Hsp60C is required in follicle as well as germline cells during oogenesis in Drosophila melanogaster

Volume 237 • Number 7 •Volume 237 • Number 7 • July 2008

DEV

ELOPM

ENTA

L DY

NA

MIC

SV

olu

me 237 N

um

ber 7 2008

Pages 1755

–1952W

iley-Liss Gary C. SchoenwolfGary C. SchoenwolfEditor-in-ChiefEditor-in-Chief

DevelopmentalDynamics

Prof.S.C.Lakhotia

New Stamp

Cover Image Hsp60C is required in follicle as well as germline cells during oogenesis in Drosophila melanogaster Surajit Sarkar, S.C. Lakhotia * Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi, India email: S.C. Lakhotia ([email protected])

*Correspondence to S.C. Lakhotia, Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India

This figure shows stage 10 (left) and stage 12 (right) wild type egg chambers stained for F-actin (red) and hsp60 (green). See Sarkar and Lakhotia, Developmental Dynamics 237:1334-1347. © 2008 Wiley-Liss, Inc.

RESEARCH ARTICLE

Hsp60C Is Required in Follicle as Well asGermline Cells During Oogenesis in DrosophilamelanogasterSurajit Sarkar† and S.C. Lakhotia*

Hsp60C gene of Drosophila melanogaster shows a dynamic spatiotemporal expression during oogenesis andseems to contribute bulk of the Hsp60 family proteins in ovarioles. Hsp60 distribution overlaps with that ofF-actin–rich membranes/structures in follicle, nurse, and egg cells throughout oogenesis. Skeletal musclefibers associated with ovarioles and in other parts of the body show patterned location of Hsp60 in A-bands.During stages 11–12, Hsp60 accumulates at junctions of nurse cells and oocyte, where a new microtubuleorganizing center is known to develop. A recessive hypomorph allele, Hsp60C1 causes complete sterility ofthe rare surviving homozygous adults. Their egg chambers show very little Hsp60C transcripts or Hsp60protein. Beginning at stages 6–7, Hsp60C1 chambers show a disorganized follicle cell layer with poor celladhesion in addition to abnormal organization of F-actin and other cytoskeletal structures in follicle, nurse,and egg cells. Additionally, expression and localizations of Hrb98DE, Squid, and Gurken proteins in nursecells and oocyte are also severely affected. Hsp60C1 homozygous follicle cell clones in Hsp60C1/� ovariolesshow disruptions in follicle epithelial and cytoskeleton arrangements. Likewise, egg chambers withHsp60C1 homozygous germline clones in Hsp60C1/� flies show abnormal oogenesis. Our results provide thefirst evidence for an essential role of Hsp60C in Drosophila oogenesis, especially in organization andmaintenance of cytoskeletal and cell adhesion components. Developmental Dynamics 237:1334–1347, 2008.© 2008 Wiley-Liss, Inc.

Key words: follicle cells; actin, MTOC; cell adhesion; cytoskeleton; striated muscles

Accepted 23 February 2008

INTRODUCTION

The Hsp60 or chaperonin family is ahighly conserved group of molecularchaperones and includes bacterialGroEL, mitochondrial Hsp60, plastidRubisco subunit binding protein, ar-chaea group II chaperonins, eukary-otic cytosolic Tcp-1 protein, and so on,and are most well known for their or-ganelle localization and roles in pro-tein folding (Ranson et al., 1998; Hartland Hayer-Hartl, 2002). In recentyears, however, they have also been

found to be involved in a variety ofnonchaperonic functions such as cellsignaling, epithelial cell remodeling/motility, apoptosis, and so on (Magu-ire et al., 2002; Gobert et al., 2004;Zhang et al., 2004; Sarkar et al., 2006;Arya et al., 2007). Besides beingpresent in the organelles (mitochon-dria or chloroplasts), the Hsp60 pro-teins are now known to be present incytosol, on cell membrane or even out-side the cell (Maguire et al., 2002;Sarkar et al., 2006).

Four Hsp60-like DNA sequenceshave been reported in Drosophilamelanogaster (www.flybase.org), whichon the basis of their putative proteinproducts are classified as members ofthe Hsp60 gene family and named, re-spectively, as Hsp60A at 10A4 poly-tene band, Hsp60B at 21D2 band,Hsp60C at 25F2 band and Hsp60D at34C1 band (Sarkar and Lakhotia,2005). As expected (Betran et al.,2002), these duplicated genes have ac-quired distinct functions (Sarkar and

Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi, India†Dr. Sarkar’s present address is Department of Genetics, University of Delhi, South Campus, New Delhi 110 021, India.*Correspondence to: S.C. Lakhotia, Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi221 005, India. E-mail: [email protected]

DOI 10.1002/dvdy.21524Published online 3 April 2008 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 237:1334–1347, 2008

© 2008 Wiley-Liss, Inc.

Lakhotia, 2005). While the Hsp60Agene seems to be the ubiquitously ex-pressed general chaperonin (Perez-gasga et al., 1999), Hsp60B has testis-specific expression and is essential forsperm individualization (Timakov andZhang, 2001). The Hsp60C gene hasbeen shown to be required for trachealdevelopment and for early stages inspermatogenesis (Sarkar and Lakho-tia, 2005), and one of the roles of theHsp60D gene seems to be in modula-tion of apoptosis (Arya et al., 2007).

In an earlier report, we (Sarkar andLakhotia, 2005) described a newHsp60C1 allele, which carries aP-transposon in the promoter regionof the Hsp60C gene and thus affectsits expression. Homozygosity for thisstrongly hypomorphic Hsp60C1 alleleresults in tracheal defects and earlyfirst instar larval lethality. Interest-ingly, �10% of the Hsp60C1 homozy-gotes survive as weak and completelysterile adults. In our previous study(Sarkar and Lakhotia, 2005), we ex-amined spermatogenesis in the raresurviving Hsp60C1 homozygous adultflies and found that, unlike the re-quirement of the Hsp60B gene for in-dividualization of sperms (Timakovand Zhang, 2001), the Hsp60C1 mu-tant allele affects early stages of sper-matogenesis. In the present study, weexamined expression of Hsp60C geneduring oogenesis and the basis for com-plete sterility of surviving Hsp60C1 ho-mozygous females.

Oogenesis in Drosophila is a com-plex developmental process involvingextensive cellular remodeling eventsand complex intercellular communica-tions. Development of egg chamberhas been subdivided into a series of 14consecutive stages characterized bydistinct morphological criteria (King,1970). A monolayer of somatic folliclecells covers the 16 germline-derivedcells (15 nurse cells and 1 oocyte) ineach egg chamber. Nurse cells and theoocyte remain interconnected bymeans of a stereotyped pattern of F-actin–based ring canals (King, 1970;Spradling, 1993). An intricate orches-tration of DE-cadherin– and �-cate-nin–dependent adhesion between oo-cyte and follicle cells, the actin- andtubulin-based cytoskeletal elementsand appropriate signaling betweenthe different cell types in egg chamberis critical for progression of oogenesis

and production of a functional egg(Peifer et al., 1993; Gonzalez-Reyesand St Johnston, 1998; Wodarz, 2002).

Our results show that the Hsp60Ctranscripts are abundant in egg cham-bers and the Hsp60C protein is a ma-jor component of Hsp60 family pro-teins in these cells. Hsp60 and F-actinshow a significant overlap/associationin follicle and nurse cells and in thecortex of early/mid stage oocytes. Fur-thermore, a remarkable concentrationof Hsp60 is seen in basal nurse cellsand at the anterior oocyte cortex instage 11–12 wild-type chambers, inthe region where the bicoid tran-scripts are known to be localized inthe oocyte cytoplasm and where a mi-crotubule organizing center (MTOC)forms during these stages (Schnorreret al., 2002). The Hsp60C1 mutants’egg chambers show little Hsp60 at anyof these sites, while the cytoskeletalelements (e.g., cell adhesion mole-cules, F-actin, tubulin, etc.) in all celltypes are severely affected. Hsp60also shows a remarkable localizationin the A-bands of skeletal muscles, in-cluding those surrounding the eggchambers and the ovarioles in wild-type but not in the Hsp60C1 mutantflies. Analysis of Hsp60C1 homozy-gous mutant cell clones in Hsp60C1/�egg chambers confirmed the essentialrequirement of Hsp60C for folliclelayer organization. Likewise, theHsp60C1 homozygous germline clonesin Hsp60C1/� flies show abnormal cy-toskeleton and other abnormalities. Itappears that in addition to the earliernoted (Sarkar and Lakhotia, 2005)role of the Hsp60C protein in trachealdevelopment and spermatogenesis,this protein is also essential for skel-etal muscle organization and oogene-sis in Drosophila.

RESULTS

Hsp60C1 HomozygousFemales Are Sterile

Ovaries of all surviving Hsp60C1/Hsp60C1 adult females were muchsmaller than in wild-type due to nearabsence of late stage follicles, withmost ovarioles showing degeneratedmaterial at their distal ends. Thenumbers of ovarioles per ovary (N �28) was, however, not affected. Otherfemale reproductive organs in

Hsp60C1 mutant flies appeared gener-ally comparable to those in wild-type(not shown). The Hsp60C1/Hsp60C1

females never laid any eggs, althougha few abnormal and collapsed eggswith patterning defects could be re-covered from the oviduct (Fig. 1).Compared with wild-type (Fig. 1A),these eggs were fragile and the major-ity of them showed poorly developedand improperly oriented dorsal ap-pendages (Fig. 1B), while a few werewithout them (Fig. 1C,D). In addition,some eggs with incomplete dumping(Fig. 1D) were also seen.

The viability as well as fertility ofHsp60C1/Hsp60C1 females was re-stored in the P-transposon excisionlines (Sarkar and Lakhotia, 2005). De-fects in oogenesis in Hsp60C1/Df(2L)cl-h2 females were similar tothose in Hsp60C1/Hsp60C1 females(see Table 1). Thus, the Hsp60C1 is aloss of function mutation and is re-sponsible for complete sterility of thefew surviving Hsp60C1 homozygousadult females.

Hsp60C Gene Is Expressedin a Stage-Specific PatternDuring Oogenesis

The Hsp60C transcripts in reproductiveorgans of adult wild-type female flieswere localized by RNA:RNA in situ hy-bridization with Hsp60C-specific ribo-probe (Sarkar and Lakhotia, 2005). Infemale reproductive organs of wild-typeflies, a strong expression of Hsp60C wasseen in the ovarioles and spermathecaebut little in calyx, oviduct, seminal re-

Fig. 1. A–D: Phase contrast images of a laidwild-type egg (A) and defective eggs recoveredfrom oviducts of Hsp60C1/Hsp60C1 females(B–D) that show patterning defects and abnor-mally located (B) or poorly developed (C) dorsalappendages. The egg in D looks like a stage 13egg chamber exhibiting a “dumpless” pheno-type. Scale bar � 100 �m.

HSP60C IS ESSENTIAL FOR DROSOPHILA OOGENESIS 1335

ceptacles, accessory glands, and thegenital chamber (not shown).

Confocal microscopic examination ofdistribution of Hsp60C transcripts indeveloping wild-type egg chambers(Fig. 2) revealed the transcripts to bemostly present in cytoplasm of follicleand nurse cells. The level of Hsp60Ctranscripts was low in the germarium

and in follicle and nurse cells of earlystage egg chambers (Fig. 2A,B). Of in-terest, while the follicle cells continuedto show a similar low level of hybridiza-tion until stage 12 of oogenesis, nursecells and the growing oocyte showed adynamic pattern. Level of Hsp60C tran-scripts in nurse cells and in oocyte in-creased until stage 10B (Fig. 2B–F), af-

ter which, a decline was seen first in theoocyte (Fig. 2E,F) followed by nursecells. Mature oocyte had little ofHsp60C transcripts (Fig. 2G), althoughsome localized hybridization signal wasseen in region where the future micro-pyle would develop (Fig. 2G).

To determine the distribution ofHsp60 protein, we stained egg cham-bers with anti-Hsp60 antibody SPA805 (StressGen, Canada), which is be-lieved to detect all the four Hsp60forms present in D. melanogaster (La-khotia et al., 2002). These prepara-tions were counterstained with tetrar-hodamine isothiocyanate (TRITC)-Phalloidin and 4�,6-diamino-2-phe-nylindole dihydrochloride (DAPI) todetect F-actin and DNA, respectively.

Hsp60 was specifically localized inthe myosin-rich A-bands of striatedmuscles that wrap each ovariole (notshown) and individual egg chambers(Fig. 3A) of wild-type flies. A compa-rable specific localization of Hsp60 inA-bands was seen in striated musclesof oviduct wall (Fig. 3B) and in otherparts of body (not shown).

Follicle cells in wild-type egg cham-bers showed abundant presence ofHsp60 in their cytoplasm. Hsp60 andF-actin overlapped along cell mem-branes between adjacent follicle cells(Fig. 3C–C�). Confocal images of lat-eral views of follicle cells, however,revealed a higher concentration ofHsp60 on the external face of folliclecells while F-actin was more abun-dant on the internal face opposingnurse cells and oocyte (Fig. 3D–D�).

Confocal images of inner optical sec-tions of wild-type egg chambers re-vealed an interesting pattern of distri-bution of Hsp60 at different stages ofoogenesis (Fig. 3E–H). Hsp60 waspresent in nurse cell cytoplasm withenhanced concentration near cell

TABLE 1. Stage 7 and Later Egg Chambers of Hsp60C1 Mutant Flies Show a Variety of Abnormalities

Genotype NNormal eggchambers

Degenerating eggchambersa

Misplacedoocytea

Binucleate/multinucleatenurse cellsa

Multipledefectsa

�/� 136 92.0% 2.2% 1.0% 1.5% 2.2%Hsp60C1/� 136 84.5% 2.2% 1.5% 2.2% 4.5%Hsp60C1/Hsp60C1 94 Nil 25.5% 8.5% 6.5% 25.5%Hsp60C1/Df(2L)cl-h2 86 Nil 25.0% 13.0% 1.5% 32.5%

a The follicle cell layer in all of these egg chambers was affected.

Fig. 2. A–L: Confocal optical sections of wild-type (A–H) and Hsp60C1/Hsp60C1 (I–L) egg cham-bers after in situ hybridization to cellular Hsp60C transcripts (bright fluorescence). The eggchamber stages are marked in each panel. H and L are high-magnification images of posteriorfollicle cells in sectional view from wild-type (H) and Hsp60C1/Hsp60C1 (L) stage 7 egg chambersto show the highly reduced levels of Hsp60C transcripts in cytoplasm of mutant follicle cells and theabsence of regular intercellular tight association between them (4�,6-Diamino-2-phenylindole di-hydrochloride [DAPI] stained nuclei appear gray in H and L). Ger, germarium; MP, micropyle region;NC, Nurse cells; OC, oocyte; FC, Follicle cell. Scale bars � 50 �m in A–G,I–K, 5 �m in H,L.

1336 SARKAR AND LAKHOTIA

membrane (Fig. 3E–E�). Its level in-creased in nurse cells from early tostage 10 chambers and its cellular dis-tribution overlapped with that of F-actin, particularly at cell membranes.Hsp60 was also seen to cover the ca-bles of actin scaffold that developaround the large polyploid nuclei innurse cells (Hudson and Cooley,2002). Compared with nurse and folli-cle cells, the growing oocyte showedless Hsp60 and F-actin, both of which,until stage 10B, were overlappinglyconcentrated along the cell membraneand cortical layer. Hsp60 also spreada little further into the oocyte cyto-plasm (Fig. 3F). In later egg chambers(Fig. 3G,H), in parallel with the distri-bution of Hsp60C transcripts (Fig. 2),Hsp60 became increasingly less abun-dant in most nurse cells and the oo-cyte, while the follicle cells continued

to show moderate levels of Hsp60along with F-actin near cell mem-branes. Beginning with stage 10B andcontinuing until early stage 13, whenmost of the nurse cells showed reduc-tion in cytoplasmic Hsp60, an increasein concentration of Hsp60, and of F-actin, was detected at the basal nursecell cytoplasm near the anterior mar-gin of oocyte (Fig. 3F,G). Interest-ingly, F-actin was absent in the ante-rior oocyte cytoplasm where Hsp60was concentrated during stages 10–12(Fig. 3F,G). Location of the redistrib-uted Hsp60 at stage 10B is in the areawhere �-tubulin is reported to be lo-calized at the beginning of stage 10Bfor development of a distinct MTOC(Schnorrer et al., 2002). In other partsof the oocyte, Hsp60 disappeared al-most completely, although F-actincontinued to be present cortically (Fig.

3G,H). Following stage 13, Hsp60completely disappeared from the ante-rior oocyte cytoplasm while on thenurse cell face, it remained confined toa small area near the nurse cell–oo-cyte junction, which would subse-quently form the micropyle (Fig. 3H).During stage 13, Hsp60 was seen ex-clusively surrounding the future mi-cropyle area along with F-actin, but inmore mature egg chambers, F-actindisappeared from this region whileHsp60 was still present (not shown).

Hsp60C1 Mutant EggChambers Show LittleHsp60C Transcripts andHsp60 Protein

Ovaries from Hsp60C1 homozygous fe-males showed very little hybridizationwith the Hsp60C riboprobe. In intact

Fig. 3. A–P: Confocal images to show localization of F-actin (red, A–P) and Hsp60 (green, A–C�,D�,E�,F–K�,L�,M�,N–P) in striated muscles surroundinga stage 9 egg chamber (A,I), oviduct muscles (B,J), and in follicle cells (C–C�,D–D�,K–K�,L–L�), nurse cells and oocyte (E–E�,F–H, M–M�,N–P) in wild-type(A–H) and Hsp60C1/Hsp60C1 (I–P) egg chambers. A–C�,D�,E�,F–K�,L�,M�,N–P show merged images. Lateral views of follicle cells are shown at highermagnification in D–D� and L–L�. Arrows in J indicate disruptions in lateral register of actomyosin fibrils in a muscle bundle. Arrow in G shows theincreased concentration of Hsp60 and F-actin at the junction of basal nurse cells and the anterior region of the oocyte during stages 11–12, wherethe anterior microtubule organizing center (MTOC) is believed to be formed (Schnorrer et al., 2002). Arrow in H marks the future micropyle region whereHsp60 gets restricted beginning with stage 13B. The mutant egg chambers (N–P) show disorganized F-actin cytoskeletal elements and very littleHsp60. Scale bars � 5 �m in A,C–E�,I,K–M�, 10 �m in B,J, 50 �m in F–H,N–P.


ovaries, some detectable signal wasseen in the associated tracheoles, butthis was significantly less than inwild-type ovaries (not shown). Sper-mathecae also showed little hybridiza-tion (not shown). The significantly re-duced levels of Hsp60C transcripts inthe mutant ovaries and spermathecaefurther confirm that the Hsp60C1 is asevere hypomorphic allele (Sarkarand Lakhotia, 2005).

Confocal microscopy confirmed sub-stantially reduced levels of Hsp60Ctranscripts in egg chambers fromHsp60C1 homozygous flies (Fig. 2I–L).The germline cells showed littleHsp60C transcripts in the very earlystages (Fig. 2I–K). During later stages,while nurse cells occasionally displayedlow levels of transcripts (Fig. 2J,K), lit-tle hybridization was detected in the oo-cyte at any stage (Fig. 2J–L). The levelof Hsp60C transcripts in follicle cellsuntil stage 4–5 in the mutant ovarieswas less severely affected (compare Fig.2A with 2I), but in later mutant eggchambers, the follicle cells also showedsignificantly reduced levels of Hsp60Ctranscripts. As seen in higher magnifi-cation images of follicle cells (Fig.2H,L), compared with the large andmany clusters of Hsp60C transcripts inwild-type follicle cells (Fig. 2H), the mu-tant follicle cells displayed smaller andfewer clusters (Fig. 2L).

The muscle layers surrounding ovari-oles, individual egg chambers (Fig. 3I),and oviduct wall (Fig. 3J) in Hsp60C1

homozygous flies showed significantlyless Hsp60 in the myosin-rich A-bands.F-actin staining revealed that the bun-dles of muscle fiber in the mutant ova-rioles were irregular and thinner thanthose in wild-type with componentfibrils of bundle often not in register(compare Figs. 3A,B and 3I,J).

The germarium and early stage eggchambers of Hsp60C1 mutant fliesshowed very little Hsp60 in follicle ornurse cells (Fig. 3K–M�) with weaksignals seen at some places near theouter membranes of follicle cells (Fig.3L–L�) and at nurse cell junctions(Fig. 3M–M�). In contrast to wild-type,the rare Hsp60C1 mutant egg cham-bers that survived (see below) to laterstages also displayed very littleHsp60. The F-actin distribution toowas severely affected (Fig. 3M–P, seelater). Among the surviving stage 10Band later mutant egg chambers, only

approximately 30% (N � 23) showedsome accumulation of Hsp60 aroundthe junction of basal nurse cells andoocyte (Fig. 3O) or the future micro-pyle region (Fig. 3P), which comparedwith wild-type chambers, was signifi-cantly less. Other regions of these lateegg chambers showed barely detect-able diffuse Hsp60 staining.

Hsp60C1 Mutant EggChambers Show Variety ofDefects in Egg Chambers

Organization of follicle and nurse cellsin wild-type, Hsp60C1, and Hsp60C1/Df(2L)Cl-h2 mutant ovarioles was com-pared (Table 1) by confocal microscopyof DAPI-stained preparations or of eggchambers from females expressinggreen fluorescent protein (GFP) -taggedHrb98DE (Morin et al., 2001). We foundthat the GFP-tagged Hrb98DE proteinshows a specific nuclear localization infollicle as well as the nurse cells of allstages of wild-type egg chambers andthus can serve as a good nuclearmarker in egg chambers. Until stage 6,the arrangement of follicle cells inHsp60C1/Hsp60C1 and Hsp60C1/Df(2L)Cl-h2 egg chambers (Fig. 4B) ap-peared generally comparable to that inwild-type (Fig. 4A). However, in allpost-stage 6 mutant egg chambers, in-stead of the regular one-cell-deep layerin wild-type (Fig. 4A; Table 1), the folli-cle cell layer at the posterior end, al-though completely covering the oocyte,was irregular and more than one celldeep (Fig. 4B). Interestingly, despitethe disorganized follicle cell layer, theidentity of polar follicle cells in majority(�98% of 55 stage 6–9 chambers) ofmutant egg chambers was not affectedas revealed by staining for Fasciclin-III(McGregor et al., 2002; not shown).

Nearly half of stage 7–8 egg cham-bers from Hsp60C1/Hsp60C1 orHsp60C1/Df(2L)Cl-h2 female fliesshowed a variety of other defects in ad-dition to the disorganized follicle celllayer (see Table 1) and in agreementwith the stage 7–8 of Drosophila oogen-esis being a developmental checkpoint(Buszczak and Cooley, 2000; Nezis etal., 2000), most of these severely af-fected egg chambers did not develop fur-ther and degenerated (Fig. 4C). The fewthat survived the stage 7 checkpoint,showed additional defects (see below).

Actin Cytoskeleton IsAffected in Hsp60C1 MutantEgg Chambers

The F-actin based cytoskeleton in mu-tant follicle and nurse cells did notshow any detectable difference fromthat in wild-type until stage 6 (notshown) except for the muscle layernoted above. Hsp60C1 egg chambersdisplayed a variety of abnormalitiesbeginning with stage 6–7 when folliclecell remodeling starts. Unlike the reg-ular monolayer of hexagonal folliclecells in wild-type (Fig. 4D), those inthe Hsp60C1/Hsp60C1 egg chamberswere highly irregular (Fig. 4E). Mostof the mutant egg chambers, includingthose developing beyond stage 7,showed much less F-actin at nurse cellborders. In some, it was abnormallyaggregated around the oocyte (Fig.4F). Nearly 28% of the 42 survivingpost-stage 8 Hsp60C1/Hsp60C1 eggchambers examined displayed bi- ormultinucleate nurse cells (Fig. 4G; Ta-ble 1), which may be due to the pooractin reinforcement of cell mem-branes. Likewise, in some mutant eggchambers, one or more of the nursecell nuclei appeared to have fallen intothe oocyte (not shown).

The F-actin cage, that forms aroundeach of the large nurse cell nucleus(see Fig. 3E) when the nurse cells be-gin to actively dump their cytoplasmiccontents into oocyte, was nearly ab-sent in the mutant stage 10 egg cham-bers (Fig. 3M). Likewise, the otheractin cables, which hold and stabi-lize ring canals during the rapid cy-toplasmic flow in wild-type, werealso absent in mutant egg chambers(not shown).

Cell–cell Adhesion IsAffected in Hsp60C1/Hsp60C1

Egg Chambers

The irregular follicle cell layer and thepresence of chambers with misposi-tioned oocyte (Fig. 4G,H; Table 1)indicate poor cell–cell adhesionin Hsp60C1/Hsp60C1 or Hsp60C1/Df(2L)Cl-h2 egg chambers. This wasconfirmed through immunostainingfor some of the cell adhesion markers.

During the cellular rearrangementsat stage 7, components of the cadherinadhesion complex, DE-cadherin, Ar-


madillo, and -catenin, are known toaccumulate along the border betweenthe oocyte and posterior follicle cells toprovide the required adhesion be-tween them, and thereby determiningthe posterior position of the oocyte(Gonzalez-Reyes and St Johnston,1998; Muller, 2000). Immunostainingof wild-type and mutant egg chambersrevealed that unlike the uniformbasal expression of DE-cadherin on allfollicle cells in wild-type egg chambersand much higher level on the two pos-terior polar follicle cells, (Fig. 4K), eggchambers from Hsp60C1/Hsp60C1

ovaries had significantly reducedstaining, particularly in the posteriorpolar follicle cells (Fig. 4L). Similarly,compared with wild-type egg cham-bers (Fig. 4M), the mutant egg cham-bers showed reduced staining for Dlg,including in the posterior polar folliclecells (Fig. 4N).

Unlike the regular distribution of-spectrin in wild-type follicle celllayer (Fig. 4O), that in Hsp60C1/Hsp60C1 egg chambers (Fig. 4P) wasuneven and disrupted. Likewise, im-munostaining for tyrosine phosphory-lation, an important regulatory eventfor formation of cell junctions (Volberget al., 1992), showed that, while folli-cle cells in wild-type showed a smoothuniform phosphotyrosine staining ofthe inner and lateral faces (Fig. 4I),those in Hsp60C1/Hsp60C1 chambersshowed uneven and irregular stainingwith missing columnar arrangement(Fig. 4J).

Hsp60C1 Mutation AffectsMicrotubules and RingCanals

Posterior follicle cells in wild-typeearly egg chambers signal the poste-rior MTOC in oocyte for microtubuleremodeling, which is responsible forthe localization of several polarity de-terminants and movement of oocytenucleus (Riechmann and Ephrussi,2001; Steinhauer and Kalderon,2006). Because Hsp60C1/Hsp60C1

egg chambers showed abnormal dis-tribution of actin and cell-adhesionproteins and aberrant arrangement ofposterior follicle cells, we examineddistribution of microtubules using an-ti–-tubulin antibody. In wild-typestage 8–10 oocyte, -tubulin staining

Fig. 4. A,B: Confocal images of 4�,6-Diamino-2-phenylindole dihydrochloride (DAPI) stainedovarioles from wild-type (A) and Hsp60C1 homozygous (B) females. C: Hsp60C1 homozygous stage9–10 chamber with degenerating nurse cells. D–F: Phalloidin-stained follicle cell layer (D,E) and eggchamber (F) from wild-type (D) or Hsp60C1 mutant (E,F) flies. G: Hrb98DE-green fluorescent protein(GFP)-expressing (green) and Phalloidin-stained (red) mid stage egg chamber from Hsp60C1

mutant female showing bi- and/or multinucleate nurse cells with misplaced oocyte and aberrantF-actin cytoskeleton. H: Combined fluorescence and differential interference contrast microscopyimage of an egg chamber with a mispositioned oocyte from a mutant fly. I,J: Higher magnificationlateral views of phosphotyrosine-stained follicle cell layer from wild-type (I) or Hsp60C1 mutant (J)females. K–P: Immunostaining for DE-cadherin (K,L) or Dlg (M,N) or -spectrin (O,P) in wild-type(K,M,O) and Hsp60C1 mutant (L,N,P) follicle cell layer (arrowheads mark the posterior pair of polarcells in K,M). Q,R: -Tubulin distribution in wild-type (Q) and Hsp60C1 mutant (R) oocytes at the10B stage. S,T: Immunostaining for HTS-RC to show status of ring canals in wild-type (S) andHsp60C1 mutant (T) ovarioles. U–X: Staining of ring canals with anti-phosphotyrosine antibody(green) and Phalloidin (red) from wild-type (U,W) and Hsp60C1 mutant (V,X) mid-stage nurse cells.Germ, germarium; NC, nurse cells; OC, oocyte. Scale bars � 10 �m in A–C,F–H,O–T,V, 5 �m inD,E,I–N,U,W,X.


was seen along the cell membrane.-tubulin was undetectable in the cy-toplasm at the posterior end while agreater concentration was seen at theanterior half, with a stronger presenceat the junction of oocyte and nursecells (Fig. 4Q). On the other hand,more than 80% (N � 25 stage 8–10egg chambers) of Hsp60C1/Hsp60C1

oocytes showed reduced and some-what abnormally distributed -tubu-lin (Fig. 4R) and in all such cases, theanterodorsal migration of the oocytenucleus was affected, indicating thatthe asymmetrical mid-oogenesis mi-crotubule array does not form prop-erly in the mutant chambers.

Status of ring canals in wild-typeand Hsp60C1/Hsp60C1 egg chamberswas assessed through staining withanti–HTS-RC, TRITC-Phalloidin, an-ti-Phosphotyrosine, or with anti-Kelchantibodies. Immunostaining with an-ti–HTS-RC, which exclusively stainsthe ring canals of developing eggchambers (Robinson et al., 1994),showed obvious differences in the ar-rangement of ring canals betweenHsp60C1/Hsp60C1 (Fig. 4T) and wild-type egg chambers beginning fromstage 6 (Fig. 4S), since approximately80% (N � 24 stage 6–11 chambers) ofstage 6 and later Hsp60C1/Hsp60C1

egg chambers showed abnormally lo-cated and/or fewer ring canals (Fig. 4T).

In wild-type egg chambers, F-actinaccumulates in the inner rim of eachring canal with phosphotyrosine at theouter rim (Fig. 4U,W). In advancedstage Hsp60C1 mutant egg chambers,some ring canals appeared abnormalwith the F-actin staining being morediffuse or nearly absent (Fig. 4V,X).Size of ring canals, however, was notsignificantly altered in Hsp60C1/Hsp60C1 egg chambers. Anti-Kelch an-tibody did not show significant differ-ences in staining of ring canals in wild-type and mutant egg chambers (notshown), except for the general defectsnoted above in the mutant chambers.

Expression and Localizationof Hrb98DE, Squid, andGurken Are Disrupted inHsp60C1/Hsp60C1 EggChambers

The heterogeneous nuclear RNA-binding (hnRNP) and other RNA-

binding proteins play significant rolesin spatial localization of the variousRNAs and proteins in oocyte (John-stone and Lasko, 2001). Because theHsp60C1 mutant egg chambers dis-played abnormalities in follicle celllayer and aberrant actin and tubulindistribution in oocyte, we examinedexpression and localization of twohnRNPs (Hrb98DE and Squid) andGurken in the mutant egg chambers.

The Hrb98DE and Squid proteinswere localized through the Hrb98DE-GFP and Squid-GFP protein trap al-leles, respectively (Morin et al., 2001).The Hrb98DE-GFP allele expressesGFP-tagged Hrb98DE in all follicleand nurse cell nuclei starting fromstage 1 of oogenesis in wild-type eggchambers (see above). However,Hrb98DE-GFP was nearly absent orsignificantly reduced in many (�78%of 39 stage 6–11 egg chambers exam-ined) nurse cell nuclei in Hsp60C1 mu-tant (Hsp60C1/Hsp60C1; Hrb98DE-GFP/Hrb98DE-GFP) egg chambers(compare Fig. 5A with 5B). Interest-ingly, no significant difference in thenuclear expression of Hrb98DE-GFPwas detected in somatic follicle cells ofwild-type and the mutant ovarioles(Fig. 5A,B).

Squid binds the gurken mRNA and

mediates its transport from nurse cellnucleus to oocyte and as reported ear-lier (Norvell et al., 2005), the Squid-GFP was nuclear in nurse cells ofstage 9–10 egg chambers in wild-type{w; �/�; Squid-GFP (44-1)/Squid-GFP(44-1)} while it localized at theanterodorsal side and posterior pole inthe oocyte (Fig. 5C). In contrast,nearly 80% (N � 32 egg chambers) ofthe mutant {w; Hsp60C1/Hsp60C1;Squid-GFP (44-1)/Squid-GFP (44-1)}stage 9–10 egg chambers showedSquid-GFP in nurse cell cytoplasm aswell (Fig. 5D); furthermore, Squid-GFP content was much less in the oo-cyte and unlike in wild-type (Fig. 5C),its localization there was never specif-ically restricted to the anterodorsaland posterior sides (Fig. 5D).

The distribution of Gurken in the mu-tant egg chambers was comparable un-til stage 6–7 to that in wild-type (notshown). However, compared with thetight localization of Gurken at the an-terodorsal corner of the oocyte in wild-type stage 10 egg chambers (Fig. 5E),the majority (�90%, N � 28 stage 7–11egg chambers) of the post-7 stage mu-tant egg chambers showed a variety ofalterations, like little Gurken in the oo-cyte but high abundance in nurse cellsor scattered distribution of Gurken

Fig. 5. A–G: Confocal images to show localization of Hrb98DE-GFP (A,B), Squid (C,D), andGurken (E–G) proteins (green fluorescence in all cases) in wild-type (A,C,E) and Hsp60C1/Hsp60C1

(B,D,F,G) egg chambers. Red fluorescence in A and B shows Phalloidin stained F-actin, while inE–G, red fluorescence shows 4�,6-Diamino-2-phenylindole dihydrochloride (DAPI) -stained nuclei(false colored). Arrow in A indicates a nurse cell nucleus with strong Hrb98DE-green fluorescentprotein (GFP) expression, which is highly reduced in case of mutant nurse cells (arrow in B). Arrowin C indicates localization of Squid in the anterodorsal region of wild-type oocyte. NC, nurse cells;OC, oocyte. Scale bars � 50 �m.


throughout the oocyte cytoplasm alongwith a greater concentration in the an-terodorsal region (Fig. 5F,G).

Hsp60C1 Mutant Follicle CellClones in Hsp60C1/� EggChambers Lose MonolayerArrangement and ShowAltered F-Actin Distribution

We generated mitotic clones of folliclecells homozygous for the Hsp60C1 mu-tant allele in Hsp60C1/� egg cham-bers by using FRT/FLP-mediated site-specific somatic recombination (Golicand Lindquist, 1989), to ascertain ifthe effects of Hsp60C1 mutation onfollicle cell arrangement and F-actinderangement were cell autonomous orresult of general weakness of the raresurviving Hsp60C1 mutant flies. TheFLP recombinase was specifically ex-pressed in follicle cells through theT155-GAL4 transgene (Duffy et al.,1998). The Hsp60C1/Hsp60C1 mitoticclones were identified by the absenceof GFP fluorescence in follicle cells. Atotal of 32 Hsp60C1/� egg chambersderived from several flies and contain-ing variable proportions of Hsp60C1

homozygous follicle cell clones wereexamined. In every case, the homozy-gous Hsp60C1 follicle cell clones,whether very small or more extensive,exhibited loose and irregular epithe-lial organization. As seen in the twostage 7 egg chambers in Figure 6A–A�and B–B�, while the GFP-expressingHsp60C1/� (or �/�) follicle cells inall clone-bearing egg chambers (N �32) displayed the regular mono-layer arrangement, the GFP-nega-tive Hsp60C1/Hsp60C1 cells, evenwhen only a few (encircled areas inthe Fig. 6) had lost the characteristictight monolayer arrangement. Thisfinding is reminiscent of the dis-rupted arrangement of follicle cellsin Hsp60C1 homozygous egg cham-bers (see Fig. 4B,C).

In addition to the altered arrange-ment of follicle cells, loss of Hsp60C inthe Hsp60C1 mutant clones also af-fected F-actin membrane cytoskele-ton. Figure 6C–C�� show surface viewsof an egg chamber bearing multipleHsp60C1 mutant follicle cell clones(encircled areas). The F-actin cy-toskeleton in each of the Hsp60C1/Hsp60C1 follicle cells was abnormal.

Interestingly, the Hsp60C1/� nursecell nuclei (GFP-positive, Fig. 6D�) inthis egg chamber showed extensiveapoptotic fragmentation (Fig. 6D),while the F-actin cytoskeleton in themand in the oocyte (Fig. 6D�) was alsoaffected. In all those egg chambers inwhich Hsp60C1 mutant clones weresmall and few, the nurse cells and oo-cyte appeared unaffected but in thosecases where a large proportion of thefollicle cells had become Hsp60C1 ho-mozygous following the FLP-inducedsomatic recombination, the nurse cellsand the oocyte were also affected tovarying extent.

Hsp60C1 Mutant Germ CellClones in Hsp60C1/� EggChambers Show Altered F-Actin Distribution and OtherAbnormalities

To further examine if Hsp60C func-tion is autonomously required ingermline cells, mutant germlineclones for the Hsp60C1 allele weregenerated in Hsp60C1/� egg cham-bers using the FLP-DFS technique(Chou and Perrimon, 1996). In theseclones, the somatic cells are wild-type(Hsp60C1/�), whereas the germlinecells are homozygous for the Hsp60C1

mutation. In each such ovary, only oneto five ovarioles contained developingegg chambers, confirming that theovoD1 mutation was not present intheir germline cells, which therefore,were homozygous for the Hsp60C1 al-lele.

The germ line clone-bearing eggchambers were stained with DAPI,Hsp60 antibody, and Phalloidin-TRITC to examine the cellular distri-bution of Hsp60 and F-actin. TheHsp60C1 homozygous nurse cells andthe oocyte in these chambers showedlittle Hsp60, although the somatic fol-licle cells had the levels expected inHsp60C1/� cells (Fig. 7). The overallarrangement of follicle cells was gen-erally comparable to those in wild-type chambers (see inset in Fig. 7A).The frequency of degenerating stage 7chambers in these mosaic ovaries(�18%, Table 2) was almost 9greater than that in Hsp60C1/� non-mosaic ovaries (2.2%, Table 1) andcomparable to that in Hsp60C1 ho-mozygotes. The surviving mosaic

chambers appeared generally normaluntil stage 7–8, although the shapeand arrangement of F-actin in severalof them was affected (Table 2; Fig.7A). Approximately 35% of stage 9–12chambers were also seen to be degen-erating (inset in Fig. 7C). Phalloidinstaining showed that the F-actin net-work in nurse cells and oocyte in mostof the surviving mid-stage mosaic eggchambers was weak and irregular(Fig. 7A–C, compare with Fig. 3F–P).The accumulation of Hsp60 proteinaround the junction of basal nursecells and anterior oocyte margin dur-ing later stages of oogenesis seen inwild-type (Fig. 3F,G) was absent inthe surviving Hsp60C1 germ line mo-saic egg chambers (Fig. 7C,D) and fur-ther unlike wild-type, the F-actin atthis junction was more on the oocyteside rather than the nurse cell side(compare Figs. 3G and 7C). Interest-ingly, like in Hsp60C1/Hsp60C1

chambers (Fig. 3M), the germ cellclone bearing egg chambers (Fig.7C,D) also failed to develop the F-ac-tin cables to hold the large nurse cellnuclei at later stages of oogenesis,which is characteristically seen inwild-type (Fig. 3E) or Hsp60C1/� eggchambers. Approximately 14% of thestage 8–9 egg chambers showed mis-placed oocyte (Table 2). In some of themosaic chambers with more severelydisorganized F-actin cytoskeleton innurse cells, the follicle cells alsoshowed abnormalities and signs of de-generation (see inset in Fig. 7C).

DISCUSSION

The present study clearly demon-strates that the Hsp60C gene ex-presses in a dynamic pattern throughoogenesis and that homozygosity forthe Hsp60C1 recessive loss of functionallele (Sarkar and Lakhotia, 2005) re-sults in severe defects in oogenesisand, thus, in complete sterility of thehomozygous mutant females. Restora-tion of fertility and other phenotypesassociated with the Hsp60C1 alleleupon excision of the P-transposon(Sarkar and Lakhotia, 2005) and asimilar or a little more exaggeratedoogenesis phenotype (Table 1) whenthis mutant allele is placed againstthe Df(2L)cl-h2 deficiency, which cov-ers this gene’s location (Lindsley andZimm, 1992), confirm that the abnor-


malities in oogenesis in the survivingHsp60C1/Hsp60C1 homozygous fe-males are indeed due to the Hsp60Cmutant allele. Because the matureegg does not show any detectableHsp60C transcripts or the protein, theHsp60C appears to have functions inoogenesis rather than having mater-nally transmitted zygotic functions.

As noted earlier (Sarkar and Lakho-tia, 2005), only approximately 10% ofthe Hsp60C1 homozygotes survive asweak and short-lived flies. Becausethe general physiology of flies isknown to significantly affect progres-sion of oogenesis, it remains possiblethat the observed defects in oogenesisin the Hsp60C1 homozygous femalesresult merely from their poor physiol-ogy. Present results, however, rule outthe possibility that defective oogenesis

Fig. 6.

Fig. 7.


in Hsp60C1 homozygous females is anonspecific effect of general weakness/poor physiology of the mutant flies.Comparison of immunostaining ofwild-type and Hsp60C1 homozygousegg chambers with the SPA805 anti-Hsp60 antibody, which is expected todetect all the four Hsp60 family mem-bers in D. melanogaster cells (Lakho-tia et al., 2002), suggests that Hsp60Ccontributes maximally to the Hsp60family proteins detected by this anti-

body in egg chambers and striatedmuscles. The remarkable cell type andstage-specific expression of Hsp60Cgene in follicle as well as germlinecells in egg chambers but not in otherparts of the female reproductive sys-tem correlates with the fact that onlythe egg chambers, but not the acces-sory gland, oviduct, etc., are severelyaffected by the Hsp60C1 mutation. Fi-nally, clonal analyses made it clearthat Hsp60C function is essential in

the follicle as well as in the germlinecells because in both kinds of mosaics,autonomous defects in F-actin cy-toskeleton and other aspects of oogen-esis were seen and in both cases, themosaic egg chambers failed to com-plete oogenesis. Therefore, it is obvi-ous that the aberrations seen inHsp60C1 homozygous egg chambersare largely due to this protein’s essen-tial functions during oogenesis ratherthan to the poor physiology of the es-capee survivors.

Abnormalities in the Hsp60C1 mu-tant egg chambers first become appar-ent after stage 5–6 in the form of mi-sorganized posterior follicle cells, andthis finding correlates well with a sig-nificant reduction in Hsp60C tran-scripts in the mutant follicle cells fromstage 5–6 onward. Although the mu-tant germ cells did not show Hsp60Ctranscripts even during stage 1–4,any ensuing abnormality in theirfunctions may not result in easily de-tectable phenotype at this early stage.The follicle cells have significant rolesin organization of egg chamber fromthe very early stage, and they are es-pecially important in converting therounded stage 5–6 chambers to an-teroposteriorly elongated later stagechambers. Therefore, any misfunc-tioning of the follicle cells at this earlystage will have cascading effects onsubsequent development of egg cham-ber.

The most significant abnormalitiesobserved in Hsp60C1 mutant or mo-

Fig. 6. A–D: Follicle cells in Hsp60C1/Hsp60C1 clones, generated by FLP-mediated somaticrecombination, in Hsp60C1/� egg chambers are irregularly arranged and show abnormal F-actincytoskeleton. A–A� and B–B� (only a part of the follicle cell layer is shown in B) are examples of smallHsp60C1/Hsp60C1 clones in two different stage 7 egg chambers (green fluorescent protein [GFP]-negative encircled areas) and in each of the clones, the monolayer arrangement of the mutantfollicle cells is disrupted; A,B show 4�,6-Diamino-2-phenylindole dihydrochloride (DAPI) fluores-cence (red, pseudo-color), A�,B� show GFP fluorescence, while A� and B� are DAPI and GFPmerged images. C–C�� show surface views of DAPI (C, red pseudo-color), GFP (C�, green),tetrarhodamine isothiocyanate (TRITC) -Phalloidin (C�, magenta pseudo-color) fluorescence of astage 8 Hsp60C1/� egg chamber with multiple Hsp60C1/Hsp60C1 mitotic clones in the follicle celllayer (encircled areas); C�� shows a merged image of C–C�. Note the disruption in regular monolayerarrangement and aberrant accumulation of F-actin in all the Hsp60C1 homozygous follicle cellclones. D–D�� show mid-plane optical section of the same egg chamber as in C–C�� displayingfragmentation (apoptosis) of nurse cell nuclei even though they are GFP-positive and, therefore,Hsp60C1/�; the oocyte area is encircled. Germ, germarium; NC, nurse cells; OC, oocyte. Scalebars � 10 �m in A–A�,C–D�, 5 �m in B–B�.

Fig. 7. Confocal images of FLP-DFS technique-generated germline mosaic egg chambers of theindicated stages stained with 4�,6-Diamino-2-phenylindole dihydrochloride (DAPI, blue), Phalloidin-tetrarhodamine isothiocyanate (TRITC; red) and immunostained with anti-Hsp60 antibody (green).Note the normal distribution of Hsp60 in follicle cells (FC, Hsp60C1/�), but its near absence in nursecells (NC) and developing oocytes (OC), all of which are Hsp60C1 homozygous. The actin filamentsin Hsp60C1 homozygous nurse cells and the oocyte (germline clones) are abnormally arranged. Theinset in A shows the surface image of the chamber shown in A to illustrate the near normalarrangement of follicle cell layer, although the nurse cells and the oocyte show somewhat abnormalactin distribution and the chamber shape is affected. The inset in C shows a degeneratingmid-stage egg chamber. The arrow in C points to the rather diffuse accumulation of F-actin atjunction of nurse cells and oocyte, while that in D points to the poorly developed actin cage in nursecell of a stage 11 chamber. Scale bars in A,C,D � 50 �m, 10 �m in D.

TABLE 2. Defects in Hsp60C1 Homozygous Mutant Germline Clones in Hsp60C�/Hsp60C1 Egg Chambers

StageTotal eggchambers

Degeneratingchambers

Surviving chambers

Nurse cells and oocyte Follicle cells

Until stage 8 38 7 No major defect, except some alterations inegg chamber shape and/or some defectsin F-actin arrangement

normal in most cases

Stage 9-10 22 10 Severe defects in arrangement of F actin innurse cells

Abnormal positioning of oocyte in 3 eggchambers

Organization affected in severelyaffected and degenerating eggchambers

Stage 11-12 8 2 Defective F-actin cageNo accumulation of Hsp60 protein at

anterior border of oocytePositioning of oocyte nucleus not affected Organization affected in severely

affected and degenerating eggchambers

Stage 12-14 6 . All abnormal showing dump less andpolarity defect phenotypes


saic egg chambers are (1) poor adhe-sion of follicle cells from stage 6–7onward, (2) membrane fragility ofnurse cells, (3) aberrations in ring ca-nals, (4) poorly developed F-actin ca-bles that cage the large polyploidnurse cell nuclei during the dumpingprocess, and (5) impaired polarity indeveloping oocyte. It is interestingthat all these abnormalities can re-sult, in a cascading manner or inde-pendently, from defective organiza-tion of the various cytoskeletalelements during the progression of oo-genesis. In this context, the overlap-ping distribution of Hsp60, F-actin,and/or -tubulin, in follicle and germ-line cells in egg chambers, especiallynear cell membranes and at siteswhere MTOCs develop, is significant.Although these different proteins didnot show absolute colocalization, thatthey were largely present in overlap-ping domains and these domains de-veloped defects in the absence ofHsp60C indicate that Hsp60C is re-quired for normal organization of cy-toskeletal components during progres-sion of normal oogenesis. It is notablein this context that an essential re-quirement of Hsp60 family proteinsfor proper folding and stability of actinand tubulin proteins and for organiza-tion of cytoskeletal elements in yeastand other eukaryotes has alreadybeen shown (Chen et al., 1994; Melkiand Cowan, 1994; Vinh and Drubin,1994; Liang and MacRae, 1997;Sarkar et al., 2006). Furthermore,Hsp60 family proteins, immunopre-cipitated with the same anti-Hsp60antibody as used by us, were found tobind most strongly with actin in C.elegans (Leroux and Candido, 1997).Considering all these, we believe thatthe Hsp60C is necessary for properorganization of the F-actin and othercytoskeletal elements in different celltypes of egg chambers. Various abnor-malities seen in egg chambers ofHsp60C1 mutant chambers thus seemto be direct or indirect consequences ofcytoskeletal abnormalities. The modeof interaction between Hsp60C andcytoskeletal components remains tobe analyzed.

The cytoskeletal elements and cad-herin-mediated differential adhesionof the follicle cells are critical for theirproliferation, migration, remodeling,fate adaptation, and for communica-

tion with nurse cells and the growingoocyte (Zhang and Kalderon, 2000;Steinhauer and Kalderon, 2006). Sev-eral actin-binding proteins are knownto be essential for maintenance of cel-lular integrity and follicle cell mor-phogenesis during Drosophila oogene-sis (Hudson and Cooley, 2002). It is,therefore, likely that absence ofHsp60C and consequent inappropri-ate organization of F-actin compo-nents effect follicle cell adhesion, withthe first effect being manifest at theposterior pole around stage 6–7, whenthe level of Hsp60C transcripts alsosignificantly declines in follicle cells(see Fig. 2). During remodeling of eggchambers at these stages, follicle cellsat the posterior pole face greaterstrain due to curvature and, therefore,the posterior polar cells require longerseptate junctions than their lateralcounterparts (Mahowald, 1972). Theweak septate junctions seen in theposterior pole cells in the mutantstage 6–7 chambers obviously do notprovide the required strong cell–celladhesion and thus result in irregularepithelium. Mutations in genes encod-ing cell adhesion molecules such asArmadillo, DE-cadherin, Merlin,-Spectrin, etc. affect follicle cell re-modeling (Oda et al., 1997; Muller,2000; MacDougall et al., 2001; Hud-son and Cooley, 2002; Steinhauer andKalderon, 2006), and the resultingphenotypes are in several ways simi-lar to those seen in the Hsp60C1 mu-tant egg chambers. In addition to theposterior group of follicle cells,Hsp60C is also essential for generat-ing and maintaining a patternedmonolayer of all other follicle cells asevidenced by the irregular arrange-ment of Hsp60C1/Hsp60C1 folliclecells in mitotic clones, irrespective oftheir location and size (Fig. 6). Mis-positioning of oocyte in some Hsp60C1

egg chambers also reflects defectiveadhesion of follicle cells with the oo-cyte. The nonautonomous perturba-tions in Hsp60C1/� nurse cells in fol-licle cell mosaics and in follicle cells ingermline mosaics at later stages of oo-genesis too reflect the need for appro-priate interactions between folliclecell monolayer and the enclosed germ-line cells (Muller, 2000).

The weak or defective F-actin net-work associated with the various cellmembranes in Hsp60C1 egg chambers

also appear responsible for ring canalaberrations, the fragility of nurse cellmembranes and for the dumpless phe-notype shown by the few survivinglate stage chambers. Defects in distri-bution and/or numbers of ring canalseven at stage 6 in Hsp60C1 mutantegg chambers (Fig. 4T) reflect defec-tive membrane dynamics duringnurse cell division and early develop-ment, presumably due to the nearcomplete absence of Hsp60C tran-scripts in germline cells from stage 1(Fig. 2). The frequent occurrence of bi-or multinucleate nurse cells or of thenurse cell nucleus falling into the oo-cyte in the mutant chambers also in-dicate fragility of nurse cell mem-branes, presumably because ofinsufficient or misorganized F-actin.It appears that, in the absence of ap-propriately continuing F-actin folding/maturation, the ring canals, even af-ter their initial assembly, may losestability and degenerate in the surviv-ing later stages.

Signaling by the polarized posteriorepithelial follicle cells is required forestablishing the posterior MTOC inoocyte and its later remodeling(Chekulaeva and Ephrussi, 2004). TheMTOC in turn is important for estab-lishing both the anteroposterior anddorsoventral egg axes in Drosophila(van Eeden and St Johnston, 1999;Riechmann and Ephrussi, 2001;Chekulaeva and Ephrussi, 2004). Thealtered distribution of -tubulin in oo-cyte in the mutant egg chambers re-flects defective remodeling of posteriorMTOC, presumably because of aber-rant signaling by the overlying disor-ganized follicle cell layer and defectivetubulin maturation in absence ofHsp60C. The remarkable similaritybetween the reported localization siteof �-tubulin (Schnorrer et al., 2002)and Hsp60 and F-actin near the basalnurse cell cytoplasm and anterior oo-cyte cortex at the beginning of stage10B, seen in the present study, is sig-nificant. Although we did not studythe distribution of �-tubulin in themutant chambers, that of F-actin atthe junction of nurse cells and oocytewas found to be affected in the mutantstage 10B-12 chambers. This findingindicates that Hsp60C plays an im-portant role in the formation of theMTOC at this region also in wild-typeegg chambers. The near absence of


Hsp60C in the Hsp60C1 mutant eggchambers may thus affect the variousMTOCs that develop in the oocyte atdifferent developmental stages. Thedefective MTOCs would affect severalmicrotubule-dependent developmen-tal processes like localization of pro-teins like Squid and Gurken, and ofthe oocyte nucleus during mid andlate oogenesis. That the disorganizedposterior follicle cells in stage 6–7Hsp60C1 mutant egg chambers affectthe posterior MTOC remodeling isalso evidenced by the fact that, ingermline mosaic egg chambers thathave a normal follicle cell layer, theoocyte nuclear position remains nor-mal (Table 2). Consequent to mislocal-ization of Squid, Gurken, and the oo-cyte nucleus in mutant chambers, eggpolarity will be affected, as reflected inabnormal dorsal appendages in thefew eggs that can be recovered. It willbe interesting to further examine ifHsp60C has a direct role also in thetransport and characteristic localiza-tion of proteins like Squid, Gurken,and so on, in oocyte.

We are not aware of any previousreport describing a patterned localiza-tion of Hsp60 on A-bands of skeletalmuscles. However, the patterned lo-calization of Hsp60 in normal skeletalmuscles and the disorganized state ofthese muscles in absence of Hsp60Cprotein are suggestive of an importantrole of this member of the Hsp60 fam-ily protein in proper organization ofactin and myosin filaments in skeletalmuscles of flies. Since normal muscu-lar organization and its neural co-or-dination have been reported to be es-sential for egg development andoviposition (Middleton et al., 2006), itremains possible that the misorga-nized striated muscle fibers that wrapgermarium and the egg chambers inHsp60C1 mutant flies may also affectoogenesis. The poor musculature inthe oviduct of the mutant flies mayhinder egg laying.

Our studies show, for the first time,that the Hsp60C protein is essentialat multiple steps of oogenesis, whichseem to relate primarily to cytoskel-etal structures in follicle as well asnurse cells. In our earlier study(Sarkar and Lakhotia, 2005) on theeffects of the Hsp60C1 mutation ontracheal morphogenesis and spermat-ogenesis, we raised the possibility

that the Hsp60C itself may also func-tion as a signaling molecule, espe-cially because such a function isknown in other systems (Gobert et al.,2004; Sarkar et al., 2006). In additionto the possible signaling activity, theHsp60C also appears to have essentialroles in organization of the various F-actin, tubulin, and other cytoskeletalelements during oogenesis. Furtherstudies will help understand how theHsp60C performs its diverse vitalfunctions in development and differ-entiation in Drosophila.

EXPERIMENTALPROCEDURES

Drosophila Stocks

The different stocks used in thisstudy, either available in laboratoryor obtained from the Bloomingtonstock center or other sources, weremaintained on standard Drosophilafood medium at 22 � 1°C. Oregon R�

served as wild-type control. Hsp60C1

is a recessive lethal loss of functionallele of the Hsp60C gene, in which aP-transposon is inserted at �251-bpposition in the promoter region(Sarkar and Lakhotia, 2005). TheDf(2L)cl-h2 chromosome carries a de-letion of the 25D06 to 25F04-05 region(Lindsley and Zimm, 1992), which cov-ers the Hsp60C locus (Sarkar and La-khotia, 2005), w; �/�; Hrb98DE-GFP(ZCL 0588)/Hrb98DE-GFP (ZCL0588) and w; �/�; Squid-GFP (44-1)/Squid-GFP (44-1) are protein-traplines in which the Hrb98DE andSquid hnRNP family proteins, respec-tively, are tagged with GFP (Morin etal., 2001). Appropriate crosses weremade to generate w; Hsp60C1/CyO;Hrb98DE-GFP (ZCL 0588)/Hrb98DE-GFP (ZCL 0588) and w; Hsp60C1/CyO; Squid-GFP (44-1)/Squid-GFP(44-1) stocks.

For generation of mitotic clones ofHsp60C1 homozygous follicle cells,w1118; P{ry�t7.2�neoFRT}40A/CyO (Xuand Rubin, 1993), and w1118; P{Ubi-GFP}33 P{Ubi-GFP}38 P{neoFRT}40A/CyO; P{GawB}T155 P{UAS-FLP1.D}JD2/TM3, Sb1 stocks (Duffy et al.,1998) were used. For generation ofHsp60C1 mutant germline clones,w1118; P{ry�t7.2�neoFRT}40A/CyO andw* P{ry[�t7.2]�hsFLP}12; P{w[�mC]�ovoD1-18}2La P{w[�mC]�ovoD1-18}

2Lb P{ry[�t7.2]�neoFRT}40A /CyOstocks (Chou and Perrimon, 1996) wereused.

Generation ofHsp60C1/Hsp60C1

Homozygous Follicle orGermline Cell Clones inHsp60C1/� Egg Chambers

Homozygous Hsp60C1 mutant folliclecell clones were generated by theGal4-directed and UAS-FLP–medi-ated site-specific mitotic recombina-tion technique (Golic and Lindquist,1989). Follicle cell clones mutantfor Hsp60C1 were generated usingthe T155Gal4/UAS-FLP transgeneinserted on chromosome 3, whichdrives expression of the site-specificyeast recombinase FLP in all thefollicle cells during oogenesis (Duffyet al., 1998). The Hsp60C1 mutantallele was recombined onto theP{ry�t7.2�neoFRT}40A carrying chro-mosome through appropriate crosses.The w1118; P{ry�t7.2�neoFRT}40AHsp60C1/CyO; �/� flies were crossedwith w1118; P{Ubi-GFP}33 P{Ubi-GFP}38 P{neoFRT}40A Hsp60C�/CyO;P{GawB}T155 P{UAS-FLP1.D}JD2/TM3, Sb1 flies. In the progeny, w1118/w1118; Hsp60C1 P{ry�t7.2�neoFRT}40A/Hsp60C� P{Ubi-GFP}33 P{Ubi-GFP}38 P{neoFRT}40A; P{GawB}T155P{UAS-FLP1.D}JD2/� female flies(non-curly wings and non-stubble) wereidentified and their egg chambers ex-amined for presence of clones ofHsp60C1 homozygous follicle cells. Themutant mitotic clones were recognizedby the absence of nuclear GFP fluores-cence, which was shown by allHsp60C1/� cells or the �/� mitoticclones. The egg chambers were stainedwith TRITC-Phalloidin and/or DAPI forexamination by confocal microscopy(see below).

Homozygous Hsp60C1 germlineclones were generated using the FLP-DFS technique (Chou and Perrimon,1996). The w1118; Hsp60C1 P{ry�t7.2�neoFRT}40A/CyO female flies werecrossed to y1 w* P{ry[�t7.2]�hsFLP}12; Hsp60C� P{w[�mC]�ovoD1-18}2LaP{w[�mC]�ovoD1-18}2Lb P{ry[�t7.2]�neoFRT}40A /CyO males.Clones were induced by exposing thirdinstar larvae at 37°C for 2 hr on 2consecutive days. Ovaries from 3–5


days adult non-curly progeny females(y1 w* P{ry[�t7.2]�hsFLP}12/w1118;Hsp60C� P{w[�mC]�ovoD1-18}2LaP{w[�mC]�ovoD1-18}2Lb P{ry [� t7.2]�neoFRT}40A/Hsp60C1P {ry�t7.2�neoFRT}40A) were dissected and exam-ined by confocal microscopy after stain-ing with DAPI, Phalloidin-TRITC, andanti–Hsp60-antibody (see below). Eggchambers from sister non-curly fliesthat did not receive heat shock duringthird instar stage were examined ascontrol.

RNA:RNA In SituHybridization With AdultFemale Reproductive Organs

An Hsp60C specific digoxigenin (DIG)-labeled riboprobe (Sarkar and Lakho-tia, 2005) was used for hybridizationin situ with cellular RNA in wild-typeand Hsp60C1 homozygous mutantadult female reproductive organs.Ovaries and the associated organswere hand-dissected from 2-day-oldfemales (wild-type and Hsp60C1/Hsp60C1) and fixed in 1:3 mixture of4% formaldehyde:heptane for 20 min.Subsequent steps for RNA:RNA insitu hybridization were carried out asdescribed earlier (Sarkar and Lakho-tia, 2005). The hybridization was de-tected either chromogenically, usingalkaline-phosphatase–conjugated an-ti-DIG antibody (Roche Applied Sci-ence, Germany) or by multiphotonconfocal microscopy (Bio-Rad Radi-ance 2100) after binding with fluores-cein isothiocyanate (FITC) -conju-gated anti-DIG antibody (RocheApplied Science, Germany).

GFP Localization andImmunostaining of EggChambers

Ovaries dissected from 2-day-old fe-male flies of the desired genotypes(and expressing GFP) were fixed in 4%paraformaldehyde in PBS (18.6 mMNaH2PO4, 84.1mM Na2HPO4, 17.5mM NaCl). After 3 10 min washingin PBTX (PBS, 0.1% Triton X-100 and0.1% bovine serum albumin), theywere mounted in antifade solution forconfocal microscopy.

The antibodies used for immuno-staining were: anti–Fas-III (1:10; Pa-tel et al., 1987), anti-Gurken (1:30;

Queenan et al., 1999), anti-Dlg (1:20;Goode and Perrimon, 1997), anti–Hts-RC (1:1; Robinson et al., 1994),anti–-spectrin (1:50; Dubreuil et al.,1987), anti–DE-Cadherin (1:20; Odaet al., 1997), all obtained from Devel-opmental Studies Hybridoma Bank(USA); the other antibodies wereMouse monoclonal Anti-Phosphoty-rosine (1:100, Santa Cruz, CA), mono-clonal anti–-tubulin-FITC Conjugate(DM1A, 1:250, SIGMA), and rabbitpolyclonal anti-Hsp60 (SPA 805,1:100, Stressgen, Canada).

For immunostaining, ovaries from2-day-old adult wild-type and Hsp60C1/Hsp60C1 females were fixed and pro-cessed for immunofluorescent stainingas described earlier (Sarkar and Lakho-tia, 2005). After binding with the de-sired primary antibody, the ovarieswere incubated for 1 hr at room temper-ature in the appropriate Alexa fluor–conjugated secondary antibody (1:200dilution, Molecular Probes, New Bruns-wick, NJ), after which the ovaries werecounterstained with DAPI (1 �g/ml,Molecular Probes) to visualize nucleus.For F-actin staining, fixed/immuno-stained ovaries were incubated withTRITC-conjugated Phalloidin (8U/ml in PBS, Sigma, St. Louis, MO)for 30 min at room temperature inthe dark. All fluorescently stainedtissues were mounted in antifade(Molecular Probes) and observedwith a Bio-Rad Radiance 2100 mul-tiphoton confocal microscope.

ACKNOWLEDGMENTSWe thank Bloomington DrosophilaStock Center and Dr. Xavier Morin(Cambridge) for fly stocks. We alsothank the anonymous reviewers andthe corresponding editor, Dr. Ken Ir-vine, for critical comments and sug-gestions. This work is supported byresearch grant from Department ofBiotechnology, Government of India,New Delhi, to S.C.L. and by the De-partment of Science and Technology,Government of India, New Delhi,through its support for the MultiphotonConfocal Microscope National Facility.S.S. was supported by a research fellow-ship from the Council of Scientific andIndustrial Research, New Delhi.

REFERENCES

Arya R, Mallik M, Lakhotia SC. 2007. Heatshock genes - integrating cell survivaland death. J Biosci 32:595–610.

Betran E, Thornton K, Long M. 2002. Ret-roposed new genes out of the X in Dro-sophila. Genome Res 12:1854–1859.

Buszczak M, Cooley L. 2000. Eggs to diefor: cell death during Drosophila oogen-esis. Cell Death Differ 7:1071–1074.

Chekulaeva M, Ephrussi A. 2004. Drosoph-ila development: RNA interference abovo. Curr Biol 14:R428–R430.

Chen X, Sullivan DS, Huffaker TC. 1994.Two yeast genes with similarity toTCP-1 are required for microtubule andactin function in vivo. Proc Natl Acad SciU S A 91:9111–9115.

Chou TB, Perrimon N. 1996. The autoso-mal FLP-DFS technique for generatinggermline mosaics in Drosophila melano-gaster. Genetics 144:1673–1679.

Dubreuil R, Byers TJ, Branton D, Gold-stein LS, Kiehart DP. 1987. Drosophiliaspectrin. I. Characterization of the puri-fied protein. J Cell Biol 105:2095–2102.

Duffy JB, Harrison DA, Perrimon N. 1998.Identifying loci required for follicularpatterning using directed mosaics. De-velopment 125:2263–2271.

Gobert AP, Bambou JC, Werts C, Balloy V,Chignard M, Moran AP, Ferrero RL.2004. Helicobacter pylori heat shock pro-tein 60 mediates interleukin-6 produc-tion by macrophages via a toll-like recep-tor (TLR)-2-, TLR-4-, and myeloiddifferentiation factor 88-independentmechanism. J Biol Chem 279:245–250.

Golic KG, Lindquist S. 1989. The FLP re-combinase of yeast catalyzes site-specificrecombination in the Drosophila ge-nome. Cell 59:499–509.

Gonzalez-Reyes A, St Johnston D. 1998.The Drosophila AP axis is polarised bythe cadherin-mediated positioning of theoocyte. Development 125:3635–3644.

Goode S, Perrimon N. 1997. Inhibition ofpatterned cell shape change and cell in-vasion by Discs large during Drosophilaoogenesis. Genes Dev 11:2532–2544.

Hartl FU, Hayer-Hartl M. 2002. Molecularchaperones in the cytosol: from nascentchain to folded protein. Science 295:1852–1858.

Hudson AM, Cooley L. 2002. Understand-ing the function of actin-binding proteinsthrough genetic analysis of Drosophilaoogenesis. Annu Rev Genet 36:455–488.

Johnstone O, Lasko P. 2001. Translationalregulation and RNA localization in Dro-sophila oocytes and embryos. Annu RevGenet 35:365–406.

King RC. 1970. The meiotic behavior of theDrosophila oocyte. Int Rev Cytol 28:125–168.

Lakhotia SC, Srivastava P, Prasanth KV.2002. Regulation of heat shock proteins,Hsp70 and Hsp64, in heat-shocked Mal-pighian tubules of Drosophila melano-gaster larvae. Cell Stress Chaperones 7:347–356.

Leroux MR, Candido EP. 1997. Subunitcharacterization of the Caenorhabditis


elegans chaperonin containing TCP-1and expression pattern of the gene en-coding CCT-1. Biochem Biophys ResCommun 241:687–692.

Liang P, MacRae TH. 1997. Molecularchaperones and the cytoskeleton. J CellSci 110(Pt 13):1431–1440.

Lindsley DL, Zimm GG. 1992. In: The ge-nome of Drosophila melanogaster. SanDiego: Academic Press.

MacDougall N, Lad Y, Wilkie GS, Francis-Lang H, Sullivan W, Davis I. 2001. Mer-lin, the Drosophila homologue of neuro-fibromatosis-2, is specifically required inposterior follicle cells for axis formationin the oocyte. Development 128:665–673.

Maguire M, Coates AR, Henderson B.2002. Chaperonin 60 unfolds its secretsof cellular communication. Cell StressChaperones 7:317–329.

Mahowald AP. 1972. Ultrastructural ob-servations on oogenesis in Drosophila. JMorphol 137:29–48.

McGregor JR, Xi R, Harrison DA. 2002.JAK signaling is somatically required forfollicle cell differentiation in Drosophila.Development 129:705–717.

Melki R, Cowan NJ. 1994. Facilitated fold-ing of actins and tubulins occurs via anucleotide-dependent interaction be-tween cytoplasmic chaperonin and dis-tinctive folding intermediates. Mol CellBiol 14:2895–2904.

Middleton CA, Nongthomba U, Parry K,Sweeney ST, Sparrow JC, Elliott CJ.2006. Neuromuscular organization andaminergic modulation of contractions inthe Drosophila ovary. BMC Biol 4:17.

Morin X, Daneman R, Zavortink M, ChiaW. 2001. A protein trap strategy to de-tect GFP-tagged proteins expressed fromtheir endogenous loci in Drosophila. ProcNatl Acad Sci U S A 98:15050–15055.

Muller HA. 2000. Genetic control of epithe-lial cell polarity: lessons from Drosoph-ila. Dev Dyn 218:52–67.

Nezis IP, Stravopodis DJ, Papassideri I,Robert-Nicoud M, Margaritis LH. 2000.Stage-specific apoptotic patterns duringDrosophila oogenesis. Eur J Cell Biol 79:610–620.

Norvell A, Debec A, Finch D, Gibson L,Thoma B. 2005. Squid is required forefficient posterior localization of oskarmRNA during Drosophila oogenesis. DevGenes Evol 215:340–349.

Oda H, Uemura T, Takeichi M. 1997. Phe-notypic analysis of null mutants for DE-cadherin and Armadillo in Drosophilaovaries reveals distinct aspects of theirfunctions in cell adhesion and cytoskel-etal organization. Genes Cells 2:29–40.

Patel NH, Snow PM, Goodman CS. 1987.Characterization and cloning of fasciclinIII: a glycoprotein expressed on a subsetof neurons and axon pathways in Dro-sophila. Cell 48:975–988.

Peifer M, Orsulic S, Sweeton D, WieschausE. 1993. A role for the Drosophila seg-ment polarity gene armadillo in cell ad-hesion and cytoskeletal integrity duringoogenesis. Development 118:1191–1207.

Perezgasga L, Segovia L, Zurita M. 1999.Molecular characterization of the 5� con-trol region and of two lethal alleles af-fecting the hsp60 gene in Drosophilamelanogaster. FEBS Lett 456:269–273.

Queenan AM, Barcelo G, Van Buskirk C,Schupbach T. 1999. The transmembraneregion of Gurken is not required for bio-logical activity, but is necessary fortransport to the oocyte membrane inDrosophila. Mech Dev 89:35–42.

Ranson NA, White HE, Saibil HR. 1998.Chaperonins. Biochem J 333:233–242.

Riechmann V, Ephrussi A. 2001. Axis for-mation during Drosophila oogenesis.Curr Opin Genet Dev 11:374–383.

Robinson DN, Cant K, Cooley L. 1994.Morphogenesis of Drosophila ovarianring canals. Development 120:2015–2025.

Sarkar S, Lakhotia SC. 2005. The Hsp60Cgene in the 25F cytogenetic region inDrosophila melanogaster is essential fortracheal development and fertility.J Genet 84:265–281.

Sarkar S, Arya R, Lakhotia SC. 2006.Chaperonins: in life and death. In:Sreedhar AS, Srinivas UK, editors.Stress responses: a molecular biology ap-proach. Trivandrum, India: Signpost. p43–60.

Schnorrer F, Luschnig S, Koch I, Nusslein-Volhard C. 2002. Gamma-tubulin37Cand gamma-tubulin ring complex pro-tein 75 are essential for bicoid RNA lo-calization during drosophila oogenesis.Dev Cell 3:685–696.

Spradling A. 1993. Developmental geneticsof oogenesis. In: Bate M, Martinez-AriasA, editors. The development of Drosoph-ila melanogaster. New York: Cold SpringHarbor Press. p 1–70.

Steinhauer J, Kalderon D. 2006. Microtu-bule polarity and axis formation in theDrosophila oocyte. Dev Dyn 235:1455–1468.

Timakov B, Zhang P. 2001. The hsp60Bgene of Drosophila melanogaster is es-sential for the spermatid individualiza-tion process. Cell Stress Chaperones 6:71–77.

van Eeden F, St Johnston D. 1999. Thepolarisation of the anterior-posterior anddorsal-ventral axes during Drosophilaoogenesis. Curr Opin Genet Dev 9:396–404.

Vinh DB, Drubin DG. 1994. A yeast TCP-1-like protein is required for actin func-tion in vivo. Proc Natl Acad Sci U S A91:9116–9120.

Volberg T, Zick Y, Dror R, Sabanay I, GilonC, Levitzki A, Geiger B. 1992. The effectof tyrosine-specific protein phosphoryla-tion on the assembly of adherens-typejunctions. EMBO J 11:1733–1742.

Wodarz A. 2002. Establishing cell polarityin development. Nat Cell Biol 4:E39–E44.

Xu T, Rubin GM. 1993. Analysis of geneticmosaics in developing and adult Dro-sophila tissues. Development 117:1223–1237.

Zhang Y, Kalderon D. 2000. Regulation ofcell proliferation and patterning in Dro-sophila oogenesis by Hedgehog signal-ing. Development 127:2165–2176.

Zhang L, Koivisto L, Heino J, Uitto VJ.2004. Bacterial heat shock protein 60may increase epithelial cell migrationthrough activation of MAP kinases andinhibition of alpha6beta4 integrin ex-pression. Biochem Biophys Res Commun319:1088–1095.


Hsp60C is required in follicle as well as germline cells during oogenesis in Drosophila melanogaster

Documents

Transcript of Hsp60C is required in follicle as well as germline cells during oogenesis in Drosophila melanogaster