Download encore, a gene required for the regulation of germ line mitosis and

Development 122, 281-290 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
DEV5022
281
encore, a gene required for the regulation of germ line mitosis and oocyte
differentiation during Drosophila oogenesis
Nancy C. Hawkins, Julie Thorpe and Trudi Schüpbach
Department of Molecular Biology, Howard Hughes Medical Insititute, Princeton University, Princeton, New Jersey 08544, USA
SUMMARY
During Drosophila oogenesis, a stem cell daughter
undergoes precisely four rounds of mitosis to generate a
cyst of 16 cells interconnected by cytoplasmic bridges. One
of the cells becomes the oocyte while the remaining 15 cells
differentiate as nurse cells. We have identified a gene,
encore, that is involved both in regulating the number of
germline mitoses and in the process of oocyte differentation. Mutations in encore result in exactly one extra round
of mitosis in the germline. Genetic and molecular studies
indicate that this mitotic defect may be mediated through
the gene bag-of-marbles. The isolation and characterization
of mutiple alleles of encore revealed that they were all temperature sensitive for this phentoype. Mutations in encore
also affect the process of oocyte differentiation. Egg
chambers are produced in which the oocyte nucleus has
undergone endoreplication often resulting in the formation
of 16 nurse cells. We argue that these two phenotypes
produced by mutations in encore represent two independent requirements for encore during oogenesis.
INTRODUCTION
of the mitotic spindle is associated with the fusome in dividing
cystocytes. Two membrane cytoskeletal proteins have been
identified as components of the fusome, α-spectrin and the huli tai shao (hts) protein, an adducin like molecule (Lin et al.,
1994). Phenotypic analysis of hts mutations have provided
evidence that the fusome plays a key role in cyst formation.
Mutations in hts abolish the fusome resulting in egg chambers
containing only a few nurse cells and which rarely form an
oocyte (Yue and Spradling, 1992).
A number of other mutations have been isolated in genetic
screens for female sterility that affect the formation of the egg
chamber. Two genes have been identified that are necessary
for proper oocyte determination. Recessive mutations in
Bicaudal-D (Bic-D) and egalitarian (egl) result in the production of egg chambers with 16 nurse cells and no oocyte
(Schüpbach and Wieschaus, 1991; Suter et al., 1989). Two loci
have been described that affect the posterior positioning of the
oocyte within the egg chamber. Females mutant for spindle-C
(spn-C) or dicephalic (dic) produce bipolar egg chambers in
which the oocyte fails to move to the posterior pole of the egg
chamber, and instead, resides in a central location and is
flanked by nurse cells on either side (Gonzalez-Reyes and St
Johnston, 1994; Lohs-Schardin, 1982). Few good candidates
exist for genes directly involved in controlling stem cell or
cystocyte divisions. Female sterile mutations in the ovarian
tumor class of genes result in tumorous egg chambers containing hundreds of undifferentiated germ cells. For the majority
of these genes, it has been suggested that they might only indirectly affect germline mitosis by disrupting germline sex determination (Pauli and Mahowald, 1990; Steinmann-Zwicky,
1992). However, one ovarian tumor mutant, bag-of-marbles
Regulation of cell division is a crucial aspect in the development of multicellular organisms. At the tip of the Drosophila
ovary, in the germarium, an invariant set of four mitotic
divisions of a stem cell daughter leads to formation of the egg
chamber. The germarium contains the germline stem cells.
Each stem cell undergoes asymmetric cell division to produce
a self renewing stem cell and a cystoblast. The cystoblast then
undergoes four successive rounds of mitosis each followed by
incomplete cytokinesis to form a cyst of 16 cells interconnected by cytoplasmic bridges called ring canals. This
invariant pattern of cell division results in a cyst containing
two cells with four rings canals, two cells with three ring
canals, four cells with two ring canals and eight cells with a
single ring canal. One of the two cells with four ring canals
becomes the oocyte, and the remaining 15 cells differentiate as
nurse cells. The 16 cell cyst then becomes surrounded by a
layer of somatically derived follicle cells. Initially, the future
oocyte occupies a central location in the egg chamber, but
when the egg chamber exits the germarium the oocyte has
assumed a posterior position in the egg chamber. This is the
first visible sign of anterior-posterior asymmetry in the developing egg chamber (for review see King, 1970; Mahowald and
Kambysellis, 1980; Spradling, 1993).
The four mitotic division are accompanied by the growth of
a cytoplasmic structure called the fusome (Telfer, 1975; Storto
and King, 1989; Lin et al., 1994). The fusome extends through
the ring canals of dividing cysts forming a branched structure
connecting the germline cells. The fusome is postulated to
control the pattern of cystocyte interconnections since one pole
Key words: encore, oogenesis, mitosis, Drosophila, bag-of-marbles
282
N. C. Hawkins, J. Thorpe and T. Schüpbach
(bam), has been implicated in directly controlling cystocyte
divisions (McKearin and Spradling, 1990; McKearin and
Christerson, 1994). Unlike most of the ovarian tumor mutants,
bam affects both male and female gametogenesis. Females
homozygous mutant for bam produce egg chambers containing
from 50 to several hundred small, undifferentiated, cells. It has
been demonstrated that the bam germ cells are mitotically
active and it has been proposed that bam is required for the differentation of the cystoblast (McKearin and Ohlstein, 1995).
We have identified a locus called encore (enc) that is
required for the correct number of cystocyte divisions.
Mutations in enc result in exactly one extra round of mitosis
in the germline. We have demonstrated that this additional
mitosis requires bam activity. In addition, an analysis of enc
mutations has revealed a requirement for enc in the process of
oocyte differentiation.
MATERIALS AND METHODS
Cytological and genetic mapping
The encBB allele was isolated after mobilization of a P[w+-lac-Z]
element (Bier et al., 1989). The P-element insertion was localized by
in situ hybridization to polytene chromosomes using standard
methods (Ashburner, 1989). A single insert was mapped to 63E on
the left arm of the third chromosome (data not shown). Consistent
with the physical mapping data enc failed to complement deficiencies
that removed the region including Df(3L)A466 (63D1,2-64B1,2)
(Kulkarni et al., 1994), Df(3L)GN34 (63D,E-64B1,2) (J. Garbe,
personal communication) and Df(3L)HR119 (63C6-63E) (Wohlwill
and Jose Bonner, 1991). A meiotic map position of 3-5.3 was determined by mapping enc relative to ru (3-0) by recombination. The outcrossed encBBr53 line was obtained by backcrossing encBB five generations to a y w stock then reestablishing the stock. For all marker
mutations and balancers see Lindsley and Zimm (1992).
Genetic screens
The encBB P-element allele was mobilized using the ∆2-3 chromosome as a source of transposase activity (Robertson et al., 1988). The
excision chromosomes were tested for lethality and female sterility in
trans to Df(3L)A466. Out of a total of 245 excision chromosomes
scored, 211 had reverted to wild type, confirming that the insertion of
the P-element was responsible for the female sterility. A lethal
excision line was generated, encr75, that failed to complement encBB
for fertility and two EMS induced lethal mutations l63Ea and l63Ec
for lethality (Wohlwill and Jose Bonner, 1991; Harrison et al., 1995).
An EMS mutagenesis was designed to isolate both lethal and
female sterile alleles at enc. st e/st e males were fed 30-50 mM EMS
as described in Grigliatti (1986) and were mated to pipe st e/TM8,
DTS4 th st Sb e females. Single st e */TM8, DTS4 th st Sb e females
were mated to encr75/dsxD Sb e males. The crosses were raised for
three days at 25˚C then shifted to 29˚C to eliminate progeny carrying
TM8, a dominant temperature sensitive balancer chromosome. The F2
generation was then scored for the absence of the non ebony (st e
*/encr75) progeny indicating that a lethal mutation had been induced
on the mutagenized chromosome. For lines that were not lethal the
entire generation was transferred to an egg laying block. Only st e
*/encr75 females were capable of laying eggs since sibling females, st
e */dsxD, were sterile due to the presence to the dsxD chromosome.
Egg lay plates were then examined for absence of eggs, the production of abnormal eggs or morphologically normal eggs that failed to
hatch. Balanced stocks for the lethal and female sterile mutations were
generated by crossing st e */dsxD st e males to Df(3L)A466/TM3, Ser,
Sb females and isolating st e*/TM3, Ser, Sb progeny.
A total of 7600 chromosomes were scored for female sterility and
14 female sterile mutations were isolated. All the alleles were temperature sensitive for the production of egg chambers with extra nurse
cells (Table 1). In addition, they all produced eggs with fused dorsal
appendages in trans to the tester chromosome encr75. This ventralized
phenotype will be described elsewhere (N. Hawkins, C. VanBuskirk,
U. Grossniklaus and T. Schüpbach, unpublished data). All of the
female sterile alleles were backcrossed to a ru st e ca chromosome to
remove extraneous lethals and to verify that all the alleles were tightly
linked to ru. A total of 8200 chromosomes were scored for lethality
and 5 lethal mutations were isolated. Two of the lethal mutations,
l(3)L5 and l(3)L1 failed to complement l63Ec (Harrison et al., 1995),
while the other 3 lethal mutations, l(3)L8, l(3)L13 and l(3)L46 failed
to complement l63Ea (Wohlwill and Jose Bonner, 1991). All of these
lethal mutations complemented encBB.
Morphological analysis of egg chambers
Females were placed in vials containing yeast for 3-5 days in
uncrowded conditions. Ovaries were then dissected in 1× phosphatebuffered saline (PBS) then fixed for 20 minutes in 2.5% glutaraldehyde in 50 mM Pipes, pH 7.4. Following fixation they were rinsed
once in 1× PBS then incubated in PBS overnight at 37˚C. Ovaries
were teased apart with tungsten needles, mounted in 50% glycerol in
PBS and examined using differential interference contrast
microscopy.
Immunocytochemistry
For Hoechst staining, ovaries were dissected in 1× PBS, fixed for 15
minutes in 8% formaldehyde in PBS, rinsed with PBS then incubated
in 1 µg/ml Hoechst for 5 minutes. After washing with PBS, ovaries
were mounted in 50% glycerol in PBS. For staining of ring canals,
ovaries were dissected and fixed as described above then incubated in
the dark with 2 units of rhodamine-conjugated phalloidin (Molecular
Bioprobes) for 20 minutes followed by extensive washing with PBS.
Ovaries were then mounted in Aqua-polymount (Polysciences, Inc.)
and visualized by confocal microscopy (BioRad MRC 600).
To analyze tumorous ovaries a single ovary was placed on a subbed
slide in a drop of 8% formaldehdye in PBS and covered with a siliconized coverslip. Gentle pressure was applied to the coverslip to
disrupt the ovary. The ovaries were fixed for 15-20 minutes. The slide
was then transferred onto dry ice and the coverslip removed with a
razor blade. A drop of 0.1 mg/ml RNase in PBS was placed on the
sample and then overlayed with a coverslip and incubated overnight
Table 1. The EMS induced enc alleles are temperature
sensitive for the extra nurse cell phenotype
% mutant egg chambers†
Alleles of
enc/Df *
D6
DD7
KK7
L32
M7
N8
OO6
Q4
R1
T2
UU3
WW1
XX1
Z3
18˚C
25˚C
4
2
2
2
1
1
1
1
0
10
1
1
1
1
30
29
62
1
39
24
63
78
67
75
49
41
17
72
*Df=Df(3L)A466.
†A total of 200-400 egg chambers were scored for each genotype. The
majority of mutant egg chambers (>95%) contained extra nurse cells while a
small fraction of egg chambers (1-2%) were bipolar or had <15 nurse cells.
encore and germ line mitosis
at 4˚C. Slides were transferred to a slide jar and washed twice for 5
minutes in PBS. Actin was visualized by covering the tissue with 100
µl of rhodamine-conjugated phalloidin and covered with a siliconized
coverslip. The slides were incubated in the dark for 20 minutes then
washed twice for 10 minutes in PBS. To stain DNA, a drop of 0.5
µM YoPro (Molecular Probes) was placed on the sample and
incubated for 7 minutes. The slides were subsequently washed in PBS
and mounted in 50% glycerol in PBS.
Whole-mount in situ hybridization
Ovaries were dissected in Ringers and the ovariole teased apart. Then
tissue was fixed in 4% paraformadehyde in PBS+0.1% Tween, 10%
DMSO and 3 volumes of heptane for 20 minutes. All subsequent steps
were carried out according to Tautz and Pfeifle (1989). Digoxigeninlabelled antisense RNA probes for bam and oskar were synthesized
using the RNA genius kit according to manufacture’s instructions
(Boehringer Mannheim). Ovaries were mounted in Aqua-polymount
(Polysciences, Inc.).
RESULTS
encore mutants affect formation of the egg chamber
We have identified a new locus called encore (enc) that is
involved in female gametogenesis. The first allele of enc,
encBB, was isolated as a recessive female sterile mutation
during a P-element mutagenesis (E. Shaddix and T.
Schüpbach, unpublished). Females homozygous for the Pelement insertion were viable but exhibited defects during
oogenesis. To examine the phenotype more closely, ovaries
were dissected from females homozygous for encBB and
stained with Hoechst to visualize the nuclei. Wild-type egg
chambers consist of 15 nurse cells and an oocyte surrounded
by a layer of somatically derived follicle cells (Fig. 1A). In enc
ovaries, three classes of mutant egg chambers were observed.
The most abundant class consists of egg chambers that contain
twice the normal number of germline cells. In these egg
chambers, a single oocyte develops at the posterior pole and
the remaining 31 cells differentiate as nurse cells (Fig. 1B). A
second less frequent class consists of bipolar egg chambers
that, in addition to exhibiting an increase in the number of
germline cells, also have a defect in the correct positioning of
283
the oocyte. The oocyte develops in the center of the egg
chamber and is flanked by nurse cells on both sides (Fig. 1C).
This phenotype has been previously described for mutants in
spn-C and dic (Gonzalez-Reyes and St Johnston, 1994; LohsSchardin, 1982). However, unlike enc, the bipolar egg
chambers present in these mutants contain the correct number
of germline cells. Finally, a small class of egg chambers are
produced that contain less than 16 germline cells. These egg
chambers fail to differentiate an oocyte and thus have only
nurse cells (Fig. 1B,D). Frequently, egg chambers with a
reduced number of nurse cells are directly adjacent to an egg
chamber with extra nurse cells. The nuclei from these two egg
chambers are comparable in size and the total number of
germline cells from the two egg chambers adds up to 32. In
Fig. 1D one egg chamber contains 8 nurse cells while the
adjacent post vitellogenic egg chamber has 23 nurse cells and
an oocyte. This suggests that in the germarium when an egg
chamber containing twice the number of germ line cells
becomes surrounded by follicle cells a small number of nurse
cells may be pinched off from the larger egg chamber and
packaged independently. Thus, our morphological observations indicate that the primary defect in enc egg chambers is a
doubling in the number of germline cells.
encBB behaves as a temperature sensitive recessive
gain-of-function allele
The encBB allele exhibits complex genetic behavior. First, the
allele is temperature sensitive. At 18˚C, 59% of the egg
chambers derived from females homozygous for encBB have
15 nurse cells, while at 25˚C only 6% of the egg chambers
dissected from homozygous females contain the normal
number of nurse cells (Table 2). Second, encBB also behaves
as a gain-of-function allele. The phenotype significantly
weakens when encBB is placed in trans to a deficiency. The
number of egg chambers at 25˚C containing 15 nurse cells
increased to 34% in hemizygous females (Table 2). Similar
results were obtained using other deficiencies (data not shown).
To determine whether this gain-of-function phenotype was the
result of modifers in the genetic background, the original encBB
stock was outcrossed several generations. In the modified
genetic background, encBB still retained it’s gain-of-function
Fig. 1. The ovarian phenotype of
encBB/encBB at 25˚C. Ovaries were
stained with Hoechst to visualize nuclei.
(A) A wild-type ovariole containing a
series of egg chambers. Each egg
chamber contains 16 germline cells
surrounded by a layer of follicle cells
(fc). The large polyploid nurse cell
nuclei (ncn) are stained intensely while
the diploid oocyte nucleus is not visible.
The oocyte (o) is located at the posterior
pole of the egg chamber. (B-D) Egg
chambers derived from encBB
homozygous females. (B) Mutant
ovarioles containing egg chambers with
extra nurse cells and egg chambers with
too few nurse cells. (C) A bipolar egg
chamber containing extra nurse cells
with the oocyte located in the center of the egg chamber. (D) An egg chamber containing 23 nurse cells (arrowhead) and an oocyte followed by
an egg chamber with only 8 nurse cells (arrow). In all panels anterior is oriented to the left.
N. C. Hawkins, J. Thorpe and T. Schüpbach
*At a low frequency (<0.5%) of the extra nurse cell egg chambers have
reverse polarity; the oocyte is located at the anterior end of the egg chamber.
†The majority of bipolar egg chambers (>99%) also have extra nurse cells.
character. However, the production of the bipolar and reduced
nurse cells classes were influenced by genetic background
since the frequency of these classes dropped to less than 2%
(data not shown).
Screen for new enc alleles
Given the unusual properties of the encBB allele, it was unclear
whether or not the defects associated with encBB represent the
loss-of-function phenotype at the enc locus. To address this
concern, we performed an EMS mutagenesis to generate point
mutations in the enc locus (see Materials and Methods). Our
goal was to determine whether the loss of enc function would
lead to female sterility or zygotic lethality, and if any other
phenotypes were associated with mutations at the enc locus. In
this screen, 14 female sterile alleles were isolated. All these
alleles failed to complement the original P-element insertion
allele encBB and therefore were new enc alleles. No lethal
alleles of enc were isolated.
Analysis of the ovarian phenotype of the EMS induced
alleles revealed that they all exhibited the same egg chamber
defects seen in encBB. Like the encBB allele, the primary defect
observed in all the alleles was a doubling in nurse cell number.
However, in contrast to the original encBB allele, the bipolar
and reduced nurse cell classes occurred at a very low
frequency, generally making up <1% of the total mutant egg
chambers. The frequency of these two classes was comparable
to an outcrossed encBB line, lending support to the idea that
these phenotypes are sensitive to genetic background. None of
the alleles produced any novel phenotypes.
A comparison of the ovarian phenotype at 18˚C and 25˚C
revealed, that like encBB, the EMS induced alleles were also
temperature sensitive, with the exception of encL32 (Table 1).
At 18˚C, mutant ovaries were composed primarily of egg
chambers with 15 nurse cells. On average less than 2% of the
egg chambers contained a doubling in nurse cell number
(except encT2/Df whose ovaries contain 10% mutant egg
chambers). However, at 25˚C, the non-permissive temperature,
the strength of the phenotype increases significantly, with
many of the alleles (e.g. encKK7, encR1) producing up to 75%
mutant egg chambers.
To determine the genetic nature of the EMS induced alleles
we compared the homozygous phenotype to the phenotype
when the alleles are in trans to a deficiency (Fig. 2). The alleles
that produced the strongest phenotype when homozygous,
encR1, encKK7, encUU3 and encDD7 all appear to behave as gainof-function alleles like the original encBB allele. In particular,
25.0
XX1
UU3
0.0
WW1
59
6
99
34
T2
3
17
1
5
R1
6
21
0
12
Q4
33
55
0
48
N8
18
25
18
25
50.0
M7
encBB/encBB
encBB/encBB
encBB/Df(3L)A466
encBB/Df(3L)A466
bipolar†
=15
nurse
cells
L32
˚C
>15
nurse
cells
enc/Df
75.0
KK7
Genotype
>15
nurse
cells*
enc/enc
DD7
% egg chambers
100.0
DDD6
Table 2. The encBB P-element insertion is a recessive
temperature-sensitive gain-of-function allele
% mutant egg chambers
284
enc alleles
Fig. 2. A comparison of the percentage of mutant egg chambers
derived from homozygous vs. hemizygous females at 25˚C. To
obtain homozgous viable stocks it was necessary to remove
extraneous lethals by recombination. Since we noticed variability in
the phenotype due to genetic background, the recombinant
chromsome for each allele was used in both the hemizygous and
homozygous combinations. A total of 200-400 egg chambers were
scored for each genotype.
although encR1 and encKK7 display only modest improvement in
phenotype in trans to a deficiency at 25˚C, there is a vast difference between the homozygous and hemizygous phenotype
at 18˚C. They are the only two alleles that result in a penetrant
extra nurse cell phenotype when homozygous at 18˚C; encR1
produces 99% mutant egg chambers while encKK7 produces
88% mutant egg chambers. In trans to a deficiency at 18˚C, both
alleles produce less than 1% mutant egg chambers (Table 1). A
large number of the alleles when homozygous produced a
weaker phenotype in which only 10-25% of the egg chambers
contained extra nurse cells. For the majority of these alleles, the
phenotype is similar or slightly stronger in trans to the deficiency suggesting that many of these weaker alleles are potentially loss-of-function alleles. However, because of the
influence of genetic background and the variability among individuals of the same genotype we cannot conclude that any of
the alleles are amorphic. A molecular characterization will be
necessary to resolve this issue.
In conclusion, we have isolated a collection of both loss-offunction and gain-of-function alleles of enc. The gain-offunction alleles produce the same phenotype as the loss-offunction alleles, suggesting that the gain-of-function alleles
behave as antimorphs and interfere with the wild-type function
of the gene. There is no indication of a zygotic requirement for
enc since no lethal enc alleles were isolated and the female
sterile alleles do not show reduced viability.
Extra round of mitosis
There are two models that can explain the doubling in nurse
cell number seen in enc egg chambers. In the first model,
instead of the follicle cells surrounding one 16 cell cyst they
would incorrectly package two 16 cell cysts together, thus
producing a 32 cell egg chamber. This would result in an egg
chamber containing four cells with four ring canals, and since
the two 16 cell cysts are independent, two oocytes should
develop. A morphological analysis of the enc phenotype
suggests that this explanation is unlikely since the doubling in
nurse cell number that occurs in enc is not accompanied by the
appearance of a second oocyte. Alternatively, a doubling in the
nurse cell number could be achieved by one extra round of
encore and germ line mitosis
285
Fig. 3. Confocal images of egg chambers derived
from wild-type and enc females at 25˚C stained with
rhodamine-conjugated phallodin to visualize ring
canals. (A) A wild-type egg chamber with four ring
canals attached to the oocyte. (B) An extra nurse
cell egg chamber derived from encBB/encR1 with the
oocyte located at the posterior pole. There is an
increase in the total number of ring canals within
the egg chamber. (C) A higher magnification view
of the oocyte in B which has five ring canals (arrow
indicates the fifth ring canal which is orientated on
its side). (D) A bipolar egg chamber from
encBB/encBB in which the oocyte, developing in the
center of the egg chamber (arrow), possesses 5 ring
canals. Scale bar, (A,B) 50 µm, (C) 20 µm,
(D) 70 µm.
mitosis in the germline. Instead of the normal four rounds of
mitosis to produce 16 cells, a fifth round of mitosis would
generate 32 cells. This second model predicts the existence of
two cells with five ring canals.
To distinguish between these two models, egg chambers
were stained with rhodamine-conjugated phalloidin to
visualize the actin-rich ring canals. In wild-type egg chambers,
we always observed four ring canals attached to the oocyte
(Fig. 3A). When enc egg chambers with extra nurse cells were
examined the oocyte invariably possessed five ring canals. In
enc egg chambers in which the oocyte is correctly positioned
at the posterior pole there is an increase in the total number of
ring canals accompanying the increase in nurse cell number
(Fig. 3A). Moreover, five ring canals are clearly visible
attached to the oocyte (Fig. 3B,C). In bipolar egg chambers
containing extra nurse cells the oocyte which develops in the
center of the egg chamber also has five ring canals (Fig. 3D).
The presence of cells with five rings canals clearly demonstrates that the doubling in nurse cell number is due to one extra
round of mitosis in the germline. Since we never observe a cell
with more than five ring canals, the number of extra divisions
is limited to one, consistent with the observation that the total
number of germline cells never exceeds 32. As in wild type,
one of the two cells with the maximum number of ring canals
develops as an oocyte and the remaining cells differentiate as
nurse cells. Therefore, the ‘correct’ cell is still determined as
an oocyte. In the bipolar egg chambers, the oocyte also has five
ring canals. Therefore, the mispositioning of the oocyte is not
a result of the wrong cell being determined as the oocyte.
Instead, it represents a failure of the oocyte to assume its
correct position in the egg chamber.
bam expression is expanded in enc mutants
To futher investigate the mitotic defect, we took advantage of
one of the few available molecular markers that is differentially expressed in dividing cysts, bam. In wild-type ovaries,
bam is expressed in the cystoblast and 2 cell cysts. This is seen
as a narrow band of staining at the tip of the germarium
immediately posterior to the stem cells (Fig. 4A) (McKearin
and Spradling, 1990). In enc ovaries, the bam-expressing
region was significantly expanded to approximately double the
width (Fig. 4B). Since the pattern of bam expression is
affected, enc has a role early in cyst development. In addition,
we can conclude that enc is a negative regulator of bam in these
additional bam-expressing cells.
bam acts as a dominant suppressor of enc
We constructed double mutants between bam and enc to place
enc action relative to bam. Mutations in bam result in the production of tumorous egg chambers containing small, mitotically active cells. Previous studies have revealed that bam
mutant germ cells are found as single cells or pairs of cells
Fig. 4. Expression of bam transcript in wild-type and mutant
germaria detected by whole-mount in situ hybridization. (A) A wildtype germarium. (B) Germarium derived from encQ4/Df(3L)A466
female. An expansion of bam expression was also observed in other
strong enc allele combinations. Ovaries were photographed at the
same magnification.
286
N. C. Hawkins, J. Thorpe and T. Schüpbach
Fig. 5. The phenotype of bam and bam enc mutants is
identical. (A,B) Egg chambers photographed under
phase contrast optics. (A) A tumorous bam∆86/bam∆86
egg chamber. (B) A tumorous encR1 bam∆86/encR1
bam∆86 egg chamber. (C-E) Confocal images of
mutant germ cells labelled with rhodamineconjugated phalloidin to visulize actin (red) and
YoPro to stain DNA (green). Both bam∆86/bam∆86 (C)
and encR1 bam∆86/encR1 bam∆86 (D) cells possess a
single actin rich structure associated with the cell
membrane (arrow). (E) A pair of encR1 bam∆86/encR1
bam∆86 cells connected by an actin rich structure
(arrow). Scale bar, (A,B) 10 µm, (C-E) 2.5 µm.
necessary for the extra round of mitosis and that the double
mutant phenotype is revealing an epistatic relationship
between the two genes.
This possibility was strongly supported by the observation
that a null allele of bam behaves as a dominant suppressor of
enc. At 25˚C, 79% of the egg chambers from encR1/Df and 66%
of the egg chambers from encBB/Df females exhibit a doubling
in the nurse cell number. When the dosage of bam is reduced
by one copy using a null allele of bam, the phenotype is greatly
suppressed. Only 15% of the encR1 bam/Df egg chambers and
5% of the encBBbam/Df egg chambers have undergone an extra
division (Fig. 6). This suppression is alleviated by reintroducing a wild-type copy of bam as a trans gene, thus conclusively
demonstrating that the suppression is due to bam and not to a
closely linked suppressor (data not shown). Since a bam
mutation dominantly suppresses the mitotic defect of enc, this
result suggests that the misexpression of bam RNA that is
observed in enc might be a cause rather than a consequence of
the extra division.
Requirement for enc in maintenance of oocyte
identity
Mutations in enc produce a second defect during oogenesis that
100.0
enc/Df
Fig. 6. A comparison
of the mutant
enc bam/Df
phenotype of enc in
females that are either
75.0
wild type for bam
(hatched) or carry one
mutant copy of the bam
50.0
gene. The graph
illustrates supression of
two strong enc
genotypes,
25.0
encR1/Df(3L)A466 and
encBB/Df(3L)A466, by
a null allele of bam,
0.0
bam∆86. The deficiency
encR1
encBB
uncovers enc but
complements bam. For this experiment more than 300 egg chambers
per genotype were scored.
% mutant egg chambers
connected by a single ring canal, possibly arrested at the cystoblast or two cell cyst stage of development (McKearin and
Christerson, 1994). The expansion of bam expression in enc
indicates that there might be a requirement for enc early in the
normal division program. Thus, we reasoned that if the encinduced extra division occurred before the arrest point for bam
mutant cells, enc bam cysts might contain more than the 1 or
2 cells observed in the bam single mutant.
The double mutant phenotype was indistinguishable from the
bam mutant phenotype. Ovaries derived from the enc-bam
double mutant contain tumorous egg chambers that appear to
be identical to the bam mutant alone (Fig. 5A,B). To determine
if the germ cells had progressed past the 1 or 2 cell cyst stage
in the double mutant, egg chambers were stained with
rhodamine-conjugated phalloidin to visualize ring canals. In
ovaries derived from both bam and the enc-bam double mutant,
each germ cell has a single focus of phalloidin staining associated with the cell membrane (Fig. 5C,D). In addition, a small
fraction of cells from both genotypes appear to be connected by
an actin-rich structure (Fig. 5E). This could potentially be a
immature ring canal suggesting that the mutant germ cells are
arrested at the two cell cyst stage. However, the majority of cells
are seen as single cells and not pairs of cells. McKearin and
Ohlstein (1995) have recently proposed that the division of bam
germ cells might be complete but that the division is accompanied by the transient formation of a ring canal. Therefore, the
focus of phallodin staining might be a remnant of this ring
canal. Alternatively, this structure might be associated with the
spectrosome. However, the spectrosome has not been shown to
contain actin and it is a cytoplasmic structure (Lin et al., 1994).
Regardless of the identity of this single actin rich structure, the
phenotype of the double mutant is identical to the bam single
mutant phenotype at this enhanced level of resolution.
There are two interpretations for this result. It is possible that
the extra division produced by enc mutations occurs after bam
arrests cyst development. Therefore, in a bam mutant development does not proceed far enough to see the effect of enc.
A more exciting interpretation is that enc mutations require
bam activity to produce an extra round of mitosis. In light of
the expansion of bam transcription in an enc mutant, it is
tempting to speculate that the misexpression of bam is
encore and germ line mitosis
indicates a role for enc in the process of oocyte determination.
In wild-type egg chambers, the nurse cell nuclei become highly
polyploid and have a large roughened appearance while the
diploid oocyte nucleus at the posterior pole of the egg chamber
remains small and smooth (Fig. 7A). Many of the enc alleles
produce a large fraction of egg chambers in which the oocyte
nucleus has an abnormal morphology. There is a range in the
severity of this phenotype. Frequently, the oocyte nucleus
appears to be moderately increased in size and no longer
smooth in appearance (Fig. 7B). Occasionally, an ‘oocyte’
nucleus is seen that is almost identical in size and morphology
to the nurse cell nuclei (Fig. 7C). Although the nucleus of the
oocyte closely resembles a nurse cell nucleus the cell retains
many features of an oocyte. It grows in size, accumulates yolk
and induces the overlying follicle cells to become columnar.
Finally, egg chambers are observed that appear to have 16
nurse cells and no oocyte. They assume a spindle like shape
and eventually degenerate prior to vitellogenesis. This oocyte
nucleus defect occurs primarily when females are raised at
18˚C. The strongest allele for this phenotype is encD6. At 18˚C,
>90% of the egg chambers derived from encD6 homozygotes
contain an enlarged oocyte nucleus and few post vitellogenic
egg chambers are seen, while at 25˚C, this defect is rarely seen
(<5%). In addition, this phenotype is seen almost exclusively
in egg chambers that have not undergone an extra round of
mitosis. This is particularly evident in examining encBB
homozygotes at 18˚C in which 38% of the egg chambers have
undergone an extra round of mitosis. The oocyte nucleus in
these egg chambers has a normal appearance. However, of the
remaining egg chambers which have the correct number of
nurse cells, approximately 65% have an oocyte nucleus defect.
287
Since this defect appears to be cold sensitive, unlike the mitotic
defect which is temperature sensitive, and occurs in egg
chambers that have not undergone an extra division, we
conclude that this represents a second, independent requirement for enc during oogenesis.
To determine if the abnormal morphology of the oocyte
nucleus is due to endoreplication, egg chambers were stained
with Hoechst to examine the polyploidization of the germline
nuclei. In wild-type egg chambers, the diploid oocyte nucleus
appears as a tiny fluorescent dot, while the nurse cell nuclei are
large and intensely stained (Fig. 7D). In enc, there is a range
in the degree of endoreplication of the ooctye nucleus. In some
instances, the oocyte nucleus is larger than a wild-type oocyte
nucleus but is still clearly distinguishable from the nurse cell
nuclei (Fig. 7E). In the most severe cases, egg chambers have
a 16 nurse cell phenotype in which it is not possible to unambiguously distinguish the oocyte nucleus from the nurse cell
nuclei (Fig. 7F).
In order to further investigate the role of enc in oocyte determination, we examined the distribution of oskar (osk) RNA,
which is enriched in the oocyte. In wild-type egg chambers,
osk is synthesized in the nurse cells and accumulates in the
developing oocyte in the germarium and remains concentrated
in the oocyte of developing egg chambers (Fig. 8A) (Ephrussi
et al., 1991; Kim-Ha et al., 1991). In all the enc alleles
examined, the early localization of osk is normal. It accumulates in the oocyte in the germarium and remains localized in
early stage egg chambers. However, in many enc egg chambers
at approximately stages 4-5 the oocyte-specific accumulation
disappears and the entire egg chamber shows weak uniform
staining (Fig. 8B). This disruption of osk localization correlates
Fig. 7. Defect in the morphology of the
oocyte nucleus in egg chambers derived
from enc females raised at 18˚C.
(A-C) Photographed with Nomarski
optics. (D-F) Stained with Hoescht to
visualize nuclei. (A) A wild-type egg
chamber containing a small, smooth
diploid oocyte nucleus. (B) An
encUU3/Df(3L)A466 egg chamber in
which the oocyte nucleus is enlarged in
size and has acquired a roughened
appearance. (C) An encOO6/Df(3L)A466
egg chamber in which the oocyte
contains an enlarged oocyte nucleus
closely resembling a nurse cell nucleus
but has still accumulated yolk. (D) A
wild-type egg chamber in which the
oocyte nucleus is a barely visible
fluorescent dot. (E) A encOO6/
Df(3L)A466 egg chamber containing a
polyploid oocyte nucleus whose ploidy
lags behind that of its sibing nurse cells.
(F) An egg chamber derived from
encOO6/Df(3L)A466 in which the oocyte
nucleus is indistinguishable from the
nurse cell nuclei. Arrows indicate oocyte
nuclei.
288
N. C. Hawkins, J. Thorpe and T. Schüpbach
Fig. 8. Localization of oskar transcript in
wild-type and enc mutant egg chambers
derived from females raised at 18˚C. (A) In
wild-type egg chambers, osk RNA is
concentrated in the oocyte at the posterior
pole. (B) In the encBB/encBB ovariole, the
early localization of osk appears normal but
then osk is distributed throughout the later
stage egg chambers. The egg chambers with
unlocalized osk had highly polyploid oocyte
nuclei. (C) A encBB/encBBegg chamber in
which osk appears to be localized normally.
(D) Same egg chamber as in C stained with
Hoecsht to visualize the nuclei. The oocyte
nucleus is partially polyploid (arrow).
with the presence of a polyploid oocyte nucleus. However, we
have observed that an oocyte nucleus which is only partially
polyploid may still retain localized osk. (Fig. 8C,D).
Thus, in addition to a requirement for enc in regulating
cystocyte divisions, it is also necessary for proper oocyte differentation. In enc mutants, the appearance of polyploid oocyte
nuclei which lose the ability to accumulate osk suggests that
there is a switch from an oocyte to a nurse cell developmental
fate.
DISCUSSION
We have identified a new locus, enc, that is required for the
proper formation of the egg chamber. Females mutant for enc
produce egg chambers with extra nurse cells. A quantitative
analysis revealed that enc egg chambers exhibit a doubling in
the number of germline cells. Although enc egg chambers
contain twice the normal number of germline cells, only a
single oocyte develops. We have shown that the oocyte now
possesses five ring canals instead of four. Since the oocyte
develops from one of two cells with the maximum number of
rings canals instead of a cell with four ring canals, it is not the
absolute number of ring canals that is a determining factor in
oocyte identity. Previously, it has been observed that oocytes
could develop from cystocytes with fewer than four ring canals
(Yue and Spradling, 1992). However, this is the first demonstration that more than four ring canals are compatible with
oocyte development. The exact doubling in the number of
germline cells, and the presence of an additional ring canal on
the oocyte has led us to conclude that the enc phenotype is due
to one extra round of mitosis in the germline. Therefore, enc
appears to be required to regulate the number of cystocyte
divisions.
The enc phenotype is unique. No other mutations have been
isolated that result in precisely one extra round of mitosis in
the germline. Mutations in genes that may have a role in regulating germline mitosis such as hu-li tai shao (hts) and bam
produce phenotypes very distinct from enc. Mutations in hts
result in egg chambers with too few nurse cells, which rarely
develop an oocyte, while mutations in bam produce an ovarian
tumor phenotype (Yue and Spradling, 1992; McKearin and
Spradling, 1990). In contrast, an extra nurse cell phenotype is
produced by mutations in the genes brainiac (brn), Notch (N),
Delta (Dl) and pipsqueak (psq), however, the mechanism
involved is different from enc. This group of mutants affect
follicle cell populations necessary for the correct separation of
adjacent 16 cell cysts, resulting in egg chambers containing
multiple 16 cell cysts (Goode et al., 1992; Ruohola et al., 1991;
Siegel et al., 1993). Unlike enc, the total number of nurse cells
in these compound egg chambers is variable and additional
oocytes often develop.
We have shown that bipolar egg chambers, which are most
prevalent in the original P-element insertion allele encBB, also
have a doubling in the number of germline cells. In these egg
chambers, the oocyte also has 5 ring canals. Thus, the bipolar
phenotype does not arise from a defect in oocyte specification.
Instead, there is an inability of the oocyte to assume its correct
position at the posterior pole of the egg chamber. In the bipolar
egg chambers, the oocyte might be impeded from assuming its
correct position in the egg chamber due to the presence of extra
nurse cells. However, in certain enc allele combinations that
produce almost 100% extra nurse cell egg chambers, the
frequency of bipolar egg chambers is very low; generally less
than 1%. Even for encBB, the frequency of this class varies
depending on genetic background. Thus, enc is not likely to be
directly involved in positioning of the oocyte.
Mutations in enc also produce egg chambers with a
reduction in the number of nurse cells. Like the bipolar class,
this phenotype was most frequent in the original P-element
induced allele, encBB, but was also observed at a frequency of
approximately 1% in the outcrossed encBB line and the EMS
induced alleles. We have determined that the reduction in the
number of germline cells is not due to a decrease in number of
cystocyte divisions. Instead, this phenotype appears to be due
to a mispackaging by the follicle cells of a small group of nurse
cells that were originally derived from a cyst that had
undergone an extra round of mitosis.
All the enc alleles are temperature sensitive for the mitotic
defect. This high incidence of temperature sensitive alleles is
highly unusual. It has been estimated that on average only 510% of EMS induced mutations are temperature sensitive
(Grigliatti, 1986). It would be remarkable if all the enc alleles
resulted in thermolabile proteins. More likely, enc could be
participating in an intrinsically temperature sensitive process.
This hypothesis is supported by the occurrence of temperature
encore and germ line mitosis
sensitive mutations in at least three other genes required for
cyst formation; ovarian tumor (otu), fs(1)1621 (=san fille) and
fs(2)B (=fes) (King, 1970; Gollin and King, 1981; Storto and
King, 1988). In the case of otu, 15 alleles were shown to be
temperature sensitive.
From our genetic analysis it remains unclear if any of our
enc alleles are null. We have generated both loss-of-function
and recessive gain-of-function alleles which produce an
identical phenotype, one extra round of mitosis in the germline.
The main difference between these two groups of alleles is the
penetrance of the phenotype. The loss-of-function alleles are
relatively weak, while the gain-of-function alleles produce
nearly 100% mutant egg chambers as homozygotes. We have
classified the gain-of-function alleles as recessive antimorphs
since the phenotype of these alleles improves when placed in
trans to a deficiency. The relatively weak phenotype produced
by the loss-of-function alleles suggests that enc function might
be partially redundant and that the antimorphic alleles produce
a ‘poison’ product that antagonizes a wild-type pathway
resulting in a stronger phenotype.
The mitotic defect of enc appears to be mediated through its
effect on bam expression. In wild-type ovaries, bam transcript
is detected in the cystobast and 2 cell cysts (McKearin and
Spradling, 1990). In enc ovaries, we have shown that the
domain of bam expression is significantly expanded. This
expansion of bam expression could merely be the consequence
of early changes in cell fate due to the reiteration of the stem
cell to cystoblast division or the cystoblast to two cell cyst
division. This might result in additional cells having acquired
a cystoblast or 2 cell cyst fate and as a consequence express
bam transcript. However, since a null allele of bam acts as a
dominant suppressor of the enc mitotic defect, the misexpression of bam transcript is not simply a consequence of the
extra division but is required to produce the extra division.
We can propose two models which could account for the
alteration in the pattern of bam expression. In the first model,
enc may be acting as a negative regulator of bam transcription.
This could occur in two ways. In enc mutants, bam could now
be expressed in later stage cystocytes, such as 4, 8 or 16 cell
cysts. Alternatively, the level of transcription from the cystoblast and 2 cell cysts may be elevated and thus detectable levels
of transcript may be present longer. In a second model, enc
could be required in the regulated turnover of bam message. If
bam message stability was increased, the transcript could
persist longer and this might be visualized as a wider domain
of bam staining by in situ hybridization.
We propose that, regardless of the molecular mechanism,
the expansion of bam transcript could result in an increase in
Bam protein levels and that the subsequent increase in Bam
protein could be responsible for the extra round of mitosis
observed in enc mutants. Although the first detectable effect
of enc mutations is on the expression of bam transcript early
in the division program, an increase in Bam protein may
result in the extra division being added onto the end of the
normal division program. In a wild-type germarium, Bam
protein is detected in two subcellular locations, the cytoplasm
and the fusome (McKearin and Ohlstein, 1995). The cytoplasmic form of Bam, BamC, is found in the mitotically
active 2, 4 and 8 cell cysts and is degraded in the 16 cell cyst.
McKearin and Ohlstein (1995) have postulated that the degradation of Bam in the 16 cell cyst may signal the cyst to
289
withdraw from the cell cycle. In enc, an increase in the levels
of BamC might result in one extra round of mitosis. Alternatively, increasing the fusomal component of Bam, BamF,
might drive the 16 cell cyst through an additional cell cycle
since the fusome has been postulated to play a role in cyst
formation (Lin et al., 1994; Telfer, 1975). However, the
presence of the fusome does not precisely correlate with
mitotically active cystocytes. It still persists in region 2 of the
germarium after the 16 cell cyst has ceased division. An
analysis of Bam protein in enc mutants should help in distinguishing between these possibilities.
It is unknown whether there is an internal counting
mechanism, possibly some tritratable factor, or signals from
the soma that instructs the 16 cell cyst to withdraw from the
cell cycle and begin differentiation. Since BamC is present in
mitotically active cysts and is degraded in 16 cell cysts it is
tempting to speculate that Bam might be part of some titratable counting mechanism whose levels are regulated by enc.
In addition to enc’s role in germline mitosis, we have shown
that there is a second requirement for enc during oogenesis in
the process of oocyte differentiation. In wild-type egg
chambers, the oocyte nucleus is arrested in meiosis I with a
DNA content of 4C, while the nurse cell nuclei begin
endoreplication as the cyst exits the germarium. In enc
mutants, we have shown that the oocyte frequently acquires a
partial nurse cell identity in that the oocyte nucleus undergoes
endoreplication. Usually the extent of endoreplication lags
behind that of the adjacent nurse cell nuclei, though egg
chambers were observed in which the oocyte nucleus was
indistinguishable from the nurse cell nuclei. This phenotype is
similar to that produced by a small subset of ovarian tumor
(otu) alleles that allow formation of a nurse cell/oocyte
syncytium. Storto and King (1988) observed oocytes derived
from weak otu alleles in which the oocyte nucleus had
undergone endoreplication. Like enc, the extent of endoreplication of the oocyte lagged behind that of its sibling nurse cells.
Two other genes have been identified that are required for
oocyte determination. Females mutant for recessive alleles in
Bicaudal-D (Bic-D) or egalitarian (egl) produce egg chambers
comprising 16 nurse cells and no oocyte (Schüpbach and
Wieschaus, 1991; Suter et al., 1989). Egg chambers develop
until mid oogenesis and then degenerate prior to vitellogenesis. In contrast to Bic-D and egl, none of the enc alleles
produce a completely penetrant phenotype for this defect
although the strongest allele, encD6, produces very few post
vitellogenic egg chambers and results in no eggs being laid.
Many other enc alleles produce an intermediate phenotype in
which post vitellogenic egg chambers are observed with a
polyploid oocyte nucleus. Since this phenotype is less
penetrant than in Bic-D or egl, enc might be involved in the
maintenance of oocyte identity as opposed to the initial establishment of oocyte identity.
A role for maintenance of oocyte identity, as opposed to
the initial establishment of oocyte identity, was also
suggested from an examination of osk localization in enc egg
chambers. We observed that osk was localized normally in
the germarium and early stage egg chambers, then at slightly
later stages, was often evenly distributed throughout the egg
chamber. Therefore, certain aspects of oocyte determination
appear initially to proceed normally, before the oocyte
switches to a nurse cell developmental fate. Hypomorphic
290
N. C. Hawkins, J. Thorpe and T. Schüpbach
alleles of Bic-D exhibit a similar pattern of osk localization
in which osk was localized normally in the germarium and
early stage egg chambers (Suter and Steward, 1991).
However, in null alleles of Bic-D , there was no oocytespecific accumulation of osk (Ran et al., 1994). Since we do
not know whether we have isolated a null allele of enc, we
cannot rule out the possibility that enc, like Bic-D and egl, is
required for the initial establishment of oocyte identity as
well as for its maintenance.
The role of enc in oocyte determination appears to represent
an independent requirement for enc and is not simply a
secondary consequence of the mitotic defect. Two lines of
evidence support this assumption. First, the oocyte nucleus
defect occurs almost exclusively in egg chambers that have not
undergone an extra round of mitosis. Second, this phenotype
is cold sensitive, unlike the mitotic defect, which is temperature sensitive. enc therefore has at least two separable functions
in oogenesis. It is required for the regulation of germline
mitosis and it is subsequently involved in the process of oocyte
differentiation.
We thank Rob Jackson, Jim Garbe, Stephen Harrison and Dennis
McKearin for providing stocks and Dennis McKearin for communicating results prior to publication. We thank Cheryl VanBuskirk for
assistance with the oskar in situs and stimulating discussions. We
acknowledge Joseph Goodhouse, of the department’s Confocal/E.M.
Core Facility, for his expert technical assistance with confocal
microscopy and helping with the computer graphics in the preparation of Figs 3 and 5. We are grateful to Robert Ray, Christopher Beh,
Elizabeth Gavis and Ken Irvine for critical reading of the manuscript
and members of the Schüpbach lab, especially Siegfried Roth and
Robert Ray, for stimulating discussions. We thank Gordon Gray for
the preparation of fly food. This work was supported by the Howard
Hughes Medical Institute and by the Public Health Service Grant
GM40558 to T. S.
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