal segment, and innervate the corresponding dermatomes and myotomes, while at cranial levels they reside primarily in the trigeminal ganglion (TG) and innervate the facial skin and musculature.
Numerous studies have focused on the unique developmental origin of sensory neurons. At trunk levels the DRG are generated from neural crest precursors, while the sensory ganglia associated with the cranial nerves, such as the TG, include contributions from the neural crest and neurogenic placodes derived from surface ectoderm (Baker and Bronner-Fraser, 2001). The earliest-expressed genes known to have an essential role in specifying the sensory lineage are the bHLH factors neurogenin1 (Neurog1) and neurogenin2 (Neurog2), with Neurog1 playing a predominant role in the TG and DRG and Neurog2 the principal factor in sensory ganglia derived from the epibranchial placodes (Fode et al., 1998; Ma et al., 1998; Ma et al., 1999). In all sensory neurons, expression of the these factors is followed by the bHLH factors Neurod1 (BETA2) and Neurod4 (Math3), which are dependent on the neurogenins (Fode et al., 1998; Ma et al., 1998; Sommer et al., 1996).
Following the neurogenic phase of sensory development, coinciding approximately with the time of exit from the cell cycle, sensory neurons of the DRG and cranial ganglia express the “pan sensory” homeodomain transcription factors Brn3a (product of the Pou4f1 gene) and Islet1 (Eng et al., 2001; Sun et al., 2008). Both factors are required to terminate the expression of the bHLH neurogenic factors (Eng et al., 2007; Eng et al., 2004; Sun et al., 2008). Brn3a has been shown to bind directly to enhancer regions within the Neurod1 and Neurod4 loci (Lanier et al., 2007), and a to negative autoregulatory region within the Pou4f1 locus itself (Lanier et al., 2007; Trieu et al., 2003; Trieu et al., 1999). As sensory maturation progresses, both Brn3a−/− and Islet1−/−sensory neurons fail to correctly express numerous markers of terminal sensory differentiation and subtype specification (Eng et al., 2004; Sun et al., 2008), demonstrating that these factors also have a direct or indirect positive regulatory role in determining sensory phenotypes.
In the present study we have examined changes in global gene expression in the trigeminal ganglion from E11.5 to E13.5, a critical period during which a majority of trigeminal neurons exit the cell cycle and initiate the expression of definitive sensory markers. In the absence of Brn3a, sensory neurons are delayed in executing the changes in gene expression characteristic of this time period. The expression of factors associated with cell proliferation is somewhat prolonged, but cell cycle exit is accomplished by E13.5 in both control and Brn3a−/− ganglia. At E13.5, Brn3a−/− TG neurons fail to repress a battery of developmental regulators which are highly expressed at E11.5 and are normally down-regulated as development progresses. Conversely, numerous genes associated with mature sensory phenotypes fail to be expressed at normal levels. Surprisingly, in the absence of Brn3a, developing TG neurons abnormally express several regulatory factors, including Nkx2.5 (tinman), Ankrd1 (CARP), Zfpm2 (Fog2), Lmcd1 (dyxin), Fgf10, Figf (VegfD), and Angpt1, which are normally associated with differentiating cardiac myocytes and/or cranial muscles rather than sensory neurons. This unexpected role for Brn3a in the repression of non-neural genes in sensory neurons illustrates that the gene expression programs regulated by transcriptional repressors may be occult and unexpected, and that unbiased screens for downstream targets are indispensible to understanding their functions.
Mice heterozygous for a Brn3a null allele (Eng et al., 2001; Xiang et al., 1996) were maintained in a C57bl/6 background. To generate tissue for microarray analysis, timed matings of Brn3a heterozygote animals were performed, and the embryos were harvested at E11.5 or E13.5. TG were removed by blunt dissection and carefully freed of adherent non-neural tissue with fine forceps. 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 using real-time qPCR from a sample of tail or hind-limb tissue harvested at the time of ganglion dissection, using PCR primer pairs targeting the POU-domain of the Brn3a gene or the neomycin resistance cassette targeted to the Brn3a locus, as previously described. For analysis of Brn3a knockout and control ganglia, TG 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 Affymetrix 430v2 GeneChip arrays were all performed according to standard protocols provided by the manufacturer (Affymetrix, Santa Clara, CA).
Microarray analysis of TG gene expression was performed on two developmental stages (E11.5, E13.5) and three genotypes (Brn3a +/+, +/− and −/−) in duplicate for a total of 12 analyses. Consistent with prior results (Eng et al., 2004), gene expression in heterozygotes correlated strongly with control ganglia and was not further analyzed. 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 (‘change p’) was performed with Microarray Suite 5.0 (MAS5, Affymetrix). Default MAS5 parameters were used for increase (I) and decrease (D) calls, which were p < 0.002 and p > 0.998 for I and D, respectively. All array values were scaled to a target value of 500 using global scaling. Microarray probe sets were related to the corresponding mouse transcripts using the NetAffx database (Affymetrix), based on the NCBI Build 36 annotation of the mouse genome.
For comparison of gene expression changes across developmental stages and between genotypes, we identified 17,351 probe sets from among the 45,037 probe sets on the array which were present in both samples of at least one stage/genotype, and had a mean expression value of at least 250 (0.5 times the scaled target value) in at least one stage/genotype. Transcripts listed in the ranked lists of changed genes (Table S1–S2, S4–S7) showed concordant I or D calls in both replicates for a given comparison (E11 WT x E13 WT, E11 WTxKO, E13 WTxKO). In cases of multiple probe sets identifying a single transcript, data are given for the probe set with the highest mean expression in any condition.
Classification of differentially expressed genes by gene ontology (GO) was performed using GeneSpring software version 9 (Silicon Genetics, Redwood City, CA). Four gene lists were created in GeneSpring with the "Arbitrary File Filter" based on significant expression differences between stages or genotypes (expression increased in development from E11.5 to E13.5, expression decreased in development from E11.5 to E13.5, expression increased in Brn3a knockout at E13.5, expression decreased in Brn3a knockout at E13.5). Each category consisted of all transcripts which exhibited concordant I or D calls in the replicate assays for a given comparison. Genes were organized into GO categories based on biological process using GeneSpring's GO browser, and GO categories enriched in our gene lists were then compiled.
Real time reverse transcription-PCR (RT-PCR) analysis was performed on samples of RNA from the trigeminal ganglia of E13.5 Brn3a+/+ and Brn3a−/− embryos harvested as described above. Samples for RT-PCR analysis were harvested independently of those used for microarray analysis. First strand cDNA synthesis was performed with a Thermoscript RT-PCR system for first strand synthesis (Invitrogen) using 1µg of total RNA according to manufacturers instructions, and real-time PCR was performed using Power Sybr Green PCR Master Mix (Applied Biosystems) and an ABI 7300 real-time PCR system. Results were analyzed according to the cycle threshold difference method (Livak and Schmittgen, 2001) Oligonucleotide primer sets used for RT-PCR appear in Table S8.
Embryos for in situ hybridization and immunofluorescence studies were generated by timed matings as described above. Embryos were fixed by immersion in 4% paraformaldehyde for 30 minutes (E11.5) or 2 hours (E13.5). Tissue was embedded in Neg50 (Richard Allan Scientific) and 20µM sections were cut on a cryostat. Rabbit and guinea pig primary antibodies to Brn3a have been previously described (Fedtsova and Turner, 1995; Quina et al., 2005).
In situ hybridization was performed on cryostat sections using digoxigenin labeled cRNA probes. Sections were pre-treated with a 10 minute digestion in 10ug/ml proteinase K followed by 10 minutes in 0.5% acetic anhydride, 0.1M triethanolamine before being hybridized at 65C overnight using 1–2ug/ml of probe. Slides were washed at 65C under stringent conditions then blocked in 10% sheep serum. Signal was detected using an alkaline phosphatase conjugated sheep anti digoxigenin antibody (Roche).at 1:2000 followed by staining in BM Purple AP Substrate (Roche). Plasmids containing cDNAs were obtained from Open Biosystems. A list of in situ probes used in this study is provided in Table S8.
Brn3a is expressed in sensory neurons at all axial levels around the time of their exit from the cell cycle, or “birthdate”, and its expression also coincides with the onset of expression of many genes which relate to specific neural functions. Thus the regulatory role of Brn3a must be understood in the context of normal developmental changes in gene expression taking place during this period. For this reason, we began the current studies by examining global gene expression in the developing trigeminal ganglion at E11.5 and E13.5, bracketing a period during which a large fraction of trigeminal neurons are born.
Microarray analysis of wildtype TG at E11.5 and E13.5 revealed extensive changes in gene expression (Figure 1). The changes were consistent with a transition from cell proliferation to terminal differentiation, and with the initiation of sensory subtype specification. Transcripts with the greatest increases in expression from E11.5 to E13.5 (Table S1) were heavily weighted toward neurotransmitters (Galanin, Th, Tac1/SP), neurotransmitter receptors (Htr3a, Htr3b, Npy5r, Grm3, Grm7), ion channels (Trpv1, Trpc4, Accn3, Scn7a), regulators of axonogenesis and synaptogenesis (Advillin, Sema3e, Syt4, Ncam2) and developmental regulators with roles in sensory specification (Ret, TrkA, Bnc1, Runx1, Hoxd1).
Genes which showed decreased expression from E11.5 to E13.5 included many factors associated with cell cycle progression, DNA replication, and mitosis (Table S2). Transcription factors associated with early steps in sensory neurogenesis were also markedly decreased in this developmental interval, including Neurog1, Neurod4, Pax3, Tcfap2b, Hes5 and Eya2, and the majority of the transcripts with the largest magnitude decreases could be assigned to one of these two classes.
We then performed a gene ontology (GO) biological process analysis to obtain a more comprehensive view of the changes in gene expression in this developmental interval. For this analysis, we defined sets of transcripts with replicated increases or decreases in expression between E11.5 and E13.5 (p < 0.002 and p > 0.998 for I and D, respectively). The gene ontology classification of the 2606 increased and 2451 decreased transcripts that met these criteria were then compared to the global set of 45037 transcripts represented on the array to determine over-represented categories. Transcripts increasing across developmental time clustered strongly in GO categories specific for neural function, including synaptic transmission, ion transport, neural development and neurotransmitter secretion (Table 1, Table S3). For transcripts decreasing from E11.5 to E13.5, the major over-represented categories included DNA metabolism, cell cycle, and cell division. (Table 1, Table S3). Together these results give a comprehensive profile of the changes in gene expression that characterize the transition from sensory neurogenesis to terminal differentiation.
We next compared gene expression in Brn3a knockout and wildtype trigeminal ganglia at E11.5 (Tables S4, S5) and E13.5 (Tables 2, ,3,3, S6, Table S7), revealing an extensive program of gene expression regulated by Brn3a. In general, the transcripts with altered expression at E11.5 were a subset of those changed at E13.5, and the direction of change was consistent at both stages. Some of these transcripts have been previously identified as downstream of Brn3a in the developing TG (Eng et al., 2004) or DRG (Eng et al., 2007), but many novel targets were identified due to improved genomic coverage by the arrays used in the present study and progress in annotation of the mouse genome.
A large fraction of the decreased transcripts in the Brn3a−/− TG represent developmental signaling molecules or transcription factors known to be associated with sensory subtype specification. Some of these changes, such as decreased expression of Runx3, TrkC, and Pou4f2/Brn3b, were evident beginning at E11.5. Other changes, including decreased expression of Bnc1, Hmx1, HoxD1 and Runx1, were not observed until E13.5, due to the later developmental expression of these factors. Loss of Brn3a also resulted in markedly decreased expression of a large number of neural-specific transcripts, including neurotransmitters (Gal, Adcyap1/Pacap, TH), receptors (Npy5r, Htr3b, Npy1r, Prokr1, Gpr149, Gpr64, Trpv1, Accn2), ion channels (Scn7a, Scn10a, Kcnab2, Kcna1) and cell surface molecules which mediate cell survival, migration and/or axon guidance (Ntrk3, Ntrk1, Rtn4rl2, Rtn4rl1). Many of these terminal differentiation genes are not significantly expressed in E11.5 control ganglia, and therefore the changes in Brn3a−/− ganglia were noted mainly at E13.5.
The set of genes with increased expression in E13.5 Brn3a−/− TG included the abnormal persistence of several transcription factors which are expressed in the early phases of sensory development, including Neurod1, Neurod4/Math3, Neurog1, Tcfap2b, Insm1, Six1, Eya1 and Eya2. Also increased are several cell cycle genes, including Cyclin B1, Cyclin D1, and Cdc20. However, the least expected finding is a marked increase in the expression of a set of genes normally associated with cardiac and cranial mesoderm development, which are examined in detail below.
The abnormal persistence of multiple classes of genes expressed in early development in the TG of Brn3a−/− mice led us to examine the global interaction between developmental changes in gene expression and Brn3a genotype. A comparative GO analysis of the transcripts altered in Brn3a−/− TG at E13.5 with changes in gene expression from E11.5 to E13.5 confirmed such a relationship (Table S3). Transcripts with decreased expression in the E13.5 Brn3a−/− TG were associated with GO categories including ion transport (including channels and receptors), cell communication (including signal transduction and neuropeptide signaling), and axonogenesis. Nearly all of these categories were also overrepresented in the GO analysis of transcripts increasing from E11.5 to E13.5 (Table 1, Table S3). Transcripts with increased expression in the E13.5 Brn3a−/− TG were associated with GO categories that included the cell cycle, cell division, DNA metabolism, and embryonic and neural development. The cell-cycle related categories were highly correlated with the GO classification of transcripts decreasing from E11.5 to E13.5 (Table 1, Table S3).
We then performed a regression analysis of transcript expression for developmental stage x genotype to examine this relationship on a gene-by-gene basis (Figure 2A). As expected from the GO analysis, the direction and magnitude of differences in gene expression levels between E11.5 and E13.5 correlated with the effects of the loss of Brn3a expression at E13.5. A large cohort of transcripts which normally increase expression as development progresses failed to reach normal expression levels in the ganglia of E13.5 Brn3a−/− mice, and conversely, many transcripts which normally decrease with developmental time were increased at E13.5 in the knockout (sectors designated “developmental delay”, Figure 2A). The relationship of the fold change in transcript levels due to developmental stage versus the loss of Brn3a expression at E13.5 can be described by a linear regression with a slope of −0.30 and R2 = 0.26. These results demonstrate a pervasive delay in the development of the Brn3a−/− TG at the transition from neurogenesis to neural differentiation. However, a large majority of the developmentally regulated genes exhibited less than two-fold changes in expression, suggesting that global developmental delay does not account for the large fold changes observed for the most strongly regulated transcripts.
We then examined the relationship between developmental changes in gene expression and Brn3a genotype for functional categories of genes, including regulators the cell cycle (including mitosis and DNA replication), mediators of axon growth and guidance, sequence specific transcription factors, and neurotransmitters/receptors. Factors mediating cell cycle progression and mitosis conformed closely to the regression line that describes the overall developmental delay in Brn3a−/− TG (Figure 2B), in that expression of genes mediating the cell cycle and DNA replication decreased from E11.5 to E13.5 and was increased in Brn3a−/− ganglia, while genes mediating cell cycle exit had the inverse relationship to both conditions. Most mediators of axon growth and guidance had increased expression from E11.5 to E13.5, as expected in maturing neurons, and also generally conformed to the linear relationship between developmental stage and Brn3a genotype.
In contrast, many genes encoding transcription factors and neurotransmitters do not conform to this linear relationship between developmental stage and Brn3a genotype. Thus many of the observed changes in these gene classes appear to represent “phenotype switching” by Brn3a−/− trigeminal neurons, which cannot be simply described as a failure to progress on schedule down the expected developmental pathway. A significant number of these transcripts (Trk receptors, Runx3, Ret, Trpa1, Hmx1) represent known markers of sensory subtypes, and the role of Brn3a in sensory subtype specification will be examined in a separate report (I. Dykes, in preparation). Finally, a subset of these transcripts encode factors most strongly associated with cardiac and cranial mesoderm development (highlighted in red).
In order to test the reliability of the gene expression changes detected by microarray, we examined the expression of 36 of the changed transcripts by quantitative RT-PCR using independent samples of E13.5 knockout and control TG tissue (Figure 3). In these assays all transcripts with >2-fold change on the microarray showed a similar direction and magnitude of change in the RT-PCR assay, including increased and decreased transcripts across all functional classes, with the exception of Mrg1, for which the change observed on the microarray was not replicated.
Persistent expression of cell-cycle associated genes raises the question of whether Brn3a−/− TG neurons exit the cell cycle at the expected time. Arguing against significantly prolonged cell division is the prior observation that Brn3a knockout TG do not have increased cell number at any stage in development (Huang et al., 1999). Because the large majority of trigeminal neurons leave the cell cycle by E13.5 (E14.5 in rat, White et al., 1995), we employed BrdU labeling at E13.5, followed by analysis at E14.5, to assess directly whether neurons were delayed in cell cycle exit in Brn3a−/−ganglia (Figure 4). Immunofluorescence for Islet1 was used to identify differentiating sensory neurons in the absence of Brn3a. Less than 2% of TG neurons incorporated BrdU at E13.5 in either control or Brn3a−/− knockout embryos, indicating that cell cycle exit is not significantly delayed in the absence of Brn3a.
This global analysis of gene expression in E13.5 Brn3a−/− TG revealed that a significant number of the most increased transcripts are not expressed in control ganglia at either E11.5 or E13.5. Unexpectedly, several of these genes are known to be expressed in the developing heart, and in some cases structures derived from cranial mesoderm, including Ankrd1 (CARP), Lmcd1, Zfpm2 (Fog2), Figf (VegfD), Tcfap2d, Irx2, Msc (MyoR), Nkx2–5 (tinman), and Actc1 (cardiac muscle alpha actin 1). In addition, we have previously shown that expression of another regulator of cardiac development, Fgf10, is markedly increased in the TG of Brn3a−/− embryos. Fgf10 is not detected by the microarrays used to generate the current data set, due to a flaw in the design of the Fgf10 oligonucleotide probe set on the arrays used for these studies (Cox et al., 2006).
Increased expression of Irx2, Msc, and Fgf10 in the TG of Brn3a−/− mice has been previously verified by in situ hybridization (Cox et al., 2006; Eng et al., 2004). The novel changes in cardiac/mesoderm gene expression detected here were verified using in situ hybridization and RT-PCR with specific primer sets (Figure 5). In situ analysis of Lmcd1 and Prox2 revealed widely distributed expression in the TG of Brn3a−/− embryos, while Eya1 appeared restricted to a subset of neurons in the periphery of the ganglion. RT-PCR assays of these transcripts, as well as Ankrd1, Zfpm2, Tcfap2d, and Nkx2.5, generally revealed increases in expression at least as large the microarray analysis.
In order to understand the developmental timing and tissue specificity of the de-repression of genes associated with cardiac/mesodermal development in Brn3a−/−embryos, we compared results for the E11.5 TG and E13.5 DRG for this set of genes (Figure 6, Table S6, S7). Tcfap2d, Irx1, Irx2, Figf, Zfpm2, and Actc1 were all abnormally expressed by E11.5 in the TG, and these changes constitute some of the largest magnitude changes observed at E11.5 (Table S5). However, increased expression of Ankrd1, Lmcd1 and Nkx2.5 was not observed until E13.5. In contrast, the expression of cardiac-related transcripts in the E13.5 Brn3a−/− DRG was minimal, suggesting that only the TG retains the potential to express this set of genes during sensory neurogenesis.
Recently, it has been reported that Brn3a is expressed from ~E10.5 onward in the developing mouse heart (Farooqui-Kabir et al., 2008). Cell-specific expression was reported from E13.0 in the atrio-ventricular (AV) valve cushions and leaflets, outflow tract, epicardium and cardiac ganglia, and at E14.5 in the intraventricular septa and ventricular myocardium. Because these results might relate to the observed regulation of cardiac genes by Brn3a in the TG, we examined the developing heart for expression of Brn3a protein (Figure S1, S2). The antibody used for Brn3a detection in these studies (Fedtsova and Turner, 1995) was generated to a region immediately N-terminal to the DNA binding POU domain, and should recognize both the reported “long” and “short” forms of Brn3a (Theil et al., 1993). The Brn3a antibody employed here gave a very strong signal in the DRG and spinal cord in the same sections used to examine heart expression (Figure S1D,H; Figure S2, B,D,F). However, in contrast to the previously reported results, we were unable to detect Brn3a protein in any of the structures examined in the previous study (Figure S1C,E,F; Figure S2A,C,E). We conclude that the dysregulation of cardiogenic genes in the trigeminal ganglion of Brn3a−/− embryos does not reflect a role for Brn3a in normal heart development, but rather an ectopic program of gene expression which must be suppressed during trigeminal neurogenesis.
Results reported here, together with earlier studies of the TG and DRG (Eng et al., 2007; Eng et al., 2004), together provide a comprehensive view of the regulatory role of Brn3a in sensory neurogenesis. First, Brn3a−/− knockout mice exhibit a pervasive delay in gene regulatory programs which characterize the transition from neurogenesis to definitive sensory differentiation. In the TG, a feature of this delay is the persistent expression of genes associated with cell division, but several factors argue against a key role for Brn3a in mediating cell cycle exit. First, expression of cell-cycle associated genes is only moderately increased in Brn3a−/− TG compared to controls. Second, significantly increased expression of cell cycle genes is not observed in Brn3a−/− DRG (Eng et al., 2007), arguing that it is not an essential mediator of cell cycle exit. Third, TG neurons are not increased in number at any stage in Brn3a−/− ganglia (Huang et al., 1999), and, as shown here, do not show persistent incorporation of BrdU beyond the usual time of cell cycle exit.
Loss of Brn3a also results in the abnormal persistence of neurogenic bHLH factors and other regulators associated with early neurogenesis. Brn3a has a well-established role in terminating the expression of Neurod1 and Neurod4 in the TG and DRG (Eng et al., 2004; Lanier et al., 2007). In the present report, profiling of gene expression across developmental time has shown that multiple transcription factors in several classes show a failure in their normal developmental down-regulation from E11.5 to E13.5 in the Brn3a−/− TG, including Tcfap2b (Ap2β), Eya1, Eya2, Insm1, Neurog1, Shox2, Six1, Zeb2 (Zfhx1b) and Nhlh2. The majority of these changes are also observed in the DRG, demonstrating a pan-sensory role for Brn3a in repressing genes associated with early sensory development.
Brn3a−/− TG exhibit decreased expression of a battery of genes associated with definitive sensory neurogenesis and subtype specification. Data from the current study confirm earlier studies showing that Brn3a is necessary for correct expression of the neurotrophin receptors TrkA and TrkC, with a particularly early and profound loss of TrkC expression (Huang et al., 1999; McEvilly et al., 1996; Xiang et al., 1996). These results also confirm markedly decreased expression of the runt-domain transcription factors Runx1 and Runx3 (Eng et al., 2007; Eng et al., 2004), which are required for the correct specification of nociceptors and proprioceptors, respectively (Chen et al., 2006; Kramer et al., 2006). These changes in gene expression at E11.5 and E13.5 are not likely to be attributable to selective death of trigeminal neurons, because significant reduction in trigeminal populations are not observed until E14.5 (Huang et al., 1999). The results presented here suggest that in the absence of Brn3a, trigeminal neurons fail to express a battery of channels and receptors associated with sensory function (Npy1r, Ntrk1/TrkA, Ntrk3/TrkC, Prokr1, Scn7a, Scn10a, Trpv1), but also exhibit increased expression of a number of markers (Htr3a, Ntrk2/TrkB, Slc6a1/Gat1, Sst/somatostatin, Trpa1,) suggesting a switch in phenotype, not simply failed maturation. A detailed analysis of phenotypic switching in Brn3a−/− sensory ganglia will be the subject of a separate report.
An unexpected finding of this analysis of global gene expression in the Brn3a−/− TG is the derepression of a set of genes which are strongly associated with development of the heart, although Brn3a−/− TG neurons retain their neuronal properties and in no sense differentiate into cardiac cells. Detailed studies over the last decade have elucidated the early morphological and molecular events of cardiac development that provide a context for these changes in gene expression. Bilateral cardiac progenitors arise within the splanchnic lateral mesoderm, condensing at the midline to form the heart tube. Early growth of the heart occurs by addition of progenitors to both anterior and posterior poles of the forming heart (van den Berg et al., 2009). The source of these progenitors is known as the second heart field which is located in pharyngeal mesoderm, including that of the pharyngeal arches (Abu-Issa and Kirby, 2007; Buckingham et al., 2005; Eisenberg and Markwald, 2004). Cardiac neural crest also migrates through the pharyngeal arches to contribute to septation of the outflow tract and aortic arch arteries (Hutson and Kirby, 2007).
Several of the genes exhibiting increased expression in the Brn3a−/− TG are associated with key events or cell populations in this developmental pathway. The homeodomain transcription factor Nkx2.5 (tinman), is first expressed in the lateral plate mesoderm, although loss of Nkx2.5 function does not arrest cardiac development until the looping stage (Biben and Harvey, 1997). Ankrd1 (originally described as CARP, for cardiac adriamycin responsive protein and also cardiac ankyrin repeat protein) is expressed in the heart tube at E8.5, and is downstream of Nkx2.5 (Jeyaseelan et al., 1997; Kuo et al., 1999). Ankrd1 is localized to the nucleus, and may be a transient repressor of some definitive cardiac muscle genes.
Zfpm2 (Fog2, friend of GATA 2) is a zinc-finger transcription factor expressed in the cardiac primordium from E8.5, and it is necessary for the development of both myocardium and cardiac vasculature (Tevosian et al., 2000). Zfpm2 interacts with Gata4 (Svensson et al., 1999), which itself is necessary for cardiac development (Kuo et al., 1997; Xin et al., 2006; Zeisberg et al., 2005). None of the Gata4/5/6 factors expressed in the developing heart exhibited increased expression in the Brn3a−/− TG, but a related factor, Gata3, was markedly increased. Because Zfpm2 can interact with Gata3 as well as Gata4 (Tevosian et al., 1999), dysregulation of Gata3 expression may intersect this pathway. Also potentially interacting with Gata signaling is Lmcd1 (LIM and cysteine-rich domains 1, dyxin), which is a LIM-domain containing protein expressed relatively late (E12.5) in the developing myocardium. Lmcd1 can bind to multiple GATA family proteins and is thought to repress GATA activity by an alternate mechanism to Fog2 (Rath et al., 2005).
In a prior study we have reported that Brn3a−/− TG exhibit markedly increased expression of another known regulator of cardiac development, Fgf10 (Cox et al., 2006). Fgf10 expression is not effectively assayed by the microarrays used in the present study (Affymetrix 430v2) due to a design flaw in the probe set for this transcript, but increased expression was detected on an earlier generation array and confirmed by in situ hybridization. Fgf10 is a marker of pharyngeal mesoderm cells which contribute to the outflow tract of the heart (Kelly et al., 2001), although the cardiac effects of Fgf10 ablation are subtle and may reflect redundancy of function in the Fgf family (Marguerie et al., 2006). We have previously concluded that a local effect of Fgf10 de-repression is not likely to account for the extensive program of altered gene expression in the trigeminal ganglion, because the necessary receptors are not expressed there (Cox et al., 2006).
Other cardiac transcripts are expressed in Brn3a−/− TG, but there is little data on their function. Irx1 and Irx2 have been identified in the ventricular septum (Christoffels et al., 2000), however, Irx2 knockouts do not exhibit a cardiac phenotype (Lebel et al., 2003), and Irx1 null mice have not been reported. Tcfap2d (Ap2δ) is expressed in the myocardial primordium at E9.5-E10.5 (Zhao et al., 2003), but its function is unknown. Figf (c-fos-induced growth factor; VegfD, Vascular endothelial growth factor D) is expressed in the atrio-ventricular cushion at E12.5 (Avantaggiato et al., 1998). Loss of Figf alone has a minimal phenotype (Baldwin et al., 2005), and Figf, the closely related VegfC, and their principal receptor VEGFR-3 appear to be involved primarily in angiogenesis and lymphangiogenesis rather than cardiac development (Haiko et al., 2008).
Some changes in gene expression in Brn3a−/− sensory ganglia may reflect the derepression of gene expression programs which are mesodermal or myogenic, but not specifically cardiac. This is consistent with recent work which has established close developmental and molecular relationships between the cranial paraxial mesoderm, which gives rise to the cranial musculature, and the splanchnic mesoderm which contributes to the heart (Tzahor, 2009). The bHLH transcription factor Msc (musculin, MyoR) is increased in the TG of Brn3a−/− mice, but unlike the majority of cardiac-associated genes, it is also de-repressed in the DRG (Eng et al., 2007). Consistent with this, Msc is normally expressed in cranial mesoderm and the anterior heart field (von Scheven et al., 2006), but is also expressed in paraxial mesoderm and developing muscles at trunk levels. Double knockouts of MyoR and the related bHLH factor capsulin result in specific loss of the muscles of mastication derived from the first branchial arch, but these mice survive until birth and cardiac abnormalities have not been reported (Lu et al., 2002). Another functionally interacting group of transcription factors which are increased in the Brn3a−/−TG include Dach2, the eyeless-related genes Eya1, Eya2, and Eya4 and the sine oculis homologue Six1. These factors have known roles in somitogenesis and skeletal myogenesis, where they interact with each other and with Pax3 (Heanue et al., 1999). Together these results suggest that in addition to repressing canonical regulators of heart development, Brn3a represses a gene expression program which relates to general aspects of mesoderm development. However, we did not observe increased expression of any of the canonical regulators of definitive myogenesis, including Myod1, Myf5, Myf6 (MRF4), or Myog (myogenin), in the Brn3a−/− TG.
Given the extensive network of transcription factors regulated by Brn3a in sensory ganglia, it is difficult to predict whether any given target is regulated by direct Brn3a binding to cis-elements within that gene locus. In prior work we have used chromatin immunoprecipitation in embryonic sensory ganglia to demonstrate direct repression of Neurod1 and Neurod4 by Brn3a, but examination of the Msc locus from −20kb to +20kb relative to the start of transcription did not reveal Brn3a binding (Lanier et al., 2007). Similar examinations of the Gata3 and Fgf10 loci also did not reveal direct interactions in the E13.5 TG (J. Lanier, unpublished results). Although this suggests indirect regulation of these targets, in some cases the relevant enhancers may lie outside the regions examined. In principle, the direct targets of Brn3a in embryonic sensory neurons could be determined by genome-wide examination of immunoprecipitated chromatin using tiled arrays or high-throughput sequencing (ChIP-chip, ChIP-seq), but application of the current technologies is precluded by the small size of the available tissue samples.
The de-repression of cardiac gene expression in Brn3a−/− trigeminal neurons leads to the question of what underlying developmental process may link the developing TG and heart. One explanation is suggested by the observation that cells contributing to the TG and the heart have a common developmental origin in the cranial neural crest. Cardiac neural crest cells originate at hindbrain levels of the dorsal neural tube, contribute to the aorticopulmonary septum, and have an indirect role in the extension of the outflow tract by the secondary heart field (Hutson and Kirby, 2007). It is possible that Brn3a helps to discriminate the crest-derived sensory neural lineage from the cardiac lineage by repressing a battery of cardiac genes. However, the cardiac genes which are de-repressed in the Brn3a−/− TG are not restricted to the cardiac structures derived from neural crest, arguing for a more general role in the repression of cardiac/cranial mesoderm genes.
A second potential connection between Brn3a and cardiac development may relate to the activity transcription factor Islet1, which is co-expressed with Brn3a in sensory ganglia (Fedtsova et al., 2003). Islet1 is expressed extensively in the developing heart (Cai et al., 2003), where Brn3a is absent, and recent work has shown that Islet1+ precursors also contribute to a subset of cranial muscles (Nathan et al., 2008). The regulatory targets of Islet1 in heart development are largely unknown, but it is possible that the repressor activity of Brn3a opposes the activity of Islet1 on a set of cardiac/cranial muscle genes during trigeminal neurogenesis. However, if this is the case, there must be a redundant mechanism for repression in the DRG, since Islet1 is also expressed in DRG neurons, and loss of Brn3a is not sufficient to activate ectopic expression of most of the dysregulated cardiac genes at trunk levels.
Perhaps the most important generalization derived from this study is that it provides striking evidence for the importance of transcriptional repression as a mechanism for generating correct neuronal phenotypes. Early models of cell-specific gene regulation, based largely on viral systems, emphasized transcriptional activation (Ptashne, 1988). Although such mechanisms are essential, it is now clear that to a large extent neuronal phenotypes are defined in development by overlapping patterns of transcriptional repression (Muhr et al., 2001), whose effects may then be stabilized by chromatin modifications (Allen, 2008). Thus to a large extent, neurons appear to be defined by what they are not, through progressive restriction of cell fate.
Targets of transcriptional repressors are occult by nature: they cannot necessarily be predicted from the normal program of gene expression for the target tissue, and are only revealed in the absence of the repressing factor. The results presented here for Brn3a in the TG illustrate the kind of unexpected programs of gene expression which may emerge when these repressor functions are disabled. Conceptually similar results have been recently obtained in the sensory ganglia of mouse embryos lacking Islet1, which unexpectedly mis-express of a set of spinal cord and hindbrain genes (Sun et al., 2008). Unbiased global assays of gene expression are thus essential to understanding repressor function, and are likely to reveal many more hidden relationships between developing cell types which may be important for applications such as the manipulation of stem cell fates.
We would like to thanks Dr. Sylvia Evans for helpful comments on the manuscript. We are also indebted to Dr. William Wachsman and Lutfunnessa Shireen of the UCSD/VA microarray core facility for assistance with microarray technology, and Gian Carlo Parico for technical assistance. Supported in part by Department of Veterans Affairs MERIT funding, and NIH awards HD33442 and MH065496 (E.E.T). E.E.T. is a NARSAD Investigator.