Copyright 0 1990 by the GeneticsSociety of America

Two Types of Genetic Interaction Implicate thewhirligig Gene of Drosophila melanogaster in Microtubule Organizationin the Flagellar Axoneme Larry L. Green,' Nurit Wolf, Kent L. McDonald and Margaret T. Fuller' Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347 Manuscript received April 30, 1990 Accepted for publication August 17, 1990 ABSTRACT The mutant nc4 allele of whirligig (3-54.4) of Drosophila melanogaster fails to complement mutations in an a-tubulin locus, a l t , mutations in a @-tubulin locus,BZt, or a mutation in the haywire locus. cy- or @-tubulin genes.The extragenic failure to However, wrl fails to map to any of the known in structural interactions with microtubule complement could indicate that the wrl product participat.es a deficiency ofwrl proteins. The whirligig locus appears to be haploinsufficient for male fertility. Both and possible loss of function alleles obtained by reverting the failure to complement between wrlnC4 and B2t" are dominant male sterile in a genetic backgroundwild type for tubulin.The dominant male sterility of the revertant allelesis suppressed if the flies are also heterozygous forBZt", for a deficiency of a l t , or for the haync2allele. These results suggest that it is not the absolute level of wrl gene product but its level relative to tubulin or microtubule function thatis important for normal spermatogenesis. The phenotype ofhomozygous wrl mutantssuggeststhat the whirligig product plays a role in postmeiotic spermatid differentiation,possibly in organizing the microtubulesof the sperm flagellar axoneme. Flies homozygous for either wrlnr4or revertant allelesare viable and femalefertile butmale sterile. Premeiotic and meiotic stages of spermatogenesis appear normal. However, in post-meiotic stages, flagellar axonemesshow loss of the accessory microtubule on the B-subfiber of outer doublet microtubules, outer triplet instead of outer doublet microtubules, and missing central pair microtubules.

M

ICROTUBULE arrays show a remarkable degree of structural andfunctional diversity. T h e mitotic spindle and the flagellar axoneme are architecturally quite different and mediate distinct functions in the cell, yet both are constructed from the same major structural proteins, the a- and @-tubulins (PIPERNO,HUANGand LUCK 1977; VALLEEand BLOOM1983). T h e key to this diversity of form and function may lie in the minor proteinsassociated with microtubule arrays. A number of microtubule-associated proteins (MAPs) havebeenidentified by biochemical approaches (reviewed in OLMSTED1986). Some MAPs such as kinesin and dynein are force and SHEETZ 1985; generating molecules (VALE,REESE PORTER andJOHNsoN 1989). Other MAPSsuch as tau and MAP2 appear tobe important for stabilizing and bundling microtubules (LEWISet al. 1989). However, a large number of additional potential MAPs have been identified. For example, the flagellar axoneme has over two hundred protein components in addition and LUCK1977), and to tubulin (PIPERNO,HUANC Drosophila embryo extracts contain 50 different tubulin binding proteins(KELLOGC,FIELDand ALBERTS

' Present address: Department of Developmental Biology, Beckman Center,B300, Stanford University School of Medicine, Stanford, California 94305-5427. Genetics 1 2 6 961-973 (December, 1990)

1989). In most cases, the in vivo functions of these proteins remain uncharacterized. A powerful genetic strategy toidentify components ofcomplex assemblies such as microtubule-based structures is to search for interacting mutations, either second-site suppressors (MORRIS,LAI and OAKLEY and DRUBIN 1989; OAKLEY 1979; ADAMS,BOTSTEIN and OAKLEY 1989), or second-site enhancers (FULLER 1986; DUTCHER, GIBBONS and INWOOD 1988; JAMES et al. 1988; STEARNS and BOTSTEIN1988; HAYSet al. 1989). InDrosophila, several extragenicmutations that fail to complement mutations in B2t, the gene encoding the testis-specific &-tubulin, have been identified (RAFFand FULLER 1984; FULLER1986; RECAN and FULLER 1988;HAYS et al. 1989; FULLERet al. 1989). One of these second-site noncomplementing mutations, nc33, has been shown by molecular analysis to be a missense mutation in a l t (HAYSet al. 1989), the locus encoding the most abundantly expressed atubulin of Drosophila (KALFAYAN and WENSINK 1982; MATTHEWSand KAUFMAN1987; MATTHEWS,MILLER and KAUF'MAN1989). Because a- and P-tubulin interact structurally to form the tubulin heterodimer, this result demonstrates that the isolation of second-site noncomplementing mutations can identify genes encoding interacting structural proteins involved in mi-

L. L. Green et al.

962

crotubule function. Another of the second-site noncomplementing mutations identified a new locus, haywire. The hay gene product appears to be required for several different kinds of microtubule-based processes during spermatogenesis, including meiosis, nuclear shaping, and flagellar elongation (RECAN and FULLER 1988; REGAN and FULLER 1990). For both and haync2,the failure to complement B2t mutations appears to be based on a poison product mechanism. Failure to complementoccurs only when a specific mutant allele of the interacting locus is present. For example, males doubly heterozygous for B2t" and haync2are sterile, but males doubly heterozygous for B2t" and a deletion of hay are fertile (REGAN and FULLER1988). Based on this apparent requirement for the presence of the defective product encoded by the haync2 allele, REGANand FULLER (1990) isolated new alleles of hay by selecting for revertants that restore fertility to the double heterozygousmales. REGANand FULLER(1990)proposed that the revertants that behave as more severe hay alleles could revert the failure to complement by a loss of function mechanism, such that thepoison product encoded by the original hay""2allele can no longer be incorporated into microtubule arrays. In this paper we describe whirligzg, a locus identified by the failure of the mutant nc4 allele to complement tubulin mutations. wrlnc4maps to 3-54.4and has a recessive male sterile phenotype in a wild type tubulin background. At least three new alleles of wrl were isolated as revertants of the failure to complement between wrlnc4and B2t". These mutants have a dominant male sterile phenotype in a genetic background wild type for tubulin or microtubule function. However,thedominant male sterility of therevertant alleles is suppressed by some mutations in B2t, a l t or hay. T h e extragenic failure to complement between wrlnc4and tubulin mutationsplus the ability of tubulin mutations to suppress dominant male sterile alleles of wrl suggest that the wrl gene product interacts with tubulin or microtubules. Phenotypic analysis of mutant alleles of wrl suggests that the wrl gene product is required for the correct architecture of the sperm flagellar axoneme. MATERIALSAND

METHODS

Fly strains and culture: Flies were raised in humidified incubators at 25" on standard cornmeal molasses agar medium. Visible mutations, deletions, transpositions, and balancer chromosomes are described in LINDSLEY and GRELL (1968) and LINDSLEY and ZIMM (1985, 1987).T h e balancer chromosome referred to as T M 3 in this paper carries the dominant visible markers Sb and Ser. T h e balancer chromosome called TM3' in this paper carries Ser but not Sb. D p ( 3 ; 3 ) E I l is a tandem duplication of 88A5-12;88D6-10 (LOCKE, KOTARSKI and TARTOF 1988). D f ( 3 L ) E ( z ) l R 2 (67E3-4;67F1-3) is a y-ray induced revertant of the gain of function mutation E(%)' at 67E (JONES and GELBART1990).

haywire maps close to E(z). Df(3L)E(z)lR2probably deletes a region that includes haywire because it fails to complement mutant alleles of hay (REGANand FULLER1990). Terminology for the tubulin loci follows the conventions agreed on for the new edition of The Genome of Drosophila melanogaster. B2t stands for thetestis-specific /3-tubulin locus at 84D. a l t stands for the a-tubulinlocus at 84B,previously designated tubA84B (MATTHEWS and KAUFMAN1987). B2t" (FULLER1986; FULLERet al. 1988) was isolated as KM24 by K. MATTHEWS. Because B2t" is recessive at 25" but dominant male sterile at 18", it was used to establish a virginizer stock as described in RECANand FULLER(1 990).T h e B2t" chromosomes used in the virginizer stock were marked with either ri and e , or ri red cv-c and jvl. B2t" virgin females used in crosses were a mixed population of B2t" homozygotes and B2tn/TM3 heterozygotes. nc4, the original mutant allele of wrl, was isolated after ethyl methanesulfonate (EMS) mutagenesis of a Ki p p chromosome in a screen for mutations that failed to complement Df(3R)Antpl7 (LEWISet al. 1980), and was originally designated EcRms4. T h e failure of wrlnc4 to complement Df(3R)Antpl7 presumably results from deletion of alt (see Table 1). wrlnc4subsequently was found to fail to complement mutations in B2t. A by wrlnr4chromosome generated by recombination was used in all experimentspresented in this paper, except where stated otherwise. Portions of the by wrlnr4chromosome on eitherside ofwrlnC4 were replaced by recombination with either ru h th st p p cu or sr ca. ru h th st p p cu wrl""'/by wrlnr4 sr ea males, homozygous only for wrlnr4 andthe intervalbetween cu and sr, were indistinguishablefrom homozygous by wrlnr4 males in fertility tests or in testis phenotype when examined by phase contrast microsco Generation of the synthetic Df(3R)wrl: Tp(3;2)ry+"PX. " ' is a transposition of 87C2-3;88C2-3 from the third chromosome to theleft arm of the second chromosome.Tp(3; l)kar" is a transposition of 87C7-D1;88E2-3 from the third chromosome to 20 on the first chromosome. T o create a synthetic deficiency for 88C2;88E3 (see Figure l), the duplication segregant of Tp(3;2)r~+"'"~' and the deficiency segregant of Tp(3;l)kar5'were brought together in the same fly by performing the following crosses. Tp(3;2)ry+"7u'" males were crossed to Sp/SM5; D ri p p B2t" e/TM3' virgin females, and a Dp(3;2)ry+"7"h'/Sp; Df(3R)ry+w7"h'/TM3' stock was established. T h e transposed segment of 3R is within an inversion on the second chromosome that covers Sp, and therefore acts as a balancer for that region of the second chromosome. Dp(3;2)ryf"70h'/Sp; Df (3R)ry+"7"h'/TM3' males were crossed to Sp/SMS; D ri p p B2t" e/TM3' virgin females. Resulting virgin Dp(3;2)ry+"70h'/SM5; D ri p p B2t" eITM3' females were mated to Tp(3;l)kar5'/MKRSmales. From the progeny of this cross, males carrying thesynthetic deficiency in genetic a background wild type for tubulin (+/K DP(3;2)r~+"'~"~'/+; Df(3R)kar51/TM3'), and males carrying the synthetic deficiency in heterozygous combination with B2t" (+/Y;D p ( 3 ; 2 ) r ~ + " ~ ~ ) ~ ' / + ; D f ( j R ) k a r Sril /pDp B2t" e ) were tested for fertility in individual mating tests. Sibling males were tested as controls (see Table 4).TOtest complementation of wrlnr4and Df(3R)wrl, +/E Dp(3;2)ry+"7"h'/+; Df(3R)kar51/D by wrlnc4males were generated by a scheme Sp/SM5; Dby wrlnr4/TM3' identical to that above except that virgin females were used in the place of Sp/SM5; D ri P p B2t" e/TM3' females. Selection of revertants: by wrlnr4/TM3males were mutagenized by feeding on a 2% sucrose solution containing 25 mM EMS as outlined in LEWISand BACHER(1968). Mutagenized males were mated to ri B2t" e virgin females and F1 B2t" +/+ wrlnc4males were testedfor fertility by mass mating

963

wrl, Microtubules and the Axoneme about 20 males to 25 B2t" virgin females per vial. If a new mutation reverted the failure to complement between wrlnC4 and BZt", the resulting F2 B2t" wrlnr4*maleswere retested for fertility by mating to B2tn/TM3 virgin females. Stocks balanced for the third chromosome were obtained male and virgin by mating the resulting F3 by wrlnC4'"/TM3 femalesiblings. T o recover dominant male sterile revertants, balanced stocks wereestablished by mating by wrlnr4"'/ TM3 virgin females to dsxMa'/TM3 males.The only fertile progeny of this crossare by w ~ l " ' ~ ~ / T M females 3 and dsxiZiac/ TM3 males, thereby regeneratingthe starting cross. T o confirm that theputative revertant was not due to acontaminating wild type fly, by wrlnC4"/TM3 virgin females were males. In all cases the presence of crossed to by wrlnC4/TM3 the mutagenized by wrlnc4chromosome was indicated because by was visible in the appropriate progeny class. Recombinationmapping and complementationtests: To map the original wrlnr4 mutation,by wrlnC4/redcu-c sbd females were mated to red m-c sbd e males. Individual male progeny carrying a thirdchromosome recombinant between the visible markers were picked and used to establish balanced stocks over TM3. T o follow segregation of the recessive male sterility associated with wrlnr4,male homozygotes from each recombinant stock were tested for fertility. To follow segregation of the failure of wrlnc4to complement BZt", recombinantlTM3 males were crossed to ri B2t" e females and F, recombinantlri B2t" e males were tested for fertility. The revertants ~ r l ~ ~ and ~ ~~ ~ r ,were l mapped by meiotic recombination with a thirdchromosome carrying the visible markers ru h th st cu sr e ca. wrlnr4"'Iwas also mapped by recombination with a chromosome carrying ru h th st p p cu sr e. Single recombinants that fell between cu (3-50.0) and sr (3-62.0) were picked and balanced stocks were established. Males were tested for cosegregation of three effects of the mutation: reversion of the failure to complement BZt", dominant male sterility in a genetic background wild type for tubulin, and failure to complement wrlnc4.Complementation with wrlnc4was assayed by cytological examination by phase contrast microscopy of developing spermatids in squashed testes. For recombination analysis, fertility of males was assayed by isolating males from females forat leastsevendays, dissecting and opening the seminal vesicles, and looking for motile sperm. For complementation tests, male fertility was assayed by mating as follows: test class males and at least ten control class sibling males (test chromosome over balancer) were mated individually with three B2t" virgin females per vial. Fertility was scored after seven to ten days. Genotypes were scored as sterile if no progeny were produced, and fertile if larvae or pupae were evident. The level of fertility was scored by estimating whether there were greater than or less than 20 pupae in the vial. Males were scored as fertile if they produced greater than 20 progeny or weakly fertile if they produced between 1to 20 progeny. Fertility of Wrlnc4mI/+ males was assayed by mass mating tests in which 20 males were mated with 25 virgin females per vial. AIthough mostvials contained no progeny, progeny were occasionally produced. If any progeny did arise, then one fertile male was assumed to have been in the vial. This procedure couldslightly underestimate the frequency of fertile males. Individual female fertility tests were performed by mating singlevirginfemaleswith three red e males per vial, and were scored as for individual male fertility tests. Cytology and electron microscopy: The stages of spermatogenesis were studied in squashes of unfixed testes examined under phase contrast light microscopy (KEMPHIJES

+/+

TABLE 1 Complementationbetween wrP' and mutant alleles of B2t or other loci important for microtubule function Allele

wr1"-4

+

F S S

wrl"'' B2t" ~ 2 t B2t" ~2t' B2tY B2t I" B2t"

s" F Fb F

s" S

Df(3R)aIt' haynr2

Df(3L)hayd

S S F

Male fertility was tested by individual male mating tests as described in MATERIALS AND METHODS. F, Number of progeny i n individual mating tests comparable to mutant/+ sibling controls. S, Males tested by individual mating produced no progeny. a About 10% of the males doubly heterozygous for wrl"" and B2t'or B2t" produced a few (lessthan 20) progeny. These numbers of progeny were reduced greatly compared to thenumberof progeny produced by sibling controls. Test males had slightly reduced fertility relative to controls. ' Df(3R)aItwas Df(3R)A41. was~Df(3L)E(z)lR2. ~ " Df(3L)hay ~ ~ ~

et al. 1980). Nuclear shaping and alignment during spermatogenesiswere studied by light microscopyof orceinstained fixed testes (LIFSCHYTZ and HAREVEN1977). Preparation of testes for electron microscopy was performed as et al. 1987. in FULLER For serial sections of testes, 400-500 serial sections (100 nm thick) were cut and picked up on 1 X 2-mm slot grids coated with 0.5% Formvar. Sections were selected for examination at 5-pm intervals (every 50 sections). Cysts containing axonemes in cross-section were identified, and individual axonemes were tracked and photographed at a magnification of 9OOOX. Tracking individual axonemes for distances greater than 40 pmwas impossiblebecauseof curvature of the testes.Within the 40 pm span it was sometimes necessary to tilt the grid up to 30" using a goniometer stage in order to achieve good cross-sectional alignment of the axonemes. Three axonemes containing outer triplet microtubules remained in good cross-section and were followed for this study. RESULTS

A recessive male sterileallele of whirligig fails to complement mutationsin several loci important for microtubule function: In a genetic background wild type for tubulin, wrlnc4 was recessive malesterile (Table 1). However, males doubly heterozygous for wrlnc4and B2t" were sterile (Table 1). wrlnc4failed t o complement B2t" in cis as well as in trans, consistent with mutations in two separate genes. Males heterozygous for a chromosome carrying B2t" a n d wrlnc4in cis and wild type (B2t" wrl""/++) produced no progeny in individual male mating tests (Table 2). T h e failure to complement betweenwrlnc4and mutations in B2t is allele specific. Males heterozygous for wrlnc4a n d B2t3, an allele that makes an unstable p2-

L. L. Green et al.

964 TABLE 2

Wrl

The failure to complement betweenB2t" and w r P occurs both in cis and trans, and is partially suppressed by a duplication of the wrl region

54.420.2

red cv-c

jvl

53.6 54.1 Genotype

Male fertility"

B2t" ~ r l " " / + + ~ B2t" w r P 4 / + Dp(3;3)EIl

0/350 12/170'

I

I

,

sbd

I

I

56.7

58.2 I

* Data are presented as number of fertile males o u t of total number of males tested by individual male mating tests as described in MATERIALS AND METHODS. Data are included for three different wild-type chromosomes, TM2, TM3 and ru h th st Pp cu sr ca. ' Of the 12 fertile males, one produced greater than 20 progeny, while the other 1 1 produced less than 20 progeny.

tubulin (KEMPHUESet al. 1982; KEMPHUES,RAFFand KAUFMAN1983), are almost completely sterile (Table 1). However, wrZnc4complements the class I1 B2t6, B2t7, B2t9 alleles but fails to complement the class I1 B2t'" and B2t" alleles (Table 1). T h e class I1 B2t alleles encode stable subunits that affect different aspects of microtubule function during spermatogenesis and thus appearto encode partially functional p2tubulin (RAFF and FULLER 1984; FULLER 1986; FULLERet a2. 1987,1988). As suggested for haync2 (REGANand FULLER1988), the allele specificity of the failure to complement may reflect functional differences among the &tubulins encoded by the different class I1 B2t alleles. wrEnC4acted as a dominant enhancer of mutations in at least two other loci important for microtubule function in Drosophila. Males heterozygous for wrlnc4 and a deletion of cult were also sterile (Table 1). wrZnC4 also failed to complement haync2(Table l), another mutation that acts as a dominant enhancerof tubulin mutants (REGANand FULLER1988). However, wrlnc4 complemented a deficiency for hay, suggesting that the failure to complement in this case requires the presence of the defective product encoded by hay"". Although wrlnC4failed to complement mutations in B2t, alt,or hay, it does not map to any of these loci. Because the recessive male sterility of wrlnC4and the failure to complement B2t" cosegregated in 74 recombinants between the visible markers cv-c (3-54.1)and sbd (3-58.2)(Figure l ) , the failure to complement B2t" and therecessive male sterility are likely to result from the same genetic lesion. T h e recombination analysis placed wrlnC4at 3-54.4 f 0.2 map units (m.u.), just distal to cv-c, and far from haywire (3-34.4), a l t (347.7) and B2t (3-48.5). The whirligig region is haploinsufficient for male fertility: Because Df(3R)red31/wrlnC4males were fertile, the deficiency does not uncover wrlnC4(Figure 1). However, whirligig is likely to lie near the distal breakpoint of Df(3R)red31 (87F12-14;88(31-3), in polytene interval 88C-D, based on the observations that Df(3R)redJl uncovered cv-c and wrlnC4mapped only

FIGURE1.-Genetic and cytogenetic map of whirligig. The genetic and cytological positions of red, m - ~jvl, , and sbd are derived from LINDSLEY and GRELL( I 968) and LINDSLEY and ZIMM (1985, 1987). Top: Recombination map showing the positions of wrl and nearby recessive visible markers in map units. Middle: Cytological map showing the positions of wrl and nearby recessive visible markers. Bottom: Cytological localization of wrl is indicated by vertical dashed lines. Deficiencies and duplications used to define the cytological position of wrl are indicated with open and closed boxes, respectively. Uncertainties in breakpoint locations are indicated by cross-hatching. Dp(3;2)ry+ is the duplication segregant from Tp(3;2)~y+"~"' and Df(3R)kar51 is the deficiency segregant from Tp(3;I)kar5'.Df(wrl) is a synthetic deficiency generated using Dp(3;2)ry+and Df(jR)kar51as described in the MATERIALS AND METHODS.

0.3 m.u. distal to cv-c. A synthetic deficiency of 886288E3, generated with the deficiency segregant from Tp(3;l)Rar5'(87C7-D1;88E2-3) andthe duplication segregant from Tp(3;2)ryf"70hr (8'762-3;8862-3)(see Figure 1, and MATERIALS AND METHODS), uncovered males the recessive wrlnc4 phenotype.Testesfrom heterozygous for this synthetic deficiency and wrlnC4 had defects in axoneme organizationidentical to those seen in wrlnc4homozygotes when examined by electron microscopy (see below), and were sterile (Table 3). As described below, heterozygotes for thesynthetic deficiency and wild type also have a male sterile phenotype. However, the testis phenotype of Of/+ was distinguishable from the homozygous wrlnC4phenotype by both light and electron microscopy (see below). These results indicate thatthe synthetic deficiency uncovers w r l . For the remainder of this study, the synthetic deficiency of 88(32;88E3 will be designated Df(3R)wrl. The chromosomal region deletedby Df(3R)wrl was haploinsufficient for male fertility. Males heterozygous for Df(3R)wrE and wild type were nearly sterile in individual male mating tests (Table 4). The seminal vesicles of Df(3R)wrl/+ males contained only a few motile sperm. Surprisingly, B2t" +/+ D f ( 3 R ) w d males possessed nearly wild type levels of fertility as compared to control class males (Table 4). Thus, B2t" acts

the

wrl,and Microtubules

965

Axoneme

TABLE 3

TABLE 4

Complementation betweenalleles of whirligig and wild-type or mutant alleles of loci important for microtubule function

The w d region is haploinsufficient for male fertility in a genetic background wild type for tubulin, but the male sterility is suppressed by B2t"

whirligig allele

Df(3R)wrl wrl"""'' W r , y 4 ~ 2

wrl"'4"'

+

wrl""

S'

Sd

ND

S ' S S ' S S ' S

B2t"

F ND F F F

Df(3R)alP hay""

F F N FD

F F

S

as a dominant suppressor of the haploinsufficiency for male fertility associated with Df(3R)wrl. T h e fertility of B2t" Df(3R)wrlmales is striking relative to the sterility of B2t" wrlnc4males (Tables 1 us. 3). Thus, the failure to complement B2t" requires the mutant nc4 allele of wrl, consistent with a poison product mechanism. Reversion of the failure to complement between wrZne4and B2t" results in dominantmale sterile alleles of wrZ: Because the failure of wrlnc4to complement B2t" appearstorequirethepresence of the defective wrlnc4gene product, revertants of the interaction could yield new alleles of wrl that no longer encode a poison product. These revertants could be new mutations in wrlnc4that either restore wild type function to the aberrant gene product, lower the level of the poison product expressed, or destroy the poisoning ability of the defective protein. Alternatively, the revertantsmight be extragenic suppressors of the failure to complement between wrlnc4and B2t". Eleven revertants of the failure to complement between wrlnc4and B2t" were obtained from l 1,000 test males following mutagenesis with EMS (see MATERIALS ANDMETHODS). The frequency of revertants induced with EMS was comparable to that obtained for revertants of haync2in a similar screen by RECAN and FULLER( 1 990). The revertants of the failure to complement between wrlnc4and B2t" fell into two classes, based on the fertility of males heterozygous for the revertant but wild type for tubulin loci. Six of the EMS-induced revertants had a recessive male sterile phenotype in a geneticbackground wild type fortubulin loci.Al-

+/+

++ B2t" +

WF

ND

SD'

(No.tested)'

+ Df(3R)wrl

ND

Male fertility was tested by individual male mating tests as described in MATERIALS A N D METHODS. F, Number of progeny in individual mating tests comparable to mutant/+ sibling controls. WF, Weak fertility. Twelve out of 52males produced less than 20 progeny in single male mating tests. The rest failed to produce progeny. S , Fifty males tested by individual mating produced no progeny. N D , not done. a Df(3R)otZt was Df(3R)A41. ' Df(3L)hay was Df(3L)E(z)ZR2. Extremely low fertility (less than ten progeny per male). See Table 4 for numbers. Males were sterile, and their axonemes had defects similar to that of wrl"" homozygotes when viewed in the electron microscope. 'Six out of 625 by ~ r l " ' ~ " ' / T M 3males produced less than 20 progeny per vial in mass mating tests of 20 males with 25 ri B2t" e virgin females. Data for wrl"""' and wrl"""' were similar to those for wrl"'""'.

+/+

Avg. No. progeny/rnale 2

Df(3L)hay'

* 7 (59) 41 * 13 (54) 4

72

++ + 14 (20)'

69 + 9

a Average number of progeny produced in individual male matings with three 8 2 1 virgin females. Individual male mating tests were performed as outlined in MATERIALS AND METHODS. Total number of males tested. wasten +/SM5; TM3'IMKRS and ten Dp(3;2)ryfu'70h'/ TM3'IMKR.Y males. B2t" +/++ was +/SM5; D ri Pp B2t" e/MKRS.

+;

++/++

though these revertants were fertile as double heterozygotes with B2tn, the fertility of the double heterozygotes was less than wild type. Based on preliminary mapping experiments, at least two of these recessive male sterile revertants appear to be extragenic towrl and therefore could be extragenic suppressors of the failure to complement between wrlnc4and B2t" (L. L. GREENand M. T . FULLER, unpublished results). We are continuing mapping experiments to determine if the other four revertants are intragenic or extragenic. Five of the EMS-induced revertants had a dominant male sterile phenotype in a genetic background wild type for tubulin. Males heterozygous for these revertants and wild type usually failed to pass sperm to the seminal vesicle, although about onein 50 males had a few motile sperm in the seminal vesicle, and about one in 100 males produced progeny (less than 20) in individual male mating tests (Table 3). These revertants were fully fertile as double heterozygotes with B2t" (Table 3). To determine if the dominant male sterile revertants behaved genetically as new alleles of wrl, three were tested for cosegregation of the dominant male sterility, the reversion of the failure to complement B2tn, and the recessive wrlnr4phenotype (see MATERIALS AND METHODS). Two sets of recombinants were picked for each revertant, one consisting of recombinants in which all of the left arm of the third chromosome plus the portion of the right arm proximal to and including cu (3-50.0) had been replaced, and the other consisting of recombinants inwhich the portion of the right arm of the third chromosome distal to and including sr (3-62.0) had been replaced. For the three revertants tested, the three phenotypes cosegregated (Table 5), indicating that the dominant male sterility, the reversion of the failure to complement, and wrl are all tightly linked. The recombination data are consistent with the hypothesis that the dominant male sterile revertants are new alleles of wrl, and they have been named wrlnC4"',~ r l " " and ~"~ Wr1nc4"3.

966

L. L. Green et al. TABLE 5

TABLE 6

The three revertants tested map to the wrl interval and cosegregate withw r F in recombination tests

A duplication in the wrl region suppressesthe dominant male sterility of and wPrvz

Phenotypes

rulwrl"" sterile" ru/+ sterile Revertant and

rulb ru2 ru3

7(F) 4(F)

mlwrl"" fertileand ru/+fertile

No. o f recombinants

6 4 4

14 11

8

Data presented as number of recombinants showing the indicated phenotype. Reciprocal recombinants were tested for each interval. (F) All recombinants that were sterile when heterozygous with wrlnr4 were fertile when heterozygous with B2t". Therefore, the revertant cosegregated with wrlnC4. a Because these revertants are dominant male sterile in a genetic background wild type for tubulin, complementation with w r y ' was tested by examining testes squashes under phase contrast microscopy for thehomozygousw r y 4phenotype. The phenotype ofwrP4/ w r y 4 in testis squashes was distinct from that of wrlnC4"/+. " rvl carried an accessory mutation that was lethal with portions of the left arm of the ru h th st cu sr e ca chromosome used in this mapping. Therefore, for r u l , 3L and the portion of 3R proximal to wrl were replaced with the ru h th st Pp cu region from the multiply marked chromosome, ru h th st Pp cu sr e. That portion of the rul chromosome distal to wrl was replaced with the sr e ca region of the ru h th st cu sr e ca chromosome as done for ru2 and ru3.

+b

Dp(j;j)E11

+"

wrl"""l

ND

S (1/20)'

wr1"'f"2

S (0/24)

(20/20) (43/43) F(48/54)d FF

D p ( 3 ; 3 ) E l l / T M 2males were crossed to either by wrl"""' sr e cal ~ " ~virgin females. Fertility of male T M 3 or by ~ r l sr~ e ~calTM3' progeny was scored by individual male mating tests and by inspection of seminal vesicles for motile sperm. Results of both tests were in agreement. Numbers in parentheses are the number of fertile malesltotal number of males tested in individual mating tests. F, Fertile; S, sterile; ND, not done. The wild-type chromosome was either T M 3 or T M 3 ' . ' The wild-type chromosome was T M 2 . ' One male produced fewer than 20 progeny. Fertility was slightly reduced compared to Dp(3;3)E11/+ control. Six of the 54 males tested had no progeny, and nine had less than 20 progeny. For the Dp(3;3)El I / + control, six of the 20 males had less than 20 progeny.

TABLE 7 Homozygotes for wrlnC4, wrln4"' or wrlndNZare viable and female fertile GenotvDe

Viabilitv" fertilitvb Female

Reversion of the failure to complement B2t" also resulted in the reversion of the failure of wrlnc4to complement other mutant loci important for micro' Data presented as number oftestclass progeny over total number of progeny recovered. tubulefunction(compareTables1 us. 3).wrlnc4"", ' Female fertility was scored by individual female mating tests. Wrlnc4~2 and ~ r were fertile l as ~ double ~ heterozy~ ~ Data are ~ presented as number of fertile females out of the total gotes with a deficiency for alt. Both ~ r and l number ~ tested. ~ ~ ~ ~ Wrlnc4rv2 ' ru h th st Pp cu wrl""lTM3 males were crossed to by w r y ' s r ca/ were fertileasdoubleheterozygotes with T M 3 virgin females to generate test progeny. hayncz (Table 3). Males doublyheterozygousfor Because T M 3 / T M 3 is lethal, 33% of the progeny are expected Wrlnc4m2 to be homozygous for wrl"". and a deletion of haywire were sterile. Howe ru h thst Pp cu wrlnC4"'/riB2t" e males were crossed to by wrl"""' ever, males heterozygous for both~ r and a ldele- ~ ~ ~ ~ ~ sr e c a l T M 3 virgin females to generate test progeny. tion of haywire were weakly fertile (Table 3). ru h th st cu ~ r calri B2t" l red~ m-c jul ~ males~ were crossed ~ ~to by ~ r l " ' ~ 'sr" ~e calTM3' virgin females to generate the test progeny. The dominant male sterile revertants may act as loss of function mutations: The similarity in genetic wrl in the polytene interval 88C2-Dl0 (Figure 1). behavior between thedominant male sterilereverThe ability of wrlnc4to act as a dominant enhancer tants and Df(3R)wrl suggests that the revertants are of tubulin mutationsis probably based on antimorphic loss offunction alleles ofwrl. Like Df(3R)wrl, ~ r l ~ rather ~ ~ ~ ~ , thanneomorphic propertiesof the mutant gene Wrlnc4m2 and ~ r l are " male ~ ~sterile ~ when ~ heterozyproduct. Dp(3;3)ElI, which apparently carries two gous with wild type. Also like Df(3R)wrl, in all three wild type copies of wrl, partially suppressed the failure cases the dominant male sterility is suppressed by B2t" to complement between B2t" and wrl"". Although (Table 3). B2t" wrl""/++ males failed to produce any progeny If the dominant male sterile revertants are loss of in single male mating tests, some B2t" wrlnc4/+ function alleles of wrl, then the dominant male sterility Dp(3;3)EII males did produce progeny (Table2). should be suppressed by a duplicationof the wild type Mutations in wrl cause defectsin the organization allele. A tandem duplication of the wrl region, of axonemal microtubules:whirligzg function appears D p ( 3 ; 3 ) E I I (88A5-12;88D6-10)(Figure1; see also to be required only for post-meiotic stages of sperLOCKE,KOTARSKIand TARTOF 1988), was tested for matid differentiation. Homozygotes for the original ability to rescue the dominantmale sterility of ~ r l ~ ~ ~ loss of~ function alleles, allele, wrlnc4, or~ two probable Wrln~4mIor ~ r l were and ~ r lMales ~ heterozygous ~ ~ ~ for ~ D, p ( 3 ; 3 ) E lI and ~ viable ~ ~ and~ female ~ , fertile wr1""4"l or ~ 7 - l were " ~ fertile ~ ~ ~(Table 6), consistent (Table 7). Spermatogenesisup toand including with the hypothesis that the dominantmale sterility is meiosis appears to be normal in mutant males. Premeiotic spermatocytes and onion stage early spermadue to loss of function mutations. Together with the tids appeared wild type by phase contrast microscopy deficiency analysis, the results with D p ( 3 ; 3 ) E lI place f

wrl, Microtubules Axoneme and the

967

I

.

.

FIGURE 2.-wrlnf'"" causes defects i n l1;~gcIlar org:lni/atioIl w i 1 h i l l c y \ 01' d v \ ( ~ l o l ) i ~~~~g) c m ~ a t i(a) d s .Phase contrast micrograph of >I squashed testis from a wild type (red c ) male. I~hgellaare organized i n long parallel arrays w i t h i n cysts (arrowheads). Rar = 20 pm. (b) Phase sr e ca) male showing disorganization contr;Ist micrograph of a squashed testis from a homozygous wrl"""" (ru h th st pf' ru r~~r/"fJn"/u~rl'r''ul of flagella within ;I cyst. Regions of disorganized flagella (arrows) and apparently normally organized flagella (arrowheads) often were found within the same cyst. Bar = 50 pnl. (c) Orcein stained testis from ;I wild type (red e ) male. Nuclei in a spermatid bundle are well-shaped and aligned (arrows). Bar = 2 0 pm. (d) Orcein stained testis from a homozygous urrlnrJ""(ru h th st pp ru wrl"r'"''/wrl"'4"" sr e ca) male showing ;Ipparently wild type nuclear shaping (arrow) but poor nuclear alignment. Bar = 20 pm. (e) Phase contrast micrograph of a testis from by wr[n'Jn'l /+ (+ = T.143) male showing vacuoles within a cyst of elongated spermatids (arrows). Flagellar elongation proceeded far, and many cysts lacked vacuolesat the time of examination (arrowheads). Bar = 2 0

pm.

testis of squashes from ~ 7 - l or~ ~ ~ ~ 'homozyarrowheads). Because elongated nuclei were observed in wrlnr4"' (Figure 2 4 , ~ 7 - l and ~ ~ wrlnr4 ~ ~ 'homozygotes and heterozygotes (data not shown), or in wrlnc4 gotes (data not shown), the perinuclear microtubules homozygotes (FULLER 1986). implicated in nuclear shaping probably function norPost-meiotic stages of spermatogenesis have similar mallyin the mutants. However, compared with wild defects in males homozygous for wrlnr4,wrlnr4"' or wrlnr4n!2 . For example, bundles of flagella in ~ r l ~ type ~ ~(Figure ~ ' 2c), nuclear alignment was poor in Wrlnc4rnI homozygotes were usually disarrayed in some or all homozygous males (Figure 2 4 and in homoregions of an elongating cyst (Figure 2b, arrow), alzygous ~ r l " ' ~and " ~ wrlnc4males (data not shown). In though elongating flagella were organized in parallel males heterozygous for the dominant male sterile linear bundles in some portions (Figure 2b, arrowallele, wrlnr4"", and wild type, flagellar elongation head. For comparison withwild type, see Figure 2a appeared normal, and the flagella within a cyst were

968

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Green et al.

FIGURE J.-Males homozygous for mutant alleles of wrl have defects in organization of the rnicrotul,ulcs of the axoneme. Electron micrographs of axonemes i n elongating spermatids. Bar = 200 nm. (A series) wild type (red e ) males: Formation of the accessory microtubule first begins as a small hook on the B-subfiber (a', arrowhead). Completion of the accessory nlicrotubule occurs in a developmental gradient wound the axoneme. The accessory microtubules most removed from the mitochondrial derivatives (a, arrowhead) complete development prior to the accessory microtubules closer to the mitochondrial derivatives (a. arrow). Densely staining material begins to form around the more developed accessory microtubules (a. arrowhead). Finally, all ofthe accessory microtubules are formed and the densely staining material condenses to form a decoration between the accessory microtubule and the B-subfiber and also a decoration on the edge of the accessory microtubule away from the B-subfiber (Sa", arrows). Note that the increase insize and staining of the crystalline bodyin the major mitochondrial derivative is a useful marker for axoneme development (compare a' us. a, small arrows). (b Series) Homozygous wrl"""" (TU h d sf pp cu W Y ~ " ~ ' " ' ' / W Y ~ " sr ~ ~ 'e" ca): ~ note that the figures are printed such that the orientation of the axonemes relative to those in the "a" series is reversed. (b' and b") Early axonemes at a stage of development corresponding to that shown in a'. Forming accessory microtubule hooks should be evident on the B-subfiber by this stage, but are absent in the mutant. Although a small projection can be seen coming from the juncture of the A- and B-subfiber (b' and b", arrowheads), these projections were in the wrong position for the nascent accessory microtubule. and were probably an early stage in the formation of the densely staining decorations that appear on mature outer doublets. An apparent cytoplasmic microtubule has invaded the axonemal space (b', arrow). Note that the axoneme in b' lacks the central pair microtubules. (b) An older axoneme at the same stage as that in panel a (compare crystalline bodies, arrows). At this stage the accessory microtubules should be obvious. Note that densely staining material (b, b', b", arrowheads) aggregates at the junction of the A- and Bsubfibers in the mutant even in the absence of the accessory microtubule. (C series) Homozygous ~ r l " " " '(TU ~ h fh st cu ~ r l " ' ~ ~ ' ~ / sr ~ re l ~ ' ~ ' " ~ ca): Triplet microtubules (arrowheads) are interspersed with doublet microtubules in the outer ring. A lone apparent accessory microtubule is attached to a B-subfiber (c, small arrow). Note that the axonemes in panels c and c' are mounted in reverse orientation to panels c" and c"'. Axoneme orientation in panels c and c' is the same as that in the "b" series. Axoneme orientation in panels c" and c"' is the same as that in the panel a series.

organized in parallel arrays (Figure 2e, arrowheads). However, cysts of late elongated spermatids developed vacuoles (Figure2e,arrows), consistent with degeneration.Heterozygotesfor either w ~ Z " ~ ~ ' " ~or Df (3R)wrZand wild type appeared similar to W ~ Z " ~ ~ ' " ~ / + heterozygotes (data not shown). Nuclear shaping and alignmentappeared wild type in wrZnc4""/+, ~ r l " ~ ~ ' "or~ Df(3R)wrZ/+ /+, males (data not shown). Males homozygous for mutationsin wrZ have defects in the structure of the spermatid flagellar axoneme. A detailed description of the development and organization of the outer microtubules of the axoneme in wild type is given in the legend to Figure 3a. Briefly, in wild type Drosophila males, the flagellar axonemes of sperm show the standard pattern of a central pair surrounded by 9 outer doublet microtubules. An accessory microtubule is attached to the B-subfiber of each outer doublet (Figure 3a, see also KIEFER 1970; TATES 1971; LINDSLEY and TOKUYASU 1980). T h e accessory microtubules are presumed to extend from the C-subfiber of the outertriplet microtubulesof the

basal body (KIEFER 1970; WARNER 1971). In wrZnr4, or W ~ Z " ~ ~ ' homozygotes, "~ the accessory microwrlncQN* tubules were almost always missing. Comparison of the axonemes from w ~ Z ~ ~ homozygotes ~ ' " ~ (Figure 3b' and 3b") with age matched wild type axonemes (Figure 3, a' and a") shows that young axonemes from the mutants lack the hooks of the nascent accessory microtubules. Although by the stage shown in Figure 3aaccessory microtubules inwild type are readily apparent, accessory microtubules were missing from more developed axonemes in the mutants (Figure3b). Occasionally, a microtubule was seen near one of the outer doubletmicrotubules in amutantaxoneme (Figure 3b', large arrow; also Figure 4). However, these probably were cytoplasmic microtubules that had invaded the axonemal region rather than bona fide accessory microtubules, because in mostcases such lone microtubules lacked the normal connections to thenearby outer doublet. Although axonemes were defective in wrZnr4"' homozygotes, the cytoplasmic microtubules surrounding the mitochondrial deriva-

wrl, Microtubules and the Axonerne

I.-Outer triplct nlicrotulmles in the :lsollclncs of honlozygotesextend for a t lexst 30 pnl.Outer triplet nlicrotubules in indivitlual asonenles were followed in sets of seri;ll sections o f testes from ru h th st cu wrl"J""/ud"J"'' sr e ra nl;lles. l'hc nlicrogl-;tphs show a representative m o n e n l e a s it progresses way from the hwal body.Note that acytoplasmicmicrotubule aplxlrentlyinv;lded the axonen1;ll region in the interval between p m e l s B and 1). then aplxtrently departed in the interval between panels c and d. (a) Sertion designated as 0 pm. (b) Section 10 pm ; I H ~from t h t in panel a.(c)Section 20 p m awav from that in Ixlnel a. (tl) Section 30 pnl away from that in panel a . Bar = 200 11111. I'l(il'RE

U'T,ll?.ln.Z

tive in elongating spermatids were present and morphologically normal in the mutant (compare Figure 3, b us. a). Males homozygous for wrlnr4"', or wrlnr4 also exhibited other defects in organization of the axoneme (Table 8). Defects include frequent lack of the central pair microtubules (compare Figure 3, b' us. b"),lackof one or more of the outer doublet microtubules, or fragmented axonemes. The number of recognizable axonemes or axoneme fragments per cross-section of a cyst was greatly below the wild type number of 64 (Table 8). Males homozygous for Wrlnr4~l , wrl n r 4 n d or wrlnr4also often had cytoplasmic inclusions in the spermatid mitochondrial derivative, a sign of poor flagellar elongation (Table 8). Despite the sterility of males heterozygous for wild type and W T ~ " ' ~ ~~" , r lor ~Df(3R)wrl, ~ ~ these ~ ' geno~ types showedno obvious consistent defect in the structure or organization of the axoneme. The number of axonemes per cystwas near the wild type number, and most axonemes looked morphologically normal. However, manycystshad a few axonemes with defects, including fragmented axonemes, missing accessory microtubules, and missing central pair microtubules (data not shown). wrlnr4may be semidominant at the ultrastructural

969

level when viewed by electron microscopy. The number of axonemes per cyst cross-section in testes from wrlnr4/+males was below that of wild type (Table 8). However, most of the axonemes observed appeared to be morphologically normal, although a fewcysts hadmany fragmented or unorganized axonemes (Table 8). Axonemes from males homozygous for W ~ I " ~ ~ " ~ often have outer triplet microtubules: I n males homozygous for ~ r l " ' ~outer ~ ' ~ triplet , microtubules were found interspersed with outer doublet microtubules in the axoneme (Figure 3c series, arrowheads). In six whole cysts examined, 2 0 4 0 % of the recognizable axonemes had at least one set of outer triplet microtubules. The number of outer triplets in an axoneme was variable (compare Figure 3, c us. c'). The positions of the outer triplet microtubules relative to eitherthe mitochondrial derivatives or the other sets of outer microtubules in the axoneme were apparently random (compare Figure 3, c, c' and c"). Outer triplet microtubules were observed in both whole (Figure 3c) and fragmented axonemes (Figure 3c'), and in axonemes both with and without central pair microtubules (data not shown). All of the outer triplet microtubules found in the axonemes of homozygousW T ~males ~ extended ~ ~ ~ for ' ~ at least 30 pm along the axoneme (Figure 4). Individual axonemes within a cyst were followed by examining serial cross-sections by electron microscopy. The outer triplet microtubules that were followed did not change to doublets, nor did any other doublet microtubules become outer triplets. DISCUSSION

Extragenic failure to complement implicates the product of the whirligig locus in microtubule func~ fails to complement mutations tion: The w ~ 1 " 'allele in the tubulin genes, B2t and a l t , and in haywirp, three loci important for microtubule function during spermatogenesis in Drosophila. Formally, wrl"" acts as a dominant enhancer of these mutations. The enhancing wrl"" allele appears to encode an antimorphic gene product. First, the failure to complement between wrZnr4and B2tn appears to require thepresence of the gene product encoded by the nc4 allele (Tables 1 and 3). Second, a duplication apparently carrying two wild type copies of wrl can partially rescue the Failure of wrlnC4to complement R2t" (Table 2), consistent with the prediction for an antimorphic gene product (MULLER 1932). One possible explanation for the failure to complement between w ~ l " and ' ~ tubulin mutants is based on structural interactions between the products of the respective genes. The defective protein encoded by ~ r l " could '~ act as a structural poison if incorporated into microtubule arrays. A similarmodel has been

L. L. Green et al.

970

TABLE 8 Males homozygousfor mutations in wrl show several defectsin the structureof the axoneme Avg. No. of whole or partial axonenles'

Genotype

~rl"'~/wrl"''' wrl"'4"i/wrl"'J"ld wrl"'4"?

w c 4 n j 2 t

wrl"'"/+/ red elred er

Range % fragmentedb

Range % missing Range % missing accessory microtubules mitochondrial central pair derivatives

21 f 9 (14) 39 ? 13 (5)

7-59 (14) 11-73 (5)

100 (14)

16 f 11 (33) 53 f 19 (58) 63.5 f 0.2 (6)

21-43 (20) 0-48 (58) 0 (6)

100 (19) 0 (58)

100 (5)

ND

33-81 (7) 17-27 (5) 0-32 (19) 0-20 (58) 0 (6)

Range 9% with defects in

19-61 3-35 0-59 0-20

(14) (5)

(30) (58)

ND

Numbers in parentheses are thetotal number of cysts examined. ND, not done. ' Number of whole or recognizable partial axonemes per cross section of a cyst f SD. Range of percent of axonemes per cyst that were fragmented or missing outer doublet microtubules. ru h th st pP cu wrl""/wrl"" sr ea. ru h th st pP cu ~ r " r ' ~ ' ' / w rsrP e~ea. ~~ ru h th st cu wrl"""" c a / ~ r l " " sr ~ 'e~ca. 'Testes from three bv wrl""lTM3 males were examined. Most cvsts of developing spermatids were apparently normal or had only a few defects. However, each testis had a few extremely disorganized cysts. 8 Data from FULLERet al. (1988).

advanced to explain the failure to complement between other nc loci and B 2 t alleles (FULLER1986; RECAN and FULLER 1988; HAYS et al. 1989; FULLER et al. 1989). Because wrlnG4maps to a locus different from any of the known a- or P-tubulin genes of Drosophila, it is unlikely to encode a tubulin protein. Instead, thegenetic interactions make whirligig a good candidate for a gene encoding a microtubule-associatedprotein. Alternatively, the wrl geneproduct could be a processing or modifying enzyme that acts o n components of microtubules, butis not itself a part of the final structure. Extragenic suppression of the apparent haploinsufficiency ofwr2 also implicates thewrl gene product in microtubule function: whirligzg appears to be haploinsufficient for male fertility. A deletion of wrl, W r l n r 4 ~ l Wrlnc47v2 , and ~ r l " ~all ~ "have ' ~ adominant male sterile phenotype in a genetic background wild type for microtubule function. The mutations responsible for the dominant male sterility behave in cosegregation analysis asif at ortightly linked to whirligig (Table 5). Both a deletion of wrl and ~ r l ~and ~ wr1"'4"3 have a dominant male sterile phenotype in a genetic background wild type for microtubule function. The dominant male sterilerevertantsarerestored to fertility by a duplication of the wrl region, consistent with the behavior of loss of function mutations at a haploinsufficient locus. The dominant male sterile revertants are unlikely all to be deletions that remove bothwrlnc4and a nearby locus that is haploinsufficient for male fertility. First, EMS is primarily a point mutagenunder themutagenesis conditions employed (ASHBURNER 1989). Second, the dominant male sterile revertants were recovered at a high frequency (1/2000). Another possibility is that reversion of the failure to complement results from lesionsin a locus tightly linked to wrl. Such mutations would have to act as both extragenic sup-

pressors of the failure to complement between wrlnr4 and B2t" and also cause dominant male sterility in a genetic background wild type for tubulin. However, the simplest explanation is that the dominant male sterile mutations reverted wrlnc4by a loss of function mechanism, and that wrl is haploinsufficient for male fertility. wr~nc4rul and ~ r l " " ' "are ~ probably not null alleles of wrl. If both were null alleles then the two mutants should have identical homozygous phenotypes. However, ~ 7 -homozygotes l ~ ~ display ~ ~ outer ~ triplet microtubules in axonemes while wrlnc4'"l homozygotes do not. ~ r l " ' ~ 'heterozygotes "~ are not fully restored to fertility by a duplication of a wild type copy of wrl, as would be expected for null a allele. Finally, Df(3R)wrll males apparently aremore fertilethaneither Wrlnc4~I /+ or ~ r l " " ~ ' " ~males / + (see Tables 3 and 4). The revertant alleles may carry two mutations in wrl: the original nc4 lesion plus a new mutation that reverts the failure to complement. The more extreme male sterility of wrlnr4'"/+ versus Df(3R)wrl/+ heterozygotes thus ~ could ~ ~ be, due to residual ~ antimorphy r l of ~the ~ nc4 lesion. Alternatively, the apparent difference in fertility could be due to differences in genetic background. Because both tubulin mutations and the hay""? mutation can suppress the haploinsufficiency associated with wrl (Table 3), it is likely to be the level ofwhirligig product relative to tubulin, other components of microtubules, or microtubule function, rather than the absolute level of whirligig product, that is important formicrotubulefunction during spermatogenesis. Mutations in a l t or B 2 t presumably lower the level of functional tubulin in the testis (FULLER et al. 1989). The haync2allele encodes a poison product that may reduce the level of microtubule function in the testis (REGAN and FULLER 1988). A deficiency of haywire partially suppressed the

+

~

thewrl, Microtubules and dominant male sterility of wrlnc4"'' but did not SUPpress the dominantmale sterility of ~ r lconsistent ~ ~ ~ with other differences between wrlnc4"" and as discussed above. ~ r l may ~ be ~ more ~ ~ sensitive ' than ~ r tothe l level~ of other ~ components ~ ~ of ~ microtubules and therefore more easily suppressed. Alternatively, wrlnc4"2 may be lesseasily suppressed because it has more extreme defects such as the abnormal persistence of outer triplet microtubules in homozygotes. The apparent requirement for specific a ratio of the wrl gene product to other components of the axoneme may be consistent with FLOOR'S(1970) "balance of components" hypothesis. In this model, many components of a complex macromolecular structure must be in the properstoichiometric ratiofor properassembly, as demonstrated for bacteriophage T 4 (SNUSTAD 1968; FLOOR 1970; SHOWEand ONORATO 1978). MEEKS-WAGNER and HARTWELL (1986) later demonstrated that the normal stoichiometry of histone dimer sets is required for high fidelity transmission of chromosomes during mitosis. These authors hypothesized that chromosome loss might originate from improper assembly of chromatin due to a stoichiometric imbalance. Myofibrillar assembly in the flight muscle of Drosophila may also be sensitive to the level of individual components. HOMYKand EMERSON(1988) found that three wild type copies of Mhc acted as a dominant enhancer of some recessive mutant alleles of loci importantfor myofibrillar function. T h e suppression of probable loss of function mutations in wrl by mutations that lower the level of microtubule function is consistent with the hypothesis that whirligig is important for the function of microtubule structures, and that the wrl gene product is a component of those structures. The wrl gene product may help organize the microtubules in the flagellar axoneme: Because homozygotes for thepossible lossof function alleles, ~ r l ~ or wrl"r4N2, are both viable and female fertile, thewrl gene product appears to be dispensable for functions outside of spermatogenesis. Meiosis and nuclear shaping appeared normal in males homozygous for wrl mutations, consistent with the hypothesis that the wrl gene product is unnecessary for the meiotic spindle and perinuclear microtubules that mediate these respective processes. However, it is possible thatthe screen for revertant alleles could select for mutations that affect only those domains in the wrl gene product important for testis- or axoneme-specific functions. Genetic and phenotypic analyses of homozygotes for a proven null allele of wrl should discriminatebetween these possibilities. The most striking effects of wrl mutations are on the structure of the flagellar axoneme. The missing accessory microtubules and the abnormalpresence of

Axoneme

97 1

outer triplet microtubules are specific structural defects observed in male sterile muta~ not ~ commonly , tions. The outer triplet microtubules observed in axonemes of ~ r homozygotes l ~are not~ simply~ short ~ extensions of basal body triplet microtubules. In organisms in which the transition has been examined, outer triplets normally change to outer doublet microtubules within 0.3-0.4 pm of the basal body (PITELKA 1974; WOLFand KYBURG1989). However, in W r l n c 4 ~ 2 homozygotes, the outer triplet microtubules

~

extend for at least 30 pm. Because the outertriplet microtubuleswere present only in homozygotes for ~ r l it is~ possible ~ ~ that ~ ~ , they result from a second recessive mutation outside of wrl. However,the only portion of the original r chromosome l ~ in these ~ homozy~ ~ mutagenized ~ gotes was the less than 12 m.u. interval between cu and sr. Thus, if outer tripletmicrotubules were a result of a second mutation, then that mutation must map close to wrl. One test that would establish closer linkage between the mutation causing the outertriplet microtubules and wrl would be to examine axonemes ~ "the ' ~ electron microscope. from D f ( 3 R ) ~ r l / w r l " ~in The presence of outer tripletmicrotubules would establish that the causative mutation is at least under Df(3R)wrl.However, the complexity of generating the synthetic Df(3R)wrl has so farprecludedobtaining this genotype. T h e wrl geneproduct may berequiredforthe organization of the outer ring of microtubules in the axoneme, or for the transition from the architecture of the basal body to that of the axoneme. In males homozygous for mutations in wrl, axonemes lack the normal accessory microtubules. In addition, axonemes r have persistent l ~ ~ from males homozygous for ~ outer triplet microtubules more characteristic of the 0 basal body. Basal bodies are organized in a9 architecture of 9 outer triplet microtubules and no ~ ~ pair ~ microtubules. ' central Accessory microtubules are absent from the basal body in Drosophila melanogaster (TATES197 1). One model to explain both the haploinsufficiency of wrl and its suppression by tubulin mutations is that the wrl gene product must be incorporated into the axoneme as it is assembled. In males heterozygous for loss of function alleles of wrl but otherwise wild type for tubulin function, the reduced amountof wrl gene product might be insufficient to keep pace with the rate of axoneme elongation. The resulting axonemes would then have subtle defectsdue to insufficient wrl geneproduct in the final structure.In males also heterozygousformutations that lower the level of tubulin or microtubule function (e.g., B2t"), the rate of axoneme elongation might be slowed enough such that the limited available wrl gene product can keep pace.

+

~

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L. L. Green et at.

We thank T. C. KAUFMAN for the original nc4 allele of whirligig. L.L.G. is grateful to NGA DANC and MICHELLESWIREN for help with mappingexperiments,mating tests and testis dissections. M.T.F. thanks E. C. RAFF for support and discussion during the initial phases of analysis of wrl"" and J. HUTCHENSfor technical assistance with the original recombination analysis of the nc4 allele. We thank BILLGELBART for notifying us about the possibility of a haploinsufficiency for male fertility in the 88C-E region. L.L.G. is indebted to CATHYL. REGAN andBARBERA ROBERTSON for discussions of science and for help in becoming a fly pusher. We thank SUSANK. DUTCHER, GEORGE GOLUMBESKI, MICHAEL KLYMKOWSKY, and MARC D. PERRY,andmembers of the FULLERlaboratory, especially BARBERA ROBERTSON, for critical comments on the manuscript. This work was supported by National Institutes of Health (NIH) Postdoctoral Fellowship GMI 1805 to L.L.G. and NIH grant R01-HD-18127 to M.T.F.

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