1 Department of Biological Sciences, University of Alberta, Edmonton, AB T6G
2E9, Canada
2 Program in Developmental Biology, Hospital for Sick Children, 555 University
Avenue, Toronto, ON M5G 1X8, Canada
* Author for correspondence (e-mail: shelagh.campbell{at}ualberta.ca)
Accepted 27 June 2005
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SUMMARY |
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Key words: Myt1, Wee1, Cdk1, Cdc25, Meiosis, Mitosis, Spermatogenesis, Oogenesis
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Introduction |
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Two related classes of Cdk1 inhibitory kinases have been identified in
metazoans: Wee1 and Myt1. Xenopus and C. elegans each
contain two Wee1-like kinases and a single Myt1 ortholog, which exhibit
distinct expression patterns during development
(Lamitina and L'Hernault,
2002; Leise and Mueller,
2002
; Murakami et al.,
2004
; Nakanishi et al.,
2000
; Okamoto et al.,
2002
; Wilson et al.,
1999
). The situation is somewhat simpler in Drosophila,
which has a single Cdk1 inhibitory kinase of each type: Wee1 and Myt1
(Adams et al., 2000
;
Campbell et al., 1995
;
Price et al., 2000
).
Drosophila Wee1 is a nuclear kinase that is essential for regulating
Cdk1 during the rapid, maternally controlled S/M nuclear divisions of early
embryogenesis; however, Wee1 is otherwise dispensable for zygotic development
(Campbell et al., 1995
;
Price et al., 2000
;
Stumpff et al., 2004
). Myt1
localizes to Golgi and endoplasmic reticulum membranes (Z.J., unpublished), as
also reported for the Xenopus and human Myt1 orthologs
(Booher et al., 1997
;
Liu et al., 1997
;
Mueller et al., 1995
). The
Myt1 kinases were originally characterized as membrane-associated
dual-specificity Cdk1 kinases that phosphorylate a threonine (T14) residue of
Cdk1, as well as the Y15 site that nuclear Wee1 kinases also target
(Booher et al., 1997
;
Liu et al., 1997
;
Mueller et al., 1995
). These
differences in Wee1 and Myt1 protein localization and target site specificity
suggest that the metazoan Cdk1 inhibitory kinases have evolved distinct cell
cycle regulatory functions required at different stages of development.
Transgenic overexpression and RNAi experiments involving Myt1 suggested
that its expression primarily affects the G2 phase of the cell cycle; however,
genetic evidence of specific functions for dMyt1 that are essential for normal
development has been lacking (Cornwell et
al., 2002; Price et al.,
2002
). Previously, biochemical studies of the prolonged `G2-like'
growth state of immature oocytes suggest that Myt1 is responsible for
inhibitory phosphorylation of Cdk1 during this stage of female meiosis in
Xenopus (Furuno et al.,
2003
; Karaiskou et al.,
2004
; Nakajo et al.,
2000
; Palmer et al.,
1998
; Peter et al.,
2002
) and in the starfish A. pectinifera
(Okano-Uchida et al., 2003
;
Okumura et al., 2002
).
Mutations affecting a C. elegans homolog have also implicated Myt1 in
the regulation of male meiosis (Lamitina
and L'Hernault, 2002
). In the present report, we describe the
isolation and characterization of mutations affecting Drosophila
Myt1. Our studies reveal that Myt1 serves regulatory functions that have not
previously been described but are important for both mitotic and meiotic cell
cycles during gametogenesis. These observations implicate Myt1 in specific
Cdk1 regulatory mechanisms that are required for coordinating cell cycle
behavior with crucial developmental transitions.
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Materials and methods |
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Molecular analysis of the Myt1 mutant alleles
To sequence genomic DNA, single flies of the appropriate genotype were
homogenized in a tube with 50 µl buffer (100 mM Tris-Cl pH 7.6, 100 mM
EDTA, 100 mM NaCl and 0.5% SDS). Proteinase K was added to a final
concentration of 100 µg/ml, and the samples were incubated at 37°C for
30 minutes. Genomic DNA (1 µl) isolated by this procedure was then used as
a template for DNA sequencing (Amersham), using oligonucleotide primers
positioned at 300-400 bp intervals on both template strands. The sequencing
reactions were repeated three times with genomic DNA isolated from different
flies of the same genotype, for confirmation.
Phenotypic analysis of the Myt1 mutants and hs-Cdk1AF expression experiments
Except as indicated, we used myt11/Df(3L)64D-F
hemizygotes as the representative myt1 mutants for our phenotypic
analysis and their myt11/TM3,Sb heterozygous siblings, as
controls. For testes preparations, 1- or 2-day-old male flies were dissected
in 1x PBS or in testis isolation buffer (TIB): 183 mM KCl, 47 mM NaCl,
10 mM Tris (pH 6.8), 1 mM EDTA (Casal et
al., 1990) plus 1 mM PMSF, then rinsed twice with the same buffer.
We followed established protocols for the testes and ovariole
immunofluorescent localization experiments
(Bonaccorsi et al., 2000
;
Mattheis et al., 2000
). The
primary antibodies and concentrations used were rabbit anti-PH3 (1/2000;
Upstate), rabbit anti-anillin at 1/300
(Field and Alberts, 1995
),
mouse anti-ß-tubulin (1/100; Sigma), mouse anti-BrdU (1/20; Jackson
Labs), mouse anti-Hts, clone C17.9C6, obtained from the Developmental Studies
Hybridoma Bank (DSHB) at 1/10 (Zaccai and
Lipshitz, 1996
), rat anti-BamC at 1/2000
(McKearin and Ohlstein, 1995
),
rabbit anti-Aly at 1/2000 (White-Cooper et
al., 2000
), rabbit (used at 1/500) and rat (used at 1/1000)
anti-Vasa (Lasko and Ashburner,
1990
), mouse anti-spectrin obtained from DSHB at 1/200
(Dubreuil et al., 1989
), mouse
anti-Eya obtained from DSHB at 1/100
(Bonini et al., 1993
), rabbit
anti-Cnn at 1/500 (Heuer et al.,
1995
), mouse anti-Mpm2 (1/200, Cell Signaling), and mouse
anti-Fas3 obtained from DSHB at 1/5 (Patel
et al., 1987
). Alexa-488 and Alexa-568 conjugated secondary
antibodies (used at 1/1000) were obtained from Molecular Probes, as was
rhodamine-conjugated phalloidin. BrdU incorporation was assayed in 1- to
2-day-old dissected testes, using published protocols
(Wolff, 2000
). For experiments
to determine the number of cells per cyst, w1118 or
myt1 mutant testes were dissected in TIB containing 8.3 µg/ml
Hoechst 33342. Whole cysts were teased out with a bent tungsten needle and
imaged using a Zeiss Axioplan 2E epifluorescence microscope, equipped with a
black and white Axiocam CCD camera. For the Cdk1-AF experiments, 1-
to 2-day-old y w; hs-Cdk1AF transgenic flies and y
w controls received 1 hour, 37°C heat shocks twice a day for three
consecutive days, prior to dissection.
Genetic tests for segregation defects during female meiosis
As female Myt1 mutants are partially fertile, a genetic cross (cross #1)
was set up between myt11/myt12 virgins (with
myt11/TM3, Sb virgins used in a control cross) and C
(1;Y)1, y, v, f, B; C(4) RM, ci, ey [R] males to detect non-disjunction
(NDJ) events during meiosis. myt11 and
myt12 were two independently isolated mutant chromosomes
with different secondary lethal mutations (allowing us to recover the viable
myt1 mutants). In this cross, non-disjunction of homologous
chromosomes was recognized by scoring for exceptional progeny: X chromosome
NDJ was represented by females with normal eyes or y,v,f, B males,
whereas 4th chromosome NDJ was scored as adults displaying the recessive
ci, ey[R] phenotype. The formula for the calculation of NDJ frequency
is 2x exceptional progeny/(2x exceptional progeny + regular
progeny).
To determine at which meiotic division(s) the X chromosome NDJ occurred in myt1 mutant females, FM7, y, B/y; myt11/myt12 females were crossed to y+ males (cross 2). NDJ at meiosis I would produce FM7,y, B/y or nullo-X eggs, whereas NDJ at meiosis II would result in either FM7,y,B/FM7,y,B, y/y or nullo-X eggs. X-chromosome NDJ events resulting in viable progeny were scored as adult females with Bar eyes and yellow bodies (from FM7,y, B/y eggs), or double-Bar eye (two copies of the B allele), yellow females (from FM7,y,B/FM7,y,B eggs) or females with normal eyes and yellow bodies (from y/y eggs). The first class is meiosis I NDJ-specific, whereas the latter two classes are meiosis II NDJ-specific progeny.
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Results |
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We isolated two myt1 mutant alleles (originally designated as
myt11 and myt12) in a genetic screen
for hemizygous mutants with phenotypic defects that could be rescued by a
P{myt1+} transgene
(Fig. 1B, Materials and
methods). These alleles exhibited markedly different viability as hemizygotes;
however, these differences were removed by out-crossing, indicating they were
due to secondary lesions. Viable hemizygous myt1 mutants
[myt/Df(3L)64D-F] exhibit bristle defects affecting the dorsal
thorax, head and eye (not shown), and are male sterile. Although myt1
females are fertile, we observed variable maternal effect lethal embryonic
phenotypes in their progeny (data not shown). Genomic sequencing of the
myt1 alleles identified identical mutations in each: a single
nucleotide deletion at position 514 (amino acid 173). The fact that EMS
mutagenesis usually causes CGTA transitions, combined with the
unlikelihood that this mutation would have occurred twice independently,
suggests that a spontaneous mutation occurred in the previously isogenized
stock we used for our screen. The myt1 mutation is predicted to cause
a frame-shift alteration in the sequence of the protein, followed by a
premature stop codon at nucleotide 689 (amino acid 232). This would truncate
the protein within the kinase domain and also delete other conserved sequence
motifs near the C terminus of the protein
(Fig. 1C), suggesting that the
mutants are likely functionally null. Moreover, myt1/Df(3L)64D-F
hemizygotes display identical phenotypes as transheterozygous combinations of
the original alleles, fulfilling classical genetic criteria that these
myt1 alleles are functionally amorphic
(Muller, 1932
).
Loss of Myt1 causes mitotic proliferation defects during spermatogenesis
The male sterility of myt1 mutants led us to look for specific
cell cycle defects during spermatogenesis, Male germline development begins
with stem cell divisions that generate gonialblasts, which then undergo four
synchronous mitotic divisions to produce cysts of 16 primary spermatocytes
(Fuller, 1993). These primary
spermatocytes remain in G2 phase for
90 hours before undergoing meiotic
divisions to produce cysts containing 64 syncytial spermatids that
differentiate into mature sperm. To analyze how loss of Myt1 function affects
these cell divisions, we used an antibody that recognizes a phosphorylated
form of histone H3 (PH3) as a marker for mitotic or meiotic cells
(Kiger et al., 2000
). In
control testes, small numbers of mitotic cells were usually seen near the tip
of the testis (Fig. 2A, white
arrow). More distally along a control testis, one often observes a single
PH3-positive meiotic cyst (Fig.
2A, blue arrow). We observed a striking increase in the numbers of
PH3-positive cells in myt1 mutants
(Fig. 2B). In addition to
clearly demarcated germline cysts, we also observed isolated PH3-positive
cells along the length of myt1 mutant testes
(Fig. 2B, green arrows), as
well as PH3-positive cells at the distal end of the testes (yellow arrow) that
were never seen in controls (Fig.
2A). These cell proliferation defects were suppressed and male
fertility was restored when a P{myt1+} transgene was
introduced into the myt1 mutant background, confirming that these
mutant phenotypes were due to a loss of Myt1 activity. The adult bristle
phenotype observed in myt1 mutants was also rescued by this transgene
(not shown).
|
|
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To test this possibility, we performed a BamC and PH3 colocalization
experiment. In controls (Fig.
4A), we never observed cysts containing more than eight cells that
were both BamC and PH3 positive, consistent with spermatogonia only undergoing
four mitotic divisions. By contrast, 30% of the myt1 mutant
testes examined (n=20) contained at least one 16-cell cyst that was
both BamC and PH3-positive (arrow, Fig.
4B), implying that these spermatogonia were undergoing an extra
round of cell division. To further test this idea, we examined germ cell cysts
by phase contrast microscopy and quantified the numbers of cells in each cyst.
As expected,
10% of the mutant cysts contain twice the expected numbers
of primary spermatocytes or spermatids, a phenotype that was never seen in
controls (Table 1). Examples of
a myt1 mutant 32-cell cyst of primary spermatocytes
(Fig. 4D,D'), 64-cell
spermatid cysts (Fig.
4F,F') and 128-cell spermatid cysts
(Fig. 4G,G') are shown,
along with corresponding 16 cell and 64 cell controls
(Fig. 4C,E). Moreover, in both
64-cell and 128-cell myt1 mutant spermatid cysts, there was
consistent evidence of variable, aberrant-looking nuclei and nebenkern
(Fig. 4F,G), which were never
seen in controls (Fig. 4E).
These observations suggest that loss of Myt1 activity affects segregation of
chromosomes and mitochondria during meiosis, in addition to the mitotic
defects described earlier.
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Each of the four major types of follicle cells can be distinguished by their cell shape and by expression of distinct molecular markers. Stalk cells have a unique disc-like shape and inter-egg chamber location (Fig. 8A, arrow); polar cells are located at the end of each egg chamber and express Fas3 before stage 9 (Fig. 8C, green); border cells maintain Fas3 expression and migrate towards the posterior after stage 9 (Fig. 8G); and stretched cells extend over the 15 nurse cells and express Eya (Fig. 8H). By these criteria, the different follicle cell types all appeared to be represented in myt1 mutants; however, unlike the controls, some of these cells were PH3 positive, suggesting that they were undergoing ectopic cell divisions (Fig. 8D-F, arrows Fig. 8J,K). Consistent with this interpretation, there were more Eya-expressing cells in the mutants than in controls by stage 9 (compare Fig. 8H,K), indicating that some of these cells were able to complete cell division. The typical `stretched' morphology characteristic of this cell type was disrupted, presumably as a result of cytoskeleton reorganization accompanying mitosis (Fig. 8H,K, arrows). We also observed ectopic PH3-positive main body follicle cells in mutant egg chambers after stage 9, long after these cells normally cease dividing (Fig. 8I,L). Thus, loss of Myt1 function causes germline-associated somatic cells to undergo ectopic cell division, in both males and females.
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Discussion |
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Overexpression studies in several systems suggest that Myt1 can influence
the timing of G2/M transitions (Cornwell et
al., 2002; Price et al.,
2002
; Wells et al.,
1999
). Loss-of-function studies now demonstrate that Myt1 serves
distinct Cdk1 regulatory functions that are essential for normal gametogenesis
and for adult bristle development (not shown). Consistent with previous
studies in other organisms that implicated Myt1 in female meiosis
(Kalous et al., 2005
;
Karaiskou et al., 2004
;
Okano-Uchida et al., 2003
;
Okumura et al., 2002
;
Palmer et al., 1998
;
Peter et al., 2002
), we
observed a marked elevation of meiotic chromosome segregation defects in the
progeny of female myt1 mutants. Female myt1 mutants are
fertile in spite of these segregation defects; however, many of their progeny
undergo variable embryonic lethality (not shown). In male myt1
mutants, loss of Myt1 function results in complete sterility. This phenotype
appears to be due to defects during both meiosis and spermatid differentiation
in male myt1 mutants. Normally, Drosophila spermatocytes
undergo a prolonged G2 phase arrest, which allows time to synthesize cellular
components required for subsequent development, before the onset of meiosis I
cell division. Oocytes do not undergo a similar growth phase, owing to
specialization of the germline nurse cells, which synthesize the mRNAs and
proteins required for egg development. Thus, unlike spermatocytes,
Drosophila oocytes almost immediately progress into prophase of
meiosis I after completing the four mitotic divisions that produce the 16-cell
cyst (Spradling, 1993
). These
differences in male and female germline development may explain the
differences in requirements for Myt1 activity that we observe. Moreover, our
data suggest that Myt1 has evolved specific functions that are important for
developmentally regulated growth phases. This hypothesis is also consistent
with the requirement for Myt1 in Xenopus and A. pectinifera
during the prolonged `G2-like' prophase arrest of early oocytes, which also
involves extensive cell growth and synthesis of proteins and mRNAs required
for subsequent embryonic development
(Furuno et al., 2003
;
Karaiskou et al., 2004
;
Nakajo et al., 2000
;
Okano-Uchida et al., 2003
;
Okumura et al., 2002
;
Palmer et al., 1998
;
Peter et al., 2002
).
In addition to confirming that Myt1 serves a conserved role in regulating meiosis, our studies of myt1 mutants also provide evidence that Myt1 serves novel functions that affect mitotic cell proliferation during both male and female gametogenesis. Mitotic index measurements of female germline cells suggest that loss of Myt1 activity influences the timing of the mitotic cell cycles that precede meiosis, although live analysis will be needed to verify this conclusion. Delays in mitosis may also contribute to this phenotype, as the observed increase in numbers of germline cysts in myt1 mutant germaria was not directly proportional to the increased mitotic index seen in germline stem cells.
A further unexpected mitotic defect associated with loss of Myt1 activity
involved ectopic divisions of germline-associated somatic cells, seen in both
males and females. As germline-associated somatic cells normally become
quiescent as they differentiate, this observation suggests a role for Myt1 in
a molecular mechanism that allows or facilitates exit from the cell cycle. An
additional mitotic defect was identified in male myt1 mutants, which
was not seen in females. Approximately 10% of the cysts undergo an extra round
of mitotic cell division before cells differentiate into primary
spermatocytes, suggesting that Myt1 also affects the fidelity or timing of
this developmentally regulated cell fate decision. However, unlike previously
described male-sterile over-proliferation mutants with cell fate defects
(Gonczy et al., 1997;
Kiger et al., 2000
;
Matunis et al., 1997
;
Tulina and Matunis, 2001
), the
majority of the myt1 mutant cysts do not undergo such ectopic mitotic
divisions. Collectively, these data suggest that Myt1 serves distinct Cdk1
regulatory functions that coordinate cell cycle behavior with important
developmental transitions. Precisely how Myt1 accomplishes these diverse
functions is unknown; however, our results are consistent with the idea that
Myt1 specifically regulates Cdk1 activity in the cytoplasm
(Liu et al., 1997
;
Wells et al., 1999
).
Strikingly, RNAi depletion of Myt1 in cultured Drosophila cells
was previously reported to cause a marked disruption of the Golgi apparatus
(Cornwell et al., 2002). This
observation suggests an intriguing possibility. As the Golgi apparatus serves
a key role in trafficking and secretion of proteins in rapidly growing cells,
perhaps Myt1 regulation of Cdk1 activity might indirectly affect the
biosynthesis and assembly of subcellular structures required for crucial
developmental transitions affected in myt1 mutants.
According to current models describing regulation of the G2/M transition,
Myt1 and Wee1 inhibit Cdk1 in the cytoplasmic and nuclear compartments during
interphase, respectively, ensuring a complete block to mitotic progression.
Once cells are ready to divide the regulatory proteins that trigger mitosis
are thought to first activate Cdk1 at the centrosomes, initiating a
self-amplifying wave of Cdk1 activation that sweeps through the cytoplasm and
into the nucleus (Jackman et al.,
2003; Kramer et al.,
2004
). This mechanism ensures that early mitotic events are
coordinated throughout the cell. Loss of Myt1 activity would be expected to
disrupt this coordination by allowing premature activation of Cdk1 in the
cytoplasmic compartment, even if Wee1 can still protect the nucleus from
active Cdk1. We are now in an excellent position to test key assumptions of
this model in vivo, using wee1 and myt1 mutants to determine
how loss of these Cdk1 inhibitory kinases affects specific cell structures and
organelles, at different stages of the cell cycle.
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ACKNOWLEDGMENTS |
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