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Biological Sciences Publications Department of Biological Sciences
1998
vasa is required for GURKEN accumulation in the oocyte, and is vasa is required for GURKEN accumulation in the oocyte, and is
involved in oocyte differentiation and germline cyst development involved in oocyte differentiation and germline cyst development
S. Styhler
A. Nakamura
Andrew Swan
University of Windsor
B. Suter
P. Lasko
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Recommended Citation Recommended Citation
Styhler, S.; Nakamura, A.; Swan, Andrew; Suter, B.; and Lasko, P., "vasa is required for GURKEN
accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development"
(1998).
Development
, 125, 9, 1569-1578.
https://scholar.uwindsor.ca/biologypub/1164
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INTRODUCTION
Segregation of the germline from the soma is a central feature
of animal development. In Drosophila, the germline is
determined through the activities of maternally expressed
RNAs and proteins which colocalize in the pole plasm at the
posterior pole of the egg (reviewed by Rongo and Lehmann,
1996). Pole cells, the progenitors of the germline, form very
early in embryogenesis, then, beginning at gastrulation, they
migrate into the interior of the embryo and ultimately associate
with the gonadal mesoderm to form the embryonic gonads
(reviewed by Williamson and Lehmann, 1996). Beginning in
larval development, germ cells proliferate and differentiate in
order to carry out spermatogenesis and oogenesis; among the
structures assembled during oogenesis is new pole plasm,
which specifies the germline for the subsequent generation of
individuals.
Genetic and molecular studies have identified numerous
genes which are required for pole plasm assembly and
subsequent posterior segment specification and germ cell
formation; many of these genes are expressed during oogenesis
and produce mRNAs and/or proteins which localize in pole
plasm or in polar granules, specialized organelles contained
within the pole plasm (reviewed by Rongo and Lehmann,
1996). Analysis of the expression of these genes supports an
early hypothesis (Mahowald, 1968) that translational control is
a major mechanism regulating Drosophila germline
development. The product of the vasa (vas) gene, a DEAD-
box-family protein which is localized in polar granules and
which shares the enzymatic functions of the translation
initiation factor eIF4A (Hay et al., 1988; Lasko and Ashburner,
1988; Liang et al., 1994), is a candidate germline-specific
translational regulator. For instance, levels of the short isoform
of OSKAR protein (OSK), a molecule central to pole plasm
assembly (Ephrussi et al., 1991; Kim-Ha et al., 1991; Ephrussi
and Lehmann, 1992), are greatly reduced in vas mutant ovaries
(Markussen et al., 1995; Rongo et al., 1995). Another pole
plasm mRNA whose translation may be activated by VAS is
nanos (nos), as nos RNA carrying an intact translational
regulation element in its 3′UTR is completely inactive in
embryos derived from vas mutant ovaries (Gavis et al., 1996;
Dahanukar and Wharton, 1996).
While the activities of pole plasm components such as VAS
have been most thoroughly studied with respect to their
function in pole cell formation and specification of the
posterior soma, clearly some genes involved in pole plasm
assembly also function in other stages of germline
development. For instance, females homozygous for either of
two strong nos alleles exhibit defects in germ cell proliferation
(Lehmann and Nüsslein-Volhard, 1991; Wang et al., 1994).
Furthermore, pole cells lacking maternal nos function fail to
complete migration and do not associate with the embryonic
gonadal mesoderm (Kobayashi et al., 1996), indicating a role
for nos in the transition from pole cell to functional germ cell.
1569
Development 125, 1569-1578 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
DEV8503
The Drosophila gene vasa is required for pole plasm
assembly and function, and also for completion of
oogenesis. To investigate the role of vasa in oocyte
development, we generated a new null mutation of vasa,
which deletes the entire coding region. Analysis of vasa-null
ovaries revealed that the gene is involved in the growth of
germline cysts. In vasa-null ovaries, germaria are
atrophied, and contain far fewer developing cysts than do
wild-type germaria; a phenotype similar to, but less severe
than, that of a null nanos allele. The null mutant also
revealed roles for vasa in oocyte differentiation, anterior-
posterior egg chamber patterning, and dorsal-ventral
follicle patterning, in addition to its better-characterized
functions in posterior embryonic patterning and pole cell
specification. The anterior-posterior and dorsal-ventral
patterning phenotypes resemble those observed in gurken
mutants. vasa-null oocytes fail to efficiently accumulate
many localized RNAs, such as Bicaudal-D, orb, oskar, and
nanos, but still accumulate gurken RNA. However, GRK
accumulation in the oocyte is severely reduced in the
absence of vasa function, suggesting a function for VASA
in activating gurken translation in wild-type ovaries.
Key words: Drosophila, RNA localization, axis patterning, vasa
(vas), oogenesis
SUMMARY
vasa
is required for GURKEN accumulation in the oocyte, and is involved in
oocyte differentiation and germline cyst development
Sylvia Styhler, Akira Nakamura
†
, Andrew Swan, Beat Suter and Paul Lasko*
Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montréal, Québec, Canada H3A 1B1
†
Present address: Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan
*Author for correspondence (e-mail Paul_Lasko@maclan.mcgill.ca)
Accepted 3 February; published on WWW 1 April 1998
1570
Similarly, various vas alleles have defects in oogenesis and lay
few or no eggs (Lasko and Ashburner, 1988, 1990; Lehmann
and Nüsslein-Volhard, 1991; Schüpbach and Wieschaus,
1991). Females trans-heterozygous for Df(2L)A267 and
Df(2L)TE116-GW18, two large deletion mutations which both
include vas, were reported to be blocked in early vitellogenic
stages of oogenesis (Lasko and Ashburner, 1988). Analysis of
whether this phenotype was caused solely by loss of vas
function has been confounded by the fact that these trans-
heterozygous deficiency lines are haploid for a large number
of genes, but that, aside from large deficiencies, a clearly null
allele of vas did not exist. Four EMS-induced alleles of vas,
vas
D1
, vas
Q6
, vas
Q7
and vas
D5
, also lead to greatly reduced
fertility, with many egg chambers blocked as for the trans-
heterozygous deficiency females (Lehmann and Nüsslein-
Volhard, 1991). The few eggs produced by females
homozygous for these alleles often lack dorsal appendages and
have the micropyle, a specialized vitelline membrane structure
normally found only at the anterior of the egg, duplicated at
the posterior (Lehmann and Nüsslein-Volhard, 1991). Again,
whether these phenotypes represent the results of a complete
loss of vas function is unknown. vas
Q6
and vas
D5
are missense
mutations which alter single amino acids of VAS and both
alleles produce substantial amounts of mutant protein (Liang
et al., 1994), so neither of these mutations is likely to be null.
For vas
D1
and vas
Q7
the molecular nature of the mutation is
unknown, but the vas coding region is unaffected in these
mutant alleles.
In this paper, we have used a new vas null allele, vas
PH165
,
a small deletion which we generated by imprecise P-element
excision, to investigate in detail the role of vas in events of
oogenesis prior to pole plasm assembly. We found that
abrogation of vas function results in defects in many aspects
of oogenesis including control of cystocyte divisions, oocyte
differentiation, and specification of posterior and dorsal follicle
cell-derived structures. Furthermore, vas
PH165
oocytes only
weakly concentrate many oocyte-localized RNAs, although
some oocyte-specific molecules, including gurken (grk;
Schüpbach, 1987; Neuman-Silberberg and Schüpbach, 1993)
RNA, remain concentrated in the oocyte in vas mutant ovaries.
However, in the case of grk, translation is severely reduced in
the absence of vas function. This provides evidence that VAS
is involved in translational control mechanisms operating in
early stages of oogenesis.
MATERIALS AND METHODS
Fly stocks
To create a null allele of vas, excision lines were generated through
the introduction of the
∆
2-3 transposase source into vas
P(ry[+])LYG2
(Rittenhouse and Berg, 1995). To do this, +/Y; vas
P(ry[+])LYG2
cn; ry
506
males were crossed to w; Bic-D
PA66
Su(Bic-D
PA66
) cn/CyO;
∆
2-
3Sb/TM3 Ser virgin females. w/Y; vas
P(ry[+])LYG2
cn/CyO;
∆
2-3Sb/
ry
506
males were then crossed to +/+; l(2)05084
P(ry[+]l(2)05084
/CyO;
ry
506
virgin females. Individual ry F
1
males representing excisions of
vas
P(ry[+])LYG2
were then individually crossed to
l(2)05084
P(ry[+]l(2)05084
/CyO; ry
506
virgin females. Balanced ry stocks
were generated and females homozygous for an excision chromosome
were crossed to Oregon R males to test for fertility. Excision lines
were screened for deletions through Southern analysis using a 1.9-kb
EcoRI genomic fragment from the vas first intron, and which includes
the vas
P(ry[+])LYG2
insertion site, as a probe. VAS protein levels from
excision lines were determined by western analysis.
vas
P(ry[+])LYG2
was provided by Celeste Berg (University of
Washington, Seattle); l(2)05084
P(ry[+]l(2)05084
/CyO was provided by
the Berkeley Drosophila Genome Project; nos
RC
, which disrupts a
splice donor site and is null for RNA and protein expression (Wang
et al., 1994; Curtis et al., 1997) was received from Takahiro Akiyama
(Azabu University, Kanagawa, Japan); and grk
HK36
, a strong grk allele
(Schüpbach, 1987) was obtained from Trudi Schüpbach (Princeton
University). The wild-type strain employed was Oregon R.
In situ hybridization and antibody staining
In situ hybridizations with DIG-labeled RNA probes and antibody
stainings were carried out on ovaries and embryos as described by
Kobayashi et al. (1998), except that DMSO was omitted from the
fixation solution used for ovaries. Primary antibodies were used at the
following dilutions: α-ORB, 1:20; α-BIC-D, 1:10; α-NOS, 1:150; α-
GRK, 1:3000; α-ADD-87, 1:20. α-NOS was pre-adsorbed with
embryos from nos
BN
females, and α-GRK was preadsorbed with
grk
HK36
ovaries. For bright-field microscopy, antibody stainings were
detected with DAB, enhanced using the Vectastain ABC or ABC Elite
kits (Vector Laboratories) and biotinylated secondary antibodies. For
confocal microscopy, antibody stainings were detected using Texas
Red-conjugated secondary antibodies (Molecular Probes). β-gal
staining of khc:lacZ ovaries was carried out as described by Clark et
al. (1994).
OliGreen/Texas Red-phalloidin staining
Ovaries were dissected in PBS and fixed for 20 minutes in a mixture
of 600 µl heptane, 200 µl 4% paraformaldehyde in PBS + 0.2%
Tween-20, and 20 µl DMSO. Following fixation, samples were rinsed
3 times with PBT (PBS + 0.1% Tween-20), incubated for 1 hour in
RNase A (100 mg/ml), and rinsed again 3 times with PBT. Ovaries
were incubated for 1 hour in OliGreen (Molecular Probes, 1:1000
dilution) and Texas Red-phalloidin (Molecular Probes), washed with
PBT, and mounted in 70% glycerol in PBS.
RESULTS
vas
PH165
is a
vas
null allele
Identification of additional vas cDNA clones has indicated the
presence of a 127-bp exon upstream of the previously reported
5′ end of vas, which extends the 5′ UTR of the gene. This exon
is separated by a large intron of 6603 bp from the remainder
of the gene (Fig. 1A,B; Berkeley Drosophila Genome Project,
unpublished results, submitted to GenBank under accession
numbers L81347, L81348, L81449, AC000466, and
AC000469). vas
LYG2
is a P-element-induced vas allele
(Rittenhouse and Berg, 1995), which we mapped to the large
first intron of vas (Fig. 1A), and which produces about 2% of
the wild-type level of VAS (Fig. 1C), a level essentially
undetectable in tissue staining experiments. Despite the very
low level of VAS in vas
LYG2
ovaries its phenotype is
hypomorphic; oogenesis in vas
LYG2
females is less severely
compromised than in Df(2L)A267/Df(2L)TE116-GW18 flies,
and vas
LYG2
females lay numerous eggs. This suggested to us
either that trace amounts of VAS are sufficient for oogenesis
to often proceed to completion, or that the more severe
phenotype observed in the double-deficiency lines results from
the effects of a reduction in dosage of a second gene which
enhances the vas phenotype.
To distinguish between these possibilities, it was necessary to
obtain a molecularly characterizable null vas allele. For this
S. Styhler and others
1571Role of
vasa
in early oogenesis
purpose, we generated a series of derivative lines by mobilizing
the P element in vas
LYG2
. Out of 201 excision events, stable lines
were generated from 129. Of these, 119 caused reversion to a
wild-type phenotype, indicating that the vas
LYG2
phenotype
results solely from the P-element insertion, and that the vas
LYG2
chromosome is free of other female-sterile mutations. Three
other derivatives were recessive lethal, and seven, although
having excised the ry
+
marker on the P element of vas
LYG2
,
remained defective in oogenesis. These were checked by PCR
and Southern blots to determine whether they carried a deletion
confined to the vas gene, and by western blots using anti-VAS
antiserum to determine whether they expressed VAS. From these
analyses, one line, vas
PH165
, was identified, which produced no
detectable VAS protein and carried a 7343-bp deletion which
removes the entire coding region of vas (Fig. 1A,C). We
confirmed the breakpoints of vas
PH165
by nucleotide sequencing,
comparing the mutant sequence to wild-type sequence provided
by the Berkeley Drosophila Genome Project. From the nature of
the vas
PH165
mutation and the fact that no VAS protein is
detectable on western blots even on long overexposures (Fig.
1C), we conclude that vas
PH165
is null for VAS. The vas
PH165
deletion is mostly limited to vas, as one of its breakpoints lies
within vas and the other is 1270 bp downstream of its 3′ end. A
nested gene may be located within the 3.5-kb third intron of vas,
which is deleted in vas
PH165
, as a 3-kb transcript present
throughout all developmental stages is detected on northern blots
using genomic probes including this intron but not with vas
cDNA probes (Lasko and Ashburner, 1988). However, any gene
other than vas which may be disrupted in vas
PH165
is almost
certainly irrelevant to the discussion below, as a vas-GFP
transgene, constructed from a vas cDNA fused to the vas
promoter, and therefore lacking any nested or 3′-flanking genes,
rescues vas
PH165
homozygotes, and Df(2L)A267/ Df(2L)TE116-
GW18 flies to fertility. The phenotypes of vas
PH165
/vas
PH165
,
vas
PH165
/ Df(2L)A267, and Df(2L)A267/ Df(2L)TE116-GW18
ovaries are essentially identical.
VAS is involved in the maintenance of germline
cysts
Upon cursory examination of vas
PH165
ovaries under the light
microscope, we noticed an obvious atrophy of the germaria as
compared with the wild-type, suggesting that fewer germ cells
were present. To investigate this more closely, we used an
antibody recognizing ADD-87 protein (Lin et al., 1994; Zaccai
and Lipshitz, 1996) which stains spectrosomes and fusomes,
specific structures present in stem cells, cystoblasts and
dividing cystocyte clusters. In vas
PH165
ovaries, a reduction in
the number of ADD-87-staining structures, and therefore a
reduction in the number of developing cysts, is readily apparent
as compared with wild-type (Fig. 2A-D). This phenotype has
high expressivity, and increases in severity with the age of the
female (Fig. 2C-D). In germaria from 7-day-old vas
PH165
females, region 1 frequently consists of only a few stem cells
and developing cysts. Posterior to these there is often what
appears to be an extended interfollicular stalk (compare Fig.
2D with Fig. 2C), likely formed by follicle cells in the absence
of cystocyte clusters. This suggests that the cystocyte clusters
remaining in the germarium have ceased to develop further and
have degenerated, while the follicle cells continue to divide.
Two other genes that function in the same pathway as vas in
posterior patterning, nos and pum, have also been implicated
in germ cell proliferation (Lehmann and Nüsslein-Volhard,
1991; Wang et al., 1994; Lin and Spradling, 1997), raising the
possibility that they also may be interacting with vas in the
Fig. 1. (A) Organization of the
vas gene. Exons (boxes) are
numbered with Roman
numerals (I-VIII), the
translational start codon is in
exon II; E, EcoRI site. The site
of the P{ry
+
, vas
LYG2
} insertion
is indicated, and the insertion
site is immediately after
position 75581 in the BDGP P1
clone DS00929 (GenBank
accession number AC002502).
vas
PH165
results from an
imprecise excision of P{ry
+
,
vas
LYG2
}, in which 7343 bp of
genomic DNA, including the
entire vas coding region, are
deleted, and replaced with 16
bp from the P element
(sequence highlighted). (B) The
5′ UTR of vas. The first 127
nucleotides make up exon I, the
6603-bp first intron follows
after nucleotide 127 (solid
triangle) and nucleotide 128
corresponds to nucleotide 76 as
reported in Lasko and
Ashburner (1988). Nucleotides 151-153 are the initiator ATG (underlined). (C; top panel) Western blot probed with α-VAS antiserum to
compare the levels of VAS in Oregon R, vas
LYG2
and vas
PH165
ovarian extracts. Extracts were loaded in various amounts as indicated at the top
of the figure. (Bottom panel) The same blot probed with α-eIF4A antiserum (Lavoie, 1995) as a loading control.
1572
germarium. pum mutants produce ovarioles which contain only
two or three clusters of undifferentiated germ cells which lack
spectrosome/fusome structures or ring canals (Lin and
Spradling, 1997), a phenotype we never observed in vas
mutants. Upon examination of nos
RC
mutant ovaries with the
α-ADD-87 antibody, we observed a phenotype which appears
similar, but somewhat more severe, than that of vas
PH165
. Many
nos
RC
ovarioles consist of a germarium with one to three cysts
(Fig. 2E), followed by an extended stalk and one to three
normal-looking egg chambers. In more extreme cases, only
remnants of spectrosome/fusome material can be detected in
the anterior of the germarium (Fig. 2F), consistent with the
conclusion that these germline cells have arrested
development.
VAS is involved in oocyte differentiation
At a low frequency (aprox. 1% for each), we observed defects
in germline differentiation and oocyte determination in
vas
PH165
ovaries, including tumorous egg chambers (Fig. 3A),
egg chambers with 16 nurse cells and no oocyte, others with
two oocytes, and again others with a mislocalized oocyte (Fig.
3B-D). Far more frequently the normal 15 nurse cells and one
oocyte are present; however, by at least two criteria the oocytes
produced in vas
PH165
egg chambers are not fully differentiated.
In wild-type development, the nurse cell nuclei endoreplicate
during pre-vitellogenic oogenesis and become highly
polyploid, whereas the oocyte nucleus remains diploid and
condenses into a tight karyosome (Mahowald and Kambysellis,
1980). However, in vas
PH165
the oocyte nucleus appears more
diffuse than does the wild-type oocyte nucleus (Fig. 3E,F).
This would be consistent either with a failure to form the
karyosome structure, perhaps involving a premature meiotic
arrest in diplotene rather than in metaphase, or an increase in
ploidy in vas
PH165
oocytes. A very similar nuclear morphology
has been observed in spindle (spn) mutant oocytes, which has
been interpreted as resulting from a delay in oocyte
determination (González-Reyes et al., 1997).
Secondly, vas
PH165
oocytes do not efficiently accumulate at
least four oocyte-localized RNAs. In wild-type ovaries, the
Bicaudal-D, orb, osk and nos RNAs all accumulate efficiently
in the oocyte within the germarium and remain concentrated
S. Styhler and others
Fig. 2. Confocal micrographs of germaria from (A) wild-type,
(B) grk
HK36
/grk
HK36
, (C) 4-day-old vas
PH165
/vas
PH165
, (D) 7-day-old
vas
PH165
/vas
PH165
, and (E,F) 1- to 2-day old nos
RC
/nos
RC
ovaries
stained for the ADD-87 protein, which marks spectrosome and
fusome structures and which is diagnostic for stem cells and
developing germline cysts. Substantially fewer foci of ADD-87 are
observed in all vas
PH165
or nos
RC
germaria as compared with the
wild-type or with grk
HK36
. Similar phenotypes were observed in
vas
PH165
/Df(2L)A267 ovaries.
Fig. 3. Phenotypes of vas-null egg chambers affecting oocyte
determination. Confocal micrographs of (A-B) vas
PH165
/vas
PH165
ovaries stained with the nuclear dye Oli-Green (Molecular Probes),
illustrating the following phenotypes: (A) tumorous germline cyst,
(B) 16 nurse cells and no oocyte. The 16 polyploid nuclei are
numbered in this panel. (C) vas
PH165
/ vas
PH165
egg chamber with two
oocytes, stained for F-actin with Texas Red-phalloidin (Molecular
Probes). (D) vas
PH165
/Df(2L)A267 egg chamber doubly stained with
Oli-Green and with Texas Red-phalloidin, illustrating a bipolar egg
chamber with its oocyte in the center. We also observed bipolar egg
chambers in vas
PH165
/vas
PH165
ovaries. (E,F) Higher-magnification
view of oocyte nuclei stained with Oli-Green; (E) wild-type,
(F) vas
PH165
/vas
PH165
. In each of these panels, an arrowhead points
to the oocyte nucleus; note the more diffuse staining in F as
compared with E, apparent in almost all vas
PH165
egg chambers.
Similar phenotypes were observed in vas
PH165
/Df(2L)A267 ovaries.
Scale bars 20 µm (A-C), 100 µm (D), 10 µm (E,F).
