symmetry breaking during drosophila...

Symmetry Breaking During DrosophilaOogenesis

Siegfried Roth and Jeremy A. Lynch

Institute of Developmental Biology, University of Cologne, Gyrhofstr. 17,D-50923 Cologne, Germany

Correspondence: [email protected]

The orthogonal axes of Drosophila are established during oogenesis through a hierarchicalseries of symmetry-breaking steps, most of which can be traced back to asymmetriesinherent in the architecture of the ovary. Oogenesis begins with the formation of a germ-line cyst of 16 cells connected by ring canals. Two of these 16 cells have four ring canals,whereas the others have fewer. The first symmetry-breaking step is the selection of one ofthese two cells to become the oocyte. Subsequently, the germline cyst becomes sur-rounded by somatic follicle cells to generate individual egg chambers. The secondsymmetry-breaking step is the posterior positioning of the oocyte within the egg chamber,a process mediated by adhesive interactions with a special group of somatic cells.Posterior oocyte positioning is accompanied by a par gene-dependent repolarization ofthe microtubule network, which establishes the posterior cortex of the oocyte. The nexttwo steps of symmetry breaking occur during midoogenesis after the volume of theoocyte has increased about 10-fold. First, a signal from the oocyte specifies posteriorfollicle cells, polarizing a symmetric prepattern present within the follicular epithelium.Second, the posterior follicle cells send a signal back to the oocyte, which leads to asecond repolarization of the oocyte microtubule network and the asymmetric migrationof the oocyte nucleus. This process again requires the par genes. The repolarization ofthe microtubule network results in the transport of bicoid and oskar mRNAs, the anteriorand posterior determinants, respectively, of the embryonic axis, to opposite poles of theoocyte. The asymmetric positioning of the oocyte nucleus defines a cortical region ofthe oocyte where gurken mRNA is localized, thus breaking the dorsal–ventral symmetryof the egg and embryo.

In Drosophila, the symmetry-breaking eventsthat establish both the anterior–posterior

(AP) and dorsal–ventral (DV) axes are com-pleted at midstage of oogenesis, long beforefertilization and egg deposition (Roth 2003).Ovarian development in Drosophila is tractable

to extensive genetic screening and geneticmosaic analysis. All stages of ovarian develop-ment starting with the divisions of germlineand somatic stem cells are accessible to in-depthmicroscopic analyses and some of themare amenable to live imaging. Furthermore,

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oogenesis in Drosophila is a slow process (10 d)in comparison to embryogenesis (1 d), allowingone to follow single steps of polarity establish-ment with great spatiotemporal resolution(Spradling 1993).

The term symmetry breaking as it will beused in the following requires some specifica-tion. All the events that are described in thisarticle, with one possible exception (oocytenucleus migration, see the following discus-sion), can be traced back to prior asymmetries.Thus, strictly speaking, they do not representsymmetry-breaking processes. However, theegg chambers or eggs that are produced inabsence of any one of these processes areindeed symmetric or lack at least one majoraxis of polarity (Fig. 1). Thus, when using theterm “symmetry breaking,” we refer to processesthat when absent result in symmetry, ratherthan to processes that establish the initialanatomical asymmetries that are presentthroughout oogenesis. The described eventsfall into two categories. Either they enhanceweak existing asymmetries or they representinductive processes transferring asymmetriesestablished within one group of cells toanother group of cells. In any case, if theprocess does not take place, existing spatialinformation is lost and the system collapsesinto symmetry.

OUTLINE OF DROSOPHILA OOGENESIS

In Drosophila, each of the two ovaries is com-posed of 16–20 independent strings of eggchambers called ovarioles (Spradling 1993).The ovarioles are the functional units of eggproduction (Fig. 2). At a given time, each ovar-iole typically contains six to seven sequen-tially more mature egg chambers (also calledfollicles) connected by somatic stalk cells. Eggchamber development has been divided into14 stages; we will refer to stages 1–6 as early,stages 7–10 as mid, and stages 11–14 as lateoogenesis. The ovariole is essentially a tubelikestructure with a long axis formed by the stringof connected egg chambers and perpendicularto it, a short axis (defining the plane of a crosssection through an egg chamber). These twoaxes of the ovariole correspond to the futureAP (long) axis and DV (short) axis of the eggand embryo.

The anterior tip of each ovariole iscomposed of the germarium, a structure thatharbors the germline and somatic stem cells(Fig. 2). The egg chambers are assembled atthe posterior end of the germarium. A single eggchamber contains a cluster of 16 germ cells,which are connected by cytoplasmic bridges,called ring canals. Only one of the 16 germcells develops as an oocyte, whereas the remain-ing 15 become nurse cells. Oocyte growth

Ventral

Dorsal

Bicoid OskarNucleus

Dorsalappendage

MicropylePosteriorAnterior

WT

gurken

Figure 1. Loss of polarity along both main-body axes in gurken mutants. Left: egg shell preparation. Right:schematic drawing of eggs showing the orientation of the main body axes, the localization of bicoid and oskarmRNA and the oocyte nucleus.

S. Roth and J.A. Lynch

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depends on the nurse cells, which become poly-ploid and transport mRNAs, proteins, andendomembrane structures (e.g., ER and Golgi)via the ring canals into the oocyte. The nursecell–oocyte cluster is surrounded by a mono-layer epithelium of somatic follicle cells, whichplay a key role in axis determination.

During midstages of Drosophila oogenesis,the future embryonic axes are specified by thelocalization of three messenger RNAs withinthe oocyte (van Eeden and St Johnston 1999;Riechmann and Ephrussi 2001) (Fig. 2). bicoidmRNA is localized to the anterior pole ofthe oocyte facing the nurse cells, whereas oskarmRNA is localized to the opposite, posteriorpole. The polar localization of these twomRNAs is maintained throughout the rest ofoogenesis and well into early embryogenesis inwhich bicoid specifies anterior (head and thorax)and oskar posterior (abdomen) regions ofthe embryonic AP axis (St Johnston andNusslein-Volhard 1992). Concomitantly withthe localization of bicoid and oskar, a thirdmRNA, encoded by gurken, accumulates in ananterior–dorsal position of the oocyte andthereby defines the DV axis of the egg. gurkenmRNA localization, however, is only transientand acts indirectly through a signaling processinvolving the follicle cells to determine theembryonic DV axis (Roth 2003; Moussianand Roth 2005). These three types of RNA

localization are the key events that lead toembryonic axis formation. Because they nor-mally take place in a stereotypic manner withregard to the ovariole architecture, one mayask which processes sense the ovariole architec-ture causing the reproducible localization oftheses mRNAs. It turns out that, by far, themost complex series of events is required toestablish AP polarity, whereas DV polarityappears to be a necessary consequence of thefinal step of AP polarization.

PRELUDE: CYST FORMATION AND THEFUSOME: GENERATING A GRADIENT OFPOLARITY BY ORIENTED ASYMMETRICDIVISIONS

The germline stem cells (GSC) at the anteriortip of the germarium undergo asymmetric divi-sions, producing one self-renewing GSC andone cell, the cystoblast, which starts differen-tiating (Fuller and Spradling 2007). It under-goes four incomplete mitotic divisions, whichproduce a cyst of 16 cells (called cystocytes)interconnected by ring canals. The divisionsare oriented, leading to a stereotypic patternof cell–cell connections: Two cells of the cysthave four, two have three, four have two, andeight have one ring canal (Spradling 1993).One of the two cells with four ring canals, alsoreferred to as pro-oocytes, will assume the

Germarium

Germlinestem cell

1 2a 2b 3 = st 1Nurse cells

Oocytenucleus

+++++

–––

––

–

Follicle cells

bcd mRNA

osk mRNA

grk mRNAFollicle

stem cell

st 2 st 4 st 6 st 8 st 9 st 10A

Figure 2. The ovariole. Top: schematic drawing of ovariole with the germarium at the anterior tip and eggchambers of increasing age. Bottom: Magnified view of germarium and stage 9 egg chamber.

Symmetry Breaking During Drosophila Oogenesis

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oocyte fate (Huynh and St Johnston 2004).Judging from the pattern of cell connections,the cyst is apparently symmetric with bothpro-oocytes, having an equal chance to assumethe oocyte fate. Moreover, all cystocytes enterprophase of meiosis I, but a synaptonemalcomplex forms only in both cells with fourring canals and to a lesser degree in the twocells with three ring canals (Carpenter 1975).Nevertheless, only the future oocyte will com-plete meiosis, whereas the other cystocytesleave the meiotic program and eventually startthe endocycles characteristic for the nursecells. These observations, together with mutantanalyses (Theurkauf et al. 1993; Huynh and StJohnston 2004), suggest that a dynamic compe-tition process takes place in which only one ofthe two pro-oocytes normally wins. Thus,the selection of one of the two cells with fourring canals looks like a typical example for asymmetry-breaking event.

A closer look at cyst formation, however,has revealed that the future oocyte appearsto be selected already at the first incompletedivision (the cystoblast division) (de Cuevasand Spradling 1998). Moreover, cyst forma-tion is accompanied by repeated cell rearrange-ments, which establish intrinsic AP polaritywithin the germline cluster and even withinthe future oocyte (de Cuevas and Spradling1998).

All of these features depend on the for-mation of the fusome, a membranous branchedstructure that runs through the cytoplasmicbridges and connects all cystocytes (Lin andSpradling 1995). In Drosophila oogenesis, thefusome is derived from a spherical structurepresent within the GSC, called the spectrosome,which is composed of membranous vesiclesand components of the submembranous cyto-skeleton, like a- and b-spectrin, ankyrin, andthe adducin-like protein Hu-li tai shao (Hts)(Yue and Spradling 1992; Lin et al. 1994;Snapp et al. 2004; Roper 2007; Lighthouseet al. 2008). During GSC division, one third ofthe spectrosome is inherited by the cystoblastand gives rise to the fusome.

The fusome orients the cystoblast and cysto-cyte divisions, because during metaphase, one

pole of the mitotic spindle is attached to thefusome (Deng and Lin 1997; McGrail andHays 1997; Grieder et al. 2000). This leads toan asymmetric division as the old fusomealways resides in one of the daughter cells,whereas the other daughter cell initially lacksfusome material (Fig. 3A). During inter-phase, however, new fusome material formswithin the cytoplasmic bridge, connecting thetwo daughter cells. The old and the newlyformed fusome of the cytoplasmic bridgeare subsequently brought into close contactand eventually fuse, resulting in the direc-tional growth and branching of the fusome (inFig. 3A, this is depicted for the second cystocytedivision).

The juxtaposition of old and newly syn-thesized fusome material is brought about bya movement of the cytoplasmic bridges towardeach other, which also moves the cystocytescloser to each other, causing the clustering ofthe later formed cystocytes at one side of thecyst. Thus, the resulting cyst structure is polar-ized (rosette shape) with the cells with fewerring canals at one and the two cells with fourring canals at the opposite site of the cyst(de Cuevas and Spradling 1998; Grieder et al.2000). Moreover, the two cells with four ringcanals are intrinsically polarized because theirring canals cluster at one side. Because thisarrangement is stabilized by adherens junctionsand maintained throughout oogenesis, it is pos-sible to predict that the part of the cell with theclustered ring canals will become the anteriorpole (Godt and Tepass 1998; Gonzalez-Reyesand St Johnston 1998a). One cell with fourring canals has the largest piece of fusomematerial going back to the fusome initiallypresent in the cystoblast (de Cuevas andSpradling 1998; Grieder et al. 2000). This cellapparently assumes the oocyte fate. Becausethe fusome is a transient structure, which dis-assembles before obvious signs of oocyte differ-entiation (the stable accumulation of oocytedeterminants, restriction of meiosis), this con-clusion is not easy to draw. Currently, it isbased on the preferential accumulation ofsome RNAs and the behavior of the centro-somes (see the following section).





OOCYTE DETERMINATION: MAKINGA DISCRETE DECISION THROUGHDIRECTED TRANSPORT

The process of oocyte determination cannot beseparated from that of oocyte differentiation.A disruption of oocyte differentiation, even atlate stages at which the oocyte is clearly dis-cernable, leads to a reversion to the nurse cellfate and the formation of egg chambers with16 nurse cells (Huynh et al. 2001b). Duringoocyte determination, two characteristics ofthe later oocyte are established in parallel.First, the oocyte needs to become the target ofnurse cell–oocyte transport, which is largelymicrotubule-based during early stages ofoogenesis (Fig. 3B). Thus, the MT network

of the cyst has to be polarized such that themajor routes of transport are directed towardthe oocyte. However, the MT-dependent accu-mulation of certain proteins and mRNAs itselfprovides also the major means of oocytedetermination (Theurkauf et al. 1993). Sec-ond, the meiotic program has to be restrictedto the oocyte and the oocyte has to success-fully go through the prophase of meiosis I andcondense its DNA to form the karyosome.Because this review focuses on spatial pattern-ing, the nuclear differentiation of the oocytewill not be pursued in more detail (for review,see Huynh and St Johnston 2004). Withregard to spatial asymmetries, the key questionis how fusome polarity gives rise to a stablepolarization of the MT cytoskeleton.

Ring canal

1 1 1

44

2 22 2

4 1

23

33

OocytePro-oocytes

B

A

Balbiani body

Fusome

Microtubules++

+

+

+

+

+ +

Figure 3. Cyst formation and oocyte determination. (A) Second cystocyte division producing a four-cell cyst. Atmetaphase, one spindle pole attaches to the old fusome. Cystokinesis is incomplete, leaving the cystocytesconnected by ring canals. Within the ring canals, new fusome material forms. The ring canals move towardeach other, leading to clustering of the cystocytes. (B) Oocyte determination. In region 2a of the germarium,the cystocytes are connected by the branched fusome. Both cells with four ring canals (pro-oocytes) showsigns of oocyte specification. Later, the fusome is replaced by a polarized microtubule (MT) networkemanating from one microtubule organizing center (MTOC) that resides in one of the two pro-oocytes. It ispositioned in the region of the Balbiani body, anterior to the oocyte nucleus.





The MT polarization of the cyst emergesgradually after the last cystocyte division. Theminus ends of MTs become enriched within thetwo pro-oocytes and eventually are all concen-trated within one of them (Fig. 3B). At thisstage, MTs can be visualized to run from thepro-oocytes through the ring canals into theneighboring pronurse cells (Grieder et al. 2000).Although the MT network spans the entire cyst,only the centrosomes within the pro-oocytesare active, whereas those in the pronurse cellsare inactivated. Remarkably, the inactive centro-somes migrate through the ring canals andcollect within the oocyte (Mahowald andStrassheim 1970; Grieder et al. 2000; Bolivaret al. 2001).

Although the centrosome migration has noapparent function (Stevens et al. 2007), itprovided an important clue for understandingthe MT organization of the cyst. Colchicinetreatment, which depolymerizes only dynamicMTs, prevents the accumulation of specificproteins and mRNAs within the oocyte, butdoes not block the migration of the centro-somes or restriction of meiosis to one cell(Huynh and St Johnston 2000; Bolivar et al.2001). Centrosome migration, however, hasbeen shown to be Dynein-dependent, implyingthe existence of colchicine-resistant MTs.Thus, the polarized MT network of early cystsis apparently composed of two types of MTs:those that are unstable and dynamic, andthose that are stable.

Indeed, the Drosophila Spectraplakin homo-log Short Stop (Shot) has been identified asa fusome component that is required forcentrosome migration and appears to stabilizeMTs (Roper and Brown 2004). Shot is a hugemultidomain protein with a carboxy-terminalMT binding domain and accumulates alongthe fusome in a polar fashion. In absence ofShot, not only the dynamic, but also stable,acetylated MTs are lost from the fusome.Thus, Shot is likely to be a key componentthat stabilizes MTs and links them to thefusome. Potentially, this occurs in polarfashion, allowing the dynein-dependent trans-port of the centrosomes and providingthe seed for the formation of a dynamic MT

network responsible for the majority of allother transport processes.

Dynein is likely to play a crucial role forsensing the weak initial asymmetries of thestable MT network and for the formation ofthe dynamic MT network. This hypothesis isbased on the observation that two dynein regu-lators, Bicaudal-D (BicD) and Egalitarian (Egl),are specifically required for oocyte determi-nation without affecting the fusome structureor the movement of the centrosomes (Suteret al. 1989; Swan and Suter 1996; Mach andLehmann 1997; Bolivar et al. 2001; Navarroet al. 2004). In the absence of either of thesefactors, oocyte-specific proteins like Orb andmRNAs like osk do not accumulate in theoocyte and, most significantly, the polarizeddynamic MT network does not form.

What role do BicD and Egl play in theseprocesses? Besides interacting with each other,each protein has been shown to bind proteinsinvolved in the function or regulation of theDynein complex: Egl to dynein light chain,and BicD to dynamitin, a subunit of the dynac-tin complex, which regulates dynein activity(Mach and Lehmann 1997; Navarro et al.2004). In addition, BicD and Egl have beenshown to recruit the dynein motor complexto mRNAs and initiate minus end-directedmRNA transport in other developmental con-texts (Bullock and Ish-Horowicz 2001). Thus,BicD and Egl likely play a critical role indynein-dependent transport of mRNAs andproteins critical for oocyte specification to thefuture oocyte.

Interestingly, BicD mRNA, BicD protein,Egl protein, and dynein themselves concentratein the oocyte in a BicD- and egl-dependentfashion. This suggests a positive feedbackmechanism generating a gradient of minusend-directed motor activity, which is moreand more focused to the future oocyte. Somefeatures of egl null mutants support a MTindependent pathway of oocyte selection(Huynh and St Johnston 2000; Navarroet al. 2004). However, the enhancement ofdynein-dependent transport toward the futureoocyte seems to be the main mechanism thatfinally enforces a sharp cell fate decision





between one cell and its neighbor. How is thispossible? In the cyst dynein, transport is self-amplifying because it moves some of its positiveregulators from the prenurse cells into theoocyte and, in an unknown fashion, polarizesthe MT network, which further focuses thetransport toward the oocyte. If the dyneincargo included proteins stabilizing MT minusends or determinants of the oocyte fate, suchproteins would be depleted from pronursecells and more concentrated in the futureoocyte, leading finally to sharp fate decisionbetween the two pro-oocytes. This process hasformal similarity to the mechanism of substratedepletion known from pattern formationtheory (Koch and Meinhardt 1994).

INTERLUDE: GERMARIUM STRUCTUREAND THE ENCAPSULATION OFTHE CYST

So far, we have looked at the germline cyst as anisolated object but have not considered itspositioning within the ovariole. The earlyevents of the formation of the fusome and theselection of the oocyte can be understoodwithout invoking interactions of the cyst withits environment as they are based primarilyon intrinsic processes within cystocytes, orinteractions among the cystocytes. For all sub-sequent steps in axis formation, interactionsbetween the germline cyst and specializedsomatic cells will be crucial. The contactbetween germline cysts and somatic cells firstarises within the germarium (Fig. 2).

The germarium has been subdivided intofour regions (Spradling 1993). Region 1 includesthe anterior tip of the germarium with the stemcell niche for the GSCs and the adjacent pos-terior zone where the cystoblasts and the divid-ing cysts reside (also see Yamashita et al. 2009).Region 2a begins when the cystocytes have com-pleted their final division. Oocyte selectiontakes place and meiosis of the selected oocyteproceeds to the pachytene stage. The folliclecell stem cells (FSC) are positioned at theborder between region 2a and region 2b(Nystul and Spradling 2007). Exactly two FSCsare positioned in fixed locations on opposite

lateral edges of the germarium. The FSCsproduce the somatic cells that start to migratebetween and establish close contacts with thecysts. At the same time, the cysts flatten tobecome one-cell thick discs spanning the wholewidth of the germarium. In region 3, the cystshave rounded up and are largely covered by fol-licle cells. The cells separating region 2b fromregion 3 cysts start to differentiate into stalkcells and polar cells (Fig. 4). In region 3, theoocyte is usually positioned posterior to thenurse cells, close to the stalk cells of the nextegg chamber. With the beginning of stalk for-mation and the positioning of the oocyte, thegeometry of the ovariole is fixed. The stalkcells define the long axis of the ovariole andthus the orientation of the AP axis of the cyst,including the future AP axis of the posteriorlypositioned oocyte. The future DV axis has tobe established within a plane perpendicular tothis axis. How are oocyte positioning and stalkcell formation coordinated?

THE FORMATION OF A POLARIZEDEGG CHAMBER: ALIGNING GERMLINEAND SOMA

One of the important prerequisites for theformation of polarized egg chambers is theestablishment of two separate populations ofsomatic cells with distinct morphogeneticprograms (the precursors of stalk/polar cellsand of the epithelial follicle cells) (Tworogeret al. 1999). The genetic mechanisms that leadto this crucial distinction are not known,neither is it known how the different behaviorof these cells is regulated. However, we havesome knowledge about the interaction of thesecells with the germline cyst that establishes eggchamber polarity.

Interestingly, AP asymmetry is establishedthrough a relay mechanism, which propagatespolarity from older to younger cysts (Torreset al. 2003; Assa-Kunik et al. 2007). In essence,this mechanism translates a temporal sequenceinto spatial order. For this relay mechanism,the stalk/polar cell precursors are the crucialplayers (Fig. 4). They are involved in a seriesof inductive interactions. First, they receive a





signal from the older germline cyst (region 3),which is transmitted backwards toward theyounger cyst, resulting in a soma–germlineinteraction with the younger cyst (region 2b).The signal propagation is largely mediated bythe Notch and the JAK/STAT pathways(Grammont and Irvine 2001; Roth 2001;McGregor et al. 2002). When propagationbegins, region 2b and region 3 cysts are sepa-rated by a pool of uncommitted stalk/polarcell precursors (Fig. 4A). In region 3 cysts,the Notch ligand Delta begins to signal to thesurrounding somatic cells. This leads to highNotch activation in stalk/polar cell pre-cursors, which are in direct contact with thegermline cyst and causes them to differentiateinto polar cells. In turn, the polar cells startto express the JAK/STAT ligand Unpaired.Unpaired is able to activate JAK/STAT signalingonly in cells that lack high levels of Notch signal-ing (Assa-Kunik et al. 2007). Thus, Unpairedcannot act on the follicle cells surrounding the

germline cyst, nor on the polar cells themselves,which all receive high levels of Notch activationfrom the germline. Therefore, Unpaired pro-duced by the polar cells can only signal in avectorial fashion to more anterior stalk/polarprecursors. Activation of JAK/STAT signalingin these anterior cells induces them to dif-ferentiate into stalk cells. Stalk cells intercalatewith each other and converge toward themiddle of the ovariole, forming a two-cell-widestalk (Fig. 4B). This stalk directly contactsthe younger cyst. At this stage, there are nointervening polar cells, as the posterior polarcells of each egg chamber are generated muchlater. The stalk cells in direct contact with theyoung cyst up-regulate the cell adhesionmolecule DE-cadherin (Godt and Tepass 1998;Gonzalez-Reyes and St Johnston 1998a; Becamet al. 2005), which is also up-regulated in theoocyte in comparison to the nurse cells. Thus,the oocyte preferentially adheres to the posteriorstalk cells. This positions the oocyte to the

Delta st 1

st 1

st 6

st 7

D

C

B

A

Anteriorfollicle cells

Polar cells

Posteriorfollicle cells

Gurken

Unpaired

Polar cellsStalk cells Unpaired

DeltaDelta

Figure 4. The role of polar and cell stalk for axisformation. The region connecting two egg chambersis schematically depicted with anterior and theyounger egg chamber pointing to the left side. (A)In region 3 of the germarium, Delta signals from thegermline to specify the anterior polar cells. These inturn express the JAK/STAT signaling ligandUnpaired and signal toward the anterior prestalkcells to induce the stalk cell fate. (B) A two-cell-wide stalk forms. Adhesive interactions between theoocyte of the younger cyst and the stalk cells tightlyposition the oocyte at the posterior pole. The firstround of oocyte repolarization takes place indicatedby the shift of mRNAs (orange crescent) from the an-terior to the posterior pole of the oocyte. (C) At stage6, a second round of Delta signaling from the germ-line induces the differentiation of the epithelialfollicle cells, which acquire competency to react to agradient of Unpaired emanating from the polar cellsand to Gurken signaling emanating from the oocyte.(D) Unpaired induces terminal cell fates: In theabsence of Gurken signaling, the three types ofanterior follicle cells form (red, orange, and yellow);in the presence of Gurken signaling, the posteriorfollicle cells form (blue).





posterior pole of the egg chamber and bringsit in line with the axis defined by the stalk.

Why is oocyte positioning a symmetry-breaking event? The pro-oocytes are alreadyin an asymmetric position with regard to thepronurse cells in stage 2a cysts. However, thisis only a weak asymmetry and most impor-tantly, it is not oriented with regard to thesomatic cells encapsulating and separating thecyst. Indeed, it turns out that the fixed arrange-ments of the oocyte–nurse cell cluster withregard to follicle and polar/stalk cells is one ofthe most important steps in axis formation. Ifthe oocyte does not establish contact with theanterior stalk cells, egg chambers develop inwhich the oocyte resides in a middle positionbetween the nurse cells. Oocyte and nurse celldifferentiation and growth proceed normally,but the oocyte never contacts the polar cells orthe neighboring epithelial follicle cells. Theseegg chambers give rise to eggs lacking both APand DV polarity. Mutants in the spindle genescause such phenotypes (Gonzalez-Reyes andSt Johnston 1994; Gonzalez-Reyes et al. 1997).

In dicephalic mutants, the oocyte is occa-sionally positioned anteriorly, rather thanposteriorly, to the nurse cells (Gonzalez-Reyesand St Johnston 1998b; Ligoxygakis et al.2001). Strikingly, under these conditions, eggchamber development proceeds in a normalfashion, just with a reverted AP axis of the egg.The eggs are even deposited and supportnormal embryonic development (Ligoxygakiset al. 2001). Thus, it is not essential that theoocyte is localized posteriorly, but rather thatit assumes an asymmetric position with regardto the nurse cells and along the axis definedby the stalk cells.

Why is it so important that the oocyteis aligned with the stalk/polar cells, eitheranterior or posterior to the nurse cells? Theanswer has to do with a signal from the oocyteto a specific group of follicle cells that them-selves are specified via polar cell signaling.Before we continue with the processes of folliclecell patterning and ooycte-to-follicle cell signal-ing, we have to turn our attention to the internalstructure of the oocyte, which changes aroundthe time of posterior oocyte positioning.

THE FIRST ROUND OF OOCYTEPOLARIZATION: THE ESTABLISHMENTOF THE OOCYTE POSTERIOR POLE

As described previously, the cyst structurealready imposes internal polarity on the pre-sumptive oocyte: The four ring canals are clus-tered at one pole. When the oocyte becomespositioned to one side of the nurse cells byexternal forces (stalk cell interaction), thisinternal polarity becomes even more pro-nounced. The anterior of the oocyte is notonly marked by the ring canals (Fig. 3B). TheMTs that grow from the presumptive oocytethrough the ring canals into the pronurse cellsemerge from an MTOC that resides at theanterior of the oocyte between the ring canalsand the oocyte nucleus (Grieder et al. 2000;Huynh et al. 2001b). Several components thatare transported in a MT- and dynein-dependentfashion to the oocyte remain close to thisanterior MTOC. These include the centro-somes, BicD, Egl, Orb, and Cup protein, oskand orb mRNAs, and the mitochondria(Huynh and St Johnston 2000; Huynh et al.2001b; Vaccari and Ephrussi 2002). An anteriormitochondrial cloud forms, which is enrichedin Golgi vesicles and overlaps with the MTOC,the localized proteins and mRNAs. Because ofits resemblance to similar structures found inoocytes of many species, including Xenopusand humans, it has been termed the Balbianibody (Fig. 3B, Fig. 4A).

In region 3, when oocyte positioning is com-pleted, a reorganization of the MT network ofthe oocyte takes place, which shifts the anteriorMTOC to the posterior pole (Huynh et al.2001b; Vaccari and Ephrussi 2002). Whetherthe MTOC disassembles anteriorly and isnewly formed posteriorly or whether it is main-tained and moves around the oocyte nucleus isnot known and can only be resolved by liveimaging. When the MTOC shifts position, theBalbiani body disassembles and the anteriorlocalized centrosomes, mRNAs, and proteinstranslocate to the posterior pole (Fig. 4B). Themitochondria of the Balbiani body dispersein the oocyte and supposedly provide all themitochondria for subsequent oocyte growth





because in later stages the nurse cell-to-oocyte transport of mitochondria seems to beblocked (Cox and Spradling 2003). A smallsubpopulation of the Balbiani body mito-chondria becomes localized to the posteriorcortex where later the germplasm will form.(Cox and Spradling 2003).

The posterior translocation of Balbiani bodycomponents leads to the first establishment of acytoplasmic region within the oocyte, whichcontinues to be present until embryogenesis. Italso defines the posterior oocyte cortex, whichlater will be crucial for the exchange of signalswith the follicular epithelium. Mutations thatprevent this first internal step of oocyte polariz-ation reveal that it also represents an importantcheck point for ovarian development (Huynhand St Johnston 2004). If it does not takeplace, the oocyte reverts to the nurse cell fateand further egg chamber development andgrowth is essentially blocked.

Despite its importance, the mechanismsinvolved in early oocyte polarization are notwell understood. The process depends on anintact MT network and requires BicD as well asdynein (Vaccari and Ephrussi 2002). Strikingly,it also depends on the par genes, a groupof genes first discovered in Caenorhabditiselegans, which has a conserved role in cellpolarization throughout animal evolution(also see McCaffrey and Macara 2009). It hasbeen shown that Drosophila homologs of allconserved par genes ( par-1, par-3, par-4,par-5, and par-6) and of aPKC are requiredfor early oocyte polarization (Cox et al. 2001a;Cox et al. 2001b; Huynh et al. 2001a; Huynhet al. 2001b; Benton et al. 2002; Vaccari andEphrussi 2002; Martin and St Johnston 2003).

In many polarized cells, like in the C. eleganszygote, the Par proteins are involved in the for-mation and stabilization of complementary cellcortex domains (Munro 2006; Goldstein andMacara 2007). A conserved ternary complex ofPar-3, Par-6, and aPKC localizes to one pole ofthe cell, whereas Par-1 is found at the oppositepole. The Par-3/Par-6/aPKC complex andPar-1 are involved in inhibitory interactions,which lead to mutually exclusive membranelocalization.

Interestingly, in Drosophila, Par-1 is local-ized to the fusome and, like other oocyte-specific proteins, becomes restricted to theoocyte in a MT-dependent manner (Cox et al.2001a; Huynh et al. 2001b; Vaccari andEphrussi 2002). First, it localizes to the anteriorpole and later relocalizes with the MTOC to theposterior pole. However, the relocalizationprocess requires par-1 itself and bazooka (baz),the par-3 homolog of Drosophila. Par-3protein is localized at the anterior pole, whereit remains when Par-1 translocates to the pos-terior pole (Huynh et al. 2001a; Vaccari andEphrussi 2002). In par-1 mutants, Par-3 doesnot remain restricted to the anterior pole.Thus, like in C. elegans zygotes and other polar-ized cells, the first internal polarization step ofthe Drosophila oocyte is accompanied by theestablishment of complementary mutuallyexclusive domains of Par proteins. However,the changes in MT polarity and the Parprotein localization seem to be interdependent,which indicates a more complex mechanism sofar unknown from other systems. In addition,it is not clear how the polarization processis initiated. Polarization coincides with theadhesive interactions between oocyte andstalk cells. Thus, it is conceivable that a signalderived from somatic cells induces repolari-zation. This view is supported by the findingthat the extracellular matrix receptor dystro-glycan is required in the germline for earlyoocyte repolarization (Deng et al. 2003).Oocytes lacking dystroglycan also show adefect in F-actin accumulation at the posteriorpole. This might hint to a similarity withother systems in which it is well establishedthat changes in the actomyosin network ini-tiate the formation of Par protein asymmetries(Murno 2006).

FOLLICLE CELL PATTERNING AND THEEMERGENCE OF AP POLARITY WITHINTHE FOLLICLE CELL LAYER

After the egg chamber leaves the germarium, itis fully surrounded by a monolayer of epithelialfollicle cells, and the posterior pair of polar cellshas formed so that each egg chamber has an





identical pair of polar cells at its terminiand thus, the somatic cells surrounding andconnecting the egg chambers are symmetric(McGregor et al. 2002). After mitotic divisionsregulated by mechanical stress of the growingoocyte and Hh signaling, the epithelial folliclecells stop dividing and begin to differentiatein response to a second round of Delta signal-ing from the germline beginning at stage6/7 (Fig. 4C) (Wang and Riechmann 2007;Zhang and Kalderon 2000; Lopez-Schier andSt Johnston 2001). How Notch controls thecell cycle switch in the follicle cells hasbeen well investigated (Sun and Deng 2007).However, for the purposes of this review, itis only important that the epithelial folliclecells during stage 6/7 become competent torespond to signals emanating from the polarcells and from the oocyte.

At stage 6/7, the egg chamber essentiallyforms an ellipsoidal structure with the polarcells defining the endpoints of the long axis(Fig. 2, Fig. 4C,D). Now, the polar cells act asorganizers of surrounding epithelial folliclecells (Grammont and Irvine 2002). Unpairedsecreted by polar cells leads to a gradient ofJAK/STAT signaling, which specifies about 200epithelial follicle cells at each end of the eggchamber to assume the terminal follicle cellfate (Xi et al. 2003). The prepattern that isestablished within the follicular epitheliumby JAK/STAT is mirror-image symmetric(Fig. 4C). This is apparent in the absence of afurther modulation of JAK/STAT signalingfrom the germline, when the terminal cells atboth ends of the egg chamber differentiateinto different types of anterior follicle cells(Gonzalez-Reyes and St Johnston 1998b). JAK/STAT signaling not only specifies the terminalfate as such, but apparently also accounts forterminal cell type diversity. Unpaired seems tofunction as a morphogen in specifying threeanterior terminal cell types as a function ofdistance from the polar cells (Xi et al. 2003)(Fig. 4D).

At the time Notch signaling providescompetency to differentiate and JAK/STAT sig-naling provides spatial patterning information,the epithelial follicle cells surround a highly

polarized germline cyst. The oocyte, positionedposterior to the nurse cells, has grown in sizeso that it just abuts the terminal follicle cells(Fig. 4C,D). MT- and dynein-dependenttransport from the nurse cells has led to theaccumulation of several mRNAs within theoocyte. In the next step of axis formation, thisinner polarity of the germline cyst is transferredto the follicular epithelium (Gonzalez-Reyesand St Johnston 1994; Gonzalez-Reyes et al.1995; Roth et al. 1995). Among the mRNAsthat have begun to accumulate within theoocyte already in the germarium is that of thegurken (grk) gene. grk codes for a transforminggrowth factor a (TGFa) homolog, a memberof the epidermal growth factor receptor(EGFR) ligand family (Neuman-Silberbergand Schuepbach 1993). At stage 6/7, Grkprotein is secreted by the oocyte and activatesthe EGFR in the adjacent terminal folliclecells (Peri et al. 1999; Queenan et al. 1999;Chang et al. 2008). Like other mRNAs thataccumulate in the early oocyte grk mRNAbecomes concentrated at the posterior cortexalready at stage 1 (Fig. 4B), and it has beensuggested that efficient secretion of Grktoward the adjacent follicle cells requires thetight localization of grk mRNA to the posteriorcortex of the oocyte (Clegg et al. 1997).Thus, posterior grk mRNA localization mightprovide a link between the early par-dependentpolarization of the oocyte and the later EGF-dependent polarization of the egg chamber(Huynh and St Johnston 2004). EGFR acti-vation in the terminal follicle cells leads to thetranscription of several immediate target genesof EGF signaling (Morimoto et al. 1996;Ghiglione et al. 1999; Reich et al. 1999) onlyin cells that have received prior Notch activationand JAK/STAT signaling (Xi et al. 2003). Thefinal outcome of this Grk signal is the inductionof the posterior follicle cell (PFC) fate.

A combination of JAK/STAT signaling andEGF activation can induce the PFC fate also inectopic positions of the follicular epithelium(Xi et al. 2003). However, almost nothing isknown about the genes downstream of thetwo pathways that specify the PFC fate. Incontrast to the three types of anterior terminal





follicle cells (AFCs) that go through a complexprogram of morphological changes, the PFCsare apparently not composed of different sub-populations and do not show obvious mor-phological differences compared with adjacentmain body follicle cells. Only genetic mosaicanalysis reveals a sharp border between the twocell types (Gonzalez-Reyes and St Johnston1998b).

Interestingly, recent work shows that besidesJAK/STAT and EGF signaling, there are twoadditional pathways required for normal cellbehavior of the PFCs, but not the main bodyfollicle cells (Riechmann 2007). First, both theupstream and the core components of theHippo tumor-suppressor pathway are involvedin controlling differentiation and cell divisionof the PFCs (Meignin et al. 2007; Poleselloand Tapon 2007). The Hippo pathway pro-motes Notch signaling and prevents multi-layering of the PFCs (Yu et al. 2008). Second,multilayering of the PFC epithelium is alsoobserved when integrin function is lost(Fernandez-Minan et al. 2007). It is notknown why the PFCs are particularly sensitiveto a loss of these two pathways. However, ithas been suggested that this might be linkedto mechanical constraints because of greatertissue curvature at the termini as comparedwith the more lateral regions (Riechmann2007).

BACK SIGNALING AND THE SECONDROUND OF OOCYTE POLARIZATION

At stages 7–9, the oocyte nucleus changes itsposition from posterior to anterior. This isaccompanied by a restructuring of the MTcytoskeleton (Theurkauf et al. 1992;Steinhauer and Kalderon 2006). The posteriorMTOC disappears and MTs begin to emanatefrom the lateral and anterior cortex, projectingthe plus ends into the center of the oocyte.Second, MT-dependent transport leads tomRNA localization to the opposite poles ofthe oocyte (Steinhauer and Kalderon 2006).So far, only the posterior cortex of the oocytewas defined as a distinct region for mRNA local-ization. Now the anterior cortex of the oocyte is

established as a separate destination for mRNAtargeting. osk mRNA is transported in aKinesin-dependent way toward the plus endsof MTs and becomes localized to the posteriorcortex (Brendza et al. 2000; Januschke et al.2002). bcd mRNA is transported in a dynein-dependent way toward the minus ends of MTsand becomes localized to the anterior cortex(Januschke et al. 2002).

When PFCs are lacking, or do not correctlydifferentiate, the oocyte nucleus does notmigrate normally and mRNA localization isaberrant (Gonzalez-Reyes and St Johnston1994; Gonzalez-Reyes et al. 1995; Roth et al.1995; Meignin et al. 2007). These observationshave led to the conclusion that the PFCsproduce a signal that is received by the oocyteand triggers changes in the MT cytoskeleton(Gonzalez-Reyes and St Johnston 1994;Poulton and Deng 2007). Because the PFCshave just been established through a germlinesignal at stage 6/7, this second signalingprocess from the soma back to the germlinehas been termed back signaling.

The molecular nature of the signal emanat-ing from the PFCs is unknown. However,there is good evidence that the signal requiresdirect contact between PFCs and the posteriorsurface of the oocyte (Poulton and Deng2007). First, mutants preferentially disruptingthe contact between PFCs and the oocytecause polarity defects in the oocyte (Martinet al. 2003). Second, if cell clones mutant forEGF or JAK/STAT signaling components donot encompass all PFCs, such that some wild-type PFCs remain, osk mislocalization isrestricted to the region of the oocyte cortexthat is in direct contact with the mutant cells,whereas osk is normally localized in regionsabutting wild-type PFCs (Frydman andSpradling 2001; Xi et al. 2003; Poulton andDeng 2006). Thus, the polarizing signal can betransmitted to the oocyte in a very local fashion.

Within the follicle cells, only three com-ponents have been found so far to be requiredfor back signaling without affecting the PFCfate. All three highlight the importance of theECM for the production of the polarizingsignal. PFC clones mutant for laminin A





(Lan) or the transmembrane tyrosine phospha-tase Dlar, a possible receptor for Lan, pre-vent proper oocyte polarization (Deng andRuohola-Baker 2000; Frydman and Spradling2001). It also has been shown that down-regulation of the Lan receptor, dystroglycan(DG), in PFCs is required for proper back sig-naling (Poulton and Deng 2006). In all threecases, loss of function or overexpression clonesin PFCs show locally restricted effects onthe oocyte cortex as described for EGF andJAK/STAT signaling components. Despite this,Lan, Dlar, and DG are likely to play an indirectrole in signal production (Poulton and Deng2006).

At present, not much is known about thenature of the signal produced by the PFCs orthe way this signal is perceived by the oocyte.Several components are required within theoocyte to establish correct MT polarity, butnot for the PFC fate. Such components mightbe a part of the intracellular pathway that trans-duces the signal. However, some of them mightalso act in parallel to the signaling process andnot be involved in providing spatial infor-mation. Candidates for back signaling factorsfall into four categories: (1) Protein kinase A(Lane and Kalderon 1994; Steinhauer andKalderon 2005), (2) Components of the Exonjunction complex (EJC) (Micklem et al. 1997;Newmark et al. 1997; Hachet and Ephrussi2001; Mohr et al. 2001) and heterogeneousnuclear ribonucleoproteins (hnRNPs) (Yanoet al. 2004; Steinhauer and Kalderon 2005),(3) Proteins involved in vesicle transport(Coutelis and Ephrussi 2007; Januschke et al.2007; Tanaka and Nakamura 2008), and (4)Par proteins and the tumor suppressor proteinLethal giant larvae (Lgl). We will focus ourdiscussion on the par genes and lgl becauseonly for these has a connection between cyto-skeletal reorganization and back signaling beenestablished.

The early requirement of the par genesmakes it difficult to investigate their functionduring later stages of oogenesis. However, byusing weak alleles of par-1, it was possible toidentify a specific requirement of par genesfor axis formation during midoogenesis

(Shulman et al. 2000). At this stage, reducedPar-1 levels lead to a lack of posterior osklocalization, which instead concentrates in thecenter of the oocyte; bcd mRNA localization isonly weakly affected (Benton et al. 2002). Thephenotype correlates with changes in the MTnetwork: The posterior MTOC does not dis-assemble and MTs emanate from all corticalregions of the oocyte projecting the plus endsto the center. Polarity defects have recentlyalso been reported for lgl mutants (Tian andDeng 2008). LGL is an evolutionary conservedWD40 domain containing protein, which actstogether with Par-1 to establish polarity in anumber of cell types (Munro 2006; see alsoMcCaffrey and Macara 2009; Prehoda 2009).Loss of lgl in the oocyte leads to partially pene-trant mislocalization of osk and bcd, as well as todefects in oocyte nucleus migration. Both Par-1(the N1 isoforms) and Lgl are localized to theposterior cortex of stage 7 oocytes and thusbelong to the earliest known markers respond-ing to the polarizing signal (Doerflinger et al.2006; Tian and Deng 2008).

It has been shown that Par-1N1 weaklyaccumulates at the posterior cortex even beforethe oocyte nucleus migrates and the MTnetwork repolarizes. The posterior recruitmentof Par-1N1 itself is not MT-dependent, but itrequires F-actin. Par-1 and Lgl form proteincomplexes and Lgl can be inactivated throughphosphorylation by aPKC (Tian and Deng2008). However, the involvement of aPKC inaxis formation during midoogenesis is stillcontroversial.

The Par-1 kinase is able to destabilize MTs.Overexpression of Par-1 prevents the formationof MTs around the entire oocyte cortex (Tianand Deng 2009). Thus, posterior localizedPar-1 might initiate the first asymmetry of theMT network by preventing MT growth fromthe posterior cortex. The mechanism by whichPar-1 controls MT stability is still controversial.In contrast to earlier reports (Doerflinger et al.2003), Tian and Deng (2009) suggest that theMT binding protein Tau is a target for phos-phorylation by Par-1. Phosphorylated Tau canno longer bind to and stabilize MTs. The taumutant phenotypes, however, are fairly weak,





suggesting that Par-1 has additional targets.Taken together, Par-1 seems to be the perfectlink between PFC derived signals and oocytepolarity, given its spatial and temporal patternof localization and its suggested proteinfunction.

However, the Par-1 mutant phenotype aswell as that of mutants in any of the other com-ponents mentioned above, with the possibleexception of the EJC protein Mago nashi, donot lead to a complete lack of oocyte polarity.Thus, it is also possible that the crucialcomponents responsible for transmitting thePFC signal have not been found so far, or thatseveral signaling processes act in parallel.Indeed, the changes of the MT network atstage 7 might be decomposed into independentelements. The stabilization of minus ends atthe lateral and anterior cortex of the oocytemight occur independent from the destabi-lization of MTs at the posterior pole inducedby the PFCs. In addition, the migration of theoocyte nucleus might require an independenttrigger. The oocyte nucleus itself has MTOCcharacteristics. Thus, its position within theoocyte has a profound influence on the overallstructure of the oocyte MT network (Guichetet al. 2001; Januschke et al. 2002; Gervais et al.2008). Januschke et al. 2006 have even pro-posed that the movement of the oocytenucleus is the actual polarizing event. Theposterior MTOC is not disassembled, butrather moves with the nucleus to the anteriorpole where preformed MTs are released fromthe nucleus and anchored to the anterior andlateral cortex. In this scenario, nuclear migra-tion does not depend on a repolarized MTcytoskeleton, but is driven by the growth ofMTs emanating from the nucleus. The mainfunction of the polarizing signal would be theinitiation of nuclear migration.

Irrespective of the steps initiating MT repo-larization at stage 7, MT polarity is furtherstabilized by feedback mechanisms occurringat two levels during stages 8 and 9. First, thepar genes establish mutually exclusive mem-brane domains, which are maintained untilstage 10A (Benton and St Johnston 2003).Par-1 kinase phosphorylates Par-3 and thereby

restricts its localization to lateral and anteriormembranes. Second, an Osk-dependent posi-tive feedback loop is initiated, which re-enforcesMT polarity (Zimyanin et al. 2007). Initially,only a weak MT gradient has been establishedon posterior Par-1N1 localization. This gradientis sufficient to initiate osk mRNA transport,first to the center of the oocyte and from thereto the posterior pole, where it can be translated.Osk protein at the posterior cortex recruitsmore Par-1 protein, which in turn re-enforcesthe MT gradient. Par-1 in turn stabilizesOsk protein through direct phosphorylation,generating a second positive feedback loop(Riechmann et al. 2002). Thus, symmetrybreaking of the MT network results frommultiple dynamic interactions, including aspatially highly restricted Osk-dependentpositive feedback loop.

So far, we have only provided a crudedescription of the of structure MT network atstage 9 when the steps of mRNA localizationtake place, which are crucial for axis formationof the later embryo. This description is largelybased on the behavior of b-Gal fusion proteinscontaining the motor domains of the minusend-directed motor Nod, which localizesanteriorly, and the plus end-directed motorkinesin, which moves to the posterior (Clarket al. 1994; Clark et al. 1997). From these obser-vations, the idea emerged that the MT networkof the oocyte is highly polarized, with minusends pointing toward the anterior and plusends toward posterior pole. Direct staining forMTs, however, did not provide a clear pictureof how the MTs are organized and often leadto conflicting results depending on fixationand staining procedures (Theurkauf et al.1992; Theurkauf et al. 1993; Cha et al. 2002;Januschke et al. 2006; Wang and Riechmann2008). The problem has not been resolved, butrecent in vivo imaging approaches show thatthe overall MT network is much less polarizedthan expected (MacDougall et al. 2003;Zimyanin et al. 2008). There seems to be onlya 20% excess of MTs with their plus end point-ing posteriorly even after completion of MTpolarity re-enforcement during stages 7 and 8,suggesting that the initial overall MT polarity





induced by the polarizing signal from PFCswas even weaker (Zimyanin et al. 2008).

THE EMERGENCE OF DV POLARITY

The posterior-to-anterior movement of theoocyte nucleus accompanies the repolarizationof the MT network at stage 7 and we havealready mentioned that it is currently impos-sible to specify the causal relationship betweenboth events (Januschke et al. 2002; Januschkeet al. 2006). In contrast, the role of the oocytenucleus in establishing DV asymmetry is wellsupported (Roth 2003). The oocyte nucleusmoves from a central posterior positiontoward the anterior side, the nurse cell–oocyte interface (Fig. 5). When the nucleusarrives at the anterior pole, it occupies a par-ticular position at the circular circumferenceof the oocyte. This position corresponds tothe future dorsal side of the egg chamber andthe later embryo. grk mRNA is targeted to the

nucleus and a second round of EGF signalingtakes place, which leads to the DV patterning ofthe follicular epithelium (Neuman-Silberbergand Schuepbach 1993; Gonzalez-Reyes et al.1995; Roth et al. 1995).

Several observations have shown thatnuclear movement is not just correlated with,but is required to establish DV polarity. grkmRNA localization to the dorsal side requirescorrect nuclear positioning. If the nucleus iswrongly positioned, it is still targeted by grkmRNA (Roth et al. 1995). This explains whylaser ablation of the nucleus causes a loss ofDV polarity of the egg chamber (Montell et al.1991). If nuclear movement fails to occur, asin mago mutants, or when the Grk signal isdelayed, eggs develop that have AP polarity inthe follicular epithelium, but lack DV polarityorthogonal to the AP axis (Micklem et al.1997; Newmark et al. 1997; Peri and Roth2000) (Fig. 5). Together, such observationsshow that nuclear movement is necessary for

Dorsal

Ventral

post.ant.

Figure 5. Oocyte nucleus migration and dorsoventral axis formation. Schematic drawings of stage 7 oocytes (left)and mature eggs. Top: Wild type. The posterior-to-anterior movement of the oocyte nucleus forces the nucleusto acquire an asymmetrical position, which determines the dorsal side of the egg and establishes orthogonalitybetween the APand the DVaxes. Middle: If nuclear movement does not occur, APand DVaxes are parallel to eachother. Bottom: In binuclear oocytes, both nuclei move to the anterior pole to adopt random position at theanterior cortex. Both nuclei induce dorsal egg shell structures.





the orthogonal orientation of the AP and DVaxes. However, they do not prove that thenucleus determines the dorsal side of the eggchamber. It is conceivable that other processesspecify this side before nuclear migration andthe nucleus moves to this prespecified position.This scenario is unlikely, however. First, theDV axes of different egg chambers within anovariole are randomly oriented with regard toeach other and with regard to the DV axis ofthe female. Second, in binuclear oocytes, bothnuclei move to the anterior cortex and inducedorsal chorion fates (Roth et al. 1999). A sta-tistical analysis shows that they choose theirposition randomly with regard to each other.This behavior is most compatible with theassumption that the egg chamber completelylacks DV asymmetry before nuclear movement.Thus, among all the symmetry-breaking stepsdescribed so far, nuclear movement mightprovide the only case in which no bias existsthat orients the process.

CONCLUDING REMARKS

The process of axis formation that we havedescribed looks overwhelmingly complex andseems to take several detours. At severalinstances, polarity is established that later islost. Thus, early AP polarity (rosette shape) ofthe germline cluster in region 2a of the germar-ium seems to be a by-product of the internalpolarization of the oocyte (clustering of thering canals). The rosette shape apparently dis-appears when the cyst flattens in region 2b.Likewise, the AP asymmetry of polar cell differ-entiation, required for oocyte positioning, islater lost, giving rise to a symmetric arrange-ment of polar and stalk cells. This symmetricarrangement is required for PFC induction byGrk. The first MT polarization of the oocyteprovides another example. This process estab-lishes the posterior pole of the oocyte and ithas been suggested that mitochondria localizedthere at stage 1 are later incorporated into thepole plasm (Cox and Spradling 2003).However, this posterior pole can only be main-tained and the pole plasm can only beassembled when the second round of MT

repolarization takes place, which reverses theearly MT polarity. Early MT polarity, however,was a prerequisite for posterior grk mRNAlocalization, which might be important forPFC induction by Grk signaling. In all of theseinstances, polarities arise transiently either as asecondary consequence or as a means ofanother step of symmetry breaking.

Another apparent detour in axis formationis represented by the reciprocal signalingbetween germline and soma. Why is polarityfirst established in the germline, then trans-ferred to the follicular epithelium and fromthere back to the germline? The enormousincrease in size of the oocyte might provide anexplanation. The volume of the oocyte in-creases by four orders of magnitude from thetime of oocyte determination to egg deposition.During these growth processes, axis infor-mation has to be accurately maintained. Thefollicular epithelium in close contact with theoocyte provides a means to store spatial infor-mation in a stable way and reuse it at a laterstage after which the oocyte might have grownconsiderably. This is definitively an issue forDVaxis formation, which we have not describedin detail, but it might also be relevant to backsignaling required for AP polarity. Using the fol-licular epithelium to store spatial informationhas another advantage. The border betweenoocyte and follicle cells provides a sharp discon-tinuity. Thus, back signaling from the folliclecells can be spatially highly confined.

Finally, most steps of axis formation duringDrosophila oogenesis do not represent truesymmetry-breaking events, but rather senseand enhance asymmetries of the ovariolearchitecture. Therefore, the question arises ofhow ovariole architecture is established inthe first place. The embryonic and larval ovaryof Drosophila consists of a growing sphericalmass of intermingled mesenchymal somaticand primordial germ cells (King 1970; Gilboaand Lehmann 2006). During late larval andearly pupal stages, somatic cells rearrangeto form stacks of disc-shaped cells (Godtand Laski 1995). The first stacks give rise tothe terminal filaments, which provide theanterior anchoring point of each ovariole.





Subsequently, cell stacks form, which give rise tothe first interfollicular stalks and to the basalstalks. The linear arrangement of these cellstacks defines the long axis of the ovariole.The type of cell rearrangement and cellrecruitment, which initiated stack formation,is known as convergent extension (Godt andLaski 1995; Stern 2004). A similar process isrequired for AP axis formation during gastrula-tion in most animals (also see Vladar et al.2009). The convergent extension processesthat establish the ovariole organization con-tinue to play a role in the adult ovary whenthe prestalk cells converge to form the two-cellwide (Fig. 4A,B) and later the one-cell wideinterfollicular stalks (Fig. 4C,D), which connectand align the individual egg chambers.

Taken together, axis formation during Dro-sophila oogenesis combines examples of mostevery process known so far to be involvedin the generation spatial asymmetries, includ-ing cell shape changes and cell migration (stalkformation), differential cell adhesion (eggchamber orientation), inductive signaling(stalk cell and follicle cell specification, back-signaling), pattern formation in epithelialsheets (JAK/STAT signaling), asymmetric celldivisions (cyst formation), cell polarization(first and second round of par-dependentoocyte polarization), and intracellular pattern-ing (oocyte migration, formation of corticaldomains for RNA localization).

To what degree is this highly intricateprocess conserved in evolution? The fixedlinear arrangement of developing egg chambersas represented in the ovariole structure seemsto be an insect invention: Within insects, itrepresents a very stable character alreadypresent in the most primitive forms, whereassuch structures are generally not found inother animal ovaries (Gutzeit and Sander1985; Buning 1994). The connection of eachovariole to its own group of germline andsomatic stem cells, the ordered sequence ofmaturation, and the intimate relation betweenthe oocyte and the follicle cells producingthe protective eggshell layers allowed forhigh fecundity and huge variety of eggshellstructures, which was likely important in

allowing insects to exploit a wide variety ofenvironmental niches. In addition, the polarorganization of the ovariole made it possibleto shift aspects of axis formation, whichtypically take place during embryogenesis, toearlier and earlier stages of oogenesis. Poste-rior cytoplasmic determinants specifying theAP axis and the asymmetric positioning ofthe oocyte nucleus correlating with dorsal sideof the egg are found already in ancestral insectorders (Sander 1976). However, there is alsoconsiderable variation in ovariole structuremainly connected to the presence or absenceand the position of nurse cells (Buning1994). Moreover, the molecular details of axisformation are highly variable even withinthe more advanced holometabolous insects(Fonseca et al. 2009). It will be interestingto see what aspects of Drosophila ovariansymmetry-breaking processes are conserved inother insect species, and whether the uniqueproperties of the ovariole structure are exploitedin different ways.

ACKNOWLEDGMENTS

We thank Jean-Rene Huynh and AntoineGuichet for excellent comments on the manu-script. J. L. was supported by a Ruth L. Kirsch-steinpost-doctoral fellowship(5F32GM078832)from the NIH. Some data reviewed here wereobtained with grants to S.R. from the DFG(SFB 446, SFB 572, SFB 680).

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2009; doi: 10.1101/cshperspect.a001891Cold Spring Harb Perspect Biol Siegfried Roth and Jeremy A. Lynch

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http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

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