1833Development 125, 1833-1843 (1998)Printed in Great Britain © The Company of Biologists Limited 1998DEV8496

Genetic dissection of sperm individualization in Drosophila melanogaster

James J. Fabrizio 1, Gary Hime 2,*, Sandra K. Lemmon 3 and Christopher Bazinet 1,†

1Department of Biological Sciences, 8000 Utopia Parkway, St. John’s University, Jamaica, NY 11439, USA2Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA3Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106,USA *Present address: Trescowthick Research Laboratories, Peter Macallum Cancer Institute, Locked Bag 1, A’Beckett Street, Melbourne, VIC 3000, Australia†Author for correspondence (e-mail: bazinetc@stjohns.edu)

Accepted 6 March; published on WWW 22 April 1998

The morphogenesis of spermatids generally takes placewithin a syncytium, in which all spermatid nuclei descendedfrom a primary spermatocyte remain connected via anextensive network of cytoplasmic bridges. A late step insperm maturation therefore requires the physical resolutionof the syncytium, or cyst, into individual cells, a processsometimes referred to as sperm individualization. Despitethe identification of specialized machinery involved in theindividualization of Drosophila spermatids (Tokuyasu, K.T., Peacock, W. J. and Hardy, R. W. (1972) Z. Zellforsch124,479-506), and of many Drosophila genes mutable to male-sterile phenotypes, little is known of the mechanisms bywhich this extensive remodeling of the cyst is accomplished.Here, the identification of a major cytoskeletal componentof the individualization complex as actin is confirmed witha simple fluorescence assay. Using rhodamine-phalloidin asa probe, the individualization complex is readily visualizedforming around bundles of spermatid nuclei at one end ofhighly elongated cysts, then translocating along the lengthof the cysts. The structure of the individualization complexin a male-sterile clathrin heavy chain (Chc) mutant isobserved to be reduced or disrupted relative to wild-type,consistent with the individualization-deficient phenotype ofthis mutant. Using the fluorescence assay, a sampling ofmale-sterile mutant phenotypes in which spermatogenesisproceeds to the assembly of highly elongated cystsdistinguishes at least four different phenotypic classes: (1)mutations (nanking class) that block or significantly retardthe assembly of the actin-based individualization complex

around the nuclear bundle, (2) mutations (dud class) inwhich the individualization complex assembles in/aroundthe nuclear bundle, but fails to translocate down the cyst,(3) mutations (mulet class) that allow the assembly of amorphologically normal individualization complex aroundthe nuclear bundle, but result in a breakdown in thecomplex after it begins to translocate down the cyst, and (4)mutations (purity of essenceclass) that allow the assemblyof a motile but morphologically altered or reducedindividualization complex. Individualization also fails in anumber of mutants with altered nuclear shape, consistentwith the hypothesis that spermatid nuclei provide a physicalscaffolding for the assembly of the individualizationcomplex. Genetic analysis suggests that a substantialnumber of additional loci with phenotypes distinguishablewith this assay remain to be identified. The large proportionof male-sterile mutations resulting in a late block tospermatogenesis, in which highly elongated cysts fail to beindividualized, suggest a substantial susceptibility of thisprocess to a broad range of cellular perturbations. Themassive reorganization of cyst cytoplasm required atindividualization is expected to be a correspondinglycomplex function requiring exquisite coordination ofmultiple cytoplasmic functions, and may account for thepreviously noted high frequency with which Drosophilagenes are mutable to male-sterile phenotypes.

Key words: Spermatogenesis, Individualization, Actin, Syncytium,Membrane, Drosophila

SUMMARY

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INTRODUCTION

Because spermatozoa are generally constructed and mawithin a germline syncytium, the final stages of spermatogenerequire the resolution of the male germline cyst into separgametes by packaging each spermatid into its own plasmembrane (Kierszenbaum, 1994), a process sometimes refeto as sperm individualization. Despite the apparent conservaof this feature of spermatogenesis, little is known of the cellumechanisms by which sperm individualization is achieved.

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Spermatogenesis in Drosophilahas been described inconsiderable detail (for recent review, see Fuller, 1993). Thspermatogenic cyst of Drosophila provides an excellentsystem for the investigation of sperm individualization,because the cysts are readily distinguished from each othand because cysts at all stages of development are pressimultaneously in the testis, where their spatial distributioreflects the stage of development. Moreover, the extremepolarized structure of the Drosophila spermatogenic cystresults in a correspondingly polarized yet synchronou

1834

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individualization process, the ultrastructure of which has bedescribed in the classic studies of Tokuyasu et al. (1972). Emature Drosophila cyst contains 64 haploid spermatiddescended by 4 mitotic divisions and a meiotic division frothe same primary spermatocyte. The mature cyst is unusually large cell (~2 mm long), with a bundle of 64 needshaped nuclei at one end and flagellar tails extendthroughout its length. Individualization is accomplished by tassembly of a cytoskeletal-membrane complex at the nucend of the cyst, which translocates the length of the cyinvesting each spermatid in its own plasma membrane simultaneously extruding most of the syncytial cytoplasfrom between sperm tails as it proceeds (Fig. 1). Tcomplex, which we refer to as the individualization compl(IC), is a coordinated array of discrete ‘investment cone(Tokuyasu et al., 1972), which move synchronously along spermatogenic cyst, each spermatid being individualized bsingle investment cone. The movement of the IC along sperm bundle causes an observable dilation of the cyst duthe accumulation of cytoplasmic material extruded out frobetween the sperm tails. The IC is then detached from mature individualized cyst when it reaches the tail end, whit is referred to as a waste bag (Tokuyasu et al., 1972).

Here we present a simple fluorescence assay for visualization of the major actin cytoskeletal component of tIC in spermatogenic cysts of Drosophila. A male-sterileclathrin mutation, Chc4, which fails to individualize spermproperly, displays a grossly altered IC distribution anmorphology by the fluorescence assay. Applied spermatogenic cysts of males homozygous for a numbemale-sterile mutations that allow the morphogenesis of higelongated cysts, the assay distinguishes several distinct claof mutant phenotypes, differing in the assembly, structure adynamics of the IC. Combined structural, genetic amolecular analysis of these mutations may allow new insiginto cellular mechanisms operating in postmeiotspermatogenesis, including individualization.

MATERIALS AND METHODS

Fly husbandryDrosophila stocks were maintained at room temperature and groon standard cornmeal, agar and molasses medium. Fly manipulawere as described by Matthews (1994) and Greenspan (19scat1ms(2)30B, ms(2)46C, ms(2)42A and tho186E P insertionmutations, previously described by Castrillon et al. (1993), weobtained from the Bloomington stock center.

ms(2)4210, ms(2) 5970, ms(2) 5720 andms(2) UK are chromosome2 mutations generated in the lab of M.T. Fuller by P-elememutagenesis using P[lacW] (Bier et al., 1989) in a white backgroundand maintained using CyO as a balancer chromosome. ms(3)3915 isa chromosome 3 mutation generated in an identical fashion maintained using TM3 as a balancer chromosome. ms(2) Hb3obtained via M.T. Fuller, is a chromosome 2 mutation generatedthe laboratory of J. Hackstein by chemical mutagenesis (Hackst1991).

Cytology and ultrastructural analysisBlebbing assay for individualization defectsTestes were dissected from adult males in Drosophila Ringers solution

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with Pipes (Castrillon et al., 1993), squashed and examined direby phase-contrast microscopy.

Phalloidin assay for the ICTestes were removed from freshly eclosed males in TB1 [15 mMpotassium phosphate (equimolar di- and monobasic) pH 6.7, 80 mKCl, 16 mM NaCl, 5 mM MgCl2, 1% PEG 6000]and dissected inTB1 on poly-L-lysine-treated slides to expose spermatogenic cyGenerally, this involves rupturing the testis at one end or the other teasing the sperm bundles out with a fine forceps. Tissue samples flattened under a siliconized coverslip and frozen in liquid nitrogeCoverslips were flicked off the slides using a razor blade and samples remaining on the slides were placed in ethanol cooled−20°C and fixed in 3.7% paraformaldehyde/PBS (130 mM NaCl,mM Na2HPO4*2H2O, 3 mM NaH2PO4*2H2O). The slides were thenwashed in PBS, blocked in PBTB (PBS, 0.1% Tween, 3% BSA) aincubated for two hours at 37°C in a solution of 6 Units/mrhodamine-conjugated phalloidin (Molecular Probes Inc., EugenOR) in PBTB. Slides were then washed in PBS, stained with DA(1 µg/ml), washed again with PBS and observed under a Nikmicrophot SA microscope with epi-fluorescence illumination. minimum of three independent preparations of each genotype wexamined using this assay, with a range of 15 (dud) to 110 (Chc4)squashes prepared from mutant males.

Electron microscopyTestes were dissected from adult males 2-5 days after eclosion prepared for electron microscopy as described by Tokuyasu et(1972), except that tissues were infiltrated and embedded in LX1resin (Ladd Research Industries, Burlington, VT). Thin sections weobserved and photographed using a Jeol 100CX transmission elecmicroscope at 80 kV accelerating voltage.

In situ hybridizationPolytene chromosome preparation and hybridization reactions wcarried out essentially as described by Pardue et al. (198Biotinylated probes containing the Drosophila white gene weregenerated by the random primer method (Feinberg and Vogelst1983) using linearized pCasPer4 (Pirotta, 1988) as template ancommercial kit (BrightStar DECAprime-Biotin, Ambion, Inc.,Austin, TX). Hybridization signals were visualized using streptavidinconjugated horseradish peroxidase (Gibco BRL, Gaithersburg, MThe peroxidase detection reaction was enhanced by the additioNiCl2 to a final concentration of 0.4 mg/ml.

RESULTS

Actin cytoskeletal dynamics of wild-typespermatogenic cystsPreviously, a dense network of ~6 nm fibrils had been localizto cone-shaped structures that form around each axonewithin the IC (Tokuyasu et al., 1972). Using rhodamineconjugated phalloidin as a probe, we have confirmed identity of these fibrils as actin fibers (Fig. 2). Simplpreparations of elongated cysts teased out of wild-type tesreveal numerous dense, coherent foci of F-actin staining (F2A). Many of these signals colocalize with the spermatnuclear bundles, while some appear to have progrescaudally along the length of the spermatogenic cyst (Fig. 2The appearance of these F-actin-rich complexes in tessquash preparations is consistent with the proposed mechanof the individualization process (Tokuyasu et al., 1972), which the IC assembles around the spermatid nuclear bun

1835Sperm individualization mutants

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and subsequently travels caudally along the length of the (Fig. 2C,D). As the IC translocates, it extrudes much of cyst cytoplasm from between the sperm tails, resulting innoticeable cystic bulge (Fig. 2E). When the IC has complethe individualization process and reaches the end of spermatogenic cyst, it is transformed into a detachable ‘wabag’ (Fig. 2F), the contents of which are presumably degrawithin the lumen of the testis (Tokuyasu et al., 1972).

Mutations disrupting the individualization processOur studies of the sperm individualization process weinitially prompted by the finding that a semilethal allele of thDrosophila clathrin heavy chain gene, Chc4, allowed the

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Fig. 1. Individualization ofDrosophilaSpermatogenic cysts.Four mitotic divisions and a meiosisfrom each primary spermatocyte,with incomplete cytokinesis aftereach nuclear division, gives rise to acyst containing a bundle of 64haploid nuclei (N) (only four areshown here for simplicity). Spermtails extend from the bundle ofneedle-shaped nuclei through thesyncytial cytoplasm of the elongatedcyst (A). Each germline cyst issandwiched between two somaticcells known as cyst cells (c.c.).Individualization involves theassembly of a membrane-cytoskeletal complex, theindividualization complex (IC), atthe nuclear end of the cyst (B) thattranslocates the length of the cyst,encasing each spermatid in anintegral plasma membrane (C) andextruding syncytial cytoplasm andother morphogenetic debris in a‘waste bag’ (WB) subsequentlyeliminated from the tail end of thecyst (D). A complex membranenetwork (represented by fine dottedlines in the drawing) extending thelength of the preindividualized cystcontains numerous cytoplasmicbridges. This membrane is eitherremoved or remodeled duringindividualization. At the end of theindividualization process, the wastebag is separated from the cyst andthe individualized sperm bundle istightly coiled before liberation fromthe two somatic cyst cells andpassage out of the testis into theseminal vesicle. Dotted lines throughthe cyst in C represent the plane ofsection observed in the electronmicrographs presented in this paper.A section to the left, or rostral sideof the individualization complex(ind.), gives a view of individualized spermatids; a section to the righsimplicity, the length/width ratio of the cysts is grossly underrepresenmelanogaster,and considerably longer in other Drosophilaspecies. SectiTokuyasu (1980), based largely on the data of Tokuyasu et al. (1972

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survival of hemizygous males and homozygous females significant frequencies (Table 1). Chc4/Y males are invariablysterile, although Chc4/Chc4 females do produce some viableeggs (Bazinet et al., 1993). Spermatogenesis is therefore only event in the Drosophilalife cycle absolutely blocked bythe Chc4 mutation. This suggests either a specialized role foclathrin-based transport in spermatogenesis, or extremsensitivity of some aspect of spermatogenesis to cytoplasmdisorganization caused by defective vesicle trafficking.

Squash preparations of Chc4 /Y mutant testes examined byphase-contrast microscopy reveal numerous elongated cybut a complete absence of motile sperm. When cysts adisrupted in such preparations, no mature individualized spe

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Dt (preind.), or caudal of the IC, affords a view of a preindividualized cyst. Forted here, since the cyst grows to approximately 2 mm in length in D.

ons through the IC itself are therefore quite rare. Adapted from Lindsley and).

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Fig. 2.Squash preparations of dissected wild-type testes stained withrhodamine-phalloidin alone (A,C,E,F) or stained with rhodamine-phalloidin and DAPI (B,D) to visualize actin fibers and nuclei,respectively. Low magnification reveals a multitude of spermatogeniccysts teased out of the testicular lumen (A,B). Actin-based ICs stainbrightly with rhodamine-phalloidin (arrows in A) and, in most cases,are found to colocalize with the spermatid nuclear bundle(arrowheads in B). However, ICs that have progressed away from thenuclear bundle (arrows in B) are also evident in the double exposure.Higher magnification of two aligned spermatogenic cysts teased outof the testes lumen reveals an actin-based IC colocalizing with thespermatid nuclear bundle in one cyst (arrowheads in C,D) and anactin-based IC traversing away from the spermatid nuclear bundle inthe other cyst (arrows in C,D). Actin-based ICs that have progresseda significant distance away from the nuclear bundle exhibit a markedincrease in volume due to the accumulation of extruded cytoplasm(arrows in E) and eventually reach the ‘waste bag’ stage (arrows inF), where the contents of the IC are presumably degraded. Bars:(A,B) 100 µm; (C-F) 20 µm.

Fig. 3. Morphology of sperm from Chc4/Y mutant males. (A) Phase-contrast view of testes squash from a 4-day-old male carrying thewild-type parental w(K)chromosome. Note the smooth texture ofsperm bundles and individual motile sperm released from maturebundles. (B) Testes squash from a 4-day-old Chc4 male. Chc4 spermbundles generally appear less tightly organized, with many structuralirregularities apparent. In still more loosely bundled mutant sperm(C), numerous bulges or blebs, sometimes regularly spaced(arrowheads), are observed on individual spermatids. In these views,the internal shaft of the spermatid generally appears to extend intactthrough the blebbed out region, suggesting that the defect resultsfrom a local disruption in membrane structure or an aberrantinclusion of cytoplasmic material from the cyst. In the mutantpreparations, some tightly bundled groups of spermatids in which theblebbing defect is not apparent, probably from less maturepreindividualized stages, are also observed (arrow). (D) View of atestes squash from a Chc4 male carrying the cloned Chc+ gene on anautosomal P-element. The morphological and motility defects arecompletely rescued by a single copy of the wild-type Chcgene. Bars,10 µm.

A B

C D

are distinguishable. Instead, their morphology is highirregular, suggestive of a large variation in the width of tsperm tails, or of a failure of sperm to be separated from eother by the individualization process. In addition, numerobulges or blebs are apparent along the length of the sperm(Fig. 3B,C).

As reported earlier (Bazinet et al., 1993), Chc4males carryingan additional copy of the cloned wild-type Chc gene are restoredto fertility. Testes squash preparations from such Chc4; P[w;Chc+] individuals are indistinguishable from those of wild-typmales, with both exhibiting numerous motile, morphologicanormal sperm (Fig. 3A,D). The spermatogenic defect in Chc4

males is therefore attributable to the clathrin mutation and some other mutation in the genetic background of these flThe pronounced ‘blebbing’ defect obvious in the sperm tailsChc4 testes preparations suggests a defect in individualization process, such that pockets of cytoplasmmaterial normally removed during the translocation of the

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may be left behind. Alternatively, the blebs may simply resufrom a random redistribution of cytoplasm upon rupture of thunfixed cysts that have failed to individualize.

Ultrastructural analysis of wild-type and Chc4

spermatogenic cystsTransmission electron microscopy of thin sections preparfrom testes of Chc4 males confirmed a defect in theindividualization process. Pre-individualized spermatogencysts, identifiable as such because of their rudimentaaxonemal decoration, light-staining mitochondrial derivativesenlarged minor mitochondrial derivatives and the presence extensive cytoplasmic ground substance between taxonemes, are evident in both wild-type (Fig. 4A) and Chc4

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Table 1. Summary of IC and nuclear bundle disruptions in the mutant genotypesDisrupted ICs

No. of Intact ICs with associated Intact ICsICs per colocalizing with scattered Intact Disrupted with associated

Viability squash intact nuclear elongated progressed progressed scatteredStock index* (avg) bundles nuclei ICs^ ICs nuclei

Mutations exhibiting disrupted individualization complexes and scattered spermatid nucleirosy 506** − 9.7 60% 0% 40% 0% 0%w(k)** − 16.6 54% 3% 36% 7% 0%Chc4 0.28 2.6 2% 93% 5% 0% 0%scat ms(2)30B 0.53 1.2 0% 50% 0% 50% 0%scat ms(2)30B/bal 1 14.5 60% 1% 38% 1% 0%crossbronx ms(2)46C 0.99 4.9 0% 92% 0% 8% 0%crossbronx ms(2)46C/bal 1 13.9 53% 0% 45% 1% 0%lie ms(2)42A 0.43 5.3 0% 66% 0% 34% 0%lie ms(2)42A/bal 1 17.8 54% 0% 45% 1% 0%dud ms(2)Hb328 0.2 7.5 46% 10% 0% 42% 2%dud ms(2)Hb328/bal 1 10.5 52% 2% 46% 1% 0%nanking ms(3)3915 0.84 0.8 8% 80%^^ 0% 0% 40%^^nanking ms(3)3915/bal 1 9 56% 6% 35% 3% 0%

Disrupted ICs Intact ICsNo. of Intact ICs with associated withICs per colocalizing with scattered Intact Altered^^^ associated

Viability squash intact nuclear elongated progressed progressed scatteredStock index* (avg) bundles nuclei ICs ICs nuclei

Mutations exhibiting altered progressed ICspoe ms(2)5970 1.05 8.6 14% 33% 0% 53% 0%poe ms(2)5970/bal 1 18.8 47% 1% 37% 1% 15%mlt ms(2)4210 1.06 13.2 58% 7% 0% 35% 0%mlt ms(2)4210/bal 1 7.8 47% 1% 50% 1% 0%

Disrupted ICs DisruptedNo. of Intact ICs with associated ICs withICs per colocalizing with scattered Intact Disrupted Ribbon-like associated

Viability squash intact nuclear elongated progressed progressed actin dot-likeStock index* (avg) bundles nuclei ICs ICs structures nuclei

Mutants exhibiting abnormal nuclear morphologymoz1 ms(2)UK 1.09 3.4 0% 31% 0% 38% 32% 0%moz1 ms(2)UK/bal 1 14.5 56% 8% 36% 0% 0% 0%moz2 ms(2)5720 1.21 4.2 0% 48% 0% 43% 9% 0%moz2 ms(2)5720/bal 1 16.5 55% 0% 43% 2% 0% 0%tho 86E 0.62 4.7 0% 74% 0% 0% 0% 26%tho86E/bal 1 11.8 55% 1% 44% 0% 0% 0%

*Viability index = no. of homozygous mutant flies observed/no. of homozygous mutant flies expected, where the number of mutant flies expected = 1/2 thenumber of balancer heterozygotes.

**These are wild-type controls, used for mutants in rosy or white backgrounds, respectively.^Progressed ICs are individualization complexes that do not colocalize with the nuclear bundle; i.e. ICs that have traversed caudally along the cyst.^^The IC and nuclear bundle disruptions are very mild in these squashes.^^^“Altered” refers to the disruption present in mlt ICs and the failure of poe ICs to enlarge.

(Fig. 4B) testes. Although only 40 flagellar axonemes are sin the mutant cyst (Fig. 4B) relative to 64 in the wild type (Fi4A), the overall cytoplasmic organization of the cysts appequite similar in the two samples.

Highly mature spermatids in sections of wild-type cysts thhave been individualized are characterized by a substanreduction in the amount of cytoplasmic ground substanbetween the axonemes, more pronounced axonemal decor(generally observable as an enhancement in radial symmetrcross-sectional views of the axoneme), the accumulationextensive amounts of dark-staining material in the mamitochondrial derivatives and a significant reduction in size the minor mitochondrial derivatives (Fig. 5A). In contrassections through many mature Chc4 mutant cysts reveal a grossdisorganization not present in immature cysts (Fig. 5B). Althouthese spermatids appear quite mature on the basis of axon

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decoration and the intense staining of the major mitochondrderivatives, extensive amounts of cytoplasmic ground substaremain between the axonemes, very irregularly distributed amothe spermatids. Gross distensions of the mitochondrderivatives, usually the minor derivative, are also commonobserved in such cysts. The cytoplasm between mutant sptails in these cysts often appears highly disorganized, withcomplex network of membranous tubules (Fig. 5BQualitatively, the appearance of Chc4 cysts changes radically latein the morphogenetic process, consistent with the hypothesis a failed attempt at individualization is the source of thpronounced loss of organization relative to wild-type cysts.

Actin cytoskeletal dynamics of Chc4 spermatogeniccystsWhile wild-type spermatogenic cysts often display th

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J. J. Fabrizio and others

ructure of elongating cysts relatively early in the preindividualizedg of mitochondrial bodies is relatively light compared to more matureig. 5), indicating that the accumulation of osmophilic material within the bodies is still in progress. Fine fibers of the axoneme are not yetd there is extensive cytoplasmic ground substance between the spermor mitochondrial derivative. mi, minor mitochondrial derivative. (B) Chc4 mutant. Bar, 2 µm.

presence of a coherent bundle of actin fibers (Fig. 2), corresponding machinery is much less frequently observedcysts from Chc4 males prepared in parallel (Fig. 6A-C; Tabl1). Infrequent ICs observed in Chc4 preparations are usuallydisorganized relative to wild-type ICs. Cysts from Chc4 malesalso reveal scattered spermatid nuclei (Fig. 6B,D) as oppoto the intact nuclear bundles present in wild-type cysts (F2B,D). When evidence of a disrupted IC is observed in Chc4

cysts, the nuclear bundle of the corresponding cyst is usualso disorganized, with numerous nuclei apparently pulled of the bundle, or scattered caudally along the cyst (Fig. 6A-Table 1). The appearance of Chc4 mutant cysts indicates thatthe IC is sometimes assembled in this mutant, but failsfunction properly. Since fertility, normal cytoskeletaappearance and intact nuclear bundle morphology are restored by the addition of a single copy of the wild-type Chcgene on a P element (Fig. 6E,F), proper assembly and funcof the IC therefore requires a fully functional Chc gene.

Assay for potential individualization defectsThe analysis of the Chc4 phenotype suggests that a simplphase-contrast examination of unfixed testis squapreparations, followed by the phalloidin assay for IC formatioand progress described above, might allow the identificationa number of different phenotypic classes from among masterile mutations that block spermatogenesis in the postmeistages. Although postmeiotic arrest is the most common cof male-sterile phenotype in Drosophila (Castrillon et al.,1993), relatively little is known about cellular factors that mabe required for the extensive morphogenetic changes obsein this stage.

We screened a collection of 74 male-sterile mutant fly stocfor late morphogenetic defects using this‘blebbing’ assay and found that six mutationsresulted in highly elongated, immobile,blebbed sperm tails. In addition, four othermale-sterile mutations previously reported toexhibit scattered spermatid nuclei (Castrillon etal., 1993) were obtained from the Bloomingtonstock center. Each of these mutations alsoresulted in the production of elongated cystsand immotile, blebbed sperm flagella. Theseten mutants exhibiting blebbing defects werefurther analyzed using rhodamine-phalloidinand DAPI to visualize the actin microfilamentcomponent of the IC and spermatid nuclei,respectively. Since the mutations exist in whiteor ry506 backgrounds, w(k) and ry506 malesalong with balancer heterozygotes were used aswild-type controls. Several different classes ofsperm maturation phenotypes have beenidentified in this screen.

Mutants exhibiting defects early in theindividualization processWhile squash preparations of wild-type testesreveal a multitude of ICs in various stages ofmaturation (Fig. 2A; Table 1), testes fromnanking mutant males reveal a general paucityof ICs (Table 1; Fig. 7A). However, when ICsare constructed in nanking ms (3) 3915 mutant

Fig. 4. Ultraststage. Staininstages (see Fmitochondrialcompleted antails. ma, maj(A) Wild type;

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testes, they appear to be tightly organized compared to thof most other mutants (see below) and are never seen displafrom the nuclear end of the cyst (Fig. 7C,D; Table 1). NankingICs often colocalize with a mildly disrupted spermatid nuclebundle (Fig. 7D), but in general, the spermatid nuclear bundmorphology is largely unaffected (Fig. 7B). These observatiosuggest a primary defect in the assembly of the IC which msecondarily prevent the translocation of the IC along thspermatogenic cyst.

Robust ICs associated with intact spermatid nuclear bundare readily observed in testes squash preparations from ms(2)Hb328 mutant flies (Fig. 7E,F), which construct a mugreater number of ICs than nanking and occasionally exhimore prominently scattered spermatid nuclei and Idisruptions (Table 1). These disrupted ICs often colocaliwith the nuclear bundle, but fragments of the complex that haprogressed away from the nuclear bundle are sometimobserved (Table 1). Intact ICs that have progressed along spermatogenic cyst are not seen in squash preparations of tefrom this mutant, suggesting that an important factor requirfor motility of the IC may be absent or defective. Defeccharacteristic of these mutations are observed much lfrequently in wild-type controls.

A mutation that allows the progress of a reduced ICalong the spermatogenic cystAn IC with a significantly reduced actin signal is built arounthe spermatid nuclear bundle in testes preparations frms(2)5970males (Fig. 8A,B). In many cases, these ICs appeto progress away from the nuclear bundle and travel ascoherent unit along the spermatogenic cyst (Fig. 8B-D; Tab1). These complexes are often observed at considera

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Fig. 5. High magnification views of sections throughmature portions of cysts from wild-type (A) and Chc4

mutant (B) testis. Wild-type cysts assume a highlyordered, sometimes paracrystalline array afterindividualization, with a significant reduction in boththe amount of cytoplasm between the sperm tails andthe size of the minor mitochondrial derivative. Mutantcysts fail to individualize properly. In this case, morespermatids than usual appear to have been encasedtightly in membrane, although grossly disorganizedexcess cytoplasmic material is seen within many of thespermatid membranes. ma, major mitochondrialderivative; mi, minor mitochondrial derivative; cn,complex tubular network. Bar, 1 µm.

Fig. 6. Squash preparations of dissected Chc4 (A-D) and Chc4 rescue(E,F) testes stained with rhodamine-phalloidin to visualize actin fibers(A,C,E), DAPI to visualize nuclei (D), or double exposed for both(B,F). Chc4 mutant cysts exhibit disrupted ICs (arrows in A-C) whichcould be responsible for the disruption of the spermatid nuclear bundleand the subsequent dragging of spermatid nuclei caudally along thecyst (arrowheads in B,D). Normal cytoskeletal appearance of the IC(arrows in E,F) and bundling of the spermatid nuclei (arrowheads in F)are restored by a copy of the Chc+ gene on an autosomal P-element.Bars: (A,B) 30 µm; (C,D) 20 µm; (E,F) 100 µm.

distances from the nuclear bundles. However, since we hnot observed the pronounced ‘cystic bulge’ associated whighly progressed wild-type ICs (Fig. 2E) in preparations froms(2)5970 homozygotes (Fig. 8C,D; Table 1), it appears ththese mutant ICs fail to undergo an increase in volumcharacteristic of wild-type ICs, which are actively extrudincytoplasm from between the sperm tails.

By introducing the 2-3 transposase source (Robertson et1988) into flies carrying the ms(2)5970mutation, we found thatthe mutation is revertible. We mapped it by in sithybridization, using the white gene to detect the P element, position 28E on the second chromosome. Complementatesting revealed that the mutation is an allele of purity ofessence (poe), mapped previously to the same cytologlocation by Castrillon et al. (1993).

IC breakdown caused by the mulet mutationSpermatogenic cysts from homozygous ms(2)4210mutantmales construct an IC which by the phalloidin assay appenormal (Fig. 8E; Table 1). However, as the complex travcaudally along the cyst, it becomes severely disrupted (F8G,H; Table 1). Nuclear scattering is notably absent in thepreparations, (Fig. 8F; Table 1). A gene product requiredmaintain the structural integrity of the IC, or to coordinate tmovement of individual investment cones within the IC, mabe defective or absent in this mutant. This phenotypcharacteristic of homozygous ms(2) 4210 males, is rarelyobserved in wild-type controls (Table 1).

The ms(2)4210mutation is revertible by 2-3 transposas(Robertson et al., 1988) and maps to cytological position 4By complementation analysis, the mutation is an allele of mulet (mlt) gene previously identified by Castrillon et a(1993).

Expressway mutantsThree of the male-sterile mutants analyzed in this screexhibit phenotypes similar to that of Chc4 (Fig. 9), in that theydisplay disrupted ICs and elongated spermatid nuclei scattealong the cyst (Castrillon et al., 1993). IC disruptions anuclear scattering are evident in the scat ms(2)30B (Fig. 9A-C), crossbronx (cbx) ms(2)46C (Fig. 9D) and long islandexpressway (lie) ms(2)42A (Fig. 9E) mutants while nearly

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absent from wild-type control preparations (Fig. 2A-D; Tabl1). Qualitatively, lie and cbx mutant testes display the moshighly disrupted ICs and the most prominent scattering spermatid nuclei (Fig. 9D,E). Testes dissected from scat

1840

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J. J. Fabrizio and others

Fig. 7. Squash preparations of nanking (A-D) and dud (E,F) mutanttestes stained with rhodamine-phalloidin to visualize actin fibers(A,C,E), DAPI to visualize nuclei (B), or double exposed for bothdyes (D,F). Spermatogenic cysts dissected from nanking mutanttestes reveal a general absence of phalloidin-staining structures (A)and non-disrupted spermatid nuclear bundles (arrowheads in B).Rarely, fairly intact actin-based ICs can be visualized at the nuclearend of the cysts (arrow in C), usually associated with a slightlydisrupted nuclear bundle (arrowhead in D). Cysts dissected from dudmutant testes usually exhibit many intact ICs (arrow in E) which co-localize with non-disrupted spermatid nuclear bundles (arrowhead inF). Bars: (A,B) 200 µm; (C-F) 25 µm.

Fig. 8. Squash preparations of purity of essence (poe)(A-D) andmulet (mlt) (E-H) mutant testes stained with rhodamine-phalloidinalone (A,C,E,G) or double exposed for rhodamine phalloidin andDAPI to visualize actin filaments and nuclei, respectively (B,D,F,H).Actin-based ICs of reduced staining intensity (arrows in A) whichco-localize with mildly disrupted spermatid nuclear bundles(arrowheads in B) are commonly observed in poe mutant testes.Actin-based ICs that have progressed along the cysts are alsocommonly observed (arrow in B). At higher magnification, theprogressed ICs appear reasonably focused or coherent, but reduced involume (arrow in C), and do not co-localize with the spermatidnuclear bundle (arrow in D). High magnification of the nuclear endof mlt mutant cysts reveals intact ICs (arrows in E) colocalizing withintact nuclear bundles (arrowheads in F). Occasionally, fairly intactICs can be seen leaving the bundle of spermatid nuclei (right side ofmicrograph F). Actin-based ICs become disrupted upon leaving thespermatid nuclear bundle (arrows in G,H). Note also the presence ofintact ICs colocalizing with intact nuclear bundles in thesemicrographs (arrowheads in G,H). Bars: (A,B,E,F) 50 µm;(C,D,G,H) 25 µm.

ms(2)30B mutant males reveal a general paucity of ICs aslightly milder disruptions of both the nuclear bundle and tIC (Fig. 9A,B; Table 1), and mild nuclear shaping defects aalso evident in these testes (Fig. 9C). Because the streaminblue fluorescent nuclei and red actin-based investment coalong the spermatogenic cysts of these mutants is reminisof time-lapse photographs of highways at night, we refer to tgeneral class of mutations as expressway mutants.

Mutations affecting nuclear morphologyTestes from homozygous tho186E males contain cysts withcomplex nuclear and IC morphologies not seen in wild-tycontrol preparations. Individual investment cones aelongated nuclei are observed scattered throughout the (Fig. 9G,H; Table 1). In addition, numerous smaller DAPsignals are observed, often colocalizing with F-actin signasuggestive of nuclear breakdown or chromosomal loss dursperm maturation (Fig. 9F; Table 1).

Some mutations allowing the assembly of highly elongatcysts appear to affect spermatid nuclear morphology earliedevelopment, since both ms(2) UK andms(2) 5720 alleles ofthe mozzarella(moz) gene display incompletely condensedirregularly shaped nuclei (Fig. 10B) as compared to wild-ty

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controls. However, a significant amount of actin staining ioften seen associated with the spermatid nuclear bundleseach of these mutants (Fig. 10A,B), suggesting thaconstruction of an IC is attempted. While distinct actin stainincharacteristic of investment cones is often observed preparations from both moz mutants (Fig. 10A,B; Table 1),unusual highly extended actin-based signals are also seen (F10C,D; Table 1), indicating that the IC may undergo a uniqumorphological change in these mutants.

1841Sperm individualization mutants

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Fig. 9. Squash preparations of scat1ms(2)30B (A-C), crossbronx (D),lie (E) and tho186E(F-H) mutant testes stained with rhodamine-phalloidin alone (A and G) or double exposed for rhodamine andDAPI to visualize actin-based ICs and nuclei, respectively(B,C,D,E,F,H). Disrupted actin-based complexes (arrows in A)colocalizing with disrupted spermatid nuclear bundles (arrowheadB) are seen in scat1ms(2)30B, as well as fragmented actin staining(arrows in C) associated with scattered spermatid nuclei (arrowhein C). Mild nuclear shaping defects are also evident (C). Severelydisrupted nuclear bundles and scattered spermatid nuclei are moobvious in crossbronx (arrowheads in D) and lie (arrowheads in E).Fragmented actin staining is commonly seen in both of thesemutations (arrows in D,E). Disrupted ICs (arrow in G) associatedwith scattered elongated spermatid nuclei (arrowheads in H) areoften seen at the nuclear ends of tho186Espermatogenic cysts. Dot-like spermatid nuclei are often seen scattered further along the cy(arrowheads in F), often in the vicinity of scattered actin comet tailike signals (arrows in F). Bars: 25 µm.

Fig. 10. Squash preparations of moz2 (A,B) and moz1 (C,D) mutanttestes stained with rhodamine-phalloidin to visualize filamentousactin (A), or double exposed for both rhodamine phalloidin andDAPI to visualize filamentous actin and nuclei, respectively (B-D).Nuclear shaping defects are prominent inmozzarellamutant testes(arrowheads in B) and the nuclear end of the cysts often displayfragmented actin staining (arrows in A,B). Hyper-extendedfilamentous actin staining is often observed along the length of thespermatogenic cysts in both alleles (arrows in C,D). Bars: (A,B) 25µm; (C,D) 50 µm.

Specificity of phenotypes assessed by deficiencyanalysis and phase-contrast observation of livepreparationsLive squash preparations of the each of the new mutants hbeen examined by phase-contrast microscopy. In both ofrelatively ‘early’ mutants, dud and nanking, irregularities areobserved in earlier stages of spermatogenesis. Both mutdisplay defects in nebenkern formation; nebenkerns appeabe lacking in many onion-stage spermatids of dud, while

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multiple smaller nebenkerns seem to be present in onion-staspermatids of nanking (data not shown). We have not yobserved any clear defect in earlier stages of mlt, poe, cbx, lie,tho, moz or scatmutant testis examined by phase-contrasmicroscopy.

Whenever possible, the phenotypes of each of the mutatioover uncovering deficiencies have been examined. Poe/Df andmlt/Df phenotypes are indistinguishable from poe/poe andmlt/mlt phenotypes, indicating that the available poe and mltmutations are null alleles.

Studies on viability reveal that Chc4, scat1ms(2)30B, liems(2)42A, tho186E and dud ms(2)Hb328are semilethal alleles,indicating that these genes are required for vital functions addition to spermatogenesis. The mlt, poe, nanking, moz andcbx mutations do not significantly affect adult viability (Table1).

DISCUSSION

In their classic ultrastructural study of sperm individualizationin Drosophila, Tokuyasu et al. (1972) identified numerouparallel arrays of 6 nm filaments in the trailing half of thespindle-shaped investment cones that mediate the proceHere we have used rhodamine-conjugated phalloidin asfluorescent probe with which to visualize this actin componeof the individualization complex at the level of the lightmicroscope. A screen of several ‘late’ male-sterile phenotypein which highly elongated cysts are produced, has allowed tdiscrimination of these ‘late failure’ phenotypes into severacategories, varying in the extent to which the actin-basecytoskeletal component of the IC is assembled, its motility anthe maintenance of its structural integrity as it translocatedown the cyst. This assay does not, however, distingui

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between genetic defects in cyst morphogenesis prior individualization and defects in the individualizatiomachinery itself. Ultrastructural analysis and subcellulocalization of the affected gene products will be requireddistinguish these two classes. Nonetheless, the simfluorescence assay described here provides an entryconsiderable advancements towards understanding extensive remodeling of cyst architecture late in tspermatogenic pathway.

As suggested by Fuller (1993) individualization may providan editing function by which defective spermatids are removfrom the developmental pathway. This is probably beillustrated in males heterozygous for the segregation disto(SD) chromosome. Within a single cyst in these malspermatid nuclei carrying a sensitive responder element (Rs)fail to condense properly, with the corresponding spermatfailing to become individualized, while spermatids carrying thomologous SD chromosome are successfully matu(Tokuyasu et al., 1977; Temin et al., 1991). Our finding diverse individualization-defective phenotypic classes arguthat such an editing function is not an active one, as this wobe expected to result in a uniform or stereotypicindividualization phenotype for most ‘late’ male-sterilmutants. Instead, each distinguishable individualizatiphenotype observed here might best be interpreted symptomatic of a different class of defect, either within genecyst architecture or the individualization complex itself, earesulting in a different form of morphogenetic derailment. Bthis view, the failure of Rsps spermatids to be individualized ismore likely a secondary or indirect consequence of incompnuclear condensation, resulting in a defective scaffoldingtemplate for investment cone assembly. More recentlyDrosophila male-sterile nuclear lamin mutant was isolated which elongated, defective cysts are produced, suggestingindividualization defect as a consequence of misshapspermatid nuclei (Lenz-Bohme et al., 1997). Thindividualization-defective phenotype of mozzarella, tho186Eand scat1ms(2)30B mutants, which also display nucleamorphology/condensation defects, are consistent with a simmechanism.

Disrupted ICs and scattered spermatid nuclei arephenotypic characteristic of crossbronx, thoand lie mutations.This phenotype may arise by one of several differemechanisms.

In the first, a defective IC may assemble around an initiaintact spermatid nuclear bundle, with the subsequmovement of this defective complex resulting in the draggiof spermatid nuclei caudally along the spermatogenic cyThis may be analogous to the involvement of F-actin bundin the correct positioning of germline nuclei in Drosophilaoogenesis, where nurse cell nuclei are anchored in place bactin cables during rapid cytoplasmic transport into the ooc(Mahajan-Milkos and Cooley, 1994a). Mutations that disruthe function of actin-bundling proteins such as quail, a villinhomolog (Mahajan-Milkos and Cooley, 1994b), singed, afascin homolog (Cant et al., 1994), and chickadee, a profilinhomolog (Cooley et al., 1992; Verheyen and Cooley, 1994),result in the displacement of nurse cell nuclei into the ricanals of the syncytium, obstructing transport of cytoplainto the oocyte (Cooley and Theurkauf, 1994). In theexamples, proper F-actin organization appears to be neces

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in order to prevent the aberrant displacement of germlinnuclei. Alternatively, in mutations such as crossbronx,spermatid nuclei may be splayed along the length of the cinitially and investment cones may assemble around eanucleus independently, giving the illusion that spermatid nuclare being dragged by defective investment cones. This maythe case in scat ms(2)30B mutant cysts, in which spermatidnuclei are frequently scattered, but in which the phalloidinbinding actin structures of the IC are seen relativeinfrequently (Table 1).

The complexity of individualization provides ampleopportunity for the direct participation of a large number ogene products and predicts a correspondingly large numbegenetic loci mutable to such a late phenotype. However, maindirect effects are also likely, as small effects on the structuand function of the individualization complex from minor locacytoplasmic disturbances could be expected to incrementcatastrophic levels over the millimeter-scale distancetraversed by the machine. For example, a subtle defect in corganization introduced before individualization in mlt mutantmales may interrupt the continuous travel of the IC as it movprocessively along the cyst, resulting in loss of coordination synchrony of individual investment cones. The consequesensitivity of spermatogenesis to otherwise subtle cytoplasmdefects of the cyst could also explain the effects of thsemilethal Chc4 allele on individualization, the high frequencywith which male-sterile alleles of lethally mutable Drosophilagenes are observed (Lindsley and Tokuyasu, 1980; Full1993) and the large proportion of late phenotypes (i.eresulting in highly elongated cysts) among male-sterimutations (Castrillon et al., 1993). Distinguishing betweemutations that directly affect the individualization process anthose that indirectly interfere with the process through a chanin general cyst organization or physiology will not be triviallarge numbers of mutants can reasonably be expected to into each class.

Tokuyasu et al. (1972) suggested that the microfilamentsthe investment cones somehow provided the motility of thindividualization complex. While the actin-based IC doeprogress caudally along the spermatogenic cyst, potenmotor protein(s) involved in its translocation remain unknownRecently, a novel minor myosin has been shown to bpreferentially localized to the Drosophila testes (Miedema etal., 1995), thus implicating itself as a candidate motocomponent of the IC. Cytoplasmic myosin II has also beelocalized to the cellularization front in the syncytial blastodermof Drosophila embryos (Schejter and Wieschaus, 1993aWhile many of the gene products involved in the cellularizatioof the syncytial blastoderm such as nullo, serendipity-α(Postner and Wieschaus, 1994) and bottleneck (Schejter aWieschaus, 1993b) appear to be blastoderm specific, conservation of function between the two cellularizatiosystems suggests the participation of a myosin motor individualization. An alternative hypothesis, motility driven byassembly and disassembly of actin filaments without the actparticipation of motor proteins, is suggested by the similariin appearance of the phalloidin signals in many of our imagto the actin-based ‘comet tail’ structures that provide motilitfor intracellular parasites such as Listeria and Shigella by thsimpler, and perhaps more primitive, mechanism (Therio1994).

1843Sperm individualization mutants

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The resolution of syncytia into individual cells is a featucommon to numerous morphogenetic pathways, including cellularization of the syncytial blastoderm in Drosophila(Schejter and Wieschaus, 1993b) and the nuclear endospof some flowering plants (Lopes and Larkins, 1993). Similarin the mammalian bone marrow, polyploid (up to 64nmegakaryocytes partition their cytoplasm into individuplatelets using reservoirs of membrane to create platedemarcation channels (Bloom and Fawcett, 1975). Furtinvestigation of Drosophilasperm individualization mayprovide insight into the mechanisms involved in resolvinindividual cells from syncytia in these and othemorphogenetic systems yet to be discovered.

We are especially indebted to Diana Bartelt for generous sharinher fluorescence microscope. We thank Margaret Fuller and AnthMahowald for encouragement and vital criticism, and Joseph Pofor expert assistance with electron microscopy. This work wsupported by NIH Grant R15 HD 31693-01 (C. B.), NSF Grant MC9305287 (S. K. L.), American Heart Association, Northeast OhAffiliate Grant 4838 (S. K. L.), and the American Cancer SocieCuyahoga County Unit (S. K. L). G. H. was supported by C. J. MarFellowship No. 937316 from the National Medical Research Counof Australia. Early stages of this work were generously supportedthe Markey Center for Developmental Genetics, Case WestReserve University.

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