Department of Biology, Emory University, Atlanta, GA 30322, USA, Program
in Biochemistry, Cell and Developmental Biology, Graduate Division of
Biological and Biomedical Sciences, Emory University, Atlanta, GA 30322,
USA
* Present address: Department of Anesthesiology, Vanderbilt University Medical
Center, Nashville, TN 37232, USA
Author for correspondence (e-mail:
bioslh{at}biology.emory.edu)
Accepted 12 July 2002
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SUMMARY |
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Key words: Cell cycle, Meiosis, Wee1p, Myt1, Spermatogenesis, C. elegans
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INTRODUCTION |
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Mitotic entry is controlled by activation of a protein complex that is
composed of the Cdc2p kinase and cyclin B
(Dunphy et al., 1988). This
protein complex accumulates during late G2 phase, but phosphorylation of
threonine-14 and tyrosine-15 on Cdc2p prevents its activation
(Coleman and Dunphy, 1994
).
Dephosphorylation of these residues constitutes the major mitotic entry signal
in eukaryotes. In fission yeast, Y15 of Cdc2p is phosphorylated by the Wee1p
and Mik1p kinases (Lundgren et al.,
1991
), while dephosphorylation is carried out by the Cdc25p
phosphatase (Russell and Nurse,
1986
; Berry and Gould,
1996
; Gautier et al.,
1991
). In metazoans, T14 phosphorylation is catalyzed exclusively
by a member of the Wee1p kinase family called Myt1
(Fattaey and Booher, 1997
).
The Myt1 kinase can also phosphorylate Y15 of Cdc2p, is about 40% identical
and 70% similar to the canonical S. pombe Wee1p kinases in its kinase
domain, contains a predicted transmembrane domain, and has a C-terminal domain
of poorly understood function. The membrane-spanning domain mediates Myt1
endoplasmic reticulum and Golgi localization in human cell culture lines when
this protein is overexpressed (Liu et al.,
1997
). Thus, Myt1 is a distinct member of the Wee1p kinase family
that appears to function in the cytoplasm to regulate Cdc2p.
We have analyzed rare dominant mutants that affect C. elegans spermatogenesis (spe mutants) and show that they contain mutations in the C. elegans wee-1.3 gene. The wee-1.3 gene is the C. elegans Myt1 ortholog and these are the first Myt1 kinase mutations recovered in any organism. These dominant spe mutants cannot perform the G2/M transition during spermatogenesis so hermaphrodites lack mature spermatozoa and exhibit, consequently, self-sterility. Genetic suppression of this self-sterility allowed recovery of loss-of-function mutants in C. elegans wee-1.3. These loss-of-function mutations reveal that WEE-1.3 is required for C. elegans embryonic and larval development. Sequence analysis of both dominant and intragenic suppressor lesions show that all dominant mutations affect a small domain in the C. elegans WEE-1.3 C-terminal region, while null suppressors affect the kinase domain. This suggests that C. elegans WEE-1.3 kinase activity is inhibited during M phase of meiosis I during spermatogenesis by a tissue-specific regulatory mechanism.
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MATERIALS AND METHODS |
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LG II: dpy-2(e8), dpy-10(e128), unc-4(e120)
(Brenner, 1974);
mab-3(e1240), eDf21 (Shen and
Hodgkin, 1988
); mIn1[dpy-10(e128) mIs14]
(Edgley and Riddle, 2001
);
mnC1[dpy-10(e128) unc-52(e444)], let-241(mn228), mnDf12, mnDf28, mnDf29,
mnDf30, mnDf57, mnDf58, mnDf60, mnDf63, mnDf71 and mnDf88
(Sigurdson et al., 1984
).
LG III: dpy-1(e1) (Brenner,
1974) and smg-6(r896)
(Hodgkin et al., 1989
).
LG IV: dpy-20(e1282ts) (Hosono, 1982).
mIs14 confers a dominant GFP+ phenotype in pharyngeal muscle, gut,
and in four- to 60-cell embryos, but only the pharyngeal marker was used
during these studies (Edgley and Riddle,
2001).
All dominant spe mutants were recovered following EMS mutagenesis
under standard conditions (Brenner,
1974) and each bears a mutation in a gene initially called
spe-37, but now named wee-1.3 for nomenclatural clarity.
wee-1.3(e1947) was isolated as previously described
(Doniach, 1986
).
wee-1.3(q89) was isolated in the laboratory of Judith Kimble and
provided by T. Schedl. wee-1.3(hc144) and wee-1.3(hc145)
were isolated by J. Varkey in the laboratory of Sam Ward.
wee-1.3(eb95) was isolated in a F1 non-complementation
screen for new recessive spe-10 alleles (W. Lindsey and S. W. L,
unpublished). wee-1.3(eb104) was isolated in a F1
non-complementation screen for new recessive fer-14 alleles (T. Kroft
and S. W. L., unpublished).
wee-1.3 suppression genetics
wee-1.3(gf) was balanced by mating wee-1.3(gf) unc-4/ dpy-2
unc-4 hermaphrodites to dpy-2 unc-4/mnC1 or mIn1 males
and picking F1 Uncs to verify the Spe phenotype. Either -ray or ENU
mutagenesis (Anderson, 1995
)
was performed prior to screening for suppression of the dominant Spe
phenotype. It was necessary to devise a mating scheme to accumulate sufficient
animals for mutagenesis because they were dominant Spe. For ENU mutagenesis,
one wee-1.3(q89gf) unc-4/mnC1 hermaphrodite was mated to four
dpy-2 unc-4/mIn1 GFP+ males on each of over 100 plates. The animals
on each mate plate were transferred daily for 4 days so that the outcross
progeny on each plate were of a similar age. When many L4 F1 were present,
they were pooled from the mate plates and mutagenized with 3.125 mM ENU for 4
hours under conditions similar to those described for ethylmethane sulfonate
(Brenner, 1974
). Mutagenized
GFP+ nonUnc hermaphrodites (
300-350) were picked to individual plates to
verify that these putative wee-1.3(gf) unc-4/mIn1GFP+ showed the
expected (unfertilized oocyte laying) Spe phenotype. Each verified
self-sterile Po hermaphrodite was mated to four dpy-2 unc-4/mnC1
males and 6968 resulting F1 Unc progeny were each picked to
separate plates. Self-fertile wee-1.3(q89;sup) unc-4/dpy-2 unc-4
hermaphrodites that segregated nonGFP Uncs and DpyUncs in the F2
were selected as candidate suppressors. They were outcrossed to dpy-2
unc-4/mIn1 GFP+ males and F1 GFP+ nonUnc progeny were picked.
Balanced wee-1.3(gf;sup) unc-4/mIn1 GFP+ were identified as
hermaphrodites that failed to segregate DpyUnc progeny.
For -ray mutagenesis, L4 hermaphrodites of genotype dpy-2
wee-1.3(e1947)/dpy-2 unc-4 were mutagenized with 1500 Rads from a
137Cs source. These hermaphrodites were crossed to dpy-2
unc-4/mnC1 males and 125 Dpy outcross progeny were each picked to a
separate plate. A single suppressor of genotype dpy-2
wee-1.3(e1947;sup)/dpy-2 unc-4 was identified as a self-fertile
hermaphrodite that segregated Dpy and DpyUnc progeny. This candidate was
crossed to dpy-2 unc-4/mnC1 males. Non-Dpy outcross progeny were
picked to plates and a balanced line of putative genotype dpy-2
wee-1.3(e1947;sup)/mnC1 was established from hermaphrodites that failed
to segregate DpyUnc progeny. No extragenic suppressors were identified in
either the
-ray or ENU screen.
smg suppression
Class 2 wee-1.3 suppressors (q89 eb60, q89 eb93 and
q89 eb87) were tested for smg suppression. wee-1.3(q89
eb60) unc-4/mIn1, wee-1.3(q89 eb93) unc-4/mIn1 or wee-1.3(q89 eb87)
unc-4/mIn1 males were crossed to dpy-1 smg-6 hermaphrodites.
NonDpy nonGFP F1 wee-1.3(q89 sup) unc-4/++; dpy-1 smg-6/++
hermaphrodites were picked. F1 hermaphrodites were allowed to
self-fertilize and the DpyUnc wee-1.3(q89 sup)unc-4; dpy-1 smg-6 and
Dpy nonUnc wee-1.3(q89 sup) unc-4/+;dpy-1 smg-6 or +/+; dpy-1
smg-6 progeny were picked. For both q89 eb60 and q89
eb93, 100% of the DpyUnc and 75% of the Dpy non-Unc show a Spe
phenotype that is identical to wee-1.3(q89)/+. q89 eb87 was
unaffected by the absence of the SMG surveillance system.
Nucleic acid methods
The chromosome II deficiency ebDf1 was mapped by polymerase chain
reaction (PCR)-based methods described previously
(Williams, 1995). Each PCR had
a control primer pair that would produce a product from the genomic DNA
present in ebDf1 homozygotes and a second pair of test primers. PCR
on wild-type embryos was carried out in parallel, and reactions were
visualized on ethidium bromide stained agarose gels. At least five PCRs were
attempted with each pair of test primers to determine if they could produce a
product from ebDf1 template DNA. This approach revealed that the left
ebDf1 breakpoint is within a 4 kb interval present in cosmid ZK1320
and the right breakpoint is within a 23 kb interval starting in cosmid ZK938
and extending through Y53C12C (data not shown). These data indicate that
ebDf1 deletes
125-150 kb on chromosome II, including the C.
elegans Myt1 ortholog, wee-1.3 (see
www.wormbase.org).
The wee-1.3 candidate gene was sequenced from each ENU induced
wee-1.3(q89;sup) suppressor. When suppressor homozygotes were viable,
individual nonGFP wee-1.3(q89;sup) unc-4 animals were picked from
wee-1.3(q89;sup) unc-4 /mIn1 GFP+ parents and used to prepare
template DNA (Williams, 1995
).
For lethal wee-1.3 null mutants, nonviable embryos were used to
prepare template DNA (Williams,
1995
). At least four independent Taq-generated PCR products were
fractionated by agarose gel electrophoresis and purified using the GeneClean
system (Bio 101, Vista, CA). Sequencing was performed at the Iowa State
University (Ames, IA) DNA sequencing facility by primer walking using standard
ABI automated sequencing. Sequence was analyzed using the DNASTAR software
package (DNASTAR, Madison, WI).
To sequence wee-1.3(gf) mutants, hermaphrodites of genotype wee-1.3(gf) unc-4/mIn1 GFP+ were crossed to dpy-2 ebDf1/mIn1 males. The nonGFP F1 hermaphrodites [wee-1.3(gf) unc-4/dpy-2 ebDf1] were picked to verify the dominant Spe phenotype. Individual animals were prepared for PCR as described for deficiency mapping. Multiple PCR reactions were pooled and sequenced, as described above, to identify the molecular lesions associated with wee-1.3(gf) mutants. All mutations were verified by sequencing the wild-type N2 strain (var. Bristol).
A wee-1.3 rescuing transgene was prepared by high-fidelity PCR
(Advantage2, Clontech Laboratories, Palo Alto, CA). A sense primer (TL49 -
5'-ATGTATTAGCATCGTTCTTTAAACCCCAACCAT-3') just outside the
predicted 3' end of Y53C12A.1 ORF and an antisense primer (TL55 -
5'-GCAAGAAAATAAAGGAGCGCAAACAAGAGT-3') just outside the predicted
5' end of the Y53C12A.6 were used to amplify a 4.3 kb fragment from N2
genomic DNA. This PCR fragment was microinjected with the dominant rol-6
(su1006) encoding plasmid pRF4
(Mello et al., 1991) into
wee-1.3(q89 eb88) unc-4)/mIn1GFP+ hermaphrodites. F1
rollers were isolated and a balanced, stable transgenic line was used to assay
for wee-1.3 rescue. This line segregates viable non-GFP Uncs that do
not have a germline. The F1 of hermaphrodites microinjected with
this and other wee-1.3-containing PCR fragments frequently died as
embryos (data not shown).
The tissue specificity of the wee-1.3 promoter was analyzed by
ligating it to the green fluorescent protein (GFP)-coding sequence
(Chalfie et al., 1994).
High-fidelity PCR (Advantage2, Clontech) with primers TL49
(5'-ATGTATTAGCATGCTTCTTTAAACCCCAACCAT-3') and TL50
(5'CAACTCGAGCATGCCTGCGGAGTGACCAAAAG-3') allowed 1 kb of sequence
5' to the transcriptional start plus the first wee-1.3 exon to
be amplified from N2 genomic DNA. These primers introduce SphI (TL49)
and Kpn1 (TL50) sites into the resulting PCR product, which was
restriction digested with these enzymes. This fragment was ligated into the
SphI/KpnI-digested GFP-encoding plasmid pPD95.77 (A. Fire,
S. Xu, J. Ahnn and G. Seydoux, personal communication) to create pSTL1. pSTL1
(10 ng/µl) was microinjected together with pMH86
(Han and Sternberg, 1991
)
dpy-20+ rescuing plasmid (100 ng/µl) into
dpy-20(e1280ts). Injected animals were grown at 25°C and
transformed animals were identified as non-Dpy F1. Stable lines
were used to determine the pattern of GFP fluorescence.
Light and electron microscopy
Light microscopy and immunofluorescence were performed as previously
described (Arduengo et al.,
1998). Post-acquisition image analysis was performed using
ImagePro (Media Cybernetics, Silver Springs, MD) and VolumeScan (Vaytek,
Fairfield, IA) software. Electron microscopy was performed as previously
described (L'Hernault and Roberts,
1995
). Figures were assembled using Adobe Photoshop 5.0 (Adobe
Systems, San Jose, CA) or Canvas 7 (Deneba Systems).
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RESULTS |
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Spermatogenesis in all dominant spe mutants arrest at an
early stage
Sperm from the dominant spe mutants were analyzed by light
microscopy. Wild-type spermatocytes progress through meiosis and
differentiation in an invariant manner
(Fig. 1A-D), with each male
producing hundreds of haploid spermatids. By contrast, all dominant
spe mutant males accumulate arrested primary spermatocytes and no
spermatids are observed (Fig.
1E-H). These mutant spermatocytes have a condensed nucleus that is
often asymmetrically located, which are characteristic of later stages of
spermatogenesis. Spermatocyte cytokinesis and condensed chromosomes aligned on
the metaphase plate have not been observed in any of the dominant spe
mutants. The same spectrum of cellular defects was observed when a dominant
spe mutation was in trans to a non-complementing deficiency (data not
shown), so this phenotype is neither dependent on the presence of the
wild-type allele nor improved by its absence. No dominant spe mutant
shows obvious evidence of earlier germline defects (data not shown).
|
Dominant spe mutants partially differentiate in the absence
of cell division
The defects associated with spe-37(e1947)/+ and q89/+
were examined by electron microscopy and both show identical ultrastructural
phenotypes. Initially, spe-37(e1947)/+
(Fig. 1I) and q89/+
(not shown) appear similar to wild type. Specifically, these mutants contain
an uncondensed, centrally located nucleus that is surrounded by a nuclear
envelope and normal ER/Golgi-derived fibrous body-membranous organelles
(FB-MOs). Terminal spe-37(e1947)/+ spermatocytes contain condensed
chromatin inside an intact nuclear envelope, FBs are not observed and MOs are
highly vacuolated (Fig. 1J).
These terminal spermatocytes polarize and place their nucleus and FB-MOs on
opposite sides of the cell, as if attempting to differentiate. However, this
attempt at differentiation occurs without karyokinesis or cytokinesis,
suggesting that these dominant spe mutations affect a gene that
coordinates the meiotic divisions with spermatocyte differentiation.
Spermatocytes in dominant spe mutants have phosphorylated
histone H3
Although spermatocytes in dominant spe mutants do not divide, they
do enter pachytene of meiotic prophase I (data not shown). Therefore, we
postulated that they are unable to complete M phase. To test this hypothesis,
wild-type and q89/+ sperm were stained with an antibody that
recognizes phosphorylated histone H3, a well-characterized marker for M
phase/chromosome condensation (Boxem et
al., 1999; Golden, 2000; Hsu
et al., 2000
; Strahl and
Allis, 2000
). In wild-type, phospho-histone H3 staining is
observed in dividing spermatocytes but not in spermatids
(Fig. 2A-D). In q89/+
males, all spermatocytes contain phospho-histone H3
(Fig. 2E-H). This suggests that
dominant spe spermatocytes arrest with condensed chromosomes,
possibly at the G2/M phase boundary of meiosis I.
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Establishing that spe-37(e1947)/+ and q89/+ are
allelic
Dominant mutants can be subjected to mutagenesis and screened for a new
recessive mutation that suppresses the dominance caused by the original
mutation (Greenwald and Horvitz,
1980; Conradt and Horvitz,
1998
). When the dominant mutation and its recessive suppressor
show tight linkage, both mutations frequently reside in the same gene. An
allelic series of such second site intragenic suppressor mutations often
reveals the null phenotype of that gene. Using this technique, nine
ethylnitrosourea (ENU) induced suppressors of q89 and one
-ray-induced suppressor of spe-37(e1947) were recovered. All
suppressor mutations genetically map very close to q89 and could be
unambiguously placed into one of three distinct phenotypic classes
(Table 1). Each suppressor
mutation functions in cis to q89, suggesting that suppression is
intramolecular in nature. Furthermore, suppressors derived from q89/+
fail to complement the suppressor derived from spe-37(e1947)/+,
indicating that q89 and e1947 are both spe-37
alleles.
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spe-37(q89)/+ G2/M meiotic arrest is partially alleviated by
Class 1 suppressors
The class 1 suppressor mutant spe-37(q89 eb61)/+, spe-37(q89
eb62)/+ and spe-37(q89 eb94)/+ hermaphrodites, unlike
spe-37(q89)/+, are self-fertile. Class 1/+ hermaphrodites have brood
sizes that are significantly smaller than those produced by wild type, and
they also produce an unusually high percentage of males
(Table 1). C. elegans
males are XO and usually arise in a wild-type hermaphrodite population by X
chromosome nondisjunction at a rate of 0.1%
(Brenner, 1974
). The higher
frequency of males suggests that an increased rate of X chromosome
nondisjunction could be occurring in Class 1/+ hermaphrodites. Alternatively,
Class 1/+ suppressor mutants might produce a high incidence of males because
some gametes lose the X chromosome by another type of meiotic aberration. All
three Class 1 suppressor homozygotes, in addition to spe-37(q89
eb62)/Df and spe-37(q89 eb94)/Df hemizygous hermaphrodites are
self-sterile, laying unfertilized oocytes and inviable embryos. Given the
increased frequency of X chromosome segregation abnormalities, autosomal
segregation might also be aberrant and thus contribute to the observed
embryonic lethality. Unlike self-sterile spe-37(q89 eb61) homozygous
hermaphrodites, spe-37(q89 eb61)/Df hemizygotes produce a few viable
progeny (mean=11 progeny; n=63); therefore two copies of
spe-37(q89 eb61) results in a more severe phenotype than one copy.
All Class 1 heterozygous, hemizygous and homozygous hermaphrodites produce
large numbers of cross progeny after mating to wild-type males. Thus, Class 1
homozygotes and hemizygotes have defective sperm but are largely or completely
unaffected in somatic development or other aspects of germline development
(Table 1).
While spe-37(gf)/+ males never complete spermatogenesis (Fig. 1E-H), the modest self-fertility shown by Class 1/+ suppressor hermaphrodites suggests that spermatogenesis is occasionally completed in these animals (Table 1). Class 1/+ mutant males contain many spermatid-like cells. Therefore, some spermatocytes in Class 1/+ suppressor mutants complete the meiotic divisions, something that never occurs in spe-37(q89)/+ mutants. However, Class 1/+ mutant testes contain many cells that show features never observed during wild-type spermatogenesis, such as 2-4 nuclei within the same cell (Fig. 3). Homozygous Class 1 mutant males contain very few sperm, but those that are present have a phenotype similar to the sperm observed in heterozygous Class 1/+ mutant males (data not shown). Overall, these data indicate that Class 1 suppressor mutants partially alleviate the G2/M meiotic block shown by spe-37(q89)/+ mutants. Perhaps Class 1 mutants reduce, but do not eliminate, the gain-of-function effects of spe-37(q89) and this explains why hermaphrodite brood size and male spermatogenesis are both different from wild type.
|
Maternal and zygotic functions of spe-37 are revealed by
hypomorphic suppressors
The Class 2 heterozygous spe-37(q89 eb60)/+, spe-37(q89
eb93)/+ and spe-37(q89 eb87)/+ mutants completely suppress
spe-37(q89)/+ and such suppressed hermaphrodites exhibit wild-type
self-fertility (Table 1). Like
Class 1 suppressors, homozygous spe-37(q89 eb60) and spe-37(q89
eb93) Class 2 suppressors are self-sterile, but they produce many more
inviable embryos than Class 1 suppressors. Homozygous spe-37(q89
eb87) hermaphrodites are self-sterile and lay many unfertilized oocytes
but do not produce inviable embryos. All Class 2 mutant hermaphrodites produce
large numbers of normal progeny after they are mated to wild-type males. These
results suggest that Class 2 alleles are not competent to support embryonic
development and at least one wild-type copy of spe-37 is required for
a zygote to develop normally.
The Class 2 suppressor mutant phenotype is partly dependent on the maternal genotype. When Class 2/+ heterozygous hermaphrodites are crossed to deficiency(Df)/+ males (where Df removes the spe-37 gene), the resulting Class 2/Df hemizygous hermaphrodites mature into adults that lay a mixture of oocytes and inviable embryos, which is like Class 2/Class 2 homozygous hermaphrodites. However, crossing sterile homozygous Class 2/Class 2 hermaphrodites to Df/+ males, results in inviable Class 2/Df hemizygous embryos that show no morphogenesis. These data suggest that a Class 2/+ mother can maternally supply enough wild-type spe-37 activity to allow her homozygous Class 2/Class 2 progeny to complete development and begin germline formation. This germline does not function normally, presumably because there is insufficient maternal endowment. When little maternal endowment occurs (Class 2/Class 2 hermaphrodites) and sperm provide no wild-type spe-37 gene, Class 2 alleles are not able to support embryonic development. These phenotypic properties are not as severe as Class 3 suppressor mutants (see below), which suggests that Class 2 suppressor mutations are loss-of-function but non-null (hypomorphic) alleles.
The many inviable embryos produced by Class 2/Class 2 homozygous hermaphrodites suggests that Class 2 mutants contain spermatozoa that are competent for fertilization. Microscopic examination of dissected heterozygous Class 2/+ or homozygous Class 2/Class 2 mutant males revealed that they contained cytologically wild-type spermatids (data not shown). These spermatids can form functional spermatozoa because hemizygous Class 2/Df mutant males sire viable progeny when crossed to wild-type hermaphrodites. As Class 2 mutants are hypomorphic, these data suggest that reduced spe-37 gene activity still allows apparently normal spermatogenesis.
Two Class 2 suppressor mutants (eb60 and eb93) are affected when
the SMG surveillance system is genetically disabled (see Materials and
Methods). The wild-type SMG surveillance system degrades mRNAs that are
defective, and a loss-of-function mutation in any of seven smg genes
eliminates this ability to detect and degrade defective mRNAs
(Mango, 2001). Heterozygous
spe-37(q89 eb60)/+ or spe-37(q89 eb93)/+, which are fertile
in a wild-type SMG background, become dominant Spe when the SMG surveillance
system is genetically disabled. Similarly, homozygous spe-37(q89
eb60) or spe-37(q89 eb93), which produce inviable embryos in a
wild-type SMG background, become dominant Spe when the SMG surveillance system
is genetically disabled. In both cases, the observed dominant Spe phenotype is
highly similar to spe-37(q89)/+. These results suggest that
spe-37(q89)-associated dominance is suppressed because eb60
or eb93 can, respectively, trigger spe-37(q89 eb60)- or
spe-37(q89 eb93)- encoded mRNA degradation by the wild-type SMG
surveillance system. Manipulation of the SMG surveillance system in the Class
2 suppressor double mutant background also provided a way to create what are
effectively spe-37(q89) homozygotes, and spe-37(q89)/+,
spe-37(q89)/spe-37(q89) and spe-37(q89)/Df (using
deficiencies that remove the spe-37 gene) all exhibit the same
phenotype. These data suggest that the spe-37(q89) mutation is
insensitive to gene dose and thus may be constitutively active or neomorphic
gain-of-function in nature [activity at an inappropriate place or time
(Muller, 1932
)].
The null phenotype of spe-37 is embryonic and larval
lethal
Like Class 2 mutants, Class 3 spe-37(q89 eb88)/+, spe-37(q89
eb90)/+ and spe-37(q89 eb91)/+ mutant heterozygous
hermaphrodites all exhibit wild-type self-fertility
(Table 1). Class 3 homozygotes
derived from a Class 3/+ mother receive a maternal endowment of SPE-37 but,
unlike Class 1 and 2 mutants, Class 3 homozygous mutants always die. Usually,
Class 3 homozygous mutant embryos die after limited morphogenesis but,
occasionally, they hatch into abnormal larvae that die. These data suggest
that in wild-type, there is limited maternal SPE-37(+) and that the zygotic
embryonic lethality of Class 3 homozygotes is the result of Class 3 alleles
that provide little or no functional SPE-37 activity.
Among individual Class 3 mutants, spe-37(q89 eb91) causes the least severe phenotype because it produces fewer inviable embryos and more inviable L1 or L2 progeny than do either spe-37(q89 eb88) or spe-37(q89 eb90) (data not shown). The spe-37(q89 eb88)/Df or spe-37(q89 eb90)/Df (where the Df removes the spe-37 gene) -associated phenotypes are identical to that shown by the respective mutant homozygotes. This suggests that these Class 3 double mutants exhibit the spe-37 null phenotype because lowering the gene dose has no effect on the observed phenotype.
A candidate gene approach reveals that spe-37 is
wee-1.3
The spe-37(e1947) -ray induced suppressor failed to
complement the chromosome II deficiencies mnDf63, mnDf58, mnDf29,
mnDf57 and eDf21 with regard to embryonic lethality and its
phenotype is similar to the Class 3 spe-37(q89 eb88) or
spe-37(q89 eb90) suppressor double mutants. The
spe-37(e1947)
-ray-induced suppressor also failed to
complement mab-3 and all ENU-induced spe-37(q89)
suppressors, so it behaved genetically like a deficiency and was named
ebDf1. As ebDf1 mapped to the same location as
spe-37(e1947), we hypothesized that it deleted the spe-37
gene and other adjacent genes. This hypothesis proved correct and the
breakpoints of ebDf1 define a region that includes the
spe-37 gene.
Although there are at least 26 predicted genes in the 125-150 kb
interval deleted by ebDf1, the only obvious cell cycle regulatory
gene in this region is wee-1.3 (see
www.wormbase.org).
As the spe-37 associated phenotype suggests it encodes a cell cycle
regulatory protein, we hypothesized that spe-37 and wee-1.3
were the same gene. This hypothesis was confirmed by showing that transgenes
containing the wild-type wee-1.3 genomic sequence could rescue Class
3 spe-37(q89 eb88) homozygotes from embryonic lethality. Although
viable, rescued transgenic Class 3 mutants are sterile because they do not
form a germline. Prior work has shown that C. elegans transgenes
frequently express in somatic tissues but show no germline expression because
of epigenetic silencing (Kelly and Fire,
1998
; Kelly et al.,
1997
). Although inconvenient, this co-suppression phenomenon
permits an assessment of whether a gene must be expressed in the germline for
normal germline development. The co-suppression phenotype suggests that
wee-1.3 expression is required for establishment and/or proliferation
of the germline.
Each of the six dominant spe-37 mutants proved to have a point mutation in the wee-1.3 gene (Fig. 4). Three of these dominant alleles (q89, e1947 and eb104) contained the same G1957A mutation that changes the encoded glycine at position 560 to an arginine. hc144 also affects glycine 560 but this mutation changes it so that glutamate (nucleotide change: G1958A) is encoded. eb95 converts the encoded glycine at position 558 to an arginine (nucleotide change: G1951A) and hc145 converts the encoded aspartic acid at position 561 to an asparagine (nucleotide change, G1960A). Remarkably, all six spe-37(gf) mutations affect a four amino acid region in the C-terminal region of wee-1.3.
|
Each of the nine ENU induced spe-37(q89) suppressor double mutants
had two mutations in wee-1.3 (Fig.
4). Each suppressor double mutant had the G1957A point mutation
(see Table 1) present in the
parental spe-37(q89) mutant. The second mutation was unique to each
suppressor mutant (Fig. 4;
Table 1). These DNA sequencing
results and wee-1.3 transgenic rescue unambiguously show that
spe-37 is the C. elegans Myt1 ortholog wee-1.3
(Wilson et al., 1999).
spe-37 suppressor mutations affect key residues in this Wee1-like
kinase and an alignment of WEE-1.3 (=SPE-37) to its orthologs helps in
interpreting some of the suppressor mutations
(Fig. 5). The Class 1
suppressors include two missense mutations (eb62 and eb94)
and one mutation (eb61) in a splice donor site
(Fig. 4;
Table 1). The eb62
suppressor mutation is the weakest Class 1 suppressor and it allows
hermaphrodites to produce broods of approx. five progeny
(Table 1). eb62 is an
I160N missense mutation in an amino acid that shows weak conservation between
wee-1.3 and its vertebrate orthologs
(Fig. 5). The eb94
suppressor mutation allows hermaphrodites to produce broods of 72. The
eb94 F103I missense mutation affects a residue that is conserved
among Myt1 orthologs (Fig. 5)
but not among other members of the Wee1p kinase family
(Wilson et al., 1999
). The
eb61 mutant permits the largest brood (
95;
Table 1) of any of the Class 1
suppressors, and it converts the fourth intron splice donor into an in-frame
codon (Fig. 4 and
Table 1). The Genemark HMM
program (Borodovsky, 1998
)
suggests that other upstream consensus splice donor sites are present (data
not shown), and the incompleteness of suppression suggests that such sites
might be used.
|
The Class 2 suppressors include two premature stop mutations (eb60 and
eb93) and one splice acceptor mutation in intron 4 (eb87;
Fig. 4A). The molecular natures
of eb60 and eb93 are consistent with their ability to be
suppressed by mutants in the SMG mRNA surveillance system, as some
smg-suppressible mutations are premature stop codons
(Mango, 2001). Both
eb60 and eb93 cause UAA ochre stop codons that would
truncate the polypeptide sequence near the C terminus. The eb87
mutation alters a conserved splice acceptor site and RT-PCR of mutant animals
shows that exon 5, which contains the transmembrane domain for WEE-1.3, is
skipped (Fig. 4B).
Additionally, the mechanism of eb87 suppression is not through a
SMG-mediated reduction in mRNA levels, as eb87 is unchanged in a
smg mutant background.
The Class 3 suppressors include two missense mutations (eb88 and
eb90) and one small deletion (eb91)
(Fig. 4;
Table 1). The eb88
suppressor results in a G245E missense mutation and eb90 results in a
H163P missense mutation; each of these mutations affects an amino acid that is
within a strongly conserved region (Fig.
5) found in all Wee1p kinases from yeast to humans
(Wilson et al., 1999). The
eb91 suppressor mutation is a 545 bp deletion that removes the first
465 bp of coding sequence and 80 bp 5' to the start codon. It is
unlikely that wee-1.3 is transcribed in eb91 suppressor
mutants because part of the promoter and the entire 5' untranslated
region, including the intron trans-splice acceptor for SL1 (A. Golden,
personal communication), are missing. Consequently, its molecular features
indicate that eb91 is a wee-1.3 null mutation.
wee-1.3 is widely expressed during C. elegans
development
The recessive lethality shown by the Class 3 spe-37(q89)
suppressors indicates that transcription of this gene is required outside the
testes. Northern hybridization experiments reveal that the
wee-1.3-encoded 2.4 kb mRNA was found in fem-1(hc17lf)
hermaphrodites, which do not make sperm, as well as fem-3(q23gf)
hermaphrodites, which make sperm but no oocytes (data not shown). Tissue
specificity of wee-1.3 was further examined by fusing the promoter
and first exon to the coding sequence for GFP and using this construct to
create transgenic worms. Seven stable transgenic lines all consistently showed
GFP during early embryonic development
(Fig. 6A,B), in the distal
region of the larval, but not adult, germline
(Fig. 6C,D), and in some larval
neurons and hypodermal cells (Fig.
6E,F). These data confirm that the wee-1.3 promoter is
active during both germline and embryonic development.
|
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DISCUSSION |
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Previous wee-1.3 RNA-interference studies (A. Golden, personal
communication) and our work both indicate that WEE-1.3 plays a crucial role
during the cell cycle in multiple C. elegans cell types. Wee1p
kinases are thought to function primarily through their phosphorylation of
Cdc2p. This suggests that the dominant spermatogenesis arrest (including the
associated lack of nuclear envelope breakdown) seen in wee-1.3(gf)
mutants occurs via Cdc2p-negative regulation because active Cdc2p is required
for both mitotic and meiotic G2/M progression in all studied eukaryotes
(Nurse, 2000). A problem with
this interpretation is that wee-1.3(gf) mutant spermatocytes have
phosphorylated histone H3, which is a known marker for active Cdc2p in the
C. elegans mitotic germline (Boxem
et al., 1999
). Phosphorylation of histone H3 is mediated by the
IpI1/aurora kinase (Hsu et al.,
2000
), and activation of this kinase is correlated with the
presence of activated Cdc2p (Boxem et al.,
1999
). Perhaps IpI1/aurora kinase requires a lower level of
activated Cdc2p than that needed for nuclear envelope breakdown and entry into
M phase.
The transmembrane region found in all Myt1 orthologs separates the
conserved, N-terminal kinase domain from a non-conserved C-terminal region
(Fig. 4). Overexpression of the
C-terminal domain in tissue culture cells causes a G2/M phase delay that
appears to be mediated by Cdc2p/cyclin
(Liu et al., 1999;
Wells et al., 1999
). The six
C. elegans wee-1.3 dominant missense mutations described here affect
a four residue motif in the C-terminal region that is not conserved between
nematodes and vertebrates at the primary sequence level, but is conserved
between C. elegans and the closely related Caenorhabditis
briggsae (Fig. 5).
Interestingly, three conserved amino acids within the wee-1.3(gf)
domain include the residues affected by each of the six spe-37(gf)
mutations.
Suppressors of the wee-1.3(gf) mutant phenotype provide some insight into the role of the Myt1 transmembrane domain. The wee-1.3(q89 eb87) suppressor double mutation encodes a WEE-1.3 protein that lacks the transmembrane domain but maintains the dominant missense mutation caused by wee-1.3(q89). The resulting suppression observed in wee-1.3(q89 eb87)/+ heterozygotes restore wild-type self-fertility (Table 1). Homozygous wee-1.3(q89 eb87) mutants survive embryogenesis and grow into self-sterile Spe adults that can produce outcross progeny after mating to wild-type males. This indicates that transmembrane localization of WEE-1.3 is required for the dominant Spe phenotype, but not required for the somatic and oocyte functions performed by this kinase. The self-sterile phenotype exhibited by wee-1.3(q89 eb87) homozygotes could indicate that membrane localization is required for WEE-1.3 to function during spermatogenesis. Alternatively, perhaps the wee-1.3(q89) dominant mutation can still affect spermatogenesis in homozygous wee-1.3(q89 eb87) suppressor mutants, but only when it does not have to compete with wild-type WEE-1.3.
Mechanism of wee-1.3(gf) dominance
The six wee-1.3(gf) mutations described in this paper specifically
affect spermatogenesis and do not require a wild-type copy of wee-1.3
to have their effect. wee-1.3(gf)/Df animals only produce the
dominant mutant form of WEE-1.3 and exhibit the same phenotype as
wee-1.3(gf)/+ animals. As the wee-1.3(gf) mutant phenotype
is not different when wee-1.3 gene dose is reduced, the dominance is
probably neomorphic/gain of function (gene activity in an inappropriate time
or place) in nature. Furthermore, these data indicate that WEE-1.3(gf) can
substitute for wild-type during embryonic and oocyte development, but not
during spermatogenesis. The WEE-1.3 protein appears to be negatively regulated
during spermatogenesis because strong hypomorphic suppressor double mutants,
like Class 2 wee-1.3(q89 eb60), mimic negative regulation and restore
spermatogenesis in both heterozygous and homozygous mutant animals. The
wee-1.3(gf) phenotype is tissue specific, and perhaps there is a
negative regulator expressed only during spermatogenesis that specifically
regulates male meiosis.
Gamete-specific cell cycle regulation has been observed in other organisms
and two cases are especially relevant to our study. The Drosophila
twine(lf) mutant fails to complete the G2/M transition during
spermatogenesis but still differentiates, which is similar to spermatogenesis
in wee-1.3(gf) mutants (Alphey et
al., 1992). Twine encodes a Cdc25p phosphatase, so
cdc25(lf) and wee-1.3(gf) mutants would both shift
cyclinB1/Cdc2p phosphorylation towards the inhibitory state. Deletion of the
mouse Cdc25b gene has no somatic effects but results in female sterility
because oocytes cannot exit meiosis I prophase arrest
(Lincoln et al., 2002
). These
data suggest that metazoan regulation of the gamete cell cycle is
fundamentally different from regulation of the somatic cell cycle, and that
studies of WEE-1.3 function in C. elegans are likely to be applicable
to Myt1 function in higher vertebrates.
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ACKNOWLEDGMENTS |
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