1 Center for Developmental Biology and Kent Waldrep Foundation Center for Basic
Research on Nerve Growth and Regeneration, University of Texas Southwestern
Medical Center, Dallas, TX 75390-9133, USA
2 San Diego VA Medical Center, University of California, San Diego, La Jolla, CA
92093, USA
3 Department of Psychiatry, University of California, San Diego, La Jolla, CA
92093, USA
* Author for correspondence (e-mail: luis.parada{at}utsouthwestern.edu)
Accepted 28 April 2003
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SUMMARY |
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Key words: TrkA, Brn3a, Sensory neuron, Transcriptional regulation, Bax
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INTRODUCTION |
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TrkA (Ntrk1 Mouse Genome Informatics), the
prototype of the Trk gene family and the receptor for nerve growth factor
(NGF), is required for the survival of nociceptive sensory and sympathetic
neurons (Martin-Zanca et al.,
1990; Smeyne et al.,
1994
). In the murine PNS, TrkA expression is confined to
neural-crest-derived sensory neurons, including trigeminal and dorsal root
ganglia, from early embryonic stages (E9.5) to adults. TrkA is also expressed
in sympathetic neurons from E16.5 onwards
(Martin-Zanca et al., 1990
;
Tessarollo et al., 1993
). In
the CNS, TrkA expression is described in only a subset of cholinergic
neurons in the basal forebrain (Holtzman
et al., 1992
). Understanding the molecular mechanisms for TrkA
expression is important to dissect the biological processes regulating the
survival and differentiation of sensory and sympathetic neurons.
One approach to identify transcriptional regulators of TrkA is to search
for known transcription factors that are expressed in sensory neurons during
mouse embryonic development and whose loss-of-function lead to alterations of
TrkA expression. The POU-homeodomain transcription factor Brn3a (Pou4f1
Mouse Genome Informatics) (Gerrero
et al., 1993; He et al.,
1989
; Ninkina et al.,
1993
; Xiang et al.,
1993
) stands out as an attractive candidate. Brn3a is expressed
early in emerging sensory neurons (Artinger
et al., 1998
; Fedtsova and
Turner, 1995
) and mutation of this gene in mice leads to abnormal
development and loss of neurons that express TrkA, TrkB (Ntrk2 Mouse
Genome Informatics) and TrkC (Ntrk3 Mouse Genome Informatics)
(Eng et al., 2001
;
Huang et al., 2001
;
Huang et al., 1999
;
McEvilly et al., 1996
;
Xiang et al., 1996
). However,
questions remain about why and how TrkA becomes downregulated in
Brn3a-null sensory neurons. Whether Brn3a is a requisite
transcriptional regulator of the TrkA enhancer or an indirect mediator of TrkA
expression is unresolved.
Previously, we identified a TrkA minimal enhancer that confers
specific expression of ß-galactosidase transgenes in PNS sensory neurons
(Ma et al., 2000). Through
mutational analysis in transgenic mice, multiple important, cis-elements were
identified. These results indicate that the tightly regulated TrkA
gene is controlled by multiple transcription factors acting in concert
(Lei et al., 2001
;
Ma et al., 2000
). However,
these studies failed to identify canonical Brn3a-binding sequences in this
enhancer. If Brn3a directly regulates TrkA transcription, the binding sites on
the TrkA enhancer remain unidentified.
In the present study, we have used genetic, biochemical and transgenic approaches to examine whether Brn3a is a direct or indirect regulator of TrkA transcription.
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MATERIALS AND METHODS |
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Genotyping
We designed novel PCR primers to genotype Brn3a-mutant mice:
wild-type 5' primer, 5'-CTTGGCTTCCACTCAGCATCTGGAGC-3';
wild-type 3' primer, 5'-CTGTATTCAGTGGAGAGAAGTGGAAACGG-3';
NEO primer, 5'-GATTCGCAGCGCATCGCCTTCTATCG-3'. PCR conditions were:
94°C, 1 minute; 55°C, 1 minute; 72°C, 1 minute; 35 cycles. The
size of the wild-type and mutant bands are 600 bp and
450 bp,
respectively. The PCR primers and conditions for Bax genotyping were
as described (Deckwerth et al.,
1996
): Bax exon primer
5'-TGATCAGAACCATCATG-3'; Bax intron primer
5'-GTTGACCAGAGTGGCGTAGG-3'; and Bax neo/pgk primer
5'-CCGCTTCCATTGCTCAGCGG-3'. PCR primers for ß-galactosidase
genotyping were: 5'-AACTGGAAGTCGCCGCGCCACTGGTGTGGG-3' and
5'-TGAACTGCCAGCTGGCGCAGGTAGCAGAGC-3'. PCR conditions were:
94°C, 30 seconds; 65°C, 30 seconds; 72°C, 30 seconds; 31 cycles.
Generation of transient transgenic embryos and X-gal staining of the
transgenic embryos were performed as described
(Ma et al., 2000
).
Purification of GST-Brn3a fusion protein
Escherichia coli BL21 competent cells were transformed with an
expression plasmid for GST-Brn3a POU-domain fusion protein
(Xiang et al., 1995). 1 liter
cell culture at OD600=0.6 was induced to express the fusion protein
with 1 mM IPTG for 4 hours. The cell pellet was resuspended in lysis buffer
and sonicated five times on ice for 10 seconds. The supernatant was purified
with Glutathone-Sepharose 4B beads (Pharmacia). Cells transformed with the
control plasmid expressing only GST were processed simultaneously.
Electrophoretic mobility shift assay (EMSA)
EMSA was performed as described (Gruber
et al., 1997) with minor modifications. Briefly, 10,000 to 40,000
cpm of 32P-labeled oligonucleotide probe was mixed with 1 µl
(0.3 µg) purified GSTBrn3a fusion protein, 5 x gel-shift buffer and 1
µg poly(dI-dC) (Boehringer). Finally, water was added to a total volume of
20 µl. The mix was incubated at 25°C for 30 minutes. For competition
assays, different amounts of cold oligonucleotides were mixed with
radiolabeled probes. Electrophoresis was done using 5% polyacrylamide gels in
0.5xTBE buffer at 25°C for 3 hours. Gels were then dried and exposed
with Kodak X-ray film.
DNase I footprint assay
DNase I footprint assays were performed as described
(Brenowitz et al., 1995) with
minor modifications. Either the 5' or 3' end of the TrkA
minimal-enhancer fragment was labeled with [
-32P]-ATP. The
labeled fragment was purified with phenol/chloroform extraction and ethanol
precipitation. Different amounts of either purified, GST-Brn3a fusion protein
or GST control protein was mixed with the labeled fragment and treated with
graduated amounts of DNase I for 2 minutes at 25°C. The reaction was
stopped by adding three volumes of cold ethanol, precipitated and run on
sequencing gel. Sequencing reactions primed from the starting position of the
labeled end were loaded onto the same gel to identify the protected
sequences.
In situ hybridization
In situ hybridization was performed as described
(Lei et al., 2001). The
templates for making antisense in situ probes were TrkA (454 bp, extracellular
domain) and NCAM (a 420 bp coding sequence). Sections were processed
simultaneously for comparison.
Immunohistochemistry
Immunohistochemistry was performed as described
(Ma et al., 2002) using the
following antibodies and dilutions: monoclonal NeuN (1:500) (Chemicon); rabbit
anti-NF200 (1:1000) (Sigma); rabbit anti peripherin (1:1000) (Chemicon); and
rabbit anti TrkA (1:1000) (Advanced Targeting System). Antigen retrieval was
performed as described by the manufacturer (BioGenex, CA) to detect TrkA
signal. Sections were processed simultaneously with the same antibody for
comparison.
Affymetrix microchip assay and analysis
Affymetrix microchip assay and analysis followed the manufacturers
instructions (Affymetrix, CA). The source of labeled probe was trigeminal
ganglia prepared from E13.5 Brn3a-null, heterozygous and wild-type
littermates.
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RESULTS |
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The proapoptotic gene Bax is required to mediate apoptosis in
sensory neurons (Deckwerth et al.,
1996). Although the nociceptive sensory neurons are normally
absent in the TrkA-/- mice at birth, these neurons survive
in TrkA-/-/Bax-/- mice
(Patel et al., 2000
). We
reasoned that if we placed the Brn3a mutation
(Xiang et al., 1996
) in the
Bax-null background, sensory neurons would be rescued, thus
permitting analysis of TrkA expression in the absence of cell death.
The genetic cross between Brn3a-null and Bax-null mice
yields nine different genetic combinations (see Materials and Methods). To
simplify our studies, we used the following four genotypes in our experiments:
Brn3a+//Bax+/ (control);
Brn3a-/-/Bax+/ (Brn3a null);
Brn3a+//Bax-/-(Bax null); and
Brn3a-/-/Bax-/- (double null). The
Brn3a+//Bax+/ mice used
as controls appear to be phenotypically normal with respect to sensory
function, fertility and longevity.
To examine TrkA expression in trigeminal ganglia, we first
performed in situ hybridization using a TrkA-specific probe with each
of the four analyzed genotypes at postnatal day 0 (P0)
(Lei et al., 2001;
Martin-Zanca et al., 1990
). As
indicated in Fig. 1A1-B4, TrkA transcripts were reduced in the two genotypes that lack
Brn3a (Brn3a-null and double-null pups). Thus, loss of
Brn3a results in the downregulation of TrkA expression,
which is unrelated to TrkA functional (trophic) dependence (n=3 for
all genotypes). As shown in Fig.
1C1-C4, NCAM mRNA is present at similar level in all four
genotypes.
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Brn3a binds the TrkA minimal enhancer
To address whether the genetic interaction between Brn3a and
TrkA is direct, we searched the minimal enhancer sequence of
TrkA for consensus Brn3a-binding sites, either
GCAT(A/T)A(T/A)T(A/T)AT (Gruber et al.,
1997) or (A/G)CTCATTAA(T/C)
(Xiang et al., 1995
). No
canonical Brn3a-binding sites could be identified. We next scrutinized the
full, 2 kb promoter/enhancer region upstream of the TrkA gene and
found no consensus Brn3a-binding sequences (data not shown).
We then turned to an empirical approach using purified GSTBrn3a
DNA-binding-domain-fusion proteins (Xiang
et al., 1995) to perform gel-shift assays with six, overlapping,
100 bp DNA fragments that cover the entire 457 bp minimal enhancer
(Fig. 4A). Only the two,
outlying, DNA fragments at the 5' and 3' ends of the minimal
enhancer were shifted by the GST-Brn3a fusion protein
(Fig. 4B), indicating the
presence of novel Brn3a-binding sites in these two regions. As controls, GST
protein alone did not cause any shift whereas an anti-GST antibody disrupted
the interaction of F1 and F6 with the GSTBrn3a protein (data not shown). To
identify the core Brn3a-binding sequences in the two 100 bp fragments, we
performed DNase I footprint analyses (Ausubel et al., 1995) with the entire
457 bp minimal enhancer using the GST-Brn3a fusion protein. As shown in
Fig. 4C and D, sequences
protected by the GST-Brn3a fusion protein were present in the 5' and
3' sequences that were included in the gel-shifted fragments
(Fig. 4A,B). The length of both
protected DNA sequences is <40 bp, providing more accurate information
about the core binding sites of Brn3a. These two protected sequences compete
successfully with the corresponding 100 bp sequences for GST-Brn3a binding in
a gel shift assay (Fig. 4E,F,
left panels), providing further evidence of Brn3a-specific DNA sequences in
the TrkA minimal enhancer.
|
Functional analysis of Brn3a-binding sites
We have previously defined several cis elements in the TrkA
minimal enhancer that are required for transcription through site-directed
mutagenesis and transgenic assays (Ma et
al., 2000). We therefore used similar methods to determine the
importance of the newly defined Brn3-binding sites for in vivo enhancer
function. The 5' and 3' core sequences defined in
Fig, 4E,F (boxed sequences)
were subjected to mutagenesis and then to transient transgenic analysis
(Ma et al., 2000
).
As previously described, the wild type TrkA minimal enhancer
drives strong and specific lacZ expression in trigeminal ganglia in >50% of
lacZ positive embryos at E13.5 (Fig.
5, E13.5 and Fig.
6A). The variability is related to transgene positional effects
(Ma et al., 2000). When the
minimal enhancer harbors the 5' Brn3a-site mutation, two out of two
(100%) blue E13.5 embryos exhibited normal expression in trigeninal ganglia
(Fig. 5 and
Fig. 6B). Mutation of the
3' Brn3a site resulted in only one out of four blue embryos maintaining
normal trigeminal expression (Fig.
5, Fig. 6C for
normal expression, and data not shown).
We next tested the functional consequences of mutating both the 3' and 5' Brn3a-binding sites in the enhancer. Of ten blue E13.5 embryos, only one exhibited appreciable expression in the trigeminal ganglion (data not shown), the remaining nine embryos either had extremely weak, or undetectable trigeminal-ganglion expression (Fig. 5, Fig. 6D, and data not shown). These results are consistent with the microarray experiments indicating that by E13.5, mutation of both Brn3a sites significantly compromised the function of the TrkA minimal enhancer in trigeminal ganglia. Mutation of each site alone has either no detectable (5' site) or weak (3' site) effect on TrkA-enhancer function.
We extended our transient transgenic analysis to E17.5 with the minimal
enhancer bearing mutations in either one or both Brn3a-binding sites. As
predicted, the wild-type enhancer drives ß-galactosidase expression
strongly and specifically in nociceptive neurons in trigeminal ganglia at
E17.5 (Fig. 5 and
Fig. 6E)
(Ma et al., 2000). Neither
mutation alone (either 5' or 3') appreciably affected the activity
of the TrkA minimal enhancer at E17.5
(Fig. 5;
Fig. 6F,G). Finally, to test if
these two Brn3a sites in the minimal enhancer are redundant for expression in
the trigeminal ganglion at E17.5, transient transgenic embryos were generated
with the minimal enhancer bearing both the 5' and 3' sites
mutations. Of eight blue embryos generated, seven showed either greatly
reduced or no detectable expression in trigeminal ganglia
(Fig. 5;
Fig. 6H,I, and data not shown).
The remaining blue embryo expressed ß-galactosidase comparably to wild
type and single-mutant embryos (data not shown). We also generated
TrkA minimal enhancer transgenic P0 pups bearing both
Brn3a-binding-site mutations, with similar results. Double mutations clearly
affected enhancer function in trigeminal ganglia
(Fig. 5 and data not shown),
with only three out of fifteen blue pups maintaining normal
ß-galactosidase expression in trigeminal ganglia. Taken together, our
transgenic results indicate that these two Brn3a sites are important for
maintaining TrkA enhancer function after E13.5, although they are
functionally redundant.
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DISCUSSION |
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Several transcription factors have been shown to function in sensory and
sympathetic gangliogenesis. For example, the bHLH transcription factors,
neurogenin1, neurogenin2 and mash1, are required for normal development of
sensory neurons (Anderson,
1999; Anderson et al.,
1997
). Because of their early expression, these factors are likely
to affect neural-precursor proliferation rather than the differentiation and
survival of postmitotic neurons (Ma et
al., 1999
). It is unclear whether these transcription factors
participate in regulating expression of TrkA.
Pou-domain proteins exert crucial effects on neural development. In the
PNS, Brn3a has received considerable attention because of its striking pattern
of expression and the dramatic phenotypic consequences of Brn3a
ablation in mice, which includes loss of sensory neurons
(He et al., 1989;
Huang et al., 1999
;
McEvilly et al., 1996
;
Xiang et al., 1996
). A key,
unresolved question has been the underlying mechanistic requirements for Brn3a
by sensory neurons. Although putative Brn3a-target genes have been proposed
(Smith et al., 1997a
;
Smith et al., 1998
;
Smith et al., 1999
;
Smith et al., 1997b
), the
relevance of these genes in vivo remains to be tested.
Brn3a-mutant sensory neurons from trigeminal ganglia have
decreased expression of TrkA, beginning at E13.5. Because
Brn3a expression appears at E9, the observed downregulation of
TrkA at E13.5 and subsequent neuronal death in Brn3a-null
mice could be caused by neuronal defects other than direct regulation of
TrkA by Brn3a (Eng et al.,
2001; Huang et al.,
1999
). Even if TrkA is regulated by Brn3a, it is unclear
whether this is direct regulation, mediated by Brn3a binding to TrkA
cis-regulatory sequences, or indirect regulation, mediated by other factors.
To investigate these possibilities, we sought to dissociate neuronal survival
from Trk-receptor expression and function.
The use of Bax-null mice permitted sensory neurons to survive in
the absence of TrkA function because Bax is required for neurotrophin-deprived
neuronal apoptosis (Deckwerth et al.,
1996). Furthermore, the sensory neurons rescued from apoptosis by
crossing into a Bax-null background are known to express normal
sensory neuron markers and have been utilized to uncouple apoptosis from other
biological events, such as peripheral axonal outgrowth and elaboration
(Deckwerth et al., 1996
;
Lonze et al., 2002
;
Patel et al., 2000
). Using a
similar approach, we were able to discern the appearance of sensory neurons in
the absence of functional Brn3a, which supports the model that Brn3a
is not required for early specification and differentiation of sensory
neurons. Instead, these neurons were present but had reduced expression of
TrkA transcripts and proteins. These results are consistent with
transcriptional regulation of TrkA expression by Brn3a.
The two Brn3a sites in the TrkA enhancer are not identical to
binding sequences described previously
(Gruber et al., 1997;
Xiang et al., 1995
). Analysis
in vitro indicates that they have lower affinity than consensus Brn3a sites,
although they are well conserved between mice and humans (data not shown)
(Gruber et al., 1997
). As
expected, mutation of both sites did not completely ablate reporter-gene
expression in sensory neurons. A reduced, but apparent, level of enhancer
activity still exists. Considering possible enhancing and suppressing effects
of genomic sequences surrounding the transgene-insertion sites, the
mutagenized enhancers might exhibit either near normal or much weaker
activity. However, the trend of reduced activity should be maintained if a
significantly large number of transgenic embryos are analyzed. Our transient
transgenic analysis fits this prediction nicely.
How does binding affinity in vitro relate to function in vivo? The
physiological environment provided by chromatin and available cofactors in a
given cell nucleus will impact on the accessibility of a transcription factor
to its DNA-binding site and, thus, the physiological affinity. In
Caenorhabditis elegans, Drosophila and mammals, there are many
examples that low affinity sites in vitro are important for transcription in
vivo. Among these are the FoxA transcription factor PHA-4 in C.
elegans (Gaudet and Mango,
2002), bicoid and dorsal morphogen in Drosophila
(Driever et al., 1989
;
Jiang and Levine, 1993
), and
HNF4 in the regulation of erythropoietin expression and Elf-1 in
lymphoid-specific-gene expression in mammals
(John et al., 1996
;
Makita et al., 2001
). In the
case of HNF4-mediated regulation of erythropoietin, transcriptional initiation
is mediated by a high affinity interaction with the retinoid acid receptors,
which is then supplanted by low affinity binding of HNF4
(Makita et al., 2001
).
Similarly, Brn3a is not required for initiation of TrkA transcription
but is required for its maintenance, beginning at E13.5. Because TrkA is still
expressed in Brn3a-null sensory neurons, although at a lower level,
other transcription factors (Lei et al.,
2001
) may also function to regulate TrkA expression. These factors
may interact with Brn3a to regulate TrkA expression in wild-type neurons.
Transcriptional cofactors also affect the activity of a transcription
factor. UNC86, a Brn3a ortholog in C. elegans, interacts with the
LIM-type homeodomain protein MEC-3 and the two proteins function
synergistically to activate the mec-3 promoter in vitro (Xue, 1992;
Xue, 1993; Lichtsteiner, 1995). In addition, both proteins are required for
the fate specification of touch cells in C. elegans through
heterodimeric binding to consensus cis-elements
(Duggan et al., 1998). Recent
experiments demonstrate further examples of how interactions between the Oct-1
POU-domain protein and SNAP190 cofactor affect DNA binding
(Hovde et al., 2002
). It
remains interesting to identify Brn3a cofactors that are important for TrkA
expression and the development of mammalian sensory neurons.
It is interesting to note that no significant differences in TrkA
mRNA levels were detected in Brn3a wild-type and heterozygous embryos
(Table 1). This finding is
consistent with our recent observation that autoregulation of the
Brn3a locus leads to suppression of haplotype insufficiency, and
results in similar levels of Brn3a mRNA in trigeminal neurons that
contain either one or two copies of the Brn3a gene
(Trieu et al., 2003). It is
also consistent with studies that show no detectable phenotype in
Brn3a heterozygotes (Eng et al.,
2001
; Huang et al.,
1999
) and validates the use of heterozygotic controls in the
present study.
Brn3a-null sensory neurons have aberrant axonal arborization at
E13.5 (Eng et al., 2001). At
this time TrkA protein levels are minimally reduced
(Huang et al., 1999
). Lack of
TrkA/NGF signaling leads to defective innervation of peripheral targets by
nociceptive neurons (Patel et al.,
2000
). Considering that Brn3a might regulate multiple target genes
in sensory neurons, it is likely that the sensory axonal defects observed in
Brn3a-null mice are the consequences of altered gene expression at
many loci, including TrkA. Identification of additional
Brn3a target genes should shed light on these complex neuronal
phenotypes.
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ACKNOWLEDGMENTS |
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