Department of Psychiatry, University of California, San Diego, and the VA San Diego Healthcare System, La Jolla, CA 92093-0603, USA
* Author for correspondence (e-mail: eturner{at}UCSD.edu)
Accepted 11 May 2004
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SUMMARY |
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Key words: Brn3a, POU-domain, Trigeminal ganglion, Microarray, Sensory neuron
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Introduction |
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Transcription factors reside permanently or conditionally in the nucleus, and are presumed to work by interacting with specific cis-acting binding sites in the vicinity of the transcription units they regulate. These `target genes' in turn mediate the effects of the transcription factor on developmental fate decisions, neuronal phenotype and cell survival. However, the downstream targets of these factors cannot necessarily be inferred from their expression patterns, because they are usually not congruent with those of other classes of neural genes, such as neurotransmitters or their receptors. In a few cases, plausible regulatory relationships have been established between neural transcription factors and their targets, but for the vast majority, no clear pathways are known. Using conventional methods applied to individual genes, establishing these transcription factor-target relationships is quite inefficient.
In principle, comparing the transcript pool of neural tissue from a wild type animal to that of an animal under- or overexpressing a given factor should yield a complete set of genes regulated in that cell type. However, because of the tremendous cellular diversity present in most regions of the nervous system, the resulting changes in gene expression in a specific cell type may be obscured by the heterogeneity of the sample. Furthermore, the changes in target gene expression may be regulated indirectly, either as downstream or compensatory effects.
We have been engaged in studies of Brn3a (Pou4f1 Mouse Genome
Informatics), a transcription factor of the POU-domain family which is
expressed in terminally differentiating neurons of the sensory peripheral
nervous system and caudal CNS. Targeted mutations in mice have shown that
Brn3a is necessary for the correct development and/or survival of neurons in
the sensory ganglia and some CNS nuclei
(McEvilly et al., 1996;
Xiang et al., 1996
). Sensory
neuron death in Brn3a knockout mice is preceded by loss of
neurotrophin receptor expression (Huang et
al., 1999
; Ma et al.,
2003
), and by markedly defective axonal growth
(Eng et al., 2001
). Despite the
success of the knockout approach in demonstrating the importance of Brn3a and
related POU factors in neural development, these experiments have yielded
little information about what genes these factors regulate, and why they are
essential for normal axon growth or neuronal survival.
In the present study, we have used microarrays to compare the patterns of
gene expression in the trigeminal ganglia of Brn3a knockout and
wild-type mice. To maximize the homogeneity of the samples and to minimize
secondary effects on gene expression, we have analyzed embryonic ganglia. At
the stage chosen for analysis, embryonic day 13.5 (E13.5), major defects in
sensory axon growth are observed in the mutant mice
(Eng et al., 2001), but the
phase of marked sensory neuron death has not yet commenced
(Huang et al., 1999
).
Our results demonstrate that Brn3a regulates a coordinated program of gene expression that defines the phenotype of developing trigeminal neurons, including the regulation of neurotransmitters, receptors, ion channels, mediators of axon growth, and other transcription factors. Many of these target genes have known roles in sensory neurons and are strong candidates for mediating the observed effects of Brn3a on axon growth and cell survival. Some of the genes regulated by Brn3a in the trigeminal ganglion are also changed in other sensory ganglia in Brn3a knockout mice, but do not appear to be altered in Brn3a-expressing CNS neurons, suggesting that the roles of Brn3a in the sensory system and CNS may be distinct.
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Materials and methods |
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Non-isotopic in situ hybridization was performed as previously described
(Birren et al., 1993). A table
of probes used and their sources appears in the Supplemental Data (Table S1,
http://dev.biologists.org/supplemental/).
Immunofluorescence for Brn3a was performed with rabbit polyclonal antisera as
previously described (Fedtsova and Turner,
1995
). Immunofluorescence for other antigens was performed with
commercially available antibodies, including rabbit anti-calretinin (Swant),
rabbit anti-galanin (Peninsula Laboratories), rabbit anti-somatostatin-14
(Peninsula Laboratories), and rabbit anti-tyrosine hydroxylase (Chemicon).
Analysis of expression array data
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 genotypes
(`change-p') was performed with Microarray Suite 5.0 (Affymetrix). Two
proprietary databases were used to relate microarray results for ESTs to the
identity of the expressed transcripts, NetAffx (Affymetrix) and GeneSpring
(Silicon Genetics). The results for those transcripts identified in both
databases were discordant in less than 1% of cases.
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Results |
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Although no differences in the phenotype of Brn3a wild-type and
heterozygous embryos have been identified
(Eng et al., 2001), tissue
samples from these genotypes were analyzed separately. The comparison of gene
expression across all three genotypes was performed to look for subtle
differences in heterozygotes, and to provide a partial replication of the
results within each experiment. All three genotypes were analyzed in two
completely independent experiments.
Trigeminal RNA were analyzed using the commercial oligonucleotide-based U74Av2 and U74Bv2 microarrays (Affymetrix). The U74Av2 array represents 12,422 transcripts, including 5993 known genes and 6429 ESTs, and the U74Bv2 array includes an additional 12,411 EST sequences. A significant number of the EST sequences present on both arrays have subsequently been related to identified genes in public and proprietary databases. Of all the transcripts represented on the U74Av2 array, 4885 were detected as `present' in both experiments in at least one of the three Brn3a genotypes, using the manufacturer's standard criteria for array analysis. The transcripts that were reproducibly present on the U74Av2 and U74Bv2 arrays were then further analyzed with respect to their relative expression in the three genotypes.
Two measures were used to compare transcript levels between samples from different genotypes, the change-p value and the fold change in the intensity of the hybridization signal. The change-p value is calculated by proprietary data analysis software (Affymetrix) using the Wilcoxon's signed rank test applied to the hybridization signals for the 16 matched and mismatched oligonucleotide probe pairs representing each transcript in the array. Change-p values <0.003 (increased expression in the arbitrarily designated `experimental' sample) or >0.997 (decreased in the experimental sample) are considered highly significant. For abundant transcripts, change-p values may be significant even when the fold change in expression is small, because for strong signals even minor relative differences may achieve statistical significance by this method. Because small relative changes in expression are not easily verified, and have uncertain biological significance, a minimum twofold increase or decrease in expression was used as an additional criterion for determining the changed transcripts of interest. More detailed information on the transcripts included and excluded by these criteria are given in Fig. S1, http://dev.biologists.org/supplemental/.
Figure 1A compares the relative expression of all present transcripts in heterozygote and knockout ganglia in one analysis using the U74Av2 array. The vast majority of the expressed transcripts fall between parallel lines designating less than a twofold change in expression. The expression values for significantly changed transcripts are located off the central axis, and the positions of selected mRNAs encoding proteins with known roles in sensory development or function are indicated.
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To test this hypothesis, we compared the expression levels of 41 increased, 62 decreased and 160 unchanged transcripts in ganglia from the three Brn3a genotypes. For both the increased and decreased transcripts, the target gene expression levels in wild-type and heterozygous ganglia showed similar differences from those of the knockout (Fig. 1B). In contrast, the wild-type and heterozygote ganglia did not significantly differ from each other for either class of target genes. These results confirm the complete suppression of a heterozygous phenotype at this stage in the trigeminal ganglia of Brn3a knockout mice.
Expression of genes previously reported to be regulated by Brn3a
Numerous genes have been previously reported to be transcriptionally
activated by transfected Brn3a in cell culture models of sensory ganglia.
These putative Brn3a targets include structural components of axons and
synapses, neurotransmitter receptors and oncogenes
(Table 4). Almost all of these
proposed downstream genes are represented on the U74A and B arrays, and were
detected (present call) in E13.5 trigeminal ganglia. However, aside from a
modest but statistically significant decrease in the neurofilament NF-H, none
of these genes were markedly affected by the loss of Brn3a expression in
vivo.
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Previous studies of Brn3a knockout mice have also revealed changes
in the expression of several genes in the sensory ganglia. A major focus of
these studies has been the neurotrophins and their receptors. We have
previously reported microarray assays showing a reduction in TrkA transcripts
in Brn3a knockout trigeminal ganglia at E13.5
(Ma et al., 2003), and our
array analysis is consistent with previous reports that the TrkA neurotrophin
receptor is decreased in these mice (Huang
et al., 1999
; McEvilly et al.,
1996
). Transcripts for the p75 NGF receptor have been reported to
be significantly decreased in mice lacking Brn3a
(McEvilly et al., 1996
),
whilst immunohistochemistry for p75 protein has been reported as unchanged in
mid-gestation knockout ganglia (Huang et
al., 1999
). In the present study, knockout levels of p75 mRNA were
approximately 40% of wild type. Transcript levels for BDNF, previously
reported to be reduced to undetectable levels at E12.5 in the trigeminal
ganglia of Brn3a null mice
(McEvilly et al., 1996
), were
found to be unchanged from controls in the present analysis. Loss of
expression of the TrkB and TrkC neurotrophin receptors has also been reported
in Brn3a null mice, but transcripts for the TrkB and TrkC receptors were not
detected (absent call) in any genotype by the probe sets designed for these
genes on the U74A array. This is an inconclusive result, which may reflect a
problem in array design.
Brn3a regulates the expression of neurotransmitter systems and other transcription factors in multiple sensory ganglia
In addition to the trigeminal ganglion, Brn3a is expressed in neurons of
the vestibulocochlear (VIII) ganglion complex, IX/X ganglion complex, and in
the dorsal root ganglia (Fig.
2B). In order to verify the gene expression changes noted in the
trigeminal array analysis, and to determine whether the trigeminal target
genes are regulated elsewhere in the nervous system, we examined the
expression of several Brn3a regulatory targets by in situ hybridization and
immunohistochemistry in E13.5 embryos.
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The expression patterns of the mediator of Ca2+ signaling,
calretinin, the neuropeptides somatostatin and galanin, and the enzyme of
catecholamine synthesis, tyrosine hydroxylase were examined in the trigeminal
ganglion by immunohistochemistry. Consistent with cell-autonomous regulation
by Brn3a, galanin and tyrosine hydroxylase co-localized with Brn3a protein in
the trigeminal neurons of control ganglia
(Fig. 2E,F), and the direction
and approximate extent of the expression changes in each of these proteins was
entirely consistent with the array results
(Fig. 2G). We also examined the
DRG and spinal cord for changes in the expression of these four proteins (data
not shown). Galanin immunoreactivity was markedly decreased in the DRG, but no
significant changes in calretinin or tyrosine hydroxylase were evident.
Somatostatin immunoreactivity accumulated abnormally in the dorsal root entry
zone of Brn3a knockout mice, a finding which may reflect either
increased expression, or the failure of sensory axons to appropriately enter
the CNS in these mutants (Eng et al.,
2001), or both. No changes were noted in any of these markers in
the Brn3a-expressing neurons of the dorsal spinal cord.
Several transcription factors were also prominent among the most changed transcripts in the array analysis. To verify the array results for the trigeminal ganglion, and examine the expression of these factors in other cranial sensory ganglia and the caudal CNS, we performed in situ hybridization for the increased transcripts GATA3, Irx1, Irx2, AP2b, MyoR, Math3 (Fig. 3A), and NeuroD1 (not shown), and for the decreased transcripts HoxD1 and Runx1 (Fig. 3B), in E13.5 wild-type and Brn3a knockout embryos. In each case the direction and magnitude of change in the in situ hybridization signal in the trigeminal ganglion correlated well with the array results.
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The transcription factors HoxD1 and Runx1 showed decreased expression in the array analysis of Brn3a knockout mice. In situ hybridization for these transcripts confirmed markedly decreased expression in the trigeminal and IX ganglion. Endogenous expression of Runx1 in the VIII ganglion appeared to be less affected.
The regulatory role of Brn3a may be distinct in the CNS
In addition to the sensory ganglia, Brn3a is expressed in specific neurons
of the CNS, residing in the habenula, midbrain tectum and tegmentum,
hindbrain, dorsal spinal cord and retina. The examination of the hindbrain
region and spinal cord by in situ hybridization (Figs
2 and
3) did not indicate any obvious
changes in the expression of neurotransmitters or transcription factors in the
CNS of Brn3a knockout mice. However, in most areas of the CNS,
Brn3a-expressing neurons have a scattered distribution, requiring methods of
detection with cellular resolution to identify changes in target gene
expression. For this reason we examined the CNS of embryos in more detail by
immunohistochemistry for the increased gene products calretinin and
somatostatin, and for the decreased gene products galanin and tyrosine
hydroxylase.
In the midbrain and hindbrain, calretinin and Brn3a are expressed in adjacent but non-overlapping cell populations (Fig. 4A,B), while in the retina, a subset of neurons cells co-express these antigens (Fig. 4D,E). The expression of calretinin was not altered in either of these regions in the absence of Brn3a (Fig. 4C,F). Similarly, somatostatin was not ectopically expressed in the CNS of mice lacking Brn3a (Fig. 4G-K). Galanin and tyrosine hydroxylase were not co-expressed with Brn3a in the CNS as they are in the sensory system (Fig. 4L,M and data not shown), and thus could not be the targets of cell-autonomous regulation by Brn3a. Taken together, the in situ hybridization and immunohistochemical data for the targets of Brn3a regulation in the trigeminal ganglia demonstrate considerable conservation of the regulatory role of Brn3a in sensory neurons at different levels of the neural axis, but suggest a distinct role for Brn3a in the CNS.
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Discussion |
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Most of the genes with profoundly changed expression can be divided into three functional categories: neurotransmitter systems and ion channels, mediators of axonogenesis/synaptogenesis, and transcriptional regulators. Each of these classes of regulated transcripts may be related to the defects in axon growth and cell survival seen in Brn3a mutant mice, but it is likely that these changes in gene expression synergise to produce the Brn3a knockout phenotype, and that no single target gene is sufficient to account for the observed defects.
Beyond explaining the sensory phenotype of Brn3a knockout mice, two interesting generalizations may be made which encompass many of the genes with altered expression. First, in the absence of Brn3a, trigeminal development is retarded, in the sense that the expression of numerous markers of a mature sensory phenotype are reduced, and the developmental expression of factors that play a transient role in the early phases of differentiation is abnormally prolonged. Second, several transcription factors are expressed outside their normal axial level in the sensory ganglia, suggesting that Brn3a acts to spatially restrict their expression.
Neurotransmitter systems and channels
The array results clearly demonstrate that Brn3a has a major role in
determining the neurotransmitter phenotype of the developing trigeminal
ganglia. Expression of the neuropeptides PACAP and galanin and the NPY1
receptor are highly dependent on Brn3a, and the rate-limiting enzyme of
catecholamine synthesis, tyrosine hydroxylase, is also significantly reduced
in Brn3a knockouts. In contrast, the expression of somatostatin and
the 5HT3A receptor are markedly increased. Studies in the developing rat have
shown that somatostatin is strongly expressed throughout the sensory ganglia
soon after neurogenesis, but by mid-gestation its expression is restricted to
a relatively small subset of sensory neurons
(Katz et al., 1992). Thus the
increased expression of somatostatin at E13.5 is very likely to represent a
failure in the normal developmental attenuation of this gene, consistent with
the idea that Brn3a knockout ganglia exhibit a pervasive maturation
defect.
Also notable are changes in the expression of sodium channels, including
Scn6 and Scn9, which are markedly decreased in Brn3a knockout
ganglia, and Scn10, which is moderately decreased (Table S2,
http://dev.biologists.org.supplemental/).
Remarkably, these changes affect only those sodium channels that appear to
have specific expression in the sensory nervous system
(Goldin, 1999;
Waxman et al., 1999
),
suggesting that Brn3a directly or indirectly coordinates expression of these
channels. In contrast, expression levels of most neurotransmitter receptors,
such as the GABA and glutamate receptors, and several classes of ion channels
with wide expression in the CNS and PNS, are unchanged. Two other markedly
changed genes, calretinin and the regulator of G-protein signaling RGS10, have
putative roles in the modulation of neurotransmitter signals mediated by
Ca2+-dependent and G-protein pathways, respectively. Altered
expression of these genes may represent primary changes, or they may occur in
an attempt to compensate homeostatically for other changes in neurotransmitter
systems.
Changes in expression of genes related to axon growth
Mice lacking Brn3a have marked defects in sensory axon growth, including
defasciculation of axon bundles and failure to innervate peripheral and
central targets (Eng et al.,
2001; Trieu et al.,
2003
). The transcripts for several proteins known to be involved
in axon growth and synaptogenesis were significantly decreased in
Brn3a null mice. Among the proteins in this category is advillin
(pervin), an actin-binding protein with specific expression in sensory and
sympathetic ganglia, which increases neurite outgrowth in cultured dorsal root
ganglia (Ravenall et al.,
2002
). Apolipoprotein E knockout mice exhibit anatomical and
functional defects in unmyelinated nerve fibers
(Fullerton et al., 1998
).
Although this has been attributed to loss of ApoE expressed in associated
glia, our results suggest that the defect may be intrinsic to sensory
neurons.
Also decreased in Brn3a knockout ganglia were transcripts for the
functionally interrelated proteins insulin-like growth factor 1 (IGF1) and
insulin-like growth factor binding protein 5 (IGFBP5). Mice lacking IGF1 have
abnormalities in sensory neurons (Gao et
al., 1999), and show defective cortical dendritic growth
(Cheng et al., 2003
). IGFBP5 is
a widely expressed protein whose role in vivo has not been clearly defined.
However, it is highly expressed in the axon terminals of developing sensory
neurons (Cheng et al., 1996
),
where it is frequently co-localized with IGF1, suggesting that it also has a
role in axon growth. Because these proteins are known to interact, relative
deficiencies in their expression may have a synergistic effect.
Another group of Brn3a-regulated proteins likely to have a role in axon growth are those involved in cell signaling and intracellular signal transduction. Transcripts with significantly changed expression include N-chimaerin, downstream of tyrosine kinase 4 (Dok4), the low affinity neurotrophin receptor p75, the small GTPases RAP (Ras family) and WRCH1 (Rho family), and Dusp6/MKP3. The expression and potential role of some of these factors in the sensory nervous system has been described; in other cases, the function of related proteins suggest that they may have significant and synergistic effects on axon growth.
Transcription factors
Loss of Brn3a results in profound changes in the expression of several
transcriptional regulators of various types, suggesting a web of
cross-regulation between genes involved in sensory neurogenesis. The
expression of a few transcription factors expressed late in sensory
development, such as Runx1, were decreased in the absence of Brn3a, but the
majority of the changes were increases, suggesting that Brn3a functions as a
repressor of transcription factors that would be temporally or spatially
inappropriate in the maturing trigeminal ganglion.
The clearest example of the role of Brn3a in restricting the spatial
expression of other transcription factors is the ectopic expression of GATA3,
Irx1, Irx2, NeuroD1 and MyoR/musculin in Brn3a knockout mice. These
factors are all expressed in the developing vestibulocochlear ganglion in
control embryos, and in the absence of Brn3a are markedly increased in the
trigeminal and IX/X ganglia, demonstrating an expansion of the expression
domain of these genes in both directions of the rostrocaudal axis. It is
likely that some of the downstream changes in gene expression in
Brn3a knockout ganglia are mediated by these factors, but current
knowledge of their role in neural development is not sufficient to predict the
effect of their mis-expression in the trigeminal ganglion. GATA3 has a known
role in the development of motor neurons originating in rhombomere 4, which
innervate the inner ear, and the inner ear itself
(Karis et al., 2001). NeuroD1
is also required for normal development of the sensory neurons of the inner
ear (Liu et al., 2000
), and
may have a cross-regulatory relationship with GATA3
(Lawoko-Kerali et al., 2004
).
Although the role of the Irx genes in sensory development has not been
described in mice, the zebrafish protein iro7, a possible paralogue of Irx1,
is required for trigeminal placode development in fish
(Itoh et al., 2002
). The bHLH
factor MyoR (musculin) is normally expressed in the developing facial muscles
of the first branchial arch, which are innervated by trigeminal neurons, but
not in the trigeminal ganglion itself (Lu
et al., 2002
). Our observation that MyoR is expressed in the
developing auditory system is the first report of the sensory expression of
this gene, and its role in neurogenesis is unknown.
Although it was not detected in the vestibulocochlear ganglion at this
stage, AP2ß showed a similar pattern of ectopic expression in the
trigeminal and IX cranial ganglia in E13.5 Brn3a knockout embryos.
AP2ß is normally expressed in the embryonic hindbrain and spinal cord,
but little is known about its role in neural development. The nervous system
of AP2ß mutant mice, which die from polycystic kidney disease,
has no obvious abnormalities (Moser et
al., 1997). However, mice lacking the related factor AP2
,
which is highly expressed in migrating neural crest and in the developing
sensory ganglia, exhibit extensive cranial abnormalities and dysgenesis of the
cranial ganglia (Schorle et al.,
1996
). There is some evidence that AP2ß is a weak
transcriptional activator, and may oppose gene activation by AP2
(Bosher et al., 1996
). Thus the
increased expression of AP2ß observed here may mimic some aspects of the
loss of AP2
.
The increased expression of Math3 and NeuroD1 in Brn3a knockout
trigeminal ganglia, together with decreased expression of the inhibitor of
bHLH function Id1, suggest a marked increase in bHLH activity in the absence
of Brn3a. Math3 and NeuroD1 have been characterized in the early development
of the trigeminal ganglion (E9.0), where both factors appear to be downstream
of the neurogenic HLH factor Ngn1 (Ma et
al., 1998). Thus the increased expression of bHLH factors in
Brn3a knockout mice may reflect the abnormal persistence of genes
normally down-regulated as sensory development progresses. Although the loss
of NeuroD1 or Math3 alone does not have an obvious effect on neurogenesis in
the trigeminal (Tomita et al.,
2000
), the increased expression of multiple bHLH genes may have a
synergistic effect in Brn3a knockout mice.
Possible mechanisms of sensory cell death in mice lacking Brn3a
Embryonic day 13.5 was chosen for gene expression analysis because it
precedes the extensive loss of sensory neurons observed at later stages in
Brn3a knockout mice, and consistent with this, we did not observe
altered expression of genes usually associated with cell death pathways, such
as caspases or bcl2-family genes. Sensory cell death in mice lacking Brn3a
occurs after these neurons normally become neurotrophin dependent, and the
decreased expression of neurotrophins and their receptors in Brn3a
knockout mice has been suggested as a cause of this mortality
(Huang et al., 1999). We have
previously reported that the expression of the TrkA neurotrophin receptor mRNA
is moderately decreased in Brn3a knockout mice
(Ma et al., 2003
). This
observation, and the decreased expression of the p75 low affinity NGF receptor
shown here, are consistent with previous results
(Huang et al., 1999
;
McEvilly et al., 1996
).
However, because the TrkA receptor is generally regarded as anti-apoptotic,
and the p75 receptor as pro-apoptotic in sensory neurons
(Huang and Reichardt, 2001
),
it is not obvious what net effect a moderate decrease in both receptors would
have on cell survival. Given the severity of the axon growth defects in
Brn3a knockout mice, another possibility is that excessive neural
death occurs because of a failure to obtain target-derived neurotrophins, but
this hypothesis has not been tested directly.
Tissue specificity of gene regulation
In the present study we have defined a set of genes regulated by Brn3a in
sensory ganglia. This represents one of the first comprehensive descriptions
of the in vivo regulatory targets for any factor regulating vertebrate
neurogenesis. Like many developmental regulators, Brn3a is expressed in a
highly specific, yet diverse set of neurons, including those of the retina,
diencephalon, midbrain, spinal cord and sensory system, leading to the
question of whether Brn3a regulates a common set of targets in these distinct
locations. In the present study we have found little evidence that the targets
of Brn3a regulation in the trigeminal ganglia are also regulated in the CNS or
in the retina. A recent analysis of the regulatory targets of the closely
related POU-factor Brn3b in the retina revealed few changed transcripts in
common with the present study, despite the fact that the retinal ganglion
cells in Brn3b knockout mice show a secondary loss of Brn3a
(Mu et al., 2004); it also did
not detect changes in the retinal target genes in sensory ganglia.
Even within the peripheral sensory system, Brn3a targets appear to be
distinctly regulated in the vestibulocochlear ganglion when compared to the
coordinated changes in expression in the trigeminal, IX, and dorsal root
ganglia. The lack of change in trigeminal target genes in the
vestibulocochlear ganglion cannot be attributed to functional redundancy of
Brn3 genes. Although Brn3b is also expressed in the vestibulocochlear system,
the loss of Brn3a expression in the vestibulocochlear ganglion also leads to
diminished expression of Brn3b, and results in significant defects in cochlear
innervation (Huang et al.,
2001). Thus it appears probable that Brn3a will have at least a
partially distinct set of regulatory targets in the auditory system.
The genes downstream from Brn3a in the sensory ganglia are very likely to
include targets that are regulated directly, and regulated indirectly by the
several other transcription factors that change expression in the absence of
Brn3a. One of the surprising features of the current study is the large number
of markedly increased transcripts in the knockout ganglia, implying direct or
indirect transcriptional repression by Brn3a. Although nearly all prior
studies of the transcriptional activity of Brn3a have proceeded from the
assumption that it is a positive regulator of gene expression, we have
recently shown that Brn3a is a direct repressor of its own expression in the
trigeminal ganglion in vivo (Trieu et al.,
2003). The recent study of the target genes of Brn3b in the retina
showed mainly decreased expression of downstream transcripts
(Mu et al., 2004
), but this
study was conducted with a retina-specific cDNA array, which would be unlikely
to include strongly increased transcripts which have low levels of expression
in the normal retina. Thus it is plausible that Brn3a, and perhaps other
factors in this class, exert their direct effects by transcriptional
repression.
Identifying the regulatory targets of neural transcription factors is an
essential component of understanding developmental pathways in the nervous
system. Here we have demonstrated an extensive program of gene regulation
mediated by one such factor. Future studies of this kind will be greatly
facilitated by the availability of more complete gene expression arrays based
on genomic sequences rather than cDNA libraries. Additional data about the
location of the transcription units in the mouse genome, and better
information about the DNA recognition properties of the various transcription
factor classes, will help to distinguish direct from secondary targets. In
addition, the confirmation of direct regulation by chromatin
immunoprecipitation may be facilitated by combining this method with array
technology or other high throughput methods
(Ren et al., 2002). These
anticipated technical advances should in principle allow the identification of
a complete set of regulatory targets for any transcription factor in any
tissue.
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ACKNOWLEDGMENTS |
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Footnotes |
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