(Received for publication, August 19, 1994; and in revised form, November 3, 1994)
From the
Genomic and cDNA clones encoding the chicken neuronal nicotinic
acetylcholine receptor 3 subunit were isolated and sequenced. The
3 gene consists of six protein-encoding exons and the deduced
protein has the structural features found in all other members of the
neuronal nicotinic acetylcholine receptor subunit family. Although they
are undetectable in most brain compartments,
3 mRNAs are
relatively abundant in the developing retina and in the trigeminal
ganglion. In situ hybridization and immunohistochemical
analysis demonstrated that in retina,
3 transcripts and protein
are confined to subpopulations of cells in the inner nuclear and
ganglion cell layers.
3 is expressed in the proximal and distal
regions of the developing trigeminal ganglion, i.e. in both
placode- and neural crest-derived neurons. Transient transfection
assays in cells freshly dissociated from selected regions of the
central nervous system at different developmental stages allowed the
identification of genetic elements involved in the neuronal-selective
expression of the
3 gene. A promoter fragment 143 base pairs in
length and containing TATA, CAAT, and other consensus sequences is
sufficient to restrict reporter gene expression to a subpopulation of
retinal neurons. This promoter is totally inactive upon transfection
into neuronal and non-neuronal cells from other regions of the central
nervous system.
Nicotinic acetylcholine receptor (nAChR) ()subunits
constitute a family of closely related proteins whose members are
assembled in various combinations at the neuromuscular junction and in
different regions of the central and peripheral nervous systems. Muscle
nAChR is a pentamer composed of four distinct subunits,
,
,
, and
; the
subunit in mammals being replaced by an
subunit during development (reviewed in Schuetze and Role(1987)
and Galzi et al. (1991)).
cDNAs encoding seven distinct
ligand-binding subunits (2-
8) and two structural
subunits (
2,
4) have been isolated from chicken neural
tissues, and most of the corresponding cDNAs have also been cloned in
the rat. In Xenopus oocytes, functional neuronal receptors are
produced upon coinjection of cRNAs or cDNAs encoding a
subunit
(
2 or
4) and any one of the
2,
3, or
4
subunits (reviewed in Deneris et al.(1991), Role(1992), and
Sargent(1993). The
7 and
8 subunits assemble into fully
functional homomeric nAChRs (Couturier et al. 1990b;
Séguéla et al.,
1993; Gerzanich et al., l994) whose properties are being
investigated by site-directed mutagenesis (Revah et al., 1991;
Bertrand et al., 1992). Recently, it was demonstrated that
neuronal nAChR have a pentameric stoichiometry, like their muscle
siblings (Cooper et al., 1991; Anand et al., 1991).
The different neuronal nAChR subunit genes are expressed in distinct
areas of the developing and adult nervous system, as demonstrated by in situ hybridization and Northern blot analysis. In the CNS,
4 and
2 mRNAs are widely distributed, whereas the other
subunit mRNAs and proteins are more restricted, yet there are many
instances of overlapping expression (Wada et al., 1989; Morris et al., 1990; Hill et al., 1993; Keyser et
al., 1993). Recent studies have shown that subunit mRNAs which are
rare in the CNS are relatively abundant in several peripheral ganglia
(Boyd et al., 1988, 1991; Couturier et al., 1990a;
Listerud et al., 1991; Corriveau and Berg, 1993). Some
neuronal nAChR mRNAs are developmentally regulated; both the
7 and
2 transcripts transiently accumulate in the avian optic tectum
between embryonic day 7 (E7) and E16 (Matter et al., 1990;
Couturier et al., 1990b).
Very little is known about the molecular mechanisms controlling gene expression in neurons. Two different cis-acting regulatory elements that mediate specific neuronal expression have been identified. One element activates promoters in neuronal cells (Yoon and Chikaraishi, 1992), whereas the other represses promoter activity in non-neuronal cells (Mori et al., 1992; Kraner et al., 1992). Although a number of transcription factors have been found in the nervous system, the identity of their target genes remains unknown (reviewed in Mandel and McKinnon(1993)), except for a transcriptional activator that binds to the regulatory sequence of several genes specifically expressed in olfactory neurons (Wang and Reed, 1993).
Because its individual
members have very different expression patterns in the chick nervous
system, the nAChR gene family is an excellent model system to study the
regulatory mechanisms that restrict gene expression to particular
classes of central or peripheral neurons. We have previously identified
the upstream sequence conferring transcriptional specificity on the
7 gene (Matter-Sadzinski et al., 1992). We describe here
the molecular cloning of the
3 gene, a member of the neuronal
nAChR gene family in the chick. We show that this gene has a highly
restricted pattern of expression within the developing central and
peripheral nervous system, and we define the 5`-flanking region
containing the regulatory sequences required for specific neuronal
expression.
Total RNA from E10 trigeminal ganglia was extracted as
described above. Double-stranded cDNA was synthetized from 5 µg of
poly(A) RNA using a Pharmacia cDNA synthesis kit and
inserted in
gt10 (Promega). Packaging (Gigapack, Stratagene) of a
fraction of the ligation reaction yielded 10
independent
virus particles. Screening performed as in Couturier et al.
(1990a) yielded two full-length cDNAs from 10
independent
plaques. The cDNAs were subcloned in pBluescript SK (Stratagene).
Partial DNA sequencing and restriction mapping established that they
were accurate transcripts of the
3 gene.
For in
situ hybridization studies, we used 45-mer antisense and sense
oligonucleotides corresponding to amino acids
Val-Tyr
of the
3 subunit.
Oligonucleotides least likely to cross-hybridize to other chick
neuronal nAChR mRNAs were selected by aligning with all known chick
nAChR transcripts. On control Northern blots of retina
poly(A)
RNA, the
P-labeled antisense
oligonucleotide hybridized (Beyer, 1991) to the same major band as the
cDNA probes. The minor mRNA band was not detected due to the very low
specific activity of radiolabeled oligonucleotides as compared to
homogeneously labeled cDNA fragments. Neither liver nor muscle
poly(A)
RNA gave detectable signals under the same
conditions.
The antisense and sense oligonucleotides were labeled to
a final specific activity of 5-10 10
cpm/pmol
by 3` tailing, using
-
S-dATP (DuPont-NEN;
1200-1400 Ci/mmol). Briefly, 4 pmol of oligonucleotide were
incubated 1 h at 37 °C with 45-50 pmol of
-
S-dATP in labeling buffer containing 100 mM sodium cacodylate, pH 7.2, 8 mM MgCl
, 1
mM
-mercaptoethanol, 200 µg/ml bovine serum albumin,
and 13 units of Terminal Transferase (Amersham Corp.) in a final volume
of 10 µl. The reaction was stopped by adding 80 µl of STE (0.1
mM NaCl, 10 mM Tris-Cl (pH 7.4), 1 mM EDTA)
and 100 µg of yeast tRNA. The probe was purified on a 2-ml Sephadex
G-50 (Pharmacia Biotech Inc.) column equilibrated with STE.
S-Labeled oligonucleotides were stored at -70 °C
and used within 2 weeks.
Plasmid plac00 was
constructed by first inserting a NotI linker into the SalI site of pCMV-nlacZ (a gift from M. J. Weber,
Université de Toulouse) and into the SmaI site of pCAT00, then the NotI-PstI fragment of
pCAT00 was replaced by the NotI-PstI fragment from
pCMV-nlacZ. Restriction fragments PS and RS were then inserted
in both orientations, and fragment NS in the correct orientation into
the blunted NotI site of plac00 immediately upstream of the nlacZ gene (Kalderon et al., 1984). pSVlac was
constructed by inserting the blunted HindIII-PvuII
restriction fragment containing the SV40 promoter at the blunted NotI site of plac00. Constructions were checked by restriction
mapping and DNA sequencing. To test whether promoter activity is
orientation-dependent, fragment PS was inserted in inverted orientation
in the lac and CAT vectors, and transfected into cells from E5 retina.
No -gal-positive cells were detected, and CAT activity was at the
background level.
For X-gal staining, the transfected cells were grown
in a four-well cluster dish (Nunc) or in a four-chamber plastic slide
(Lab-Tek) for 48 h. The culture medium was then removed and the cells
were rinsed with PBS (pH 7.2) and fixed in 2% formaldehyde, 0.4%
glutaraldehyde (prepared in PBS) for 5 min at room temperature. They
were rinsed twice with PBS and stained for 3 h at 37 °C in PBS
containing 1 mg/ml X-gal, 4 mM KFe(CN)
, 4 mM K
Fe(CN)
, and 2 mM MgCl
. The cells were rinsed once with PBS and either
mounted in fluoromount (Fisherbiotech) or stored in the fix solution
(2% formaldehyde, 0.4% glutaraldehyde) at 4 °C.
Figure 1:
Partial restriction map of the neuronal
nAChR 3 gene. The overlapping recombinant phage 1, 11 and 17 (arrows) define a unique region of the chicken genome
extending across 13 kbp and containing the
3 gene. Numbered boxes
locate the exons encoding the subunit. In addition to coding sequences (filled boxes), exons 1 and 6, respectively, contain 5`- and
3`-untranslated sequences (hashed boxes). H, HindIII; P, PstI; R, EcoRI; Bg, BglII; Ba, BamHI sites.
Two full-length 3
subunit cDNA clones were isolated from an embryonic trigeminal ganglion
library (see ``Materials and Methods''), using a genomic
probe encoding the subunit-specific cytoplasmic region of the protein.
Both cDNA clones were checked by restriction site analysis and partial
DNA sequencing to ensure that they were accurate transcripts of the
3 gene.
Figure 2:
Nucleotide and deduced protein sequences
of the chicken 3 gene. Nucleotides are numbered from the
translation start site on the proximal 5`-flanking region of the
3
gene. The double underline identifies the oligonucleotide used
for primer extension analysis and the transcription initiation site is
marked with an arrow. The TATA and CAAT boxes are underlined; CACCC boxes are overlined; E-boxes and
the AP-1 motif are labeled (
) and (ˆ), respectively. Lower case nucleotide symbols indicate donor and acceptor
sites of intervening sequences; dashed underline, postulated
transmembrane regions TM1-TM4. The coding sequence contains 52.5%
AT. The mature
3 subunit (residues 1-435) has a calculated
molecular weight of 50036 daltons and has potential N-linked
glycosylation sites at positions 28, 143, and 432 (*). Cysteines 130
and 144 and intervening sequence are highly conserved in all known
subunits of ligand-gated ion channels belonging to the nAChR
superfamily.
The 3 protein has three potential N-linked
glycosylation sites (N28, N143, and N432), the first and second of
which are conserved in several neuronal nAChR subunits. Two cysteine
residues that correspond to cysteine 128 and 142 (
1 subunit
numbering) (Nef et al., 1988) are also present in the protein
but the subunit lacks the pair of vicinal cysteines at position
192-193 that is the hallmark of all
subunits and forms part
of the ACh-binding site (reviewed in Galzi et al.(1991)).
Although
3 is thus operationally defined as a beta subunit,
alignment of its amino acid sequence with those of all other known
avian neuronal nAChR subunits (Table 1) reveals that
3
aligns significantly better with most of the
than with the
subunits (e.g. 68.2% amino acid identity and 11.3% amino acid
similarity with the
5 subunit). When the alignment was made
between
3 and related members of the superfamily in other species,
the highest percentage of identity (83.6) was obtained with the rat
3 subunit (Deneris et al., 1989) and with the goldfish
n
2 (74.9) and n
3 (77.0) subunits (Cauley et al.,
1989, 1990).
Figure 3:
Northern blot analysis of 3
transcripts in chick retina and trigeminal ganglion. A,
poly(A)
RNA (500 ng/lane) isolated from E12 liver,
optic tectum, retina, and E10 trigeminal ganglion was fractionated by
gel electrophoresis, blotted to nitrocellulose, and hybridized with a
P-labeled
3 probe (genomic fragment NcoI-EcoRI) encoding part of the cytoplasmic region
of the protein. Exposure times for the left and right panels were 24 h
and 10 days, respectively. Arrowheads locate the 28 and 18 S
rRNA bands. B, total RNA (500 ng/lane) isolated from the
retina at different stages of development (the embryonic day is
indicated by numbers and the newly hatched and adult stages
are indicated by H and A, respectively) were
hybridized to the
3 probe as in A. Exposure time was 10
days. Lower panel, methylene blue staining of the
blot.
Figure 6:
Localization of the 3 transcription
start site. A, molecular map of the 5`-flanking region of the
3 gene. The extent of the three restriction fragments used for
Northern blot analysis is indicated by the horizontal lines below the map. The box indicates the position of coding (filled box) and non-coding sequences (hashed box) in
exon 1. H, HindIII; P, PstI; Ns, NsiI; R, EcoRI; Pv, PvuII; S, SphI. B, Northern blot
analysis was performed to map the 5`-end of the
3 gene.
P-Labeled fragments 1, 2, and 3 were hybridized to 1
µg of poly(A)
RNA from E12 retina. C,
mapping of the transcription initiation site by primer extension.
Poly(A)
RNA (2 µg) from E12 retina (R)
and total RNA from primary cultures of chick embryo fibroblast-like
cells (F) were hybridized to a
P-end-labeled
synthetic oligonucleotide complementary to nucleotides -15 to
+4 from the 5`-flanking region of the
3 gene (double
underline in Fig. 2). The extended fragment synthesized by
reverse transcriptase is marked (*) and maps a single transcription
initiation site at position -111 of the translation start site.
Adjacent is a dideoxy sequencing ladder (GATC) of a
3
genomic clone primed with the same
oligonucleotide.
Figure 4:
Localization of 3 transcripts and
protein in the retina. Darkfield photomicrographs of neonatal (P1)
retina sections hybridized with
3 oligonucleotide antisense (A) and sense (B) probes. The sections were exposed
for 3 weeks. In the retina,
3 transcripts are detected in the
ganglion cell layer (GCL) and in the inner half of the inner
nuclear cell layer (INL). The
3 protein was detected (C) by antiserum to the
3 cytoplasmic domain
(FP
3in). Bound antibodies are visualized using biotinylated goat
anti-rat IgG and avidin-biotinylated HRP complex. Anti-
3
immunolabeling is present in the GCL and the INL. Note the presence of
heavily labeled cells within the GCL (arrows) and along the
inner edge of the INL (arrowheads). Bar, 50
µm.
To determine which retinal cells accumulate detectable
levels of the 3 protein, antiserum to FP
3in was used on
sections of neonatal retinae (P1). The antibodies specifically labeled
subpopulations of cells from the GCL and INL (Fig. 4C).
The anti-
3 antibody failed to label the inner plexiform layer
(IPL) although that layer contains processes from ganglion and amacrine
cells.
These restricted distributions of hybridization and
immunoreactivity suggest that 3 transcripts and the
3 protein
accumulated in the large majority of ganglion cells and in a subset of
amacrine neurons.
Figure 5:
Localization of 3 transcripts and
protein in the trigeminal ganglion. In situ hybridization of
E8 trigeminal ganglion sections with
3 oligonucleotide antisense (A) and sense probes (B). Sections were exposed for
four weeks. Note the stronger labeling in the distal part of the
ganglion. Immunohistological localization of the
3 protein in E10
trigeminal ganglion (C) by an antiserum to the
3
cytoplasmic domain (FP
3in). Bound antibodies are visualized using
biotinylated goat anti-rat IgG and streptavidin-biotinylated
phycoerythrin complex. Anti-
3 immunolabeling is present in some
but not all neurons. In D, a similar section was processed as
in C, except ,that normal rat serum replaced the antibody to
FP
3in. Bars, 70 µm (A and B), 60
µm (C and D).
Immunohistochemistry on E8 and E10
trigeminal ganglion reveals 3 immunoreactive sites throughout the
ganglion. Not all cell bodies are labeled (Fig. 5C),
and immunoreactive cells from the distal and proximal regions of the
ganglion show stronger labeling at E10 than at E8.
We next determined the approximate
5`-end of the 3 transcript. Restriction fragments 1, 2, and 3 (Fig. 6A) were used as radioactive probes to detect
3 transcripts in neuroretina RNA blots (Fig. 6B).
The two mRNA species were only detected upon hybridization with
fragment 3, suggesting that the transcription initiation site of both
mRNAs is localized within the 128-bp immediately upstream of the
initiator ATG. The precise transcription initiation site of
3 mRNA
was mapped by primer extension. A
P-labeled 19-mer
synthetic oligonucleotide complementary to nucleotides +4 to
-15 (relative to the translation initiation site, Fig. 2)
was used in the reaction as described under ``Materials and
Methods.'' A single extended product was detected with
poly(A)
RNA from retina, demonstrating the presence of
a single initiation site 111 bp upstream of the ATG (Fig. 6C).
Figure 7:
The 5`-flanking region of the 3 gene
contains regulatory elements which confer neuronal specificity. A, schematic structure of the chimeric plasmids tested for
activity in transient transfection assays. Restriction fragments
beginning 128 bp upstream of the initiator ATG and extending 5` to a
distance of -199 (PvS); -271 (RS);
-343 (NS); -850 (PS) and approximately
-2 kbp (HS) were fused to the CAT gene in pCAT00, as was
a fragment extending from -271 to -850 (PR).
Fragments RS, NS, and PS were also fused to the bacterial
-galactosidase gene (lacZ) in plac00. B, comparison of
the restriction fragment promoter activities in neural cells, CEFs, and
CHO cells. Constructs were transfected into neural cells dissociated
from E5, E8, and E13 retinae, E5 and E10 optic tecta, and into glial
cells, CEFs and CHO cells. 48 h after transfection, cells were
processed for CAT assay. The CAT activity obtained with transfection of
pSVCAT is arbitrarily set at 100 for each cell type. All other CAT
activities are given relative to this value. Activity of the SV40 early
promoter in arbitrary CAT units per µg protein was similar in all
cell types at all stages of development (for details see
Matter-Sadzinski et al., 1992).
In these different cell systems, promoter activity was determined by
CAT assay or X-gal staining 48 h after transfection. In all cell types
at all the studied stages, the SV40 promoter drove -gal expression
in 10-15% of the cells. As shown in Table 2, the constructs
driven by fragments PS, NS, and RS all yielded about the same fraction
of
-gal positive cells in E5 and E13 retinal cells. In contrast,
no labeled nuclei were ever detected in E5 and E10 optic tectal cells,
astroglia, and CHO cells (frequency <10
of
-gal positive cells obtained upon transfection by the SV40
promoter).
Although the fragments PS, NS, and RS were active in
similar fractions of transfected retinal cells, Fig. 7B demonstrates that these fragments have very different activity
levels in CAT assays. The constructs containing the CAT gene fused to
fragments HS, PS, NS, and RS were expressed at significant levels in
cells from E5 retina, being about 3-fold higher in the RS construction.
The promoter activity of fragments PR and PvS was at the background
level. In E8 and E13 retina, the constructs were much less active than
at E5, yet they maintained the same rank-order in potency. Putting
together the -gal and CAT assays, we conclude that the much
reduced CAT activity measured in E8 and E13 retina results from a
reduced reporter gene expression in a stable number of cells. This
decrease coincides with the period of development when the majority of
retinal cells withdraw from the mitotic cycle. In agreement with the
-gal results, none of the constructs expressed detectable CAT
activity in E5 and E10 tectal cells, astroglia, CEFs or CHO cells.
Thus, the RS fragment (143 bp) contains regulatory elements sufficient
to restrict promoter activity to the retina, i.e. to the only
region of the CNS where expression of the
3 gene was detected.
The deduced sequence of the 3
protein has features found in all neuronal nAChR subunits. Lack of the
two adjacent cysteines corresponding to Cys-192 and -193 of the muscle
1 subunit classify this protein as a
subunit. We did not
succeed in reconstituting a functional receptor upon injection into
Xenopus oocytes of
3 cDNA either alone or in pairwise combinations
with
2,
3,
4 or
6. (
)
5 also fails
to yield functional receptors when injected alone or in pairwise
combination with any of the
subunits (Couturier et al.,
1990a). Likewise, the cognate rat
3 and
5 cDNAs fail to
reconstitute functional receptors in oocytes (Boulter et al.,
1990). However, recent immunoprecipitation experiments using subunit
specific monoclonal antibodies indicate that some native neuronal
nAChRs may assemble from more than two subunits and that the
5
subunit appears capable of participating in several distinct neuronal
nAChR subtypes (Conroy et al., 1992; Vernallis et
al., 1993). We speculate that the
3 subunit perhaps
participates in similar co-assemblies to form functional receptors in
the nervous system. Alternatively,
3 may require an unidentified
-subunit for assembly, or it may require a processing step that
does not take place in oocytes.
As illustrated in Table 3, the
avian 3 and
5 subunits share amino acid substitutions in
functionally important regions which are otherwise highly conserved in
all other members of the neuronal nAChR family. The same amino acid
substitutions are also found in the rat
3 and
5 subunits as
well as in the goldfish na2 and na3 subunits. The changes at positions
93 and 94 may have important effects on ligand binding cooperativity,
as suggested by mutagenesis analysis at the corresponding sites of the
1 and
7 subunits (reviewed in Changeux et al.,
1992). Changes in the region TM1-TM3 may affect channel
properties as TM2 lines the channel (Giraudat et al., 1987;
Leonard et al., 1988) and as rings of charged amino acids on
either side of TM2 play an important role in ion selectivity (Imoto et al., 1988). Moreover, mutations of L247 in TM2 of the
homomeric
7 channel (L251 in
1 numbering) profoundly modify
the properties of the channel (Revah et al., 1991; Bertrand et al., 1992).
In neonatal retina the 3 transcripts are localized in the inner
half of the INL and in the GCL. Immunohistochemical analysis
demonstrates that the
3 transcripts are indeed translated and that
the protein product mainly accumulates in the INL and GCL. The
localization of the transcripts and protein suggests that in the retina
the
3 gene is mainly expressed in ganglion and amacrine cells. In
the trigeminal ganglion at E8,
3 transcripts are present mainly in
the distal and proximal regions of the ganglion, being clearly more
abundant in certain large cells of the distal part. Cells that form the
avian trigeminal ganglion arise from the neural crest and placodal
epithelium. Neural crest cells are concentrated in the proximal and
central parts of the bilobed ganglion while placode-derived cells are
restricted to the distal aspects of each lobule (Hamburger, 1961).
Based on these observations, we suggest that
3 transcripts are
present in cell subpopulations derived from both embryonic origins.
In addition to the appropriately spaced TATA and CAAT boxes, fragment RS contains one copy each of the CACCC and CANNTG (E-box) motifs. These motifs are known to regulate transcription in other genes (Myers et al., 1986; Murre et al., 1989). In particular, muscle nAChR transcription is regulated, at least in part, by the interaction between E-boxes and myogenic factors that are members of the HLH family of transcription factors. Finer mapping and mutagenesis studies are in progress to determine the number and function of the discrete cis-acting elements present in this regulatory region.
The 7 and
3
promoters exhibit contrasting behaviors during development of the
central nervous system. At early stages of embryogenesis, the
7
promoter is widely active in undifferentiated tissues of the developing
CNS, and as development proceeds its activity becomes restricted to
particular neuronal phenotypes (e.g. retinal ganglion cells)
(Matter-Sadzinski et al., 1992). In contrast, activity of the
3 promoter is restricted early on to a subset of neuroepithelial
cells of the retina.
As judged by expression of two
reporter genes, both promoters are very active in neuroepithelial cells
of the retina on embryonic day 6, although the mRNAs encoded by these
two genes are either very sparse (
3) or undetectable (
7) at
this early stage of development. This apparent discrepancy between
transcriptional activity and mRNA levels may reflect a process of
post-transcriptional regulation resulting in delayed accumulation of
the transcripts at early stages of development. In transfection
experiments, activity of both the
3 and
7 promoters declines
sharply in the retina between E5 and E13. In differentiated retina
(E13), the
3 and
7 promoters remain active, respectively, in
about 10% and 1% of the cells, and that is consistent with RNA blot
analysis showing that
3 transcripts are much more abundant than
7 transcripts at this stage of development.
Although the
7 and
3 genes use different strategies to achieve their
stringent neuron specificity within the nervous system, both genes
begin to be expressed in the retina several days before establishment
of functional connections. The significance of this early expression
remains unknown, but it suggests that the two corresponding receptor
subunits may mediate regulation of cell differentiation during retina
neurogenesis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X83739 [GenBank]and X83740[GenBank].