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INTRODUCTION |
Long before synaptic networks are fully established, electrical
activity present in developing neurons regulates neuronal differentiation (1-4). In particular, electrical activity mediated by
the NMDA1 class of glutamate
receptors is required for normal neuronal development. Loss of NMDA
receptor function during development increases neuronal cell death (5),
prevents the formation of precise neural circuits (6, 7), diminishes
respiration and feeding (8-10), and has been implicated in fetal
alcohol syndrome (11) and schizophrenia (12). NMDA receptor function
can also regulate neuronal proliferation (13) and migration (14).
Despite the importance of NMDA receptors for normal development and
adult brain function, knowledge of molecular mechanisms regulated by NMDA receptors in developing neurons is rudimentary.
Evidence from development as well as adult models of learning and
memory indicate that regulation of gene expression is an important
strategy that can be used to mediate changes in neuronal structure and
function. Developmental studies suggest that mRNA abundance is
rate-limiting for the accumulation of functional neurotransmitter
receptors on neurons during development in vivo (15).
Changes in gene expression also accompany activity regulated synaptic
plasticity events in the developing visual system (16), as well as at
the neuromuscular junction (17). Transcription factors have recently
been shown to play a critical role for the development of synaptic
connectivity (18-20). Similarly, transcription may be required for
activity dependent long term changes in synaptic strength in mature
nervous systems (21-23). Finally, the electrical activity of neurons,
including NMDA receptor function, has been shown to regulate gene
expression in the adult hippocampus (24-27), a structure critical for
the establishment and maintenance of memories (28-30).
To test the hypothesis that NMDA receptor-dependent
regulation of gene expression is required for, and directs, molecular and cellular mechanisms for neuronal development, we have designed a
screen based on the neural circuit that connects highly specialized whiskers (mystacial vibrissae) found on the snout of the mouse to their
synaptic targets in the brain stem. These whiskers are richly
innervated by pseudounipolar sensory neurons of the trigeminal ganglion
that project centrally to the brain stem trigeminal complex. Here
synaptic inputs from the trigeminal ganglion that correspond to the
mystacial vibrissae are organized in a pattern that matches their
topographic organization on the face. This pattern of whisker representations, called barrelettes at the level of the brain stem, is
relayed and reiterated in the thalamus and finally the cerebral cortex,
and requires NMDA receptor function for normal development (8, 10, 31,
32). The whisker representations are comparable to other sensory maps,
for example, for touch, hearing, and vision, that are found in humans,
and serve as a powerful mammalian model for highly patterned synaptic
development and organization in vivo (33).
By using a unique combination of NMDAR1 knockout mice (8),
the trigeminal system of whisker representations, and DNA array analysis, we have discovered a group of three NMDA
receptor-regulated genes (NARG1, NARG2, and
NARG3) that share striking regulatory features. In the brain
stem, where barrelettes are present in wild-type but absent in NMDA
receptor knockout mice, these NARGs are expressed at
approximately 2-fold higher than normal levels in NMDAR1
knockout animals. Comparison of NARG expression among NMDAR1 knockout, wild-type, and heterozygous brain stems
indicates dose-dependence in terms of NMDA
receptor-dependent regulation of these genes. In the adult,
all three NARGs are expressed at low levels, but with
similar spatial distribution. During brain development these genes peak
in expression around the time of birth, with highest levels in regions
where proliferation and migration are still occurring. Expression is
dramatically down-regulated during early postnatal development at the
same time that NMDAR1 is up-regulated. The results support
the hypothesis that NMDA receptor-dependent regulation of
gene expression plays a crucial role for neuronal maturation during
mammalian brain development.
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EXPERIMENTAL PROCEDURES |
RNA Isolation--
All tissue dissection for RNA preparation was
performed under ice-cold saline. For prenatal brains, the dam was
anesthetized by metofane and embryos were quickly removed. For P0 mice,
animals were anesthetized briefly on ice, decapitated, and the tissue was rapidly dissected. Older animals were anesthetized by metofane prior to decapitation and dissection. Total RNA was isolated by homogenization and extraction with an acid guanidinium
thiocyanate/phenol/choloroform mixture as previously described
(34) with modifications (Atlas Pure Total RNA Isolation Kit,
CLONTECH). RNA yield, concentrations, and quality
were determined by OD260 and corroborated by ethidium bromide staining in formaldehyde agarose gels. Genotyping was performed
by PCR as previously described (35).
DNA Array Analysis--
Total RNA was extracted from P0 mouse
brain stems and used to prepare wild-type (+/+) and NMDAR1
knockout (
/
) first-strand cDNA probes that were hybridized to
nylon and glass cDNA arrays. For nylon array analysis
(CLONTECH catalog number 7741-1), first-strand 32P-labeled cDNA probes were prepared and the membranes
processed according to the manufacturer's instructions. Specific
signal was detected for 297 out of 588 genes represented. Of these, 161 genes yielded signals at least 2-fold above background; expression was
compared between wild-type and knockout for these genes. Three independent experiments were performed, and no significant differences were observed in signals obtained using wild-type and knockout probes.
For microarray analysis (Mouse GEM 1, Incyte), RNA from brain stems of
10 wild-type and 10 knockout P0 mice was extracted in parallel, and the
genotypes of the final wild-type and knockout RNA preparations were
reconfirmed by RT-PCR. The RNA samples were processed by Incyte on GEM
1, which contains a total of 8,734 elements, including at least 7,634 unique genes/clusters, controls, and a number of partially
characterized elements (ESTs). Taken together, our array experiments
analyzed about 8,500 genes. Assuming that the number of the mouse genes
is no more than about 60,000, i.e. similar to human
estimates (36), our screen included 10-15% of all expressed mouse genes.
RNase Protection--
RNase protection experiments were carried
out as previously described (15). Briefly, total RNA (10-20 µg) was
annealed to a molar excess of 32P-labeled single-stranded
antisense RNA probe for 5 min at 85 °C in 80% formamide, 40 mM PIPES buffer (pH 6.7), 0.4 M NaCl, and 1 mM EDTA, and the hybridization was continued for 12-18 h at 45 °C. Single-stranded RNA was digested by incubating for 60 min
at 15 °C with 4 µg/ml DNase-free RNase (Roche Molecular
Biochemicals) and 0.4 µg/ml RNase T1 (Roche Molecular Biochemicals).
The RNases were then inactivated by treatment with proteinase K and
SDS, and the RNA was extracted with phenol/chloroform, precipitated with ethanol, dissolved in loading buffer containing 80% formamide, and electrophoresed in 5% acrylamide containing 8 M urea.
The dried gels were exposed at
70 °C to Kodak BioMax MS film, and signals were quantified directly from the gels using a Molecular Dynamics Storm 860 PhosphorImager (except for one NARG1 experiment in
which a scanned autoradiograph was quantified using NIH Image). Probes
used for RNase protection are as follows: NARG1, nucleotides 19-291 of
AA474587; NARG2 nucleotides 102-365 of AA472833; NARG3, nucleotides
5-283 of AA473329.
In Situ Hybridization--
In situ hybridization
analysis was performed as described (37, 38). Briefly, cryostat
sections (12 µm) were cut, air dried, fixed for 30 min in 4%
paraformaldehyde, dehydrated with ethanol, and stored in
80 °C.
Sections were thawed, digested by proteinase K, acetylated, and
dehydrated with ethanol. The sections were hybridized at 57 °C for
16 h with 5 × 106 cpm/ml of
35S-labeled riboprobe, followed by digestion with 50 µg/ml RNase A at 37 °C for 30 min, and washed in a series of SSC
solutions of increasing stringency, ending with 0.1 × SSC, 1 mM dithiothreitol at 60 °C for 30 min. The slides
were exposed to Kodak BioMax MS film and were subsequently coated with
NTB-2 emulsion and developed after 22-25 days. The sequence of the
riboprobes are the same as those used in the RNase protection assays.
For all samples, corresponding sense riboprobe was used as control and
found to yield only background levels of silver grains. For comparisons of knockout and wild-type brains, sections were placed on the same
slide and processed identically.
Northern Hybridization--
A mouse poly(A)+ RNA
Northern blot (OriGene) containing RNA from multiple adult tissues was
hybridized with 1 × 106 cpm/ml of
32P-labeled cDNA probe in ULTRAhyb buffer (Ambion) at
42 °C for 16 h. The highest stringency wash was 0.1 × SSC, 0.1% SDS, at 42 °C for 15 min. The probes used were: NARG1,
the 630-base pair SmaI/KpnI fragment from IMAGE
clone 805455; NARG2, the 609-base pair
HindIII/BseRI fragment from IMAGE clone 804793;
NARG3, the 563-base pair DraI fragment from IMAGE clone 805306.
NARG1 (mNAT1) cDNA Clones--
To obtain cDNA clones
that contain sequence upstream of the NARG1 EST (AA474587), an arrayed
mouse testis cDNA library (OriGene) was screened by PCR. PCR
primers were designed based on the EST sequence: 5' primer,
5'-CAGTACGTGTGTTGTGTTTCCA-3'; 3' primer, 5'-TTCAAGCCATGGTAGAGTTTTC-3'.
The clones obtained from this screening are conventional (not PCR
clones), and our detailed analysis of the complete mNAT1 cDNA
sequence is forthcoming. Although the longest NARG1 cDNA clone
characterized lacked the 5' most 54 base pairs of coding sequence, this
was obtained from EST sequences (AA163290 and AA561496). A partial
mNAT1 cDNA has previously been reported (39).
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RESULTS |
A Screen for NMDA Receptor-regulated Genes in Developing
Neurons--
Brain stems, including nucleus principalis, nucleus
interpolaris, and nucleus caudalis, were dissected from neonatal mice produced from NMDAR1 heterozygous crosses. These nuclei
receive synaptic input from the whisker pad via the trigeminal
ganglion, and contain whisker representations in wild-type mice that
are absent in NMDAR1 knockouts. In initial experiments,
duplicate nylon membrane arrays containing previously characterized
mouse cDNAs were hybridized with wild-type (+/+) and
NMDAR1 knockout (
/
) cDNA probes (Fig.
1A). None of the 588 genes
represented met our follow-up criteria of at least a 2-fold change in
signal between wild-type and NMDAR1 knockout.

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Fig. 1.
Comparison of gene expression between
wild-type (+/+) and NMDAR1 knockout ( / ) brain stem
from neonatal mice. A, nylon cDNA array
analysis; 588 mouse cDNAs. None of these genes met our follow-up
criteria of at least a 2-fold change in signal between wild-type and
knockout (signals were averaged from three independent experiments).
These results also serve as a control; none of the 15 stress response
genes and 61 cell death genes present on the array were regulated by
the absence of NMDAR1. First-strand 32P-labeled cDNA
probes derived from total RNA preparations were used for these
experiments. Identities of the genes on the array are given in
CLONTECH's product analysis certificate, catalog
number 7741-1. B, microarray analysis; 8734 mouse elements.
Five genes were found that demonstrated differences of between 2- and
2.5-fold; the sectors of the array that contain these elements are
shown with the black outline removed from the elements of interest.
Fluorescently-labeled first-strand cDNA probes from wild-type (+/+;
Cy3) and NMDAR1 knockout ( / ; Cy5) brain stem were
prepared and hybridized to a mouse GEM (custom service by Incyte). The
identities of the elements present on the GEM are available (Mouse
GEM1; Incyte).
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The number of genes detected on the nylon array represents less than
1.0% of the mouse transcriptome. To increase the scale of the screen
to include 10-15% of the mouse transcriptome, probes from wild-type
and NMDAR1 knockout brain stem were hybridized to a mouse
gene expression microarray containing a total of 8,734 elements.
NMDAR1 wild-type and knockout brain stem probes detected 8,598 elements, i.e. over 98% of the cDNAs present on
the array. Approximately 5,300 elements were detected at sufficient
levels to allow comparison between NMDAR1 wild-type and
knockout gene expression. Of these, five elements were found that
demonstrated differences of between 2- and 2.5-fold (Fig.
1B). We initially checked the validity of the microarray
results by semi-quantitative RT-PCR (not shown) and then by RNase
protection analysis (Fig. 2). Three of
the five original elements identified on the microarray were confirmed
as NMDA receptor-regulated genes: NARG1, NARG2, and NARG3. In addition, heterozygous RNA yields signals for
NARG1 and NARG3 (signals were too weak to address
NARG2 in heterozygotes) that are intermediate between those
obtained in homozygous knockout and wild-type preparations (Fig. 2),
indicating that NMDA receptor-dependent regulation of these
genes is dose-dependent.

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Fig. 2.
RNase protection using
32P-labeled cDNA antisense riboprobes confirms that
NARG1, NARG2, and NARG3 are expressed
at approximately 2-fold higher than normal levels in the absence of
NMDAR1. The predicted sizes (nucleotides) of undigested probes
(arrowheads) and protected species (arrows) are
indicated. Autoradiographs from three experiments (horizontal
bars) for NARG1 and NARG3 are shown, as well
as for one NARG2 experiment. Undigested probe and tRNA
controls were performed in all experiments, although only one set of
these controls per NARG is shown here. Quantification of
knockout/wild-type signal ratios obtained by RNase protection are as
follows; NARG1: P0 brain stem, 1.7 ± 0.3 (n = 4), P0 brain (not shown), 1.4 ± 0.1 (n = 4); NARG3: P0 brain stem, 2.5 ± 0.8 (n = 4), P0 brain (not shown), 1.4 ± 0.1 (n = 5). RNase protection signals for NARG2
in the brain stem were very low and were not quantified. However, in
all experiments that compared NARG2 signal between wild-type
and knockout brain stem, higher signal was observed in knockout samples
(n = 3; a typical experiment is shown). Quantification
of P0 whole brain signals for NARG2 indicates a level of
regulation of about 2-fold (knockout/wild-type ratios were 1.9 and 1.6 in two independent determinations), which is comparable to the level of
regulation observed on the cDNA microarray (2.2-fold).
Up-regulation of NARG expression also occurs in
heterozygotes (n = 7: 5 for P0 brain stem, including 2 for NARG1 and 3 for NARG3; 2 for P7 brain,
including 1 for NARG1 and 1 for NARG3). Moreover,
signals for heterozygous samples were intermediate between wild-type
and knockout. This indicates that the observed regulation by NMDA
receptors is dose-dependent. GenBankTM
accession numbers of the ESTs that correspond to these genes
are: NARG1, AA474587; NARG2, AA472833; NARG3, AA473329.
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NARG1, NARG2, and NARG3 Share Similar Spatial Expression
Patterns--
In situ hybridization confirmed up-regulation
of NARG1 and NARG3 in NMDAR1
knockouts, and revealed similar spatial distribution for
NARG1 and NARG3 expression in the neonatal brain
(Fig. 3; unfavorable signal to noise
ratios have thus far prevented in situ hybridization
analysis for NARG2). Highest levels of expression were
observed in the ventricular zone, ganglionic eminence, cortex, olfactory bulb, and basal ganglia. At the time of birth these regions
contain numerous proliferating and migrating cells.

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Fig. 3.
In situ hybridization analysis
using 35S-labeled riboprobes on sagittal sections from P0
mouse brains demonstrates that NARG1 and
NARG3 are expressed at higher levels in
NMDAR1 knockouts ( / ) than in wild-types
(+/+). In wild-types, significant expression of NARG1
is observed in the ventricular zone and intermediate zone
(arrowheads), basal ganglia and ganglionic eminence
(G), cortical plate (arrows), and olfactory bulb
(OB). NARG1 is also expressed in the cerebellar
cortex and the caudal inferior colliculus, just rostral and superior to
the cerebellum (C). In wild-types NARG3 displays
a similar expression pattern to NARG1 in the telencephalon,
and shows little signal in more caudal regions of the brain. Sense
controls yielded background levels of signal (not shown).
BS, brain stem. Scale bar, 1 mm.
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To determine whether these genes share similar spatial expression
patterns in other parts of the body, we performed Northern blot
analysis on multiple tissues from the adult mouse. Although levels of
expression were found to be low in all adult tissues examined, similar
patterns of expression were observed for these three genes (Fig.
4).

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Fig. 4.
Northern blot analysis of NARG
expression in adult mouse tissues. Two µg of
poly(A)+ RNA are present in each lane. Markers (top to
bottom) are the same for each blot: 9, 6, 5, 4, 3, 2.5, 2, and 1.5 kilobases, respectively.
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NARG1, NARG2, and NARG3 Are Transiently Expressed in the Developing
Brain--
To begin to understand the physiological significance of
NARG1, NARG2, and NARG3 in the developing brain, we performed RNase protection analyses on whole brain RNA extracted on embryonic day 13 (E13), postnatal day 0 (P0), P7, and P35. These three NARGs display virtually identical patterns of expression: relatively low on
E13, high on P0, and very low thereafter (Fig.
5). This pattern is in contrast to that
observed for NMDAR1, which is expressed at lowest levels at
E13, and expression steadily increases as the brain matures (40) (Fig.
5, bottom panel). The three NARGs are
down-regulated as NMDAR1 is up-regulated, precisely as would be predicted by results that demonstrate higher NARG
mRNA levels in NMDAR1 knockout neural tissue than in
wild-type.

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Fig. 5.
NARG1, NARG2, and NARG3
are transiently expressed at high levels in the neonatal
brain. RNase protection analysis using 32P-labeled
single-strand antisense RNA probes was performed on wild-type brain
total RNA extracted at various stages of development. Riboprobes
complementary to NARG1, NARG2, and NARG3 were
synthesized, isolated, annealed with the indicated RNA sample, digested
with RNases, and fractionated by gel electrophoresis. Autoradiographs
of gels are shown, with the predicted sizes of the undigested probes
(arrowheads) and protected species (arrows)
indicated. Probe, undigested probe; E13,
embryonic day 13; P0, postnatal day 0; P7, postnatal day 7;
P35, postnatal day 35.
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NARG1 Is the Mammalian Homologue of a Yeast N-terminal
Acetyltransferase That Regulates Entry into Go Phase of the
Cell Cycle--
To determine the identity of NARG1, NARG2, and NARG3
we have isolated and sequenced a number of cDNA clones. Because the
ESTs on the microarray correspond to 3' regions of the transcripts, we
screened libraries to obtain cDNA clones that contain upstream coding regions. Sequence analysis of partial cDNA clones indicates that NARG2 and NARG3 represent previously unknown
genes. BLAST searches revealed that NARG1 is the mammalian
homologue of N-terminal acetyltransferase 1 (NAT1), a gene that was originally discovered in yeast (Fig.
6). Therefore, we will henceforth refer
to NARG1 as mNAT1. Null mutants of nat1 in S. cerevisiae display reduced acetyltransferase activity,
derepression of a silent mating type locus, and failure to enter
Go (41).

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Fig. 6.
NARG1 is the mouse homologue of yeast
NAT1. Alignment of NARG1 (mouse) amino acid sequence with NAT1
from various species. The first 137 out of ~850 amino acids are
shown. Over the entire amino acid sequence, NARG1 is 46% homologous
(28% identical) to Saccharomyces cerevisiae NAT1.
Xenopus: GenBankTM accession number AF247679.
Drosophila: AE003512. S. cerevisiae: X15135. The
portion of NARG1 shown here is contained in AA163290.
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DISCUSSION |
Despite the role of electrical activity and NMDA receptors in
regulating neuronal development including migration, proliferation, survival, and synaptic plasticity, the question of what genes are
regulated by NMDA receptor function in developing neurons has not been
systematically addressed. To test the hypothesis that NMDA
receptor-dependent electrical activity of neurons directs gene expression to facilitate activity dependent neuronal development, we have performed a screen based on the neural circuit that connects the highly specialized whiskers found on the nose of the mouse to their
synaptic targets in the brain stem. We have identified a group of three
genes, NARG1, NARG2, and NARG3, that share
striking regulatory features including NMDA
receptor-dependent regulation, a transient surge in
expression early in brain development, low levels of expression in the
adult, and similar spatial distribution. All three of these genes are
expressed at higher than normal levels in NMDAR1 knockout
brains, and at least two of them display dose-dependence in terms of
regulation by NMDA receptor function. During neonatal development all
three NARGs are sharply down-regulated at the same time that
NMDAR1 is up-regulated. The results indicate that maturation
of NMDA receptor function in developing neurons plays a role in
down-regulating this group of genes, and support the hypothesis that
NMDA receptor-dependent regulation of gene expression is
required for, and directs, molecular and cellular mechanisms for
neuronal development.
Genetic compensation versus direct regulation is a
consideration in any screen for genes regulated as a consequence of
transgenic manipulation. We have addressed compensation on three
levels. First, among ~8,500 genes examined, only three NMDA
receptor-regulated genes have been identified as a result of
NMDAR1 knockout. The results indicate a degree of
specificity that argues against large scale induction of compensatory
mechanisms. Second, we have investigated genes known to be involved in
stress response (15 genes; Fig. 1) and cell death (61 genes; Fig. 1) by
using nylon membrane array analysis. None of these genes were
significantly regulated by the lack of functional NMDA receptors in the
mouse neonatal brain stem. Third, to address the possibility that the
regulation observed for NARG1, NARG2, and NARG3
could be attributed to the fact that NMDAR1 knockout mice
die within 24 h of birth, we have examined the expression of these
genes in heterozygous animals, which are viable and grossly
indistinguishable from their wild-type littermates. (Knockout mice
appear healthy at birth, and are dissected well before the onset of
morbidity, which starts after 10-24 h (42).) The results demonstrate
mRNA levels for these genes in heterozygotes that are intermediate
between those found in wild-type and knockout brain stems. Previous
reports have also demonstrated graded effects of NMDAR1 gene
dosage on the survival of both hippocampal neurons and Purkinje cells
in cultures prepared from wild-type, heterozygous, and
NMDAR1 knockout mice (43, 44). Taken together, these
findings argue against genetic compensation and suggest that NMDA
receptor-dependent regulation of NARG1, NARG2,
and NARG3 is specific and dose-dependent.
Previous screens for neuronal genes regulated by electrical activity
have focused on overall levels of activity rather than NMDA
receptor-specific activity. Three independent studies have used
electrical stimulation (25), the
-aminobutyric acid-A antagonist
metrazol (26), and the glutamate analogue kainate (24), respectively,
to induce hippocampal seizures in adult rats. The rationale of these
experiments is that the exceedingly high levels of electrical activity
associated with seizures should robustly induce synaptic plasticity
events and therefore allow large numbers of candidate genes to be
identified. The only previously published screen for genes regulated by
electrical activity in developing neurons in vivo used a
differential cloning paradigm based on the activity dependent
segregation of retinal ganglion cell synaptic inputs into eye-specific
domains in the cat visual system (38). By blocking spontaneously
occurring action potentials in the developing lateral geniculate
nucleus and comparing gene expression with controls, class I major
histocompatibility antigens (MHC I) were identified. The MHC I
invariant light chain,
2-microglobulin, as well as a
component of a receptor complex for MHC I, CD3
, were also found to
be expressed by subsets of central nervous system neurons. These data,
as well as recent functional analyses, support the novel hypothesis
that MHC I proteins facilitate activity dependent synaptic plasticity
in developing neurons (38, 45). The MHC I results also demonstrate the
viability of the strategy of identifying important genes for neuronal
development by virtue of regulation by physiological levels of
electrical activity in the developing brain.
Taken together, previous screens have identified ~400 activity
regulated genes that could play a role in synaptic plasticity and other
activity regulated events in neurons. It is somewhat surprising,
therefore, that our screen of 10-15% of the mouse transcriptome
identified only 3 genes, two of which are novel. If this sample of the
transcriptome is typical, it follows that by saturating the present
screen we would have identified on the order of 30-45
NARGs, most of which would be expected to be novel. Although
this is far fewer genes than would be predicted based on adult
hippocampus seizure models, the strong agreement between microarray and
RNase protection analyses (~2-fold regulation for NARGs1-3 was measured by both approaches) make it unlikely
that the microarray results greatly underestimated the number of genes regulated by the loss of NMDAR1. Moreover, an examination of
microarray analysis studies indicates that, under physiologically
controlled conditions, on the order of 0.1-2% of genes are regulated
by 1.8-fold or more (46). This level of regulation is consistent with
the findings reported here.
There are, however, a number of potential explanations for the apparent
discrepancy between our results and those obtained using seizure
models. First, it is possible that more genes are regulated by overall
action potential activity than those that are regulated by NMDA
receptor function alone. Second, it has been shown that regulation of
expression of a given gene by electrical activity is a specific
property that can change among brain regions as well as during
development (47, 48). It is plausible, therefore, that a greater number
of genes are regulated by electrical activity in the adult hippocampus,
where almost all activity regulated neuronal genes have been
discovered, than in the developing brain stem. An additional
possibility is that the number of genes regulated by normal
physiological levels of electrical activity is lower than the number of
genes that can be regulated by seizures. Finally, the set of genes
examined on the nylon array and the microarray did not contain a number
of genes known to be regulated by electrical activity, e.g.
neurotrophins, and therefore may not be statistically representative of
a more complete sampling. It is clear that additional analyses of NMDA
receptor-regulated gene expression in developing neurons are needed to
resolve this issue.
Another issue that should be addressed in future experiments is whether
or not the electrical function of NMDA receptors is solely responsible
for NMDA receptor-dependent regulation of gene expression.
While pharmacological and transgenic studies indicate that this is
likely to be the case (e.g. Refs. 24, 25, and 49), there is
evidence that NMDA receptors contribute to organizing a network of
postsynaptic proteins that connects the synapse to the cytoskeleton
(50, 51). In addition to disrupting NMDA receptor-mediated electrical
activity, knockout of the NMDAR1 subunit is also expected to disrupt
this structural function. This could potentially cause downstream
changes, including the regulation of gene expression.
The unusual regulatory features shared by NARG1, NARG2, and
NARG3 may reflect a common function in developing neurons.
NARG1, NARG2, and NARG3 are transiently expressed
at high levels in P0 brain, expression is highest in neurons that are
just exiting or about to exit the cell cycle, and NARG1 (mNAT1) is the
mammalian homologue of a yeast N-terminal acetyltransferase that
regulates entry into the Go stage of the cell cycle (41).
In addition, pharmacological studies that have established a role for
NMDA receptors in cell cycle regulation during the final stages of dentate gyrus maturation in the rat hippocampus (13, 52). Taken
together, the results suggest that these genes may facilitate the
transition from proliferation of neuronal precursors to differentiation of neurons, in part via NMDA receptor-dependent regulation.
In the present study we report the first specific genes that are
regulated by normal levels of NMDA receptor function in developing neurons in vivo. The results support the hypothesis that
NMDA receptor-dependent regulation of gene expression is
required for, and directs, molecular and cellular mechanisms for
neuronal development. Moreover, the identification of NMDA
receptor-regulated genes provides the insight and tools needed to begin
to understand the relationship between NMDA receptor function, gene
expression, and ultimately neuronal differentiation. Because
NARG1, NARG2, and NARG3 share regulatory
features, these genes are likely to be regulated by common
cis-regulatory elements and transcription factors that can
be identified by promoter analysis. Future studies of these and other
NMDA receptor-regulated genes should make an important contribution to
the insight needed to obtain an integrated understanding of molecular,
anatomical, and electrophysiological principles that guide neuronal development.