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The role of vasa during oogenesis

Sylvia Stybler

Oepartment of Biology

McGili University, Montreal

Marcb 1998

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Abstract

The Drosophila melanogaster gene vasa is known to be necessary for the establishment of a functional pole plasm, as weIl as for the completian of oogenesis. Ta further elucidate its role~ we have created a null mutation of the vasa gene and examined

vasa-null avaries for defects. A.nalysis of these avaries has revealed that "'asa is involved in various aspects of oogenesis~ including the growth of germ-line cysts. oocyte differentiation. anterior-posterior egg chamber patteming, and dorsal-ventral follicle patteming. In addition, vasa-nuJl oocytes fail ta show efficient accumulation of various localized RNAs., such as Bicaudal-C~ Bïeal/dal-D, egl, enc, orb~ oskar. and nanos, but

•

•

Résumé

Dans Drosaphila me/anogaster. le gène, vasa, est necessaire pour l'établissement d'une

plasme polaire fonctionnel, et aussi pour la conclusion d'oogenèse. Pour mieux comprendre

ce rôle, nous avons produit une mutation nulle de

vasa

et nous avons examiné les ovocytes

nulles-vasa pour des défauts. L-analyse des ovaires démontre que

vasa

joue un rôle dans

plusieurs aspects d'oogenèse, comprennant l'augmentation des cystes de la lignée

germinale, la differenciation des ovocytes, l'établissement antéro-postérieur, et

l'établissement dorsa-ventral. En plus, les ovocytes nulles-vasa ne démontrent pas

l'accumulation efficiente de plusieurs ARNs localisés, comme

Bicaudal-C, Bicauda/-D, egl.

enc. orb, oskar, et nanas, mais accumulent le ARN gurken. Intéressentement,

l'accumulation du protéine GURKEN dans l'ovocyte est réduit sevèrement't et celle de

BICAUDAL...C est diminuée considérablement dans les ovocytes

nulles-vasa.

Ces

resultats suggèrent un rôle pourvasa dans l'activation de traduction des ARNs spécifiques

Table of Contents

ABSTRACT 11

RÉSUMÉ .••...•...•...•.••..•.••.•••••••.•.••....•••...••.••.•....•.•.•.•.•••.•.••••••.••.•.•.•...•....•..•..•.•..•.•.••••.•••.•.•••••••.••• 111 TABLE OF CONTENTS .••••.••...•..•.•..••..•...•••••..•.••.•..•••...••••.•••••.•••••.•••..••••••••••.•••••••.•••••••••••.••••.••.•••.••..• IV

PREFACE _..• VI

CONTRIBUTION OF AUTHORS VIII

ACKNOWLEDG MENTS IX

CHAPTER ONE: LITERATURE REVIEW ••..••••.•.•..•.••••••....•••..•..•••••.•••••..•.•.••.•..••...•.••...•.•••••.•.•.•...•••.•••• 1

1.OOGENESIS IN DROSOPHIL-\ 2

/./ .., Reviel\' 2

1.1.1 Formation ofa 16-ccll Cysl 2

1.1.2 Early Oogenc:sis : The Gennarium 3

1.1.3 Laie Oogenesis ; The: Vitellarium 3

/.2 Fornlation afthe OoC)·te ./

1.2.1 Asymme:tric Inherilance of the: Spe:crrosome: .t

1.2.2 Oocyte De:te:rmina[ion .t

1.2.3 Oocyte DitTerentialion 6

1.2." Follicle CcII~ligralions 6

/.3 Establishment ofPolarit}· -;

1.3.1 .-\nlerior-Postcrior.~xis " 7

1.3.2 The Anlcrior Determinant.bcd 8

1.J.3 Dorsal-Ventral Axis 9

I.JA grk and lop/DER Requircd for Antc:rior-Posterior Axis 10

2.ANTEROPOSTERIOR PATIERNlNG ANDGEPJ-,.ILINE DEVELOPMENT 11

2./ Posteriar Classafl~faternQI-EffectGenes 11

2.2.-lssembly a/the Pole Plasm /2

2.2.1 oskis Under Translational Control 12

2.2.2 stauEncodes an RNA-binding Prolcin 13

2.2.3 VAS is a DEAD-box Protcin 14

2.2..t The Postc:rior Determinant. nos 14

CHAPTER TWO: INVESTIGATING THE ROLE

or

J'ASA DURING OOGENESIS _..•• 16

INTRODUcnON 17

MATERlAlSAND METHODS 19

REsULTS 20

DISCUSSION 22

FIGURES 23

CHAPTER THREE : J'AS15 REQUlRED FOR GRK ACCUMULATION IN THE OOCYTE, AND IS INVOLVED IN OOCYTE DIFFERENTIATION AND GERM LINE CYST DEVELOPMENT _24

SUMMARY 25

INTRODUCTION 26

FIGURES 52

ACK.NOWlEDGE~tENTS 60

CHAPTER FOUR: BIC-C PROTEIN IS REDUCED IN VAS NULL OVARIES...•...61

Preface

In accordance vlith thesis specifications from The Faculty of Graduate Studies and Research, the following statement is reprinted.

Candidates have the option of including, as part of the thesis, the text of one or more papers submitted or ta be submitted for publication, or the clearly-duplicated text of one or more published papers. These texts must be bound as an integral part of the thesis.

If this option is chosen. connecting texts that provide logical bridges bet\veen the different papers are mandatory. The thesis must be \mtten in such a \vay that it is more than a me:-e collection of manuscripts; in other \vords. results of a series of papers must he integrated.

The thesis must still conform to aU other requirements of the "Guidelines for Thesis Preparation". The thesis must include: a table of contents. an abstract in English and French. an introduction \vhich clearly states the rationale and objectives of the study, a revie\v of the literature, final conclusion and summary, and a thoreugh bibliography or reference list.

AdditienaI materi

al

must be provided where appropriate (e.g. in appendices) and in sufficient detail to allow a c1ear and precise judgement to be made of the importance and originality of the research reported in the thesis.

Contribution of Authors

Chapter Three is the manuscript fonn of a paper which

bas

been accepted for publication, entitled"vas is required for GRK accumulation

in

the oocyte, and is involved

in oocyte ditIerentiation and genn line cyst development." The following people are responsible for the work reported

in

the designated figures:

Akira Nakamura: lst exon sequence and Vasa westerninChapter 3, Figure IB,C Andrew Swan: ail confocaI microscopy, stainings

in

Chapter 3, Figures 2,3, 6A-B Paul Lasko: contributed greatly to the writing orthe paper and the assembly of the figures

Chapter Four is part of the work which has been submitted for publication in a paper entitled "Premature translation of

oskar

in oocytes lacking the RNA..binding

Acknowledgments

•

•

•

1

~

Oogenesis in Drosophila

1.1 A Review

In Drosophila melanogaster. the process of oogenesis results with the fonnation of a gamete containing factors necessary for the initiation and maintenance of metabolism and development in the ~mbryo. As embryonic gene expression begins only at the blastoderm stage. such factors must be matemally supplied and are thus crucial to the development of the embryo.

1.1.1 Formation of a 16-cellCyst

The female Dros0l'ltila possesses two avaries consisting of approximately 16 ovarioles each. The avariait: ltself represents an egg assembly line in which the growing egg chambers are moved posteriorly as

a

result of rhythmic contractions of ovarian muscles. This structure may be further separated into two parts: the germarium and the vitellarium. The gennarium ~ontains a smaIl population of oogonic stem ceIls which proliferate and differentiate to l'orm cystoblasts. which in tum undergo four consecutive mitotic divisions. resulting in 16progeny cells. Due to incomplete cytokinesis. these ceUs

1.1.2 Early Oogenesis : The Germarium

A tubulin cytoskeleton fonns such that a single microtubule organizing center

(MTOC) is established in the oocyte and extends through the ring canals into the nurse

cells" thus joining ail 16 cells. Surrounding the numerous cysts found in region 2a of the

germarium are mesodermal derivatives called follide cells. As the cysts are moved

posteriorly into region 2b, these follicle cells begin ta maye inwardly and \vill separate

the cysts into individual components. The cysts spend about {Wo days between leaving

the gennarium and entering the process of vitellogenesis. During this time. the nurse cells

become large and polyploid, enabling them ta synthesize large quantities of proteins and

RNAs, which are transferred through the ring canals and into the oocyte during later

stages of oogenesis.

1.1.3 Late Oogenesis : The Vitelfarium

The vitellarium is composed of a series of progressively older egg chambers at

various stages of vitellogenesis, numbered from stages 1-14 by King, 1970. During the

tirst 7 stages of vitellogenesis" the oocyte and nurse cells are of similar size and are

surrounded by a monolayer of follicle cells. Stages 8-10 mark the beginning of rapid

oocyte growth as the oocyte begins the uptake of yolk proteins and a series of migrations

take place such that the oocyte is surrounded by the follicIe cells.

Once the oocyte is encircled by fanicle cells" it must be covered by an eggshell

and other specialized structures. During the final stages of vitellogenesis, the follicle cells

t\\"O dorsal appendages are formed to facilitate embryonic respiration. A protuberance called a micropyle is also formed at the anterior, which possesses an opening through

which sperm may enter during fertilization. The nurse and follicIe cells \vill then

degenerate~leaving behind the mature egg.

1.2 Formation of the Oocyte

1.2.1 Asymmetric Inheritance of the Spectrosome

Of the 16 cystocytes contained within a single cystoblast, one is singled out ta acquire the active MTOC and is thus destined to become the oocyte. Germ cells carry a sphericai cytoplasmic structure called the spectrosome~ which contains alpha-spectrin and the adducin-like protein" Hu-Ii tai shao (Hts: Lin et al.. 1994b). During the tirst cystoblast division" the spectrosome is inherited by only one of the two daughter cells and grows from this cystocyte into the other cells during subsequent divisions. The end result is a branched structure called the fusome which disappears after the fourth division (Lin and Spradling, 1997). In hlS mutants, which fail ta form the spectrosame, genn cell division proceeds abnormaIly and an oocyte is rarely formed (Yue and Spradling, 1992; Lin et al.,

1994b). This asymmetric inheritance of the spectrosome seems ta therefore detennine which of the two daughter cells will become the oocyte.

1.2.2 Oocyte Determination

Recessive mutations in either of these genes results in the failure to accumulate oocyte-specific mRNAs in a single celt, resulting in female sterility due to the formation of egg chambers containing 16 nurse cells and no oocyte. A similar phenotype is obtained through treatment with microtubule-depolymerising drugs, suggesting this net\vork is

involved in the determination of an oocyte. In addition, the microtubule network is not formed in Bic-D mutants and is not well maintained in egl mutants (Theurkauf et al.,

1993). These findings support a model in which mRNAs are transported along the microtubule net\vork into the MTOC-containing cell of the cyS!, thereby designating it ta become the oocyte.

Bic-D has been sho\vn ta encode a protein with similarity to the coiled-coil

1.2.3 Oocyte Differentiation

Upon entering germarial region 2a, cystocytes must begin their differentiation into nurse cells or the oocyte. Mutations in the ovarian tumour mutant,bag-ofmarbles (barn),

result in foUides consisting mainly of mitotically active unditTerentiated ceUs, implicating barnas a signal for ineomplete cytokinesis (McKearin and Spradling, 1990; McKearin and Ohlstein. 1995). In contrast to this tumourous follicle phenotype, mutations in encore (enc) cause one extra round of eystocyte divisions" resulting in 32-cell foHicles (Ha\vkins et al.. 1996). [n addition" expression ofbam is expanded to double the \vildtype width in germarium region 1 ofencmutants. suggesting that encfunctions to restrict barnexpression, thus indicating a role in early cyst development.

1.2.4 Follicle Cell Migrations

Until stage 6 of oogenesis, the follicle cells surrounding the egg chamber are of a relatively uniform size and shape. During stages 7-8, the pasterior follicle eells begin ta become increasingly columnar. while thase at the anterior begin ta flatten. These eells then migrate posteriorly, while a group of specialized cells, the border cells, squeeze through the nurse cells, and by stage 10, come together to caver the anterior end of the oocyte.

In Bicaudal-C (Bic-C) mutants, oogenesis proceeds nonnally until stage 10, a

establishment of anterior-posterior polarity (Mohier and Wieschaus~ 1986; Ashburner et

al.~ 1990; Schüpbach and Wieschaus~ 1991; Mahone et al., 1995).. Females heterozygous

for Bic-C alleles produce embryos with various anterior-posterior patteming defects,

including bicaudal embryos, and have mislocalizedoskar (osk) and nanas (nos) RNAs at

their anterior end (Mahone et aI.~ 1995). As Bic-C RNA encodes a protein with five RNA-binding domains of the KH type. these findings suggest a role for Bic-C in the localization of posterior RNAs.

1.3 Establishment of Polarity

1.3.1 Anterior-Posterior Axis

Anterior-posterior asymmetry is tirst visible in the gennarium upon oocyte detennination, at which point the oocyte assumes its position at the posterior of the egg chamber. The dicephalic.. armadillo.. spindle-C (spn-C), and home/ess (hls) genes

function in the posterior positioning of the oocyte within the egg chamber~as mutations in these genes resuIt in improper positioning of the oocyte (Lohs-Schardin~ 1982; Peirer etaI.~ 1993; Gonzalez-Reyes and St Johnston, 1994; Gillespie and Berg~ 1995). Inspn-C

mutants, this results in bipolar egg chambers in which bicoid (bcd) mRNA localizes to

both poles of the oocyte, andosk mRNA to the center (GonzaIez-Reyes and St Johnston,

1994). Mutations in h/s~ aIso known as spn-E, give a similar oocyte displacement

putative helicases, and is suggested ta function indirectly in the localization of specifie

RNAs required for embryonic polarity.

Similar to the hls phenotype, maternai effect mutations in 0018 RNA-binding

(orb) produce ventralised eggs and affect the localization of mRNAs in the oocyte

(Chrïsterson and McKearin, 1994; Lantz et al." 1994; Roth and Schüpbach, 1994). The

orbgene is expressed only in the germ Hne and encodes a protein with similarity ta the

RNA recognition motif (RRM) family of RNA or single..stranded nucleic acid-binding

proteins, suggesting it may act as a component of the cellular machinery responsible for

the asymmetric distribution ofmRNAs \vithin the oocyte (Lantz et al., 1992).

Once in its posterior position. the oocyte induces the adjacent follicle cells to

adopt a posterior fate. rather than a default anterior one (Gonzâlez-Reyes and St 10OOston..

1994). These p05terior follicle cells \vill later signal back ta the oocyte, causing a

polarization of its microtubule cytoskeleton" 50 that the minus end lies at the anterior pole

and the plus end at the posterior. This polarized microtubule cytoskeleton then directs the

localization of the anterior and posterior determinants, bicoid (bcd) andosk. respectively.

After fenilization, the bcd transcripts are translated, resulting in the production of an

anterior-to-posterior gradient of BeO protein (Frigerio etal., 1986; Berleth et

aI.,

1988; Driever and Nüsslein-Volhard, 1988; St Johnston etal., 1989).

1.3.2The Anterior Determinant, bcd

(Frigerio et al., 1986; Berleth et al., 1988), and fonns an anterior-to-posterior concentration gradient over the anterior t\vo-thirds of the early embryo. The presence of a homeodomain \vithin the BeD protein suggests it is responsible for the direct regulation ofdownstream zygotic target genes.

One such target is the gap gene, hunchback (hb), which is required for the proper development of the head and thorax (Bender et al., 1987; Lehmann and Nüsslein-Volhard. 1987b; Tautz et al ... 1987). The expression ofhb transcript is dependent upon Bed.. as it does not form in bcd mutant embryos, and its expression expands posteriorly when the gene dosage of bcd is increased (Schroder.. et al .• 1988; Tautz.. 1998; Struhl et aL.. 1989). The 5" region of hunchback cantains three strong and three weak BCD-binding sites (Driever and Nüsslein-Volhard.. 1989a), and can drive the expression of a reporter gene in a BCD-dependent manner. indicating that BeD is a transcriptional activator ofhb

(Schroder, et al., 1988: Struhl et al., 1989; Driever and Nüsslein-Volhard, 1989b). A long poly(A) tail has been found ta be essential for bcd function, as its removal is associated with1055of bcd

RNA.

translation (Lieberfarb et al., 1996).

1.3.3 Dorsal-Ventral Axis

Thegrkgene encodes a transforming gro\vth factor (TGF)-alpha-like protein that

contains an epidermal growth factor (EGF) repeat (Neuman-Silberberg and Schüpbach., 1993). Its rnRNA is specifically localized to the posterior cortex of the oocyte during early stages of oogenesis.. and then upon migration of the oocyte nucleus, transiently localizes to the anterior cortex of the oocyte and becomes restricted to the antero-dorsal corner (Neuman-Silberberg and Schüpbach. 1993). This asymmetric localization will serve as a source of localized GRK protein to activate the overlying foIlicIe eells on the dorsal side (Roth et al.. 1995). The TOPIDER protein is the Drosophila homolog of the EGF receptor.. suggesting that GRK protein binds to TOPIDER in the adjacent foIlicIe cells.. thus activating a signal transduction pathway resuIting in the determination of dorsal fate (Neuman-Silberberg and Schüpbach. 1993; Schüpbach and Wieschaus.. 1991).

1.3.4 grkand top/DER Required for Anterior-Posterior Axis

The tàilure of the oocyte nucleus ta migrate ingrkandtop/DER mutants suggests the involvement of a polarized microtubule network in this process. Eggs laid by grk females often possess a second micropyle at the anterior pole., a structure normally round

al the posterior (Schüpbach, 1987), due to a duplication of anterior follicle cells at the posterior pole of the egg chamber (Gonzâlez-Reyes et al., 1995). Furthermore, kinesin-heta-gal fusion protein, which contains the motor domain of the plus-end-directed microtubule motor kinesin and localizes to the posterior pole of wildtype oocyte,

localizes to the middle of these mutant egg chambers, suggesting thatgrkand top/DER

anterior-dorsal follicie cells are induced, the formation of the anterior-dorsal-ventral axis depends upon the prior polarization of the anterior-posterior axis.

2. Anteroposterior Patterning and Germline Deve/opment

Localization of various RNAs to the oocyte occurs near the MTOC, at the posterior of the oocyte.. suggesting that transport is occurring along microtubules from the nurse cells to the oocyte. In support of this view.. a microtubule-depolymerising drug, colchicine, prevents the accumulation of RNAs to the OOC}1e (Theurkauf et al., 1993; Pokrywka and Stephenson. 1995). Many of these posteriorly-Iocalized factors form a specialized region of c}1oplasm termed the pole plasm, which subsequently \vill fonn the progenitors of the germ line.. the pole cells (Mahowald, 1968; Illmensee and Mahowald,

1974; Okadaetal... 1974).

2.1 Posterior Class of Maternal-Effect Genes

The pole plasm contains t\vo localized signais: the posterior determinant which controls abdominal development and the second signal which contrais the development ofthe pole cells. Mutations

in

genes affecting this posterior group result in abdominal panerning defects and deletions, but the majarity of these genes are also required for the formation of the pole plasm.

abdominal deletions and detècts in pole plasm formation (Boswel1 and Maho\vald~ 1985; Lehmann and Nüsslein-Volhard~ 1986; Schüpbach and Wieschaus~ 1986; Lehmann and Nüsslein-Volhard, 1987a; Manseau and Schüpbach~ 1989; Boswell et al., 1991), thus displaying a grandchildless phenotype. The two remaining genes~ nanas (nos) and pumilio (pum). are required specifically for abdominal formation (Lehmann and

Nüsslein-Volhard~ 1987a. 1991).

2.2 Assembly of the Pole Plasm

The assembly of the pole p[asm occurs in an ordered cascade. such that the initial localization ofosk rnRNA and STAU protein is dependent on the functions of the capu.

spir. and mago genes (Kim-Ha et al., 1991: Ephrussi et al." 1991: St Johnston et al..

1991). The next component ta lacalize is the protein product of the vas gene, which then

recruits TUD pratein, nos mRNA.. and pum mRNA" with each step depending on the function of the preceding genes (Lasko and Ashburner, 1990; Raff et al., 1990; Wang and Lehmann.. 1991; MacDonald, 1992; Bardsley et al.~ 1993). Excluding pum. these genes ail function in abdominal development due ta their role in localizingnos mRNA to the pole plasm~ where it serves as a localized source for a posterior to anteriorNOS protein gradient (Ephrussi and Lehmann~ 1992; Smith et al., 1992; Barker et al., 1992; Wang et al., 1994).

2.2.1 osk isUnd., TranslatlonalControl

point~ its translation is repressed by BRUNO protein~ which binds to bruno response elements (BREs) in theosk3'UTR (Kim-Ha et al., 1995). Upon posterior localizatioo, the

repression of theosk rnRNA is lifted~ and therefore mutations which preventosk mRNA

localization also lack OSK protein as this translational repression is not removed (St

Johnston and Nüsslein-Volhard, 1992; Newmark and Boswell, 1994; Christerson and

McKearin~ 1994). QSK activity is regained when BRUNO-mediated repression is

eliminated. indicating that localization activates translation through the alleviation of

repression.

Two isoforms of OSK protein existe synthesized from two different initiation

codons (Rongo et al., 1995: ~[arkussen et aL 1995). The shorter isotorm is involved in

pole plasm assembly and its levels are greatly reduced in vas mutants (Markussen etai. 1995; Rongo et aL 1995), suggesting a possible role for VAS in the translational

activation ofosk.In addition. OSK has been sho\\'TI both in vitro and through a yeast

two-hybrid analysis to interact with VAS (Breitwieser et al., 1996), an interaction which is

essential for pole plasm assembly.

2.2.2stau Encodes an RNA-binding Protein

Thestau gene encodes an RNA-binding protein which functions in 10caIizingosk

andbcdRNAs to their respective ends of the egg chamber.

In

addition to their abdominal

defects and lack of pole ceIls, mutations instauresult inembryos possessing minor head defects (Schüpbach and Wieschaus, 1986a, 1986b), due to a block in the anterior

results in increased amounts of STAD protein, as \vell as the bcd or osk RNAs,

suggesting that these molecules maye as a camplex (Ferrandon et al., 1994).

2.2.3 VASis a DEAD-box Pratein

The VAS protein is a member of the DEAD·bax family of putaùve RNA

helicases, similar ta eukaryotic initiation factor 4A.. and has been shown.. in vifro. ta

funetian as an ATP-dependent RNA helicase (Lasko and Ashbumer, 1988: Hay et al...

1988; Liang etal... 1994). VAS protein is expressed bath in the nurse cell nuclei and in their cytoplasm as early as germarial region 2a. Later in oogenesis<t it is transparted into

the oacyte and localizes ta the posterior tip (Lasko and Ashburner<t 1990; Hay et al..

1990). The protein remains at the posterior pole until early embryogenesis, \vhen it is

incorporated inta the pole cells.

2.2.4 The PasterforDeterminant, nos

Function of the

nos

gene is critical for specifying the pasterior somalie structures

of the egg (Wang and Lehmann, 1991; Lehmann and Nüsslein-Volhard" 1991; Wharton

and Struhl, 1991). NOS protein prevents the expression of HUNCHBACK (HB) protein

from maternaI hb rnRNA by repressing translation through nanas response elements (NREs) in the hb 3'UTR (Hülskamp et al., 1989; Irish et al., 1989; Struhl, 1989;

Wharton and Struhl, 1991). The HB protein then aets as a morphogen

in

the repression of

abdominal gap genes (Hillskamp et al., 1990, Struhl et al., 1992), thereby allowing their

[n addition to its role in hb represssion during early embryogenesis..

nos

has an eartier role in oogenesis. The

nos

transcript is detectable ooly in females and early embryos, and females mutant for various

nos

alleles lay very fe\\· eggs. Upon doser examination, mutant ovarioles appear to undergo normal oogenesis once it has been initiated, however, few cysts are produced as a result of defective stem cell proliferation (Lehmann and Nüsslein-Volhard.. 1991: Wang etal.. 1994).

pum is a posterior group gene similar to nos.. with its mRNA localized to the pole plasm in early embryos (MacDonald.. 1992; Barker et a1.. 1992). In

pum

mutants..

nos

Introduction

MaternaI mRNAs are deposited in the oocyte and are used at different times

during oogenesis and embryogenesis. For this reason~ it is vital that their translation be regulated such that their expression is activated at appropriate tirnes and in the appropriate places. Consequently, the regulatory mechanisms used for this purpose are of great significance and interest.

The product of the vas gene has been implicated in the translational activation of oskandnos(Markussen et al... 1995; Gavis et al... 1996).. suggesting it may play a role in the translation of additional RNAs. In females homozygous for deletions whose region of overlap uncovers the vas gene~ development of the germ line usually becomes abberant upon oocyte differentiation and a severely reduced production of eggs is obtained~

suggesting that

vas

possesses a role in early oogenesis in addition ta its well-defined function in pole plasm assembly (Lasko andAshbumer~ 1990).

To elueidate the role of

vas

during oogenesis, four

vas

alleles were examined for defects in oogenesis. Attempts were made to discover genes which, \vhen placed inIrons

Materials and Methods

Fly Stocks

Most fly strains used have been previously described: vasPD, Schüpbach and

Wieschaus~ 1986; vasD1 , vasD5, spnC422, spnC660 Tearle and Nüsslein·Volhard, 1987;

VQSLYG2, Rittenhouse and Berg, 1995; Df(2L)A267, Ashburner et al., 1982;

Df(2L)TEl16-G~V18. Lasko and Ashburner~ 1988;orbF3-13, Lantz et al., 1994; grkHK36,

Schüpbach, 1987; arerQB, Schüpbach and Wieschaus~ 1991. Fly stocks were ordered from the Bloomington Stock Center. The \vildtype strain employed was Oregon R.

HoecbstlRhodamine-Phalloidin Staining of Ovaries

Results

vas

aUefes show various phenotypes

Fourvasalleles were examined for oogenic phenotypes: vasPD,vasLYG2,vasD1,

and vasD5. The ovaries of homozygous females were dissected and stained \vith HoechstlRhodamine-PhaIloidin and were examined using confocaI microscopy. The examined aIleles sho\ved a \Vide range of phenotypes. vasD5 appeared phenotypically most severe~ as the ovaries sho\ved an obvious atrophy and consisted mainly of degenerating cysts (Figure 1F). Occasional eysts developed past early stages and often possessed an oocyte mislocalized to the anterior end (Figure Il). vasD1 displayed a less severe phenotype~ although mast egg chambers degenerated upon stage 6 of oogenesis (Figure 1E)~ while egg chambers developing past this stage often had misiocalized oocytes (Figure 11). Older femaIes possessed degenerating ovaries much like those in

vasD5 females. In contrast, the ovaries of vasLYG2 pragress ta later stages, although

Many dicephalic chambers are praduced (Figure 1D). Early chambers display a tumourous phenotype (Figure le). Upon eloser examination using confocal microscapy, further defects are seen, such as egg chambers containing 16 nurse ceIls and no oocyte (Figure 1H). vas

PD

appeared to he the least affected, as the avaries examined appeared normal (Figure 18,0)and produce numerous eggs.

Contribution of anotber locus affectsvasphenotype

oogenic phenotypes. Df(2L)A26ï showed a low hatching frequency (Table 1) when placed in trans to all alleles, as \vell as \vhen placed over a wildtype chromosome. In contrast, Df(2L)TEl16-G~V18 sho\ved

a

relatively high hatching frequency except when placed intrans togrkHK36,in which case the hatching frequency was 66% compared to 79% when placed over the \vildtype chromosome. These results suggest that the reduction in dosage of a gene u.'1covered by the Df(2L)A267 _{deletion contributes to the observed} phenotype. Both deficiencies sho\ved no hatching when placed over various vas alleles (Table 1).

Table 1: Hatching frequencies in eggs producedbyvasdeficiencies Allele Df(2L)A276 Df(2L)TEI16-GWI8

°IctHatched (total eggs) %Hatched (total eggs)

Discussion

To determine the l'ole ofvas during oogenesis~ \ve examined various vas alleles for defects during this process. The examined alleles sho\v a wide range of phenotypes of

differing severity, suggesting that trace amounts of VAS are often sufficient for the

completion of oogenesis. The vasD5 mutation has been found to be a nucleotide

substitution ofGG.I\ by GA.A. in the 425 amino acid stretch \vhich is highly conserved among DEAD-box proteins, resulting in the loss of RNA-unwinding ability (Liang et aL..

1994). The mutations in vasDJ and l,'asPD remain unknown, although no amino acid

substitutions have been found in their coding region (Liang et al., 1994), and vasLYG2

results from a P..element insertion which we mapped to the tirst intron (Chapter 3, Figure

1). As such a wide range of phenotypes is seen ana these alleles still produce varying

amounts of VAS protein, it appears that small amounts of VAS are sufficient to allow for

completion of oogenesis.

A further attempt was made to discover genes which genetically interacted with

\·as.Two vasdeficiencies, Df(2L)A26ïand Df(2L)TEl16-GW/8,were placed in iransto

various alleles of genes possessing oogenic phenotypes. Varying results were obtained,

suggesting that another gene uncovered by the deletion was contributing to the witnessed

phenotypes. Taken together, these results confound any attempts to detennine the l'ole of

vasduring oogenesis, as no allele was a true null allele ofvas.

In

order to determine the

Figures

Figure 1.Homozygous avaries ofvas alleles stained\vithHoechst(A·F) and

Chapter Three :

vas is required for GRK

accumulation in the oocyte, and is involved in

oocyte differentiation and germ line cyst

Summary

TheDrosophila genevasa is required for pole plasm assembly and function, and aIso for

compLetion of oogenesis. To investigate the raIe ofvasa in oocyte development, we generated a ne\v null mutation ofvasa, which deletes the entire coding region. Analysis

ofvasa-null ovaries revealed that the gene is involved in the growth of germ-Hne cysts.

[n vasa-null ovaries, gennaria are atrophied.. and contain far fewer developing cysts than

do \vild-type germaria; a phenotype similar to, but less severe than, that of a null nanas

allele. The null mutant also revealed roles for vasa in oocyte ditTerentiation.. anterior-posterior egg chamber panerning, and dorsal-ventral follicle patteming, in addition to its bener-characterized functions in posterior embryonic patterning and pole cell specification. The anterior-posterior and dorsal-ventral paneming phenotypes resemble those observed ingurken mutants. vasa-null 00C)1eS fail to efficiently accumulate many localized RNAs, such as Bicaudal-D, orb. oskar. and nanas, but still accumulate gurken

RNA. However.. GRK. accumulation in the oocyte is severely reduced in the absence of

Introduction

Segregation of the germ line from the soma is a central feature of animal development. In

Drosophila..

the germ [ine is determined through the activities of matemally-expressed RNAs and proteins \vhich colocalize in the pole plasm at the posterior pole of the egg (reviewed in Rongo and Lehmann, 1996). Pole cells, the progenitors of the gcrm line.. form very early in embryogenesis, then, beginning at gastrulation.. they migrate into the interior of the embryo and ultimately associate with the gonadal mesodermto form the embryonic gonads (reviewed in Williamson and Lehmann.. 1996). Beginning in larval development. germ cells proliferate and differentiate in arder to carry out spermatogenesis and oogenesis; among the structures assembled during oogenesis is ne\v pole plasm.. which specifies the germ line 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 rnRNAs and/or proteins which localize in pole plasm or in polar granules, specialized organelles contained within the pole plasm (reviewed in Rongo and Lehmann, 1996). Analysis of

factor eIF4A (Hay, et al., 1988; Lasko and Ashburner~ 1988; Liang, et al.~ 1994), is a candidate germ-line-specifie translational regulator. For instance, levels of the short

isofonn of OSKAR protein (OSK), a molecule central to pole plasm assembly (Ephrussi etal.~ 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 nanas (nos), asnos R.l\l.~carrying an intact translational regulation element in its 3'UTR is completely inactive in embryos derived from vasmutant ovaries (Gavis, etal.~ 1996; Dahanukar and Wharton, 1996).

While the activities of pole plasm components such as VAS have been most thoroughly studied \vith respect ta their function in pole cell formation and specification of the posteriorsoma~clearly sorne genes involved in pole plasm assembly aIso function in other stages of genn line development. Forinstance~females homozygous for either of t\vo strongnos aHeles exhibit defects in germ cell proliferation (Lehmann and Nüsslein-Volhard, 1991; Wang, et al.~ 1994). Furthermore~ pole cells lacking maternai

nos

function fail to complete migration and do not associate with the embryonic gonadai mesoderm (Kobayashi, et al., 1996), indicating a raIe fornos in the transition from pole cell to functional germ cell. Similarly, various vas alleles have defects in oogenesis and

lay few or no eggs (Lasko and Ashbumer, 1988, 1990; Lehmann and Nüsslein-Volhard, 1991; Schüpbach and Wieschaus, 1991). Females trans-heterozygous forDf(2L)A267and

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 ofvas

did not exist. Four EMS..induced alleles ofvas~ vasDl~ vasQ6~ vasQ7, and vasD5,also lead to greatly reduced fertility~ with many egg chambers blocked as for the trans..

heterozygous deficiency females (Lehmann and Nüsslein..Volhard~ 1991). The feweggs producedby 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 ofvas function is unknown. vasQ6and l:asD5are missense mutations \vhich alter single amino acids of VAS and both aIleles produce substantial amounts of mutant protein (Liang, et aL, (994), so neither of these mutations is likely to be null. For vas

D

! and vasQ7 the molecular nature of the mutation is unknown. but the vas coding region is unaffected in these mutant alleles.

[n this paper, we have used a new vas null allele, vasPH165, a small deletion

which \ve generated by imprecise P-element excision~ to investigate in detail the role of

vasin events of oogenesis prior to pole plasm assembly. We found that abrogation ofvas

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

Materials and Methods

Fly stocks

To create a null allele of vas, excision !ines were generated through the introduction of the

&-3

transposase source into vasP(ry[+}JLYG2 (Rittenhouse and

Berg~

1995). Tado this. +IY; vasP(ry[+])LYG2 en; ry506males were crossed to w;

Bic-d'A66SufB ic-d'A66) cn/CyO: &-3SbITAl3 Ser virgin females. wlY; vasP(ry[+]JLYG2

~6

-cnlCyO:LJ2-3SblryJ males \vere then crossed to +/+;!(2j0508-1P(ry[+]1(2jOJ08-1ICyO:

rySD6 virgin females. Individual ry fI males representing excisionsofvasP(rY[+]JLYG2

Viere then individually crossed to /(2jOS08-1Pfry[+j/(2)0508-1ICyO; rySD6 virgin females. Balancedry stocks were generated and females homozygous for

an

excision chromosome \vere crossed to Oregon

R

males to test for fertility. Excision lines were screened for

deletions thraugh Southem analysis using a 1.9 kbEcoRI genomic fragment from the vas

tirst intron., and which includes the vasP(ry[+]JLYG2 insertion site., as a probe. VAS protein levels

from

excision Hnes were determined by Western analysis.

vasP(ry[+J)LYG2 was provided by Celeste Berg (University of Washington,

(Schüpbach~ 1987) was obtained from Trudi Schüpbach (Princeton University). The

\\ild-type strain employed was Oregon

R.

In silu bybridization and antibody staining

In situ hybridizations with DIG-labeled RNA probes and antibody stainings \vere carried out on ovaries and embryos as described in KobayashL et al. (1998)~ except that DMSO was omitted from the fixation solution used for ovaries. Primary antibodies were used at the following dilutions: a-ORB~ 1:20; a-BIC-O. 1: 10; a-NOS~ 1: 150: a-GRK~

1:3000; a-ADD-87. 1:20. a-NOS was pre-adsorbed \vith embryos trom nosB1Vfemales. and a-GRK \vas preadsorbed with grkHK36 ovaries. For brightfield microscopy, antibody stainings \Vere detected \vith DAB~ enhaneed using the Vectastain ABC orABC

Elite kits (Veetor Laboratories) and biotinylated secondary antibodies. For eonfoeai microscopy, antibody stainings were detected using Texas Red-conjugated secondary antibodies (Molecular Probes). ~-GAL staining ofkhc:lacZ ovaries was carried out as

described in Clark, et al. (1994).

OIiGreenlRhodamine-phalloidin staining

Ovaries were dissected in PBS and fixed for 20 minutes in a mixture of 600

ml

Results

vas'H16Sisa vasnull allele

Identification of additionalvas cDNA clones has indicated the presence of a 127-bp exon upstream of the previously-reported 5' end ofvas, which extends the 5' UTR of

the genet This exon is separated by a large intron of6603 bp from the remainder of the gene (Fig. lA..B; BerkeleyDrosophila Genome Project.. unpublished results, submined to GenBank under accession numbers L81347.. L81348" L81449.. AC000466.. and AC000469). VQSLYG2 is a P-element-induced "'as allele (Rinenhouse and Berg, 1995), which \ve mapped to the large first intron ofvas(Fig. lA)" and which produces about 2% of the \\'ild-type level ofVAS (Fig. 1Cl,a leveI essentially undetectable in tissue staining experiments. Despite the very lo\v level ofVAS in vasLYG2 ovaries its phenotype is hypomorphic; oogenesis in vasLYG2 females is less severely compromised than in

Df(2L)A26ï/Df(2L)TEl16-GfV18 flies, and vasLYG2 females Iay 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 etTects of a reduction in dosage of a second gene which enhances thevasphenotype.

stable Hnes \vere generated from 129. Of these, 119 caused reversion to a wild-type

phenotype~ indicating that the vasLYG2 phenotype results solely from the P-element insertion, and that the vasLYG2 chromosome is free of other female-sterile mutations. Three other derivatives were recessive lethal and seven, although having excised thery+

marker on the P element of vasLYG2, remained defective in oogenesis. These were checked by PCR and Southem blots ta determine whether they carried a deletion contined to the vas gene, and by Western blots using anti-VAS antiserum ta determine whether they expressed VAS. From these analyses, one Hne, vasPHl65, was identified, which produced no detectable VAS protein and carried a 7343-bp deletion \vhich removes the entire coding region ofvas(Fig. lA. C). We confirmed the breakpoints of

vasPH165 by nucleotide sequencing, comparing the mutant sequence to wiId-type

sequence provided by the Berkeley Drosophila Genome Project. From the nature of the

vasPH165 mutation and the fact that no VAS protein is detectable on Western blots even

on long overexposures (Fig. lC), we conclude that vasPHI65 is null for VAS. The

vasPH165deletion is mostIy limited to vas, as one of its breakpoints lies within vasand

the other is 1270 bp downstream of its 3' end. A nested gene Mayhe located within the 3.S-kb third intron of vas, which is deIeted in vasPHl65, as a 3..kb transcript present

throughout aIl developmental stages is detected on Narthem blots using genomic probes inc1uding this intron but not with vas cDNA probes (Lasko and Ashbumer, 1988). However, any gene other than vas which may be disrupted in vasPHJ65 is almast certainly irrelevant ta the discussion below, as a vas-GFP transgene, canstructed fram a

fertility. The phenotypes of vasPHJ65/ vasPHJ65! vasPHJ65/ Dj(2L)A267, and

Dj(2L)A267/ Dj(2L) TEl 16-GfVJ8 ovaries are essentiaIly identical.

VAS is involved in the maintenance of germ-line cysts

Upon cursory examination ofvasPH165 avaries under the light microscope, we noticed an obvious atrophy of the germaria as compared with the wild-type, suggesting

that fe\\'er germ cells were present. To investigate this more closely, \ve used an antibody

recognizing ADD-87 protein (Lin.. et al .. 1994; laccai and Lipshi~ 1996) which stains spectrosomes and fusomes. specifie structures present in stem cells. cystoblasts. and

dividing eystocyre clusters. In vasPHI65avaries. a reduction in the number of

ADD-8?-staining structures, and therefore a reduction in the number of developing cysts. is readily

apparent as compared with \vild-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

PH

165 females. region 1 frequently consists of only a few stem cells and developing cysts. Posterior to these is often what appears ta be an extended

interfollicular stalk (compare Fig. 2D with Fig. 2e), likely fonned by follicie celis in the absence of eystocyte clusters. This suggests that the cystocyte clusters remaining in the

germarium have ceased ta develop further and have degenerated, while the follicie cells

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 germarium. pum mutants produce ovarioles \vhich contain only two or three clusters of undifferentiated germ cells

which Jack spectrosome/fusome structures or ring canals (Lin and Spradling, 1997), a phenotype we never observed invasmutants. Upan examination ofnos

RC

mutant avaries with the a-ADD-87 antibody. we observed a phenotype which appears similar, but some\vhat more severe.. than that of vasPH165. Many nosRC ovarioles consist of a germarium \vith one ta three cysts (Fig. 2E), follo\ved by an extended stalk and one to three normaJ-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 germ line cells have arrested development.

VAS is involved in oocyte differentiation

At a low frequency (-1% for each), we observed defects in germ Hne differentiation and oocyte determination in vasPHJ65 ovaries, including tumorous egg chambers (Fig. 3A), egg chambers with 16 nurse cells and no oocyte, athers with t\VO oocytes, and again others with a misJocaJized oocyte (Fig. 3B-D). Far more frequently the normaJ 15 nurse cells and one oocyte are present; ho\vever, by at Ieast two criteria the oocytes produced in vasPHl65 egg chambers are not fully differentiated. In wild..type

oocyte nucleus appears more diffuse than do wild-type oocyte nuclei (Fig. 3E,f). This \vould 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 invasPH165oocytes. A very similar nuclear morphology has been observed in

spind/e (spn) mutant oocytes, \vhich has been interpreted as resulting from a delay in

oocyte determination (Gonzalez-Reyes, et al., 1997).

Secondly, VQSPH165 oocytes do not efficiently accumulate at least four

oocyte-localized RNAs. In wild-type ovaries. the Bicaudal-D. orb, osk.. and nos RNAs aIl

accumulate efficiently to the ooc)i1e within the germarium and remain concentrated

therein throughout the early stages of oogenesis (Ephrussi.. et al., 1991~ Kim-Ha.. et al.,

1991: Suter and Steward.. 1991: Lantz.. et aL. 1992; Wang, et al... 1994; Fig.

~A..B..D..EJ.J,M). Oocyte accumulation of these RNAs is much less pronaunced in

"'osPHI65 egg chambers in which Bic-D RNA localizatian is essentially undetectable

(Fig. 4C), and the concentration oforb RNA in the oocyte is onJy slightly above the levels in the nurse ceUs (Fig. 4F). In vasPH165 egg chambers, osk RNA is aIso not

observed to accumulate in the aOC)1e in the germariurn or tirst vitellarial stages of development. Rather,oskRNA tends ta concentrate in a somewhat diffuse manner in the

Many oocyte-specifie RNAs are still translated, and accumulate in vas-null oocytes despite defects in RNA localization

Despite the poor localization oforb RNA, ORB protein accumulates efficiently in

vasPH165oocytes, although somewhat later in oogenesis than in the wild-type (compare

Fig. 4H with Fig. 40). NOS protein expression is aIse broadly similar to \vild-type in

VQSPH165 ovaries. The reduced number of developing cysts in vasPH165 germaria

complicates the analysis. but the peak ofNOS protein expression in 4-to-8-cell cysts at the posterior ofregion 1 (Wang etaL~ 1994) remains apparent in vasPHl65 (Fig. 4P~Q). OSK protein is not detectably translated in early oogenesis in \vild-type norin vasPH165

egg chambers(Kim-Ha~etal... 1995; Rongo.. etal.~ 1995; data not shown).

In wild-type oocytes. BIC-D and EGALITARIAN (EGL) proteins co-Iocalize in the oocyte cytoplasm~ in a posterior crescent from stages 2-7 (Suter and Steward~ 1991; Mach and Lehmann~ 1997; Fig. 5A.. C and D). In vasPH165, BIC-O and EGL are efficient1y translated and are localized to the oocyte. Ho\vever, the (wo proteins are tightly concentrated in a focus which, in higher magnification in both brightfield and confocal microscopy, appears to be adjacent ta the oocyte nucleus (Fig. 58, E, and data not sho\\'n).

var

HI6S

Most

vasPH165 egg chambers develop into germ-line cysts containing 15 nurse

cells and a posteriorly-Iocalized oocyte. These appear normal until about stage 6, whereupon aIl the nuclei undergo pycnosis and further developrnent ceases (Fig. 6A). Approximately 70% of aIl vasPH165 egg chambers examined terminate development in this manner. A minority (-200/0) ofvasPH165 oocytes continue developing beyond stage 6; most of these are blocked at stage 9-10, after which the oocyte often loses its integrity al the anterior end. and nurse cell nuclei invade the region of the egg chamber normally occupied only by the oocyte (Fig. 68). Finally, we found that a smaIl number of oocytes produced by vas

PH

165 females complete oogenesis. but that these frequently have duplicated micropyles at bath the anteriar and posterior ends (Fig. 6C). These eggs also often have dorsal appendage defects consistent \vith ventralizatian of the chorion: of 73 eggs analyzed.. 14 (19%) lacked dorsal appendages; 48 (660/0) had a single fused dorsal appendage; and only Il (15%) had two dorsal appendages.

vasactivity is required for efficient accumulation ofGRK protein

The phenotypes of late-stage

vasPH165 oocytes suggest that the GRK signalling

pathway (GonzaIez-Reyes, et al., 1995; Roth, et al., 1995) may he inactive in these avaries., and led us to investigate the expression and distribution of

grk

RNA and protein

in vasPH165. Unlike Bic..D, orb, osk., and nos RNA, localization of grk RNA to the

area in

vasPH165

oocytes (Fig. 7B), similar to the BIC-DIEGL localization pattern in these oocytes (Fig.

5).

Later in oogenesis, grk

RNA

becomes anteriorly localized in both wild-type and

vasPH

165 oocytes, although in the mutant its distribution may extend funher ventrally(Fig. 7e and D).

Despite the relatively normal accumulation ofgrkR.NAinl'as?H165oocytes, and unlike the other proteins discussed above. the effects of a loss of vas activity are striking

\'~ith regard ta GRK protein accumulation; essentially no localized GRK is observed in

vasPHJ65oocytes (Fig. 7E and F). Furthermore.. as measured on Western blots.. the level

ofGRKprotein is greatly reduced in

vasPH

J65ovaries as compared with wild-type (Fig. 7G). Upon overexposure of such Western blots a smal1 amount ofGRKcan hedetected in both grkHK36 and vasPH165 extracts (Neuman-Silberberg and Schüpbach.. 1996; data

not

shawn). As grk is required for specification of dorsal and posterior follicle structures, the duplicated micropyles and dorsal appendage defects found in

vasPHJ65

eggs (and in eggs produced by other vas aIleles; Lehmann and Nüsslein-Volhard, 1991; Schüphach and Wieschaus, 1991) are likely to be caused by the reduced level of GRK in

vas

mutants. These results also suggest that VAS may activate GRK translation in wild-type

oocytes.

We examined the organization of

the

microtubule cytoskeleton in

vasPHJ65

oocytes using

a

khc:lacZ

reporter gene construct (Clark, et al., 1994). In

the

minority of

Discussion

Partially redundant roles for VAS in early oogenesis

We have isolated a

Dull

allele ofvas, vasPH165, and its analysis reveaIs that VAS

is involved in many stages of oogenesis, including cystocyte differentiation, oocyte differentiation, and specification of anterior-posterior polarity in the developing cysts. Ho\vever. the phenotype ofvasPH165 is inconsistent \vith a total failure of any of these processes. indicating that VAS has a partially redundant role in bringing them about. Sorne vas-null egg chambers. namely those with (\VO oocytes or \vith mislocalized

OOC)1eS (Fig. 3e.D) are similar to those produced in spn-A. spn-B, spn-C.. spn-D, and

homeless (spn-E) mutants (GonzaIez-Reyes and St Johnston.. 1994: Gillespie and Berg,

1995~ Gonzalez-Reyes et al... 1997). Furthermore, the vas-null oocyte nucleus appears to

he very similar to those observed inspn mutants (Fig. 3F.. Gonzalez-Reyes et aL, 1997),

and defects in anterodorsal accumulation ofgrk mRNA in stage 9 have aIso been reponed

forspn mutants (Gillespie and Berg, 1995; Gonzâ1ez-Reyes etal., 1997). It is likely that

l'as and the spn genes have overlapping functions in oocyte determination, and it is

note\\"onhy in this context that homeless, which, like vas, is required in the genn line,

encodes a DE-H-box protein somewhat related toVAS. A role for thespn gene praducts

in translational activation of grk has been suggested (GonzaIez-Reyes et al., 1997). Construction of multiple mutants withvasPH165 and spn mutations will be important in

vasand nos are bath required for germ line cyst development

We have observed a decrease in the number of developing germ line cysts in

vas

PH165 avaries, and a similar, but more severe, phenotype occurs in ovaries from

femaies null fornos function (Fig. 2; Wang, et al., 1994; Curtis, et al., 1997). Because of

the similarities between this aspect of the ltaS and nos mutant phenotypes, and because VAS has been implicated in transIational activation ofnos in the pole pIasm (Gavis~ et

al.~ 1996; Dahanukar and Wharton~ 1996), it is tempting to speculate that VAS and NOS

functian in the same path\\'ay ta promote germ line cyst deveIopment, and that VAS ma)' activate (but nat be absalutely essential for) translation of NOS in this stage of germ line development as weIl. Our immunostaining for NOS neither supports nor refutes this possibility. While we see no obvious Iack of NOS in vasPH165germaria (Fig. 4Q), it is presently impossible to detect differences in NOS level in stem cells or cystoblasts.. as even in \vild-type avaries these cells are not detectabIy stained with NOS antibody (Wang, etal.~ 1994; Fig. 4Q). The higher Ievel of NOS in 4-8 cell cysts, which appears to be unaffected by vasPH165~ would not he responsible for cyst development't as it occurs at a later developmental stage than that at which the cysts are blocked innos

RC

ovaries.

The reduction in the number of developing germ Hne cysts we observe in

vas

and

nos

null mutants could resuIt from a failure of the pole cells to migrate ta the gonad

possibilities~\ve do not think the defect involves pole cell migration, as we never observe completely agametic avaries in vas or nos mutants. Agametic ovaries are observed even

in cases \vhere pole cell migration is incompletely blocked, such as in the progeny of

AS-Pgc

females (Nakamura, et al., 1996). Direct assessment of the role of zygotic VAS in pole cell migration is confounded by the fact that maternai VAS perdures until embryonic stage 15-16~ long after pole cell migration is complete.

Different threshold levels of VAS are required for its various activities

In wild-type tlies. vas is abundantly expressed in the female germ line~ and.. while VAS is coneentrated at the posterior pole of the oocyte from stage 10 ofoog~nesis.. easily deteetable levels of VAS are present uniformly throughout the early embryo (Lasko and

Ashbumer~ 1990). These uniforrn levels are sufficient for ectopie posterior somatie structures to develop in circumstances where OSK is present outside of the pole plasm, for instance in females overexpressingosk or earrying the P[osIcBRE-] transgene (Smith,

et al., 1992; Kim-Ha.. et al.., 1995). For embryos produced by females earrying aBic-D

number of eggs than do vas

PHI

65 homozygotes, and sho\v defects in oogenesis at lo\ver penetrance and of less severity than for the null (S. S. and P. L., unpublished

observations).

RNA localization is dispensable for localizing proteins to the early oocyte

In vasPH165 avaries, grk RNA and the BIC-DIEGL complex still localize

efficiently to the oocyte~ but these molecules are concentrated in a tight focus rather than being distributed more \videly in the oocyte cytoplasm. This suggests that in wild-type egg chambers, loealization of these molecules may occur in two steps: a vas-independent step, \vhich coneentrates them in the oocyte. and a second step \vhich distributes them to a specifie region \vithin the oocyte, which depends direetly or indirectly on vas activity. i\lternatively, loss of vas function results in the formation of a nover structure or structures in the oocyte cytoplasm which trap grk RNA and the BIC-DIEGL complex. The distribution of BIC-D and EGL is unaffected ingrkHK36 ovaries~ indicating that the

abnormaI loealization ofthese two proteins invasPH165 oocytes is not a consequence of the failure to accumulate GRK in vasPH165(data not sho\\'n). We have found that

vas-null mutations affect sorne of the same processes as do Bic-Dandeglmutations; namely, oocyte determination (in rare 16-nurse-cell and t\Vo-oocyte egg chambers) and establishment of dorsal-ventral polarity (Swan and Suter, 1996; Mach and Lehmann~

1997; this paper). [n this context, it is possible that these vas phenotypes are a result of the altered distribution of BIC-DIEGL we observed. The altered distribution ofgrkRNA within the early oocyte is similar ta that abserved in maelstrom (mael) mutants whieh

affect the organization of oocyte microtubules (Clegg, etal., 1997). However, unIike in

oocytes (Fig. 7) suggests that the organization of the microtubule cytoskeleton is at least

partiaIlymaintained.

Is VAS a direct activator ofGRK translation?

genes aubergine and encore have been implicated as essential for efficient GRK

accumulation (Wilson't et al., 1996; Ha\vkins, et al., 1997).

Reduction of GRK activity explains only a subset ofvas phenotypes

Duplication of anterior vitelline membrane structures occurs in strong grk mutant

alleles. and GRK signalling has been implicated in the specification of posterior polar follicIe cells (Gonzâlez-Reyes. et al.. 1995; Roth. et al... 1995). We demonstrate in this paper that many of the oocytes that complete oogenesis in a loss-of..function vas mutant have duplicated micropyles and/or fused or reduced dorsal appendages.. and four other\'as

alleles (PfV72.. QSlï. AQB3. RG53) have previously been shown to produce eggs \'fith fused and reduced dorsal appendages when hemizygous over Df(2L)osp29 (Schüpbach and Wieschaus, 1991). AIl of these phenotypes resemble those observed ingrk mutants,

making it likely that these aspects of the vas mutant phenotype are brought about by a

reduction of GRK activity, particularly since \ve show that GRK accumulation is negligible in vas-null oocytes. However, aspects of the vas-nulI phenotype which

manifest themselves earlier in oogenesis, such as the reduced number of developing cysts, the defects in RNA localization to the oocyte, and the aberrant localization of BIC-O and EGL \vithin the oocyte, are not found in grk mutant ovaries (Fig. 2; S.S. and P.L.,

involved in establishing cytoskeletal polarity in the early cyst and/or in localizing RN.~s

Addendum

In addition to the RNA stainings described in the Results of Chapter 3, in situ RNA hybridizations were performed for bcd. egl and enc (Figure 8A-F). In wildtype, bcd RNA localizes to the oocyte during early oogenesis, and then moves to the anterior of the oocyte (Figure 8A). In later stage vasPH165 egg chambers with nurse cells remaining on either side of the oocyte, the bcd RNA is seen to localize to both ends of the egg chamber (Figure 8B). Egg chambers possessing a properly-localized oocyte show the wildtype bcd localization pattern, although localization appears somewhat weaker (data not shown).

egl RNA localizes to the oocyte and is concentrated at the posterior cortex unti 1

stage 7, at which point it is localized in an anterior ring at the nurse cell-oocyte boundary

(~fach and Lehmann, 1997: Figure SC). In the vas null, the transcript localizes in much the same manner. however, it remains somewhat diffuse (Figure 80), consistent with the localization of various other RNAs shown in Figure 4.

The \vildtype

enc

RNA colocalizes with grk (Hawkins et al., 1996; Figure SE), however, unlike grk RNA.. its localization pattern is not weil maintained in vasPHJ65 (Figure 8F). Instead, the 10calization of enc is rather faint and diffuse.

and reflect the wildtype localization pattern (Figure 8G-J). Occasionally, ENC is seen to accumulate in a tight focus, much like the localization of BIC-D and EGL (Figure SH). Again, this suggests that vas may be involved in distributing these proteins within the oocyte.. or 10ss of vas function may result in the formation of a structure in the oocyte

cytoplasm which binds various proteins. The localization of ENC is normal in grkHK36

oocytes (data not shown). indicating that the abnormal distribution of this protein in

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CACTAGATTT TTGGTACTTT TAACAGATCC TTTTTGGTTT 40

TGCGTTGCGC GAAGTGATCT GAACTTATCA AAGTTTGTAA 80 GGTAATACAT AAAGTGAAAA AGAATTAATT TGCTCTTGAA 120 AGGCAGCfCA AATTAAAAAA AAATATCAAT ATG • . . . 153

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~~---Figures

Figure l. A) Organization of the vas genet Exons (boxes) are numbered \vith Roman numerals (I-VIII)~ the translationaI start codon is in exon II; E, EcoRI site. The

. f th P{ + LYG2}. . . . d d th· . . .

site 0 e ry, vas Insertion IS plcture , an e insertion site IS

immediately after position 75581 in the BOGP Pl clone 0800929 (GenBank accession number AC002502). vas

PH16j results from an imprecise excision of

P{ry+, vasLYG2 }. 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\"as. The tirst 127 nucleotides comprise exon 1.

the 6603-bp tirst intron tàlIows 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 blct probed with a-VAS antiserum to compare the levels of VAS in

•

Figure 2. Confocai micrographs of germaria from A) \vild-type, D) grkHK36/grkHK36,

C) four-day-oid vasPH165/ vasPH165, D) seven-day-oid vasPH165/ vasPH165,

and E,F) one-to-two-day oid nosRCI nosRC avaries stained for the ADD-87

protein, which marks spectrosome and fusome structures and which is diagnostic for stem cells and developing germ-line cysts. Substantially fewer foci of ADD-87 are ùbserved in aIl vasPH165 ornosRC germaria as compared with the

wild-type or Similar phenotypes \vere observed in

Figure 3. Phenotypes ofvas-null egg chambers atTecting oocyte determination. Confocai micro-graphs of A-D) vasPH165/ vasPH165ovaries stained with the nuclear dye Oli-Green (Molecular Probes), illustrating the follo\ving phenotypes: A)

tumorous germ-Hne cyst~ B) 16 nurse cells and no oocyte. The 16 polyploid nuclei are numbered in this panel. C) vas

PH

165/vasP

H

165egg chamber \vith two oocytes, stained for F-actin \vith Texas Red-phalloidin (Molecular Probes).

0) vasPH165/Df(2L}A267 egg chamber doubly stained with Oli-Green and ~ith

Texas Red-phalloidin.. illustrating a bipolar egg chamber \vith ilS oocyte in the center. We aise observed bipolar egg chambers in vasPH165/ vasPH165ovaries.