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Development 115, 395-402 (1992) Printed in Great Britain © The Company of Biologists Limited 1992 395 Gonad formation and development requires the abd-A domain of the bithorax complex in Drosophila melanogaster SUSAN CUMBERLEDGE 1 -*, JANOS SZABAD 2 and SHIGERU SAKONJU 1 1 Howard Hughes Medical Institute, Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA 2 Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, POB 521, Hungary •Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA Summary The abdominal-A {abd-A) gene, a member of the bithorax complex, is required for the correct identity of parasegments (PS) 7 through 13. Mutations in iab-4, one of the cis-regulatory regions of abd-A, transform epidermal structures of PS 9 and also cause loss of gonads in adultflies.Here, we describe a developmental and molecular analysis of the role of iab-4 functions in gonadal development. In flies homozygous for a strong iab-4 allele, gonadogenesis is not initiated in the embryo because the mesodermal cells fail to encapsulate the pole cells. Flies homozygous for weaker iab-4 mutations sometimes form ovaries. The ovary-oviduct junctions are abnormal, however, and egg transfer from the ovary to the uterus is blocked in the adult. To localize the sites that require iab-4 function, we have analyzed animals chimeric for the mutant and wild-type cells. These chimeras were generated by three kinds of transplan- tation experiments: pole cells, embryonic somatic nuclei or larval ovaries. Our results suggest that iab-4 is required in the somatic cells of the gonadal primordia, but not the germ line. In addition, the formation of functional ovary-oviduct junctions and egg transfer also requires iab-4 functions in the somatic cells of the ovary and in at least one additional somatic tissue. Key words: Drosophila, gonad formation, bithorax complex, abd-A, iab-4, chimera. Introduction The bithorax complex (BX-C) of Drosophila melano- gaster contains three homeotic genes - Ultrabithorax, abdominal-A and Abdominal-B - that specify the identity of two thoracic and eight abdominal segments. abdominal-A (abd-A) is required for determining the correct identity of parasegments (PS) 7 through 13, corresponding to the abdominal segment (A)l posterior compartment through A8 anterior compartment (for review see Peifer et al., 19S7 and Duncan, 1987). Mutations in abd-A not only cause homeotic transform- ations of epidermal structures, but also affect other tissues such as muscle, fat body, gonads and the CNS (Lewis, 1985; Karch et al., 1985; Busturia et al., 1989). It has been proposed that abd-A functions as a molecular switch, selecting different developmental pathways by regulating the expression of different sets of downstream or 'realizator' genes (Lewis, 1964; Garcia-Bellido, 1975). In support of this model, recent molecular analyses have shown that the abd-A encodes a homeodomain protein (Karch et al., 1990; Macias et al., 1990) which can function as a transcription factor both in tissue culture cells and in the fly (B. Appel and S. Sakonju, unpublished results). Yet the molecular events between the initial activation of abd-A and the development of its target tissues and organs remain uncharted. One way of approaching this problem is to focus on a particular morphogenetic pathway, identify the steps in the pathway and then determine which of these steps are disrupted by mutations within abd-A. We have chosen to examine the role of abd-A during the formation and subsequent development of the gonadal primordia. Mutations in the abd-A domain fall into two classes: the abd-A alleles and the iab alleles. The abd-A~ alleles disrupt the homeobox-containing transcription unit and are homozygous embryonic lethal (Busturia et al., 1989; Karch et al., 1990; B. Appel, M. Lamka and S. Sakonju, in preparation). They cause a strong trans- formation of PS7-9, and a weaker transformation of PS10-12, towards PS6. The iab~ alleles disrupt the cis- regulatory regions of abd-A and cause transformations in subsets of PS7-9 through altered expression of the abd-A gene (Busturia et al., 1989; Karch et al., 1990; Simon et al., 1990). In this work, we have focused on mutations that affect the iab-4 cis-regulatory region located about 20 kb upstream from the start site of abd-

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Page 1: Gonad formation and development requires the abd-A domain …dev.biologists.org/content/develop/115/2/395.full.pdf · Gonad formation and development requires the abd-A domain of

Development 115, 395-402 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

395

Gonad formation and development requires the abd-A domain of the

bithorax complex in Drosophila melanogaster

SUSAN CUMBERLEDGE1-*, JANOS SZABAD2 and SHIGERU SAKONJU1

1 Howard Hughes Medical Institute, Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA2Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, POB 521, Hungary

•Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA

Summary

The abdominal-A {abd-A) gene, a member of thebithorax complex, is required for the correct identity ofparasegments (PS) 7 through 13. Mutations in iab-4, oneof the cis-regulatory regions of abd-A, transformepidermal structures of PS 9 and also cause loss ofgonads in adult flies. Here, we describe a developmentaland molecular analysis of the role of iab-4 functions ingonadal development. In flies homozygous for a strongiab-4 allele, gonadogenesis is not initiated in the embryobecause the mesodermal cells fail to encapsulate the polecells. Flies homozygous for weaker iab-4 mutationssometimes form ovaries. The ovary-oviduct junctionsare abnormal, however, and egg transfer from the ovaryto the uterus is blocked in the adult. To localize the sites

that require iab-4 function, we have analyzed animalschimeric for the mutant and wild-type cells. Thesechimeras were generated by three kinds of transplan-tation experiments: pole cells, embryonic somatic nucleior larval ovaries. Our results suggest that iab-4 isrequired in the somatic cells of the gonadal primordia,but not the germ line. In addition, the formation offunctional ovary-oviduct junctions and egg transfer alsorequires iab-4 functions in the somatic cells of the ovaryand in at least one additional somatic tissue.

Key words: Drosophila, gonad formation, bithoraxcomplex, abd-A, iab-4, chimera.

Introduction

The bithorax complex (BX-C) of Drosophila melano-gaster contains three homeotic genes - Ultrabithorax,abdominal-A and Abdominal-B - that specify theidentity of two thoracic and eight abdominal segments.abdominal-A (abd-A) is required for determining thecorrect identity of parasegments (PS) 7 through 13,corresponding to the abdominal segment (A)l posteriorcompartment through A8 anterior compartment (forreview see Peifer et al., 19S7 and Duncan, 1987).Mutations in abd-A not only cause homeotic transform-ations of epidermal structures, but also affect othertissues such as muscle, fat body, gonads and the CNS(Lewis, 1985; Karch et al., 1985; Busturia et al., 1989).It has been proposed that abd-A functions as amolecular switch, selecting different developmentalpathways by regulating the expression of different setsof downstream or 'realizator' genes (Lewis, 1964;Garcia-Bellido, 1975). In support of this model, recentmolecular analyses have shown that the abd-A encodesa homeodomain protein (Karch et al., 1990; Macias etal., 1990) which can function as a transcription factorboth in tissue culture cells and in the fly (B. Appel and

S. Sakonju, unpublished results). Yet the molecularevents between the initial activation of abd-A and thedevelopment of its target tissues and organs remainuncharted. One way of approaching this problem is tofocus on a particular morphogenetic pathway, identifythe steps in the pathway and then determine which ofthese steps are disrupted by mutations within abd-A.We have chosen to examine the role of abd-A duringthe formation and subsequent development of thegonadal primordia.

Mutations in the abd-A domain fall into two classes:the abd-A alleles and the iab alleles. The abd-A~ allelesdisrupt the homeobox-containing transcription unit andare homozygous embryonic lethal (Busturia et al., 1989;Karch et al., 1990; B. Appel, M. Lamka and S.Sakonju, in preparation). They cause a strong trans-formation of PS7-9, and a weaker transformation ofPS10-12, towards PS6. The iab~ alleles disrupt the cis-regulatory regions of abd-A and cause transformationsin subsets of PS7-9 through altered expression of theabd-A gene (Busturia et al., 1989; Karch et al., 1990;Simon et al., 1990). In this work, we have focused onmutations that affect the iab-4 cis-regulatory regionlocated about 20 kb upstream from the start site of abd-

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396 S. Cumberledge, J. Szabad and S. Sakonju

A transcription unit. Flies carrying deletions or chromo-somal breaks that separate the iab-4 region from theabd-A transcription unit are fully viable. In these flies,the fourth abdominal segment (A4) is transformedtowards the third (Lewis 1978; 1985; Karch et al., 1985).In addition, Lewis and co-workers (Lewis 1978; 1985;Karch et al., 1985) have also noted that iab-4 mutationscause sterility through a loss of adult gonads.

Here we describe a detailed analysis of the role ofiab-4 functions in gonadal development. The male andfemale reproductive systems have been studied exten-sively in Drosophila (for reviews see Lindsley andTokuyasu, 1980; Mahowald and Kambysellis, 1980;Babcock 1971a, b). The two gonadal primordia, eachcomposed of a cluster of 8-10 germ cell precursorssurrounded by a layer of mesodermal cells, are firstformed during mid-embryogenesis in the ventral meso-derm A5 region (Poulson, 1950; Underwood et al.,1980; Hay et al., 1988; Lasko and Ashburner, 1990).The somatic cells of the gonadal anlagen originate in theprimordia at approximately the fourth abdominalsegment (Szabad and Nothiger, 1992); however, thesegmental boundaries of the mesoderm, relative to theectoderm, are shifted posteriorly by a half segment.Thus, by the time of gonadogenesis, these cells arelocated at the level of A5 (Tremml and Bienz, 1989).

In females, the presumptive ovaries continue to growwith little differentiation until the pupal stage. Then themesodermally derived cells differentiate into a varietyof cell types, including the epithelial cells of theovarioles, the peritoneal sheath, the basal stalk cellsand the posterior calx (King et al., 1968; King, 1970).Concurrently, the genital discs, derived from ectoder-mal cells of A8-A10, are also developing, forming theuterus, the main oviduct and two lateral oviducts,(Poulson, 1950; Babcock, 1971a). After pupariation asthe lateral oviducts grow, they meet and fuse withovaries, thus completing the internal duct system.Transfer of mature oocytes from ovaries throughoviducts to the uterus in adult females requires thecoordinated functions of several organs, including thenervous system, muscular network of the ovarianperitoneal sheath and contractile fibrillae in the epi-thelial sheath of the ovarioles (Bodenstein, 1950).

Development of testes proceeds at a faster rate(Cooper, 1950). By the second instar, the testes aremuch larger and more differentiated than their femalecounterparts. By the early stages of pupal development,the seminal vesicles (derived from the male genital disc)connect with the testes.

In this paper, we first identified the morphologicalprocesses during gonadogenesis that are affected bylesions in the iab-4 region of abd-A. By constructingchimeras, we then investigated which of the tissues thatconstitute the reproductive organs are affected by iab-4mutations. Our results suggest that iab-4 functions arerequired in the somatic cells, but not in the germ cells,of the gonads. For the establishment of correct ovary-oviduct connections and egg transfer in females, iab-4functions are required in the gonadal somatic cells aswell as at an additional site(s) outside of the ovaries.

Materials and methods

Antibody stainingFor staining with anti-vasa antibody, which specifically labelspole cells (Hay et al., 1988), embryos were collected from aniab-^^/TMS, ftz-lacZ stock. This TM3 balancer chromosomehas an insertion of lacZ fused to theftz promoter, allowing theidentification of genotypes in embryos (constructed andkindly provided by Sarah Smolik-Utlaut). Homozygousembryos were identified, when double labeling with anti-bodies directed against vasa and /3-galactosidase, as those notshowing the ftz-lacZ pattern. Immunodetection methods wereas described in Boulet et al. (1991). Briefly, embryos weredechorinated in a hypochlorite solution, washed and fixed in4% paraformaldehyde in PBS/heptane (1:1) for 20 minutes.The aqueous layer was replaced with methanol and theembryos were shaken until the vitelline membrane wasremoved, and then washed several times in methanol andrehydrated in PBS. After washing several times in PBS-BT(0.1% BSA, 0.1% Triton X-100 in PBS), embryos wereincubated overnight in primary antibody diluted in PBS-BT,and then washed multiple times in PBS-BT. Biotinylatedsecondary antibody binding (Vectastain ABC Kit;.Vector-labs) and subsequent HRP immunochemistry was carried outas per manufacturer's directions. After staining, the embryoswere dehydrated in ethanol and mounted in methyl salicylate.Anti-vasa antibody was used at a 1:5 dilution, and anti-/3-galantibody (Cappel) at 1:2,000. The anti-vasa antibody wasgenerously provided by Y. N. Jan.

The monoclonal antibody, DMabd-A.2, directed againstabd-A protein was prepared and kindly provided by DianeMattson and Ian Duncan (Kellerman et al., 1990). For thewild-type expression pattern, Canton-S embryos were col-lected and labeled with the antibody at a dilution of 1:2,000.Histochemical staining procedures were the same as describedabove for the anti-vasa antibody. To observe the abd-Aexpression pattern in an iab-4 mutant, embryos were collectedfrom iab-r^/TMS, ftz-lacZ stock and the mutant embryosidentified as described above.

Germline chimerasPole cell transplantations were performed as described byLehmann and Nusslein-Volhard (1987). Donor embryos(Table 1) were collected from the following cross: iab-4302/TM6, Tb X iab-4302/TM3, Sb. Host female embryos wereobtained by crossing ovo°'/Y males to +/+ (Ore-R) females.F! ovoD1/+ females do not deposit eggs due to germlinedefects (Busson et al., 1983; Komitopoulou et al., 1983). Hostmale embryos were obtained from crossing X*Yy su(wa) w°males to +/+ (Ore-R) females. The Ft XO males are steriledue to germline defects (Marsh and Wieschaus, 1978). Bothdonor and host eggs were collected for one hour and allowedto age at 25°C for 1.5 hours. Embryos were then dechorinatedin a sodium hypochlorite solution, washed, desiccated andcovered with 10S Voltalef oil. The pole cells from the donorembryos were collected and transplanted into the posteriorpole of the host embryos. Transplantation procedures wereperformed at 18°C. Surrogate embryos were allowed todevelop at 25°C. After hatching, the genotype of thetransplanted germline cells was determined by crossing thehost flies to wild-type flies, and scoring the phenotype of theF2 progeny. Genetic markers and standard chromosomes areas described in Lindsley and Grell (1968).

Larval ovary transplantationsOvary transplantations were carried out according to the

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abd-A and gonad formation 397

procedures of Clancy and Beadle (1937). Larvae were raisedon standard cornmeal yeast media. Wandering third instarswere collected from the walls of the bottles. Host and donorlarvae were washed in 70% EtOH, etherized and dissected insterile PBS. Genotypes of the donor and host larvae are listedin Table 4. After implantation of the ovaries, chimeric animalswere raised at 25°C and screened for fertility. Analogousexperiments with male third instar larvae were not performedsince, by third instar, the testes are much larger than ovaries,making transplantations unfeasible.

Nuclei transplantationsNuclei transplantations were performed as described bySantamaria (1986). In the first experiment, donor embryoswere derived from a cross between +/+ (Ore-R) females andFs(3)Apc, mwh e/TM3, Sb Ser males. Fs(3)Apc is a dominantfemale sterile mutation that alters follicle cell functionsleading to a specific mutant phenotype (Szabad and Hoff-mann, 1989; Erdelyi and Szabad, 1989). In the secondexperiment, donor embryos were obtained from flies homo-zygous for two copies of a hsp70promoter-lacZ transgene (Bg9.6.1; Lis et al., 1983). Host embryos were derived from iab-4302/TM3, Sb stock. Nuclei were removed from donorembryos, 1-2 hours after egg laying, and then injected into 0-1hour host embryos at 38% egg length, at the site of thepresumptive A4 segment. After hatching, adults werescreened for fertility by mating to Canton-S flies. Eachexperimental animal was heat-shocked at 37°C for 1 hour,allowed to recover for 30 minutes at 25°C, and then dis-sected. The abdominal cavity and reproductive organs wereremoved, fixed in 4% paraformaldehyde in PBS for 10minutes, washed in PBS, stained for /S-galactosidase activityas described in Lis et al. (1983), and scored using a compoundmicroscope.

Results

Gonadal defects caused by iab-4 mutant allelesBased on the penetrance of sterility, the iab-4 allelescan be placed into the phenotypic series: iab-4302, iab-4166 > iab-44-5DB > iab-445 (Lewis, 1985). For example,>99% of males or females homozygous or hemizygousfor a strong iab-4~ allele, iab-43 , are sterile, while83% of the iab-445 hemizygous males and 92% of theiab-445 hemizygous females are affected. We began ouranalysis by comparing the reproductive organs dis-sected from wild-type adult flies with those from iab-43O2/iab-4302 and iab-445/iab-4302 adults (Fig. 1). 84% ofiab-4302I iab-4302 females and 91% of males lack gonads(Fig. IB, E). The associated sexual organs, all deriva-tives of the genital discs, appear to be normal, with theexception of the lateral oviducts in females. While thelateral oviducts are always present, they often appearstunted (Fig. IB) compared to those of the wild-typefemale (Fig. 1A). The male seminal vesicles, however,appear to be fully formed (Fig. IE) as in the wild-typemale (Fig. ID). Dissection of iab-4302 and iab-4166

hemizygotes gave similar results (data not shown).Females carrying one copy of a weaker iab-4 allele

usually form one or two ovaries. Their attachment tothe lateral oviduct, however, is often defective (Fig.1C). For example, about a half the expected number of

ovaries were present in the twelve iab-441'/iab-4302

females dissected; of those present, two thirds were notattached to the oviduct. The free-floating ovaries wereoften facing anterior, rather than the normal posteriordirection. Both attached and free-floating ovaries werecomposed of 14-18 ovarioles (normal ovaries usuallycontain 15-20 ovarioles) and fully mature oocytes.Often the mutant ovaries contained more matureoocytes than wild-type ovaries (compare Fig. 1A andC). In two of the three cases where the ovaries wereattached, partially degenerated eggs were observed inthe lateral oviduct and the oviduct-ovary junctions wereabnormal. No eggs were ever observed in the uterus,and all twelve females were sterile. It appears that thesterility is caused, at least in part, by the disruption ofnormal egg migration, perhaps due to an abnormaljunction between the gonad and the genital duct or todefective neuronal or muscular functions. All otherassociated sexual organs derived from the genital discsappear to be normal.

Male iab-445/iab-4302 flies displayed an analogousphenotype: often only one testis was present (Fig. IF).Approximately 42% of iab-445/iab-43 males dissectedcontained at least one pigmented, mature testis withsperm; yet only 22% were fertile. We suspect thatsperm transfer has been affected. However, unlikepreviously reported cases of defective sperm trans-mission, the seminal vesicles in these males were notdistended (Lindsley and Tokuyasu, 1980).

These results suggest that lesions in the iab-4 regionof abd-A affect at least two steps in gonadal develop-ment. First, the formation and/or development of maleand female gonads is disrupted. Second, attachment ofthe presumptive ovaries to the lateral oviducts isaltered. Migration of germ cells from the gonadsthrough the reproductive ducts may also be inhibited,although we have observed this only for females. Thesephenotypes could be the manifestation of a singleprimary defect, or several independent malfunctions.

Gonadal defects in the embryoTo determine when, and in which tissues, iab-4+ isrequired for gonad formation and differentiation, wehave examined the consequences of loss of iab-4function at several stages in development. We began bymonitoring gonadogenesis in both wild-type and iab-4~embryos. To follow pole cell migration and subsequentformation of the gonadal primordia, embryos werecollected at various times, fixed and then stained with amonoclonal antibody that recognizes the pole-cell-specific vasa antigen (Fig. 2). In wild-type embryos, thepole cells move dorsally into the posterior midgutprimordium during gastrulation (stage 6 of Campos-Ortega and Hartenstein, 1985), pass through theposterior midgut walls and, by the germ-band-shorten-ing stage, align along the wall of the body cavity•adjacent to the primordia of abdominal segments 5through 8 (Fig. 2A). By stage 14, the mesodermal cellsencapsulate a cluster of pole cells thereby forming arudimentary gonad in the ventral A5 region (Fig. 2B;Poulson, 1950; Underwood et al., 1980; Hay et al.,

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398 5. Cumberledge, J. Szabad and S. Sakonju

1988). Pole cells not enclosed within the gonadalprimordia gradually die. By stage 16, the mesodermalsheath of the gonad consists of approximately 27-37cells (Sonnenblick, 1941).

In iab-4302 embryos pole cell migration is unimpededuntil late stage 13/early stage 14. The pole cells moveinto the ventral mesoderm along the body cavity, butthe somatic cells do not form a sheath around them andsubsequent development of the gonadal primordia isarrested (Fig. 2C). The germ cells remain dispersedthroughout the mesoderm, and do not form a cluster inthe A5 region; instead, they appear to die gradually.Our results indicate that iab-4302 function is notnecessary for germ cell migration to the ventralmesoderm, but is required for the formation of themesodermal gonadal sheath.

abd-A expression in gonadal mesodermIt is not known if iab-4 functions autonomously in themesoderm; therefore it is of particular interest toascertain if abd-A is expressed in the presumptivegonadal soma and if the abnormal behavior of thegonadal anlage in iab-4~ embryos correlates with achange in expression of abd-A in these cells. Thepattern of abd-A protein expression in the developingembryo has been described previously (Macias et al.,1990; Karch et al., 1990). abd-A protein is found in acomplex pattern in parasegments 7 through 13 in thenuclei of the epidermis, the nervous system, themesoderm and the amnioserosa. Both the above groupshave reported that the pattern of epidermal expressionin iab-4" embryos is similar to wild type. Karch et al.(1990) have also found that the intersegmental neuronsand the pericardial cells lack detectable expression inthe mutant.

Since these studies were not focused on expressionspecifically during gonadogenesis, we have used amonoclonal antibody directed against abd-A protein tocompare abd-A expression in wild-type and \ab-4~embryos in the mesodermal gonadal anlagen just beforeand during gonad formation. In late stage 13 wild-typeembryos, low levels of abd-A antigen are detected inthe mesoderm in parasegments 8-12, the region wherethe pole cells are found just prior to gonad formation.By stage 15, the gonads are clearly visible as adorsolateral cluster of cells at A5. Anti-abd-A antibodyweakly stains the somatic cells that surround thedistinctly large pole cells (Fig. 3). Intersegmentalneurons stain strongly with anti-abd-A antibody at thisand later stages (Fig. 3), but not at stages prior to theformation of the gonads. In iab-4302 and iab-^-503

embryos, the spatial distribution of the abd-A protein inPS7-13 mesoderm was indistinguishable from the wildtype (data not shown). We have also found that theintersegmental neurons no longer express abd-A pro-tein, confirming the earlier observation (Karch et al.,1990). Thus the mutant phenotype of the mesodermalcells cannot be readily correlated with a concomitantqualitative change in abd-A expression. It is possible,however, that we failed to detect subtle spatial ortemporal changes.

Table 1. Pole cell transplantations

(A) Female germ-line chimerasHost cross: +/+ females x ovoDI/Y males

Donor crossNo. fertile

females Germ cell genotype

iab-43a2[TM6, Tb xiab-4302/TM3, Sb

14 3 iab-43O2/TM3, Sb4 iab-4301/iab-4302

4 TM6, Tb/TM3, Sb3 iab-^/TMd, Tb

(B) Male germ-lineHost cross: +/+

Donor cross

chimerasfemales x X"

No. fertilemales

•Yy su{W)W males

Germ cell genotype

iab-43a2/TM6, Tb xiab-4302/TM3, Sb

2 iab-43O2/TM3, Sb3 iab-4302'/iab-4302

1 TM6, Tb/TM3, Sb2 iab-4302/TM6, Tb

The genotype of the donor pole cells was determined by matingchimeric females (A) to +/+ males and chimeric males (B) to+/+ females.

Somatic versus germ-line requirements for iab-4+

functionThe inability of iab-4302jiab-4302 embryos to formgonads could reflect a requirement for iab-4 functioneither in the somatic tissue, or the germ cell precursors,or both. Using the technique of pole cell transplan-tation, we have constructed and analyzed germ-linechimeras to examine if iab-4 function is needed in thegerm line (Table 1). Pole cells from iab-4302/iab-4302

embryos were transplanted into surrogate ovoD1/+female or X/O male host embryos, which cannot makefunctional gametes of their own. When female iab-4302

homozygous pole cells are transplanted into host femaleembryos (Table 1A), or male iab-4302 homozygous polecells are transplanted into host male embryos (TableIB), the chimeric embryos develop normally andmature into fertile adults. Therefore, normal develop-ment of ovaries and testes does not require iab-4+

function in the germ cells.

Rescuing the iab-4 mutant phenotype bytransplantation of somatic nucleiSince iab-4 function is not required in the germ cells, itmust be required in somatic cells for gonad formation.To determine which somatic cells require iab-4 func-tion, we constructed and analyzed somatic chimericanimals. iab-4+ nuclei were transplanted into host iab-4302 embryos along the ventral midline, in the presump-tive mesoderm region, at 38% egg length, where cells ofthe fourth abdominal segment form (Campos-Ortegaand Hartenstein, 1985). Donor cells were marked usingtwo independent methods. In the first experiment, 50%of the donor embryos carried the follicle cells-specificmarker mutation Ape (Szabad and Hoffmann, 1989; seeMaterials and methods). A total of 8 iab-4302 homo-zygous females were recovered (Table 2). Four of these

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i

F

Fig. 1. Morphology of adult reproductive organs from wild-type and iab-4302 homozygous flies. Adult females (left) andmales (right) were dissected and the reproductive tissues were stained with methylene blue and photographed. (A) Wild-type (Canton S) female. Black arrowhead points to an egg in the lateral oviduct, and white arrowhead to an egg in theuterus. (B) iab-4302 homozygous female, neither of the two ovaries are present. (C) iab-445/ iab-4302 female, only one ovaryis formed. Arrowheads point to eggs lodged in the main oviduct and at the ovary-oviduct junction. (D) Wild-type (CantonS) male. (E) iab-4302 homozygous male; the testes are not formed. (C) iab-445/ iab-4302 male, only one testis is present, o,ovaries; d, main oviduct; /, lateral oviduct; u, uterus; t, testes; v, seminal vesicles; a, accessory glands; e, ejaculatory duct.

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B

Fig. 2. Migration of pole cells in wild-type and iab-4302

homozygous embryos. Optical section of wild-type stage 12(A), wild-type stage 13 (B) and iab-4302 homozygous stage14 (C) embryos stained with anti-vasa antibody. Anterior isto the left. Arrowheads in C show pole cells that are notencapsulated in the mutant.

Fig. 3. Distribution of abd-A protein in a stage 13 CantonS embryo. The embryo was stained using monoclonalantibody, DMabd-A.2, as described in Materials andmethods. The embryo has been dissected and flattened toreveal interior tissues and is shown anterior to the top.abd-A protein is detected in the somatic cells surroundingthe gonad (go), the intersegmental neurons of A4-A7(arrowheads), in the ventral cord (vc) and in epidermalcells overlaying the gonad and intersegmental neurons. Theabd-A expression in the gonadal soma is distinct but weakand is not always clear in photos taken from a single planeof focus.

9o

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abd-A and gonad formation 399

Table 2. Rescue of the gonadless phenotype in iab-4302 homozygous female embryos by nuclei transplantations

Donormarker

Control*Apc/+ or

+/TM3hsplO-lacZ

Control hostiab-4W2/TM3

Total Chimera

17 n.d.

65 9

Total

2148

22

None

1844

17

Total

282

3

One

Non-att

251(6)

1(16)

Mutant host iab-4302/iab-4302

Number of ovaries

Att

31(9)*

2 (2); (10)*

BothTotal non-att

2 12 1 (13)(17)

2 0

11

0

Two

Bothatt

(3)01)

One non-att+one att

00

2 (1)(5); (15)(16)

Art, attached to oviduct; Non-att, not attached to oviduct; n.d., not determined; numbers in parenthesis, number of ovarioles;underlined numbers, cells of donor genotype evidenced; *, eggs derived from these ovaries; *, number of ovaries in non-injected iab-4302

females.

females did not have ovaries. One female developed anovary and another female had two ovaries, with all theeggs and egg primordia showing the Ape mutantphenotype. These Ape egg-containing ovaries wereattached to the oviduct, although eggs were depositedfrom only one of them. We take these females to beexamples of rescue of the gonadless iab-4302 phenotype.The other two host females carrying non-Ape eggs mostlikely originated through leakiness of the iab-4302

mutant phenotype since their ovaries were not attachedto the oviduct (Table 2).

In the second experiment, the iab-4+ donor nucleiwere marked with a hsplO promoter-lacZ transgene.The chimeras were first tested for fertility, then theywere dissected and their internal abdominal organs(including the gonads when present) were stained for/S-galactosidase (/3-gal) activity. Among 65 control hosts{iab-4302/TM3, Sb females), nine (14%) containedovaries that stained for yS-gal. It should be noted,however, that only varying fractions of the eggprimordia stained blue in these control females. 22 iab-4302 homozygous females were recovered (Table 2).Five of the 22 females developed one or two ovaries. Infour of these females, the somatic cells of at least one ofthe ovaries were labeled with /3-gal (Table 2). In thesefemales, unlike the control females, the entire ovaries,including all the ovarioles, stained blue. All the blue-staining ovaries were also attached to the oviducts.Three iab-4302 homozygous females had one ovary thatdid not stain with /?-gal activity. These ovaries were not

attached to the oviduct, and may have resulted from theleakiness of the iab-4 allele since approximately 13% ofiab-4302 homozygous females form unattached ovaries.The correlation between the appearance of ovaries thatare connected to oviducts and the presence of iab-4+

cells in these gonads suggests that iab-4* function isrequired in these cells.

All four /S-gal-labelled ovaries were attached to theoviduct and one female was able to deposit eggs. In theothers, eggs were never transferred to the uterus. Thisobservation suggests that iab-4+ function is requirednot only for the formation of the gonadal soma but alsofor egg transfer at a site outside of the ovary. In the oneegg-laying female, the ovary and oviducts stainedpositive for yS-gal activity. The oviduct staining mayhave been a consequence of follicle cell debris, whichoften accumulates in the genital ducts.

Nuclei transplantation experiment was also carriedout in males (Table 3). In this experiment, the donoriab-4* cells were labeled with hsp-lacZ transgene. Aftertransplantation, testes developed in five (28%) of the 18host iab-4302 homozygous males. /J-gal-labelled testicu-lar sheet cells were detected in one testis of each of twomales, one of whom gave rise to offspring.

In the two cases where complete rescue was ob-tained, the somatic tissue of the gonads also containediab-4+ cells, strongly supporting the model wherebyiab-4 function is necessary in the mesoderm. Yet, thismay not be sufficient to obtain complete rescue ofsterility. Three chimeric females and one chimeric male

Table 3. Rescue of the gonadless phenotype following transplantation of iab-4+ nuclei into iab-4302 homozygousembryos

Donormarker

ControlhsplO-lacZ

*, presence of cells with

Total

14318

the donor

Absent

12913

genotype was

145

evidenced

Total

113

in these

Testis

One

Non-att

63

two chimeras.

Present

Art Total

5 30 2

One of them gave

Two

Bothnon-att

00

rise to offspring.

Bothatt

32*

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400 S. Cumberledge, J. Szabad and S. Sakonju

Table 4. Larval ovary transplantations

Genotype of chimeras

y v f mal /yvf mal ovaryin iab-4™2 / iab-4302 host

yvf mal /yvf mal ovaryin iab-44' / iab-445 host

iab-4166 / DpP5 ovaryin ovoD 1 + host

iab-4451 iab-445 ovaryin ovoD1 1 + host

No. of chimericadult females

10

4

8

5

No. with donor No. with eggsovaries attached in lateralto host oviduct oviduct

6

3

T

4b

'In seven cases, one host ovary and one donor ovary were attached tobIn all four cases, one host ovary and one donor ovary were attached.

0

4

7

1

the lateral

No. with eggsin uterus

0

0

7

1

oviducts; in one

No. ofNo. with fertile

stored sperm chimeras

9 0

2 0

n.d. 7

5 2

case, both host ovaries were

% fertile

0

0

87.5

20

attached.

produced gonads that stain with /S-gal, but they werenot functional. These results, in combination with ouranalysis of adult phenotypes, suggests that formation ofcompetent gonad-duct connections may require iab-4function in additional somatic cells.

Formation of functional ovary-oviduct junctionsBecause the genital disc and the gonads developindependently and establish connection only afterpupariation, it is possible to transplant gonads of onegenotype into larval hosts of a different genotype. Weconstructed ovarian chimeras to test which tissuesrequire iab-4+ function in order to form competentovary-oviduct junctions. First, we implanted iab-4+

larval ovaries into iab-4302 or iab-445 homozygous larvaelacking ovaries (Table 4). The chimeric animals wereallowed to develop into adults, the females were testedfor fertility, and then dissected. As shown in Table 4,iab-4+ ovaries grew and developed in iab-4302 or iab-44

hosts. They appeared to undergo normal differentiationand attached to the lateral oviducts, yet all of thechimeric females were sterile. Stage 14 oocytes could beseen in the lateral oviducts, but never in the uterus. Thissterility is not caused by the failure of the females tomate since sperm was found in the sperm receptacle(Table 4). Therefore, these results suggest that iab-4+

function is required for egg transfer and/or oviposition.

The transplantation operation itself does not inter-fere with the formation of the lateral oviducts, nor doesit impair egg movement: when control iab-4+ ovarieswere transplanted into ovoD1/+ larvae (ovoD1/+ fe-males do not form functional ovaries of their own),eight of nine chimeric larvae grew into fertile adults(Table 4). In only one case did the ovary fail to attach tothe lateral oviduct. These results indicate that at leastone aspect of ovarian activity - normal egg transfer fromthe lateral oviducts to the uterus - requires iab-4+

function in the somatic cells outside of the ovaries.By performing the reciprocal experiment, we also

tested whether formation of ovary-oviduct junctionsrequires iab-4+ function in the somatic tissues of theovaries. Since iab-445 is a leaky allele, some iab-445

homozygous larvae contain gonads. When these iab-445

ovaries are transplanted to an iab-4+ host, they appear

to develop normally (Table 4), but the chimeric ovary-oviduct junction is deformed. In a few cases, eggdeposition appears to be normal, although in most casesthe eggs never migrate to the uterus (Table 4). In thesechimeric animals, the structures derived from thegenital discs, as well as the surrounding muscle andnervous tissue, are iab-4+; only the ovaries are deficientin iab-4 function. Together these results indicate thatdevelopment of functional ovaries requires iab-4+

function in the somatic cells of the ovary and at a secondsite in the animal.

Discussion

We have undertaken a developmental analysis of iab-4mutants in order to identify the primary lesion(s) fromwhich the gonadal defects arise. By analyzing theembryonic and adult gonadal phenotypes of iab-4~females, we have found that iab-4+ function is requiredfor three steps: (1) encapsulation of pole cells by nearbymesodermal cells during embryogenesis, (2) formationof functional oviduct-ovary junctions during pupaldevelopment and (3) normal egg transfer through theoviduct in the adult. Disruption of egg transfer may be aconsequence of abnormal ovary-oviduct junctions ormay represent an independent defect. Male iab-4" flieshave the same embryonic defect as females and mayhave analogous pupal and adult defects; many iab-4^5

males form testes, yet are infertile, suggesting thatsperm transfer is faulty.

We have shown by analyzing germline chimeras thatiab-4 funtion is not needed in pole cells to form gonads,suggesting that iab-4 functions strictly in somatic cells.Our nuclei and larval ovary transplantation exper-iments confirm that the formation of ovaries requiresiab-4+ activity in the somatic cells of the gonads. Inaddition, to develop functional ovaries iab-4 activity isrequired at a second site. This site is apparently outsideof the ovarian cells since wild-type ovaries cannotdevelop into functional adult ovaries when transplantedinto iab-4~ larvae. Previous gynandromorph analyses(Szabad and Fajszi, 1982) have identified three foci inthe Drosophila female nervous system that control eggtransfer, from the ovaries through the oviduct to the

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abd-A and gonad formation 401

uterus, and egg deposition. These foci map to moreposterior blastoderm regions than the adult brain.Normal egg laying requires communication between theovaries, the oviducts and the uterus, and is governed byvarious hormonal and neuronal cues. A most likely siteof the abd-A requirement outside of the ovaries is in theneuronal cells. These neuronal cells may develop fromthe neuroectodermal region at the level of A4 of theblastoderm located between the mesoderm and ecto-derm. In this regard, it is interesting to note that one ofthe few disturbances of abd-A expression patterns iniab-4~ embryos, observed by Karch et al. (1990) andconfirmed in our study, is the loss of expression in theintersegmental neurons. We did not detect abd-Aexpression in these neurons prior to the formation ofgonads. Therefore, abd-A in the intersegmental neur-ons may contribute to the proper functioning of thegonads in later stages but perhaps not for the initialformation of the gonads. A second, less likely,possibility is that abd-A is also required for genital discdevelopment. With the availability of antibodies di-rected against abd-A protein, it should now be possibleto determine the expression patterns in the pupal andadult neurons, as well as the genital structures.

There is a long-standing question as to whetherdetermination of the insect mesoderm is autonomous orif it is governed by the ectoderm (see Lawrence andJohnston, 1984, for a discussion). For example, it hasbeen suggested that some segmental muscle patterns inDrosophila are determined by Abd-B activity in theassociated neurons and not in the myoblasts themselves(Lawrence and Johnston, 1986). This led us to ask ifabd-A expression in the mesodermal gonad anlage isrequired for formation of the gonadal primordia. Twolines of evidence suggest that it is acting autonomously.First, the gonadal primordia are formed before thenervous system is fully developed (stage 14). At thistime, the mesodermal cells that become incorporatedinto the gonads are already expressing abd-A protein.Second, analyses of chimeric animals generated bynuclei and larval ovary transplantation experimentssuggest that iab-4+ function is required in the ovarianmesoderm. In the nuclei transplantation experiment inwhich the donor cells were labeled with the hsp70promoter-lacZ construct, the entire follicle cell popu-lation stained for /S-galactosidase activity. Similarly,with the donor cells marked with an Ape mutation, alleggs showed the Ape phenotype. Had donor cells of theneuroectoderm origin induced mesodermal cells toinitiate gonadogenesis, patches of the host (iab-4~) cellswould have been expected in the ovaries. The absenceof the host cells in the rescued ovaries, therefore,argues that iab-4+ function is required autonomously inthe mesodermal cells.

If iab-4+ is acting autonomously in the mesodermalgonad anlage, why do we not detect a correspondingloss of abd-A expression in mutant embryos? Mostlikely explanation is that we did not detect subtle spatialor temporal differences in the pattern of expression.The iab-4 deletions affect cis-regulatory regions andare partial loss-of-function alleles which are not fully

penetrant. Stronger mutations decrease the frequencywith which gonads are formed, but not the amount ofsomatic tissue formed. In the rare iab-4302 escaperswhere an ovary is present, it contains the correctnumber of ovarioles, whereas other mutations (e.g.some oscar" alleles) are known to reduce the number ofovarioles formed (Szabad and Nothiger, 1992). Theseobservations are consistent with the idea that athreshold level of abd-A activity triggers a decision inthe mesoderm to form an ovary in an 'all or nothing'manner. Perhaps small changes in the level of abd-Aexpression cause the cells to drop below the threshold.

A variety of lacZ enhancer trap lines have beenobtained recently whose /5-gal expression patterns arerestricted to specific parts of the reproductive system(Fasano and Kerridge, 1988). It may be possible toidentify genes whose spatial and temporal expressionpatterns correlate with the tissues affected by the iab-4mutations. Such genes would be good candidates forthe realizator genes acting downstream in the pathway.It is not known if abd-A regulates the same downstreamtargets in, for example, the mesodermal gonad anlage,the pupal ovaries and the associated neurons. Havingthe same 'selector' gene act at multiple steps and indifferent tissues may help to coordinate the develop-ment of complex organ systems.

We thank Ed Lewis for providing many of the stocks used inthis work, John Lis for the hsp70-LacZ line, Y. N. Jan for anti-vasa antibody and Diane Mattson and Ian Duncan for anti-abd-A antibody. We also thank Anne Boulet, Brad Johnsonand Rolf Ndthiger for helpful comments on the manuscript.

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(Received 24 February 1992)