Copyright © 1998 by the Genetics Society of America
Genetics 148: 361–372 (January, 1998)
An Altered Intron Inhibits Synthesis of the Acetylcholine Receptor
a
-Subunit
in the Paralyzed Zebrafish Mutant
nic1
Diane S. Sepich,
1Jeremy Wegner, Sherry O’Shea and Monte Westerfield
Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403 Manuscript received July 22, 1997
Accepted for publication September 30, 1997
A B S T R A C T
The acetylcholine receptor (AChR), an oligomeric protein composed of five subunits, is a component of the postsynaptic membrane at the vertebrate neuromuscular junction that plays a central role in synap-tic transmission. The zebrafish mutation nic1 blocks the expression of functional and clustered nicotinic muscle AChRs. To understand the mechanisms underlying this lack of AChRs, we characterized the molec-ular defect in nic1 mutants. Our results suggest that the mutation affects the gene coding for the a-subunit of the AChR. Southern blot hybridization and DNA sequence analyses showed that the nic1 AChR a -sub-unit gene lacks part of intron 6 where the splicing branchpoint normally forms. Several lines of evidence suggest that this deletion blocks normal splicing; most nic1a-subunit mRNAs retain intron 6 and are larger and less abundant than wild-type, some nic1a-subunit mRNAs are internally deleted, and wild-type a -sub-unit mRNA rescues nic1 mutant cells. The nic1 mutation reduces the size of an intron, which prevents effi-cient splicing of the pre-mRNA, thus blocking synthesis of the a-subunit and assembly of AChRs. By this route, the nic1 mutation leads to paralysis.
viewed in Hall and Sanes 1993), and such clusters fail to form in the absence of innervation (Dahm and Landmesser 1991; Liu and Westerfield 1992). Nor-mal clustering also requires the presence of all four subunits to form assembled receptors, although single subunits have clustered in a highly expressing cell line (Maimone and Merlie 1993).
To examine further the roles of the a-subunit during the formation of neuromuscular junctions in vivo, we previously screened for nonmotile mutants and iso-lated the g-ray-induced nic1 zebrafish mutation ( West-erfieldet al. 1990). The nic1 mutant embryos are para-lyzed but otherwise develop normally. We showed that although nic1 mutant muscle cells contract in response to direct electrical stimulation, they lack functional AChRs as indicated by (1) an absence of AChR clusters identified by a-bungarotoxin binding or binding of an-tibodies that recognize the b, g, or d subunits and (2) their lack of response to the acetylcholine agonist, car-bachol (Westerfieldet al. 1990). From analysis of ge-netic mosaic embryos, we showed that the mutation acts autonomously in muscle cells (Sepichet al. 1994); mutant muscle cells fail to cluster AChRs even when in-nervated by wild-type motoneurons, whereas wild-type muscles cluster AChRs when innervated by nic1 mutant motoneurons. Based on these results, we reasoned that the nic1 mutation may directly affect expression of one or more of the AChR subunit genes.
Here we provide evidence that the nic1 mutant phe-notype is the result of a mutation in the gene coding for the a subunit of the AChR. We demonstrate that
Corresponding author: Monte Westerfield, Institute of Neuro-science, University of Oregon, Eugene, OR 97403.
E-mail: [email protected]
1Present address: Cell Biology and Neuroanatomy, University of
Minnesota, Minneapolis, MN 55455.
A
CETYLCHOLINE receptors (AChRs) are oligo-meric proteins composed of four types of poly-peptide subunits assembled in the ratio of a2bgd. The AChR a-subunit (AChRa) is expected to play a critical role in normal assembly, clustering, and function of the AChR. Expression of the subunit proteins in Xenopus oocytes has shown that all four subunits are required to form receptors that respond normally to ACh ( Mish-inaet al. 1984). Although only the a-subunit is indis-pensable for a-bungarotoxin binding, the absence of any of the other subunits reduces binding. Mutational analysis of the subunit genes expressed in transfected COS cells demonstrated that the N-terminal domains of the a and d subunits are required for surface expres-sion of AChRs and for the formation of ad het-erodimers, an early step in the assembly of AChRs (Verrall and Hall 1992). Further, a cytoplasmic loop on the a subunit is required either in the early associa-tion of a and g subunits or in the final assembly of abd and ag subunits (Yu and Hall 1994).(re-362 D. S. Sepich et al.
the phenotype is linked to a restriction fragment length polymorphism (RFLP) in the a-subunit gene and that both the RFLP and mutant phenotype map to the same location on the genetic map. We then show through sequence analysis that the nic1 mutant a -sub-unit gene lacks a specific part of intron 6 where the branchpoint may normally form during splicing. The deletion may block splicing of this intron. Consistent with this interpretation, we find that the a-subunit AChR mRNA is larger in mutants than in wild-type. Fur-ther, nic1 mutant muscle cells express intron-contain-ing AChRa mRNA and other incorrectly spliced tran-scripts with partial deletions of the coding sequence. We find that providing nic1 mutant cells with wild-type AChRa mRNA rescues the mutant phenotype.
Our results suggest that a zebrafish mutation that prevents expression of functional a-subunits blocks ex-pression of AChRs, thus providing strong support for the central role of the a-subunit. Furthermore, our analysis of the nic1 mutation suggests that a reduction in the size of an intron can decrease the efficiency of splicing of the a-subunit gene to a level that blocks as-sembly of the oligomeric AChR protein, leading to pa-ralysis of the embryo.
M AT E R I A L S A N D M E T H O D S
Fish stocks: We obtained diploid homozygous mutant em-bryos by crossing fish heterozygous for the nic1b107 ( Wester-fieldet al. 1990) mutation. Haploid mutant embryos were
ob-tained by previously described methods (Streisinger et al.
1981). Embryos were staged according to hours (h) or days (d) postfertilization at 28.58 or by number of somites. Em-bryos and adults were maintained with standard conditions (Westerfield 1995).
Identification of fish heterozygous for nic1: Fish heterozy-gous for nic1 were identified either by examination of the progeny from individual crosses or by PCR amplification of intron 6 from the individual’s genomic DNA.
DNA was extracted from caudal fins clipped from anesthe-tized adult fish. Fins were placed in 50 ml of digestion buffer (1.5 mm MgCl
2, 50 mm KCl, 0.3% Tween 20, 0.3% NP-40, 10
mm TRIS, pH 8.3) heated at 988 for 10 min and then cooled
to 558. We added 150 mg of proteinase K (Boehringer Mann-heim, Indianapolis, IN) and digested the fins at 558 for 60 min and then heated them at 988 for 10 min to inactivate the protease. The resulting DNA solution was diluted approxi-mately 25-fold in water and stored at 2208. The DNA (1–5 ml) was denatured in the presence of PCR primers (40 ng) in a total volume of 10 ml for 5 min at 988 (primers sequence 59-CGG GCT TGT GTT TTA CCT GC, corresponding to sequence GLVFYLI at amino acid 229 in the protein, and 39-GAG GTG GAC GGG ATC AAC TCG, corresponding to se-quence ELIPSTS at amino acid 262). PCR contained 10 ml of DNA primer, 13 Vent buffer (New England Biolabs, Beverly, MA), 6 mg of bovine serum albumin (New England Biolabs), 0.25 mM dNTPs (Boehringer Mannheim), and 0.2 ml of Amp-litaq polymerase (Perkin-Elmer, Norwalk, CT) in a total vol-ume of 30 ml. Reactions were amplified in an MJ Research (Waltham, MA) thermocycler, with program 3step (948 for 45 sec, 36 cycles of 928 for 45 sec, 608 for 45 sec, 728 for 1.5 min, 728 for 5 min) and held at 48 until collected.
Isolation of zebrafish AChRa cDNA: We screened 1.3 3 106 clones from a 32h embryonic zebrafish cDNA expression
library (lgt11, a gift from Kai Zinn, Stanford University) with
32P-labeled random hexamer-labeled probes (Feinberg and
Vogelstein 1984). The probes were made from two HinfI
fragments from the Torpedo AChRa cDNA (a gift from Toni Claudio, Yale University). The coding sequence corresponds
to the ACh-binding site and transmembrane region III. Filters were hybridized in 53 SSC, 50 mm sodium pyrophosphate,
53 Denhardt’s, 100 mg/ml calf thymus DNA, 100 mg/ml yeast tRNA, with 2.2 3 105 cpm/ml of probe at 548 for 2 days,
washed twice in 53 SSC with 0.1% SDS at 548, twice in 23 SSC with 0.1% SDS at 548, and exposed to film for 2–5 days at
2708 with intensifying screens. Three overlapping clones were recovered and subcloned into the EcoRI site of pBlue-Script II KS(2) (Stratagene, La Jolla, CA).
The sequence of the longest clone (pAChRa or p3a1) was determined. The sequence was assembled and the encoded protein sequence deduced (Genetics Computer Group, Uni-versity of Wisconsin). Alignments with sequences from other species were made using Lasergene (DNAstar, Madison, WI).
Isolation of wild-type AChRa gene: We screened 4.6 3 105
clones from a zebrafish genomic library (lDASH II, a gift from Barbara Jones and Martin Petkovich, Queen’s
Uni-versity, Kingston, Ontario) with a 32P-labeled random
hex-amer-primed zebrafish AChRa cDNA (pAChRa). Filters were hybridized with 5 3 105 cpm/ml probe in 53 SSC, 50 mm
so-dium pyrophosphate, 53 Denhardt’s, 100 mg/ml calf thymus DNA, 100 mg/ml yeast tRNA at 628 for 2 days, then washed to 0.253 SSC, 0.1% SDS at 658. Filters were exposed for 6 days at
2708 with an intensifying screen. Three clones were isolated. We determined the DNA sequences for exon/intron junc-tions surrounding introns 5 and 6 (numbered by analogy to the human gene; Noda et al. 1983).
Isolation of nic1 mutant AChRa gene:Oligonucleotide prim-ers were synthesized (Biotech Services, Univprim-ersity of Oregon; primers a59-GCT GCA GAT CGT GGT GCT TCA C, and a39 -GCT GCA CCA CAG AGA TGC TGA) to regions of the AChRa cDNA flanking the RFLP. An approximately 2.0-kb product was amplified from genomic DNA isolated from nic1 mutant haploid embryos or from DNA isolated from adult fish heterozygous for the RFLP in a 100 ml reaction mix of 100 pmol of each primer, 200 mM dNTPs, 13 Replinase buffer (New England Nuclear, Dupont) and 1 unit of Replinase. PCR amplifications were obtained in a MJ Research thermocy-cler. The resulting PCR products were size fractionated on an agarose gel. Ends of the DNA products were repaired with T4 DNA kinase and DNA fragments were self-ligated (Kaufman
and Evans 1990) and digested with PstI. PCR products were
cloned into the PstI site of pBlueScript II KS(1) (Stratagene). Colonies were screened by hybridization with an AChRa cDNA. Subclones (p14.2ampmut and p15ampmut) were re-covered from material amplified from the DNA of two het-erozygous fish and were partially sequenced.
Sequencing: DNA was sequenced by a modification of the method of Chen and Seeburg (1985); double-strand
se-quencing mixes were prepared with Sequentide (New En-gland Nuclear, Dupont). Some of the sequence was deter-mined using automated DNA sequencers at the Central Services Laboratory of the Center for Gene Research and Bio-technology at Oregon State University at Corvallis and at the Research Services of the Molecular Genetics Facility at the University of Georgia (GenBank accession numbers: U70435, U70436, U70437, U70438) or at the University of Oregon Bio-tech Lab.
In situ hybridization: AChRa antisense riboprobe was
kit 1 (Boehringer Mannheim). In situ hybridizations were per-formed as previously described (Oxtoby and Jowett 1993).
Embryos were frozen and sectioned. Nuclei were labeled with 1% neutral red after in situ hybridization.
Mapping: The nic1 mutant phenotype was mapped with
the random amplified polymorphic DNA (RAPD) method used to establish the zebrafish linkage map (Postlethwait et
al. 1994). In brief, fish heterozygous for the nic1 mutation were crossed to Darjeeling zebrafish to give nic1/Dar F1
het-erozygotes. Haploid progeny were obtained from the F1
fe-male fish and were sorted into motile and nonmotile (wild-type and nic1 mutant) pheno(wild-types. DNA was isolated and pooled from a batch of about 50 wild-type or nic1 mutant hap-loid embryos. Approximately 150 RAPD primer sets that were used to establish the map were tested against the pooled wild-type or nic1 mutant DNA. Primer sets that appeared to be linked to the mutation were tested against a second batch of DNA. Primer sets that still appeared to be linked were then tested against about 50 individual wild-type and about 50 nic1 mutant haploid embryos. Once a linkage group was found, all primer sets within that group were tested. The number of re-combinant embryos was used to calculate recombination dis-tances (LINKER program, Postlethwait et al. 1994).
Map-maker (Lander et al. 1987; Postlethwait et al. 1994) was
used to prepare a linkage map.
RNA isolation, Northern blot analysis, and RNase protec-tion: RNA was isolated from zebrafish embryos and adults by an acid–phenol method (Chomczynski and Sacchi 1987).
Poly(A)1 RNA for Northern blots was isolated using the Mi-crofast Tract kit (Invitrogen, San Diego, CA). Blots were hybrid-ized overnight in 53 SSC, 53 Denhardt’s, 50% formamide, 0.1% sodium dodecyl sulfate (SDS) and 100 mg/ml salmon sperm DNA (Sambrook et al. 1989), washed to 0.53 SSC at
room temperature and exposed to film at 2708, with an inten-sifying screen for 49 hr or washed to 0.13 SSC at room tem-perature and exposed for 7 days.
RNase protection experiments were performed with the RPA II kit (Ambion, Austin, TX). A 320 bp RsaI fragment from the nic1 mutant gene (p14.2ampmut) was cloned into the SmaI site of pBlueScript II KS(1). Antisense riboprobe was transcribed from the T7 promoter of XhoI-digested plasmid. Antisense zebrafish MyoD1 (Weinberg et al. 1996) was
tran-scribed with T7 polymerase from XbaI-digested plasmid. Ribo-probes were synthesized with the Maxiscript kit (Ambion) and purified by gel electrophoresis. Total RNA (20 mg) was hybridized with about 50 kcpm at 428 overnight. Gels were dried onto paper and exposed to film on an intensifying screen at 2708 for 12 hr to 10 days. Relative abundance of mRNAs was estimated by comparison to the MyoD1 control.
RT-PCR: RNA was prepared from 70 wild-type and 70 nic1 mutant embryos at 50h using Tripure isolation reagent (Boe-hringer Mannheim). RNA (1 mg) was reverse transcribed (RT) and subsequently amplified using a CapFinder PCR cDNA synthesis kit (CLONTECH). The following primers were used for amplification of a-subunit cDNAs from wild-type and nic1 mutant cDNA samples: (forward, exon 5 primer A) GAG GTG GAC GGG ATC AAC TCG, (forward, exon 6 primer B) GGG CTT GTG TTT TAC CTG CCC; (reverse, exon 7 primer C) CGA GTT GAT CCC GTC CAC CTC, (reverse, exon 8 primer D) TGG TTA TGG GAG AGT GGT AAG. PCR conditions were 948 for 1 min (first cycle only), 928 for 30 sec, 608 for 30 sec, 758 for 90 sec, cycle 30 times, 758 for 5 min. DNA frag-ments were subcloned into the pCR2.1 vector using the TA cloning kit (Invitrogen). Samples were sequenced by the Uni-versity of Oregon sequencing facility.
Southern blot analysis: DNA was extracted from zebrafish by a modification of the method of Blin and Stafford
(1976). Adults were anesthetized by chilling with ice. Tissues
were quick frozen in liquid nitrogen and ground with pestles (Kontes, pellet pestle) in extraction buffer (embryos) or in a frozen mortar and pestle (adults) and dispersed in extraction buffer (50 mm EDTA pH 8.0, 0.5% sarcosyl, and 0.2 mg/ml
proteinase K). The tissue was incubated at 508 for 1–3 hr, ex-tracted three times with phenol/chloroform/isoamyl alcohol, ethanol precipitated, and resuspended in sterile H2O. Blots
were prepared according to Sambrook et al. (1989). Gels
were blotted onto positively charged nylon membranes (Boeh-ringer Mannheim) or Hybond-N membranes (Amersham, Ar-lington Heights, IL).
Blots were hybridized with either 32P-labeled random
hex-amer-labeled probes or digoxygenin-labeled (Boehringer Man-nheim) probes made from all or part of the AChRa cDNA. Radioisotope-labeled probes were hybridized in 53 SSC, 50 mm sodium pyrophosphate, 53 Denhardt’s, 100 mg/ml calf
thymus DNA, 100 mg/ml yeast tRNA, and 0.2% SDS buffer at 658 for 16–48 hr, washed in 0.53 SSC at 628, and exposed to film on an intensifying screen at 2708 for 3–7 days. Digoxyge-nin-labeled probes were hybridized overnight at 658 in 53 SSC, 0.1% N-lauroylsarcosine (Sigma), 0.02% sodium lauryl sulfate, 1% blocking reagent (Boehringer Mannheim), with the addition of 50 mg/ml heparin (Sigma), and subsequently washed in 0.13 SSC, 0.1% SDS at 658. Bands were revealed with anti-digoxygenin–alkaline phosphatase antibodies and Lumiphos (Boehringer Mannheim) according to the manu-facturer’s instructions and exposed to film for 3 hr to over-night. Seven probes were used for Southern blot analysis: a full-length cDNA, a partial cDNA whose 39 extent is coinci-dent with the most 39 probe of Figure 2D, three fragments shown in Figure 2D, and two BamHI fragments from the 59 end of the cDNA.
Western blot analysis: Western blot analysis was conducted essentially as described previously (Hatta et al. 1991).
Wild-type or nic1 mutant embryos at 3–4d were solubilized in 3.5% SDS. Proteins were separated by electrophoresis on a 10% polyacrylamide gel and blotted to polyvinylidene difluoride sheets (Towbin et al. 1979). After blotting, the sheets were cut
into strips that contained the equivalent of approximately 100 embryos each. The strips were probed with a primary anti-body (MAB 149; Sargent et al. 1984) specific for the AChRa
protein.
Expression of wild-type AChRa cDNA in nic1 mutant
em-bryos: The AChR cDNA was subcloned into a plasmid
(Klein-Hitpass et al. 1986) containing estrogen-responsive
elements (ERE) from the frog vitellogenin A2 gene to form plasmid pEREaAChR. Embryos at the two- to four-cell stage were coinjected with the pEREaAChR (30 ng/ml) and pKCR2-ER (15 ng/ml), which contains the human estrogen receptor cDNA driven by an SV40 promoter (Breathnach
and Harris 1983; Green et al. 1986). To activate AChR
ex-pression, embryos were transferred at 6 or 24h to medium containing 1027mb-estradiol.
After the embryos grew 28h to 3d, their clustered AChRs were labeled as previously described (Westerfield et al.
1990). In brief, they were soaked in 40 mM tetramethyl-rhodamine a-bungarotoxin (R-BTX; Molecular Probes, Eugene, OR) in 15% dimethyl sulfoxide in L-15 medium at 2–128 for 15–30 min. Embryos were rinsed three to five times in L-15 medium. Rhodamine-labeled AChR clusters were visualized with a Zeiss UEM microscope, Videoscope KS 1381 intensify-ing tube, and MTI series 68 video camera. Images were aver-aged (8–16 individual images) using a Macintosh IIci com-puter (Apple) and Axovideo software (Myers and Bastiani
with Photoshop software (Adobe). Digitized fluorescence im-ages were processed only by subtracting the background be-low a minimum intensity (threshold pixel value). This mini-mum intensity level was selected by eye. Composite images were made by adding fluorescent images to Nomarski images.
R E S U LT S
The paralyzed phenotype of nic1 mutation is linked to an RFLP in the AChRa gene: The zebrafish mutation nic1 blocks AChR function (Westerfield et al. 1990) and acts autonomously in muscle cells (Sepich et al. 1994). Because the mutation produces no other obvi-ous phenotypes and lacks embryonic lethality, we sus-pected the mutation might specifically block the syn-thesis of AChRs rather than block a general cellular function that would indirectly cause paralysis. To exam-ine AChR synthesis, we cloned the zebrafish AChRa cDNA. The longest clone of 2.2 kb spans the coding se-quence and includes approximately 150 base pairs (bp) of untranslated 59 leader and approximately 700 bp of untranslated 39 region, ending in a 26 bp poly(A) tail.
The conceptually translated protein sequence (Fig-ure 1) is similar at the amino acid level to the AChRa proteins of other vertebrates: 77% identical to Xenopus 1A (Hartman and Claudio 1990), 78% to Xenopus 1B (Baldwin et al. 1988), 77% to Torpedo californica (Noda et al. 1982), 77% to mouse (Boulter et al. 1985), and 79% to chicken (Conti-Tronconi et al. 1988).
We compared the Southern blot patterns of the AChRa gene from haploid nic1 mutant and wild-type sibling embryos. We sorted haploid progeny from in-dividual heterozygous female fish (nic1/1 ) according to phenotype, extracted DNA from pools of phenotypic nic1 mutant or wild-type embryos, and hybridized the resulting Southern blots with probes from the zebrafish AChRa cDNA. Mutant embryos had RFLPs in the AChRa gene for restriction enzymes HindIII, BglII (Figure 2A), and PstI (not shown). As expected, the Southern blot pattern in wild-type sibling embryos matches that of the clonal line, C29 (Figure 2) and C32 (data not shown), the backgrounds on which the nic1 mutation was generated.
The AChRa gene and the nic1 mutation map togeth-er: We mapped (Postlethwait et al. 1994) both the nic1 mutant phenotype and the AChRa gene. The nic1 mutant phenotype is linked to four markers on linkage group VI of the zebrafish map (Figure 2C); nic1 is 39.8 cM from SSR.26, 8.5 cM from M12.350, 22.4 cM from N5.675, and 29.6 cM from U6.850. The map distances we report differ slightly from the published linkage group VI. Presumably this reflects the effect of genetic background on recombination frequency or statistical variation owing to the small number of embryos used to map the nic1 locus.
We mapped the AChRa gene using a polymorphism present in nic1 mutants. PCR primers to sequences
flanking intron 6 amplified an approximately 220-bp product from the DNA of haploid nic1 mutant em-bryos, but not from their haploid wild-type siblings. Us-ing the same embryos from the previous mappUs-ing cross, we amplified the 220-bp product from all 46 hap-loid phenotypically nic1 mutant embryos, but not from their 48 haploid wild-type siblings. Thus, the AChRa gene is located within 0–2 cM of nic1.
nic1 mutation is a deletion: The differences in the Southern blot pattern between haploid mutants and their wild-type siblings can be explained by deletion of a region containing a HindIII site (Figure 2D). The nic1 mutant Southern blot pattern shows an 8.1-kb HindIII fragment, whereas two fragments of 6.2 and 3.5 kb ap-pear in wild-type (Figure 2B). Digestion with restriction enzymes PstI (not shown) or BglII (Figure 2A) also re-vealed a single fragment in nic1 mutants that is smaller than in wild-types. These differences are consistent with a deletion.
We identified the region of the gene that corre-sponds to the RFLP. Most of the nic1 mutant AChRa gene is indistinguishable from wild-type; probes from the 59 or 39 ends of the cDNA showed identical patterns in the genomic DNA from nic1 mutants and wild-type siblings (see Figure 2B, most bands are identical). The differing polymorphic fragments correspond to a sin-gle region of the AChRa gene. Probes hybridizing to the coding region between the ACh-binding site and transmembrane domain I detect the 6.2-kb HindIII fragment in wild-type DNA (Figure 2D, the two more 59 probes). Probes spanning transmembrane domains II and III detect the 3.5-kb fragment in wild-type (Figure 2D, most 39 probe), whereas both sets of probes detect the nic1 mutant 8.1-kb fragment. The smaller DNA fragments in nic1 mutants suggest that the nic1 muta-tion resulted from the loss of approximately 1–3 kb of the AChRa gene, where it codes for transmembrane domains I to domain II.
The differences we observed between the sequences of the wild-type and nic1 mutant AChRa genes were confined to intron 6. As expected from the Southern blot analysis (Figure 2B), the nic1 mutant gene lacked approximately 1 kb of intron 6 present in the wild-type gene (Figure 3), reducing the intron to 110 bp. Wild-type and nic1 mutant sequences are identical for the first 39 bp and almost identical for the last 71 bp of in-tron 6. We found single basepair mismatches at
nucle-otide (nt) 42 (C→G) and at nt 45 (G→A) of the nic1 mutant intron 6.
Neither the wild-type nor the nic1 mutant intron contains a polypyrimidine tract or potential branch-point in the typical location, adjacent to the 39 splice junction. Both introns have long (60–120 bp) runs of poly(GT) adjacent to the 39 splice site. The wild-type in-tron has several polypyrimidine tracts (Figure 3, bars above sequences) near potential branchpoints (Figure
Figure 1.—Interspecies comparisons of the conceptual translation of the zebrafish AChRa cDNA. Comparison with AChRa
3, arrows) at 150–250 bp upstream of the 39 splice site. Although there are several potential branchpoint aden-osines in the nic1 mutant intron, all are within 44 bp of the 59 splice site and thus may be too close for efficient splicing (Fu et al. 1988; Smith and Nadal-Ginard 1989; reviewed in Smith et al. 1989). Northern blot analysis (data not shown) supported this interpretation; AChRa mRNA in nic1 mutants is larger than in wild-type siblings. nic1 mutants make mutant AChRa mRNA: The differ-ences between the nic1 mutant and wild-type AChRa genes suggest that the nic1 mutant gene makes an AChRa mRNA that retains intron 6. We tested this pre-diction using an RNase protection assay. We designed a riboprobe (Figure 4) that would protect intron 5 (pre-dicted to be spliced out in all cases), exon 6 (pre(pre-dicted to be retained in all cases), and intron 6 (predicted to be retained only in mutants). Wild-type AChRa mRNA protected a fragment of about 239 nt, corresponding to exon 6. RNA from embryos carrying the nic1 mutant gene protected two fragments: the 239-nt exon 6 frag-ment and an approximately 290-nt fragfrag-ment corre-sponding to exon 6 and intron 6. Both RNAs in nic1 mutants are reduced in abundance relative to myoD mRNA, which was used to indicate the total quantity of muscle mRNA (data not shown). The smaller fragment protected in nic1 mutants is present at approximately 25% of the level observed in wild-type embryos. The in-tron 6-containing fragment is present at about 15% of the amount observed for the wild-type protected frag-ment in wild-type embryos.
To obtain further proof of the mutant nature of the nic1 AChRa mRNA, we amplified RNA from mutant and wild-type embryos by RT-PCR using four sets of primers that span intron 6 (Figure 5). The largest band amplified from mutant nic1 RNA by each primer pair was 110 bp longer than the corresponding product from wild-type RNA (Figure 5, asterisk). Sequence anal-yses of these products showed that they all contained the 110 bp nic1 mutant intron 6 (Figure 3), demon-strating that these nic1 transcripts fail to splice the mu-tant intron. Amplification of nic1 mumu-tant RNA with two of the primer pairs, AD and BD, revealed additional products (Figure 5, solid line) that appeared consistently in each RT-PCR. Sequence analyses of these products showed that they represented incorrectly spliced tran-scripts which lacked parts of the coding sequence. Out of a total of 15 sequenced clones, none corresponded to the wild-type sequence; all were incorrectly spliced. Clones were of six types that retained intron 6 or spliced exon 6 to exon 8 and four types that spliced out parts of the sequence from exons 6 to 8.
nic1 mutants express reduced amounts of AChRa pro-tein: The splicing defects demonstrated by our RNase
as solid white boxes. Fragments that are the same in nic1 mu-tants and wild types are shown as dashed line boxes. Fragment sizes are indicated in kilobases.
Figure 2.—The nic1 mutation and the AChRa gene are
protection (Figure 4) and RT-PCR (Figure 5) analyses predicted that the nic1 mutant gene produces trun-cated a-subunit proteins at reduced levels. To examine this possibility, we compared AChRa proteins from wild-type and nic1 mutant embryos by Western blot anal-ysis (Figure 6). We probed blots of proteins isolated from whole embryos with an antibody (MAB 149) that has been shown to recognize an epitope in the intracel-lular domain near the C-terminal of the AChRa protein (Sargent et al. 1984). The antibody revealed a band at about 50 kD in wild-type, but not in nic1 mutant pro-tein, consistent with the mutant producing reduced amounts of AChRa.
Early expression of the AChRa gene differs between mutants and wild types: We used RNA in situ hybridiza-tion to examine levels and patterns of expression in vivo. The same tissues express AChRa mRNA in nic1 mutant embryos and their wild-type siblings; however, there are distinct differences in abundance and cellular localization of the message.
At 30h, muscle cells in both mutant and wild-type embryos express high levels of the AChRa mRNA. Transcripts are most abundant in muscle cells near the lateral surface of the myotome (Figure 7, A and B), which may include the precursors of the adult slow muscles (Devoto et al. 1996). Abundance of the mRNA in lateral nic1 mutant muscle cells is comparable to or exceeds that in wild-type cells and appears to be more closely associated with nuclei than it is in wild-type mus-cle cells (Figure 7, C and D). Wild-type embryos also ex-press AChRa mRNA in the cytoplasm of more medial muscle cells, whereas, consistent with the RNase protec-tion studies (Figure 4), cytoplasmic expression of the mRNA is reduced in nic1 mutants (Figure 7, B and D).
Wild-type AChRa mRNA rescues the nic1 mutant phe-notype: If the mutant lacks AChRs because of an absence of the a-subunit mRNA, then supplying the wild-type mRNA should restore AChRs. We tested this prediction by injecting nic1 mutant and wild-type embryos, at the two- to four-cell stage, with a plasmid expressing the
wild-Figure 3.—The nic1
mutant AChRa gene lacks intron sequence and makes an mRNA that is larger than in wild types. Wild-type gene sequence ap-pears in upper line and nic1 mutant sequence below. Splice junctions are de-noted by boldface GT at 59 junction (consensus, (C/ A)AG GTRAGT) and AG at the 39 junction (YnNYAG
type AChRa cDNA. At 28h, 2d, or 3d, we examined the surviving embryos for the presence of clustered AChRs by labeling with rhodamine–a-bungarotoxin. We then fixed the embryos and examined them for AChRa RNA
by in situ hybridization. Out of 68 injected embryos, 75% were phenotypically wild type, as expected; they could swim and contained many labeled AChR clusters in a wild-type pattern. Seventeen embryos were identi-fied as nic1 mutants because they didn’t swim. These em-bryos had rhodamine–bungarotoxin labeling on some of their muscle cells, unlike uninjected nic1 mutants, which never show detectable levels of bungarotoxin la-beling (Westerfield et al. 1990). Fifteen of these em-bryos had both rhodamine–bungarotoxin labeling on muscle cells and muscle cells that contained high levels of AChRa mRNA, and we could identify individual muscle cells that contained both the wild-type AChRa mRNA (Figure 8, top) and AChR clusters (Figure 8, bottom). The patchy appearance of these rescued cells is probably the result of the mosaic distribution of the injected cDNA (Westerfield et al. 1992). In two 48h embryos, the injected AChRa cDNA rescued nerve– muscle communication; each embryo had a muscle cell that contained AChRa mRNA, that clustered AChRs, and that contracted, whereas neighboring cells did not. Contractions are never observed in uninjected nic1 mu-tants at this stage (Westerfield et al. 1990). The rescue of AChR clustering and neuromuscular function by supplying a source of wild-type mRNA supports the in-terpretation that the paralysis in nic1 mutants results from an absence of wild-type a-subunit mRNA.
Feeble movements may indicate leakiness of the mu-tation: Some mutant embryos recover slightly from pa-ralysis, beginning at 2–3 days of development. Compared to movements in wild-type siblings, the movements in nic1 mutants are fewer and slower. Because the feeble movements resemble movements in wild-type siblings and are provoked by touching the embryos, we speculate that nic1 mutant embryos may have a few functional AChRs. We have been unable to detect AChR clusters with rhodamine–a-bungarotoxin labeling in weakly mo-tile mutant embryos, suggesting they are too few in num-ber to be detected by this method. Alternatively, the fee-ble movements could result from depolarization of dying muscle cells. This seems unlikely, however, because weakly motile nic1 mutant embryos can live for up to 12 days and we observe no obviously abnormal cell death.
Figure 4.—Embryos that carry the nic1 mutant gene make
an AChRa RNA that retains intron 6. (Left top) Schematic representation of riboprobe. Exon is represented by open box, introns by thin lines, vector sequences by thick lines. (Left bottom) Predicted protected fragments. (Right) RNase protection analysis. Mutant nic1 and wild-type siblings (wtsib) make an RNA that retains intron 6, whereas unrelated wild-types (wt) make only fully spliced AChRa mRNA.
Figure 5.—The nic1 mutant AChRa gene is incorrectly
spliced. (Top) Schematic diagram of part of the AChRa pre-mRNA in wild types (wt) and nic1 mutants (nic1). Solid lines indicate coding sequences of exons 5–8 and the intron 6 se-quence from the nic1 mutant (exons 5–8 are truncated). In-trons (not drawn to scale) are indicated by dashed lines. Primers used for PCR amplification of RT RNA are indicated by half arrows and letters. Scale bar, 50 bp. (Bottom) RT-PCR amplification products from wild-type (WT) and nic1 mutant RNA using primer pairs AD, AC, BD, and BC, whose binding sites are indicated by the schematic above. Amplification products from the bands indicated with asterisks (*) were se-quenced and correspond to the nic1 mutant transcripts with unspliced intron 6. Amplification products from the bands dicated with dashes (-) were sequenced and correspond to in-correctly spliced transcripts with internal deletions of the cod-ing sequence. Size markers in kilobases.
Figure 6.—Expression of
AChRa protein is reduced in nic1 mutants. Western blot of proteins extracted from wild-type (wt) and nic1 mutant (nic1) embryos probed with mab 149 (Sargent et
al. 1984), which is specific for the intracellular domain of the AChR
D I S C U S S I O N
The nic1 mutant phenotype is linked to a deletion in the AChRa gene: Our results provide several lines of evidence that implicate the AChRa gene as the locus of the nic1 mutation. First, the nic1 paralyzed phenotype is linked to the AChRa gene. The nic1 paralyzed phe-notype maps to the same location as the PCR marker of the small nic1 mutant intron 6, placing the mutation within 0–2 cM of the AChRa gene (Figure 2C). Second,
our Southern blot analyses (Figure 2, A and B) demon-strate that the paralyzed nic1 mutant phenotype is linked to an alteration in the AChRa gene and suggest that ap-proximately 1.5 kb of the AChRa gene containing a HindIII site is absent from nic1 mutants. Third, direct comparison of the corresponding regions of the wild-type and mutant genes (Figure 3A) shows that a se-quence is missing that reduces the size of intron 6 in nic1 mutants without disturbing coding sequence or splice junctions.
The nic1 mutation makes unspliced AChRa tran-scripts: The structure of intron 6 of the AChRa in the
Figure 7.—The expression pattern of the AChRa differs
between wild-type sibling and nic1 mutant embryos. Trans-verse sections at trunk levels of 30-hr wild-type (wt) (A) and nic1 mutant (B) embryos. AChRa expression is revealed by the blue reaction product. Nuclei are stained with neutral red. AChRa mRNA is less abundant in the nic1 mutant medial myotome. Side view of whole-mount wild-type (C) and nic1 mutant (D) embryos. AChRa mRNA is more closely associ-ated with nuclei in nic1 mutant embryos. Scale bar, 50 mm.
Figure 8.—Clustered AChRs can be rescued in nic1
nic1 mutant suggests how the mutation may act; an es-sential interaction between the splice junction and splicing branchpoint may be sterically inhibited. Splic-ing requires the presence of conserved sequence ele-ments at the exon/intron borders (59 and 39 splice junctions), an element within the intron where the branchpoint forms, and an adjacent polypyrimidine tract (Smith et al. 1989). The branchpoint and polypyrimi-dine tract are usually near (18–37 nt upstream) the 39 splice junction but can be much farther upstream. In the first step of splicing, a guanosine at the 59 splice junction makes a covalent bond with an adenosine in the splicing branchpoint. Efficient splicing requires a minimum separation of 50 or more nucleotides be-tween the 59 splice site and the branchpoint. In the splicing of SV40 small-t antigen pre-mRNA, deletions that reduced the separation to less than 46-nt reduced or blocked splicing (Fu et al. 1988). In a-tropomyosin pre-mRNA, the normal inhibition of splicing of exon 2 is relieved by increasing the separation from 42 nt to 51–59 nt (Smith and Nadal-Ginard 1989). The nic1 mutant intron is small (110 nt) with several potential splice branchpoints near the 59 splice junction. The po-tential branchpoint farthest downstream from the 59 splice junction is 44 nt away, which is potentially too close for efficient splicing. The potential branchpoint that most resembles the canonical sequence is located 24 nt downstream of the 59 splice junction, too close to support splicing. Splicing efficiency and the use of a particular branchpoint are also improved by an adja-cent polypyrimidine tract (Reed 1990; Roscigno et al. 1993). In the wild-type intron, there are polypyrimidine tracts mixed with potential branchpoints at 140–240 nt upstream of the 39 splice site. In the nic1 mutation, can-didate branchpoints lack these downstream polypyrimi-dine tracts. Consequently, the potential branchpoints in nic1 mutant AChRa may be unable to engage in splicing.
In our RNase protection experiments, we found that most of the RNA in nic1 mutants retains intron 6 (Fig-ure 4). A smaller number of transcripts retain exon 6 and splice out intron 6. Sequence analysis of RT-PCR products (Figure 5) demonstrated that nic1 mutant em-bryos can remove intron 6 only by splicing out flanking coding sequence and consequently make no detectable wild-type mRNA. We also found the amount of AChRa message reduced in nic1 mutants (Figures 4 and 7). The amount of intron 6-containing mRNA in nic1 mu-tants and the messages with internal deletions are much reduced from wild-type mRNA levels, suggesting that splicing efficiency is lower or that the unspliced and incorrectly spliced RNAs are unstable.
There are several possible consequences of an spliced pre-mRNA. First, if the remaining intron is un-recognized, the RNA may leave the nucleus unspliced. The translation of a nic1 mutant mRNA that contains intron 6 would end at an frame stop codon at the
in-tron/exon junction, producing a truncated protein. The predicted truncated protein includes the extracel-lular domain, transmembrane domain I, and 35 novel amino acids encoded by the intron. Such a truncated protein might block assembly of functional receptors as a dominant negative mutant. A mutant a-subunit pro-tein that was engineered with a truncation after trans-membrane domain I was shown to bind to the d -sub-unit and block the assembly of the receptor (Vernall and Hall 1992). Because the nic1 heterozygote is with-out apparent phenotype, such as weakness or fatigu-ability, we have no evidence for the translation of an unspliced RNA that acts as a dominant negative.
Second, if the intron sequence remains unspliced, the RNA may be retained within the nucleus until it is degraded. We see a close association of AChRa mRNA with nuclei in nic1 mutant embryos (Figure 7). The re-duced amount of AChRa RNA in mutants (Figures 4 and 7) is consistent with efficient breakdown of the un-spliced mRNA, either in the nucleus or the cytoplasm. The mislocalization and reduced levels of AChRa mRNA in nic1 mutants are presumably responsible for the reduced expression of AChRa protein (Figure 6).
Third, the RNA may be spliced inefficiently to yield low levels of functional mRNA. Our RNase protection experiments showed both spliced and unspliced AChRa RNA (Figure 4), and our sequence analysis of the RT-PCR products (Figure 5) showed that all spliced transcripts include deletions of coding sequence. Thus, cryptic splicing, forced by the small nic1 mutant intron 6, forms internally deleted products. Some of these products, however, may code for functional or partially functional receptor subunits. This interpretation may explain the partial recovery of the paralyzed phenotype we see in older embryos.
Interpretation of the nic1 mutant phenotype: The nic1 mutant is a paralyzed embryo that later often shows weak contractions of a few muscles. The behavioral re-covery in nic1 mutants may indicate expression of a few transcripts from the mutant locus that code for func-tional AChR a-subunits. An alternative explanation for the functional recovery is that muscles may be activated by nonmuscle AChRs. Corriveau et al. (1995) have shown that embryonic chick muscles express substan-tial amounts of neuronal AChRs containing a 7-sub-units, as well as other neuronal AChRs. If zebrafish muscles also synthesize neural a7-subunit-containing receptors, then these receptors could theoretically me-diate movement in nic1 mutant embryos.
deletion of sequences within intron 6. (3) AChRa mRNA in nic1 mutant embryos is longer than in wild-types, consistent with a retained intron. (4) The nic1 mutant embryos express a-subunit mRNAs that retain intron 6 or are internally deleted and express reduced amounts of a-subunit protein. (5) Our in situ hybridiza-tion experiments showed that a-subunit mRNA is much reduced in abundance in nic1 mutant embryos, consis-tent with an unstable mRNA. (6) The absence of clus-tered and functional AChRs is rescued in nic1 mutant embryos by supplying wild-type AChRa mRNA. Conse-quently, partial loss of intron sequences leads to paraly-sis of the nic1 mutant embryo.
We thank Jon Lindstrom for the generous gift of AChR
antibod-ies, Ruth Bremiller and Michelle McDowell for help with
histol-ogy, Frankie Kimm and Ellen Johnson for help with fish, and
Charles Kimmel for comments on the manuscript. The ERE
con-struct was developed with help from Uwe Strähle. This work was
supported by National Institutes of Health grants NS21132, HD22-486, and GM7257.
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