chromosome damage and early developmental arrest ...rex (ribosomal exchange) is a genetically...

Copright 0 1994 by the Genetics Society of America

Chromosome Damage and Early Developmental Arrest Caused by the Rex Element of Drosophila melanogaster

Leonard G. Robbins* and Sergio Pimpinellit”

*Genetics Program and Department of Zoology, Michigan State University, East Lansing, Michigan 48824-1312, and tlstituto di Genetica, Uniuersita di Bari, 70126 Bari, Italy

Manuscript received February 15, 1994 Accepted for publication July 8, 1994

ABSTRACT Rex (Ribosomal exchange) is a genetically identified repeated element within the ribosomal DNA

(rDNA) of Drosophila melanogaster. Rex has a semidominant maternal effect that promotes exchange between and within rDNA arrays in the first few embryonic mitoses. Several of Rex’s genetic properties suggest that its primary effect is rDNA-specific chromosome breakage that is resolved by recombination. We report here that rDNA crossovers are only a small, surviving minority of Rex-induced events. Cytology of embryos produced by Rex-homozygous females reveals obvious chromosome damage in at least a quarter of the embryos within the first three mitotic divisions. More than half of the embryos produced by Rex females die, and the developmental arrest is among the earliest reported for any maternaleffect lethal. The striking lethal phenotype suggests that embryos with early chromosome damage could be particularly fruitful subjects for analysis of the cell biology of early embryos.

R X’S maternal effect causes mitotic rDNA exchange in paternally derived chromosomes (ROBBINS

1981). Inter-array recombination takes place in embryos whose mothers are Rex and whose fathers contribute a chromosome, referred to as a target chromosome, that has two rDNA arrays. An example of a mating that gen- erates these mitotic events, and the events themselves, are diagrammed in Figure 1, with an attached-XY chromosome shown as the target.

With appropriate markers, “spiral” exchanges (Figure 1A) yield phenotypically distinct offspring. Although more difficult to detect, “hairpin” exchanges (Figure 1B) yield chromosomes that contain both recombination products, making them especially informative for unraveling the mechanism of Rex-induced exchange. Analysis of the pairs of recombinant arrays indicates that Rex-mediated exchanges are often unequal, having corresponding duplications and deficiencies in the two recombinant arrays. They are also often non-reciprocal; many recombinant arrays have substantial deletions, without any corresponding duplication in the other exchange product (RASOOLY and ROBBINS 1991). The deletions always map to the site of exchange, and are detectable as bb mutants, deletions of molecular markers, or deletions of a distinct function (&(Rex), see below) (ROBBINS 1981; RASOOLY and ROBBINS 1991).

Rex events are rDNA specific in two respects. First, Rex promotes exchange in every tested target that has two nucleolus organizers, but does not induce exchange in chromosomes with two heterochromatic blocks if even one block lacks rDNA (SWANSON 1987). Second, all of the

’ Present address: Dipartimento di Genetica e Biologia Molecolare, Univer- siti di Roma “La Sapienza,” P. A. Mor0 5, 00185 Roma, Italy.

Genetics 138: 401-411 (October, 1994)

crossovers are recombinant for rDNA molecular markers (RASOOLY and ROBBINS 1991; P. CRAWLEY and L. ROBBINS, unpublished data).

Rex also causes heritable copy number changes in single rDNA arrays. This is similar to the rDNA magnification phenomenon first seen in bb/Ybb- males (RITOSSA 1968), but differs from that situation in that Rex’s maternal action causes these intra-array events. Rex/rDNA-deficiency females crossed to bb2/Y males yield about as many bb’ magnified products as do bb2/ Ybb- males crossed to normal-X/rDNA-deficiency females (S. HAWLEY, unpublished results; P. CRAWLEY and L. ROBBINS, manuscript in preparation). Thus, the Rex target is the rDNA, not the peculiar chromosomes used to detect spirals and hairpins.

Maternaleffect suppressors of Rex (Su(Rex) ) are present in many laboratory stocks (RASoowand ROBBINS 1991) and in wild populations (BOUSSAHA 1991). Both Rexand one X-linked Su(Rex) map within the rDNA, but both of them map at more than one site along the molecular map and are, therefore, repeated (RASOOLY and ROBBINS 1991; rDNA mapping procedures have been re- viewed in WILLIAMS and ROBBINS 1992). Thus, Rex and this Su(Rex) are either rDNA variants or non-rDNA sequences within the rDNA array. One autosomal Su(Rex) has also been found (SWANSON 1984; RASOOLY and ROBBINS 1991 ) .

Dosage experiments indicate that Rex is a neomorph or strong hypermorph, not a loss of function (RASOOLY

and ROBBINS 1991). Thus, the Rex function must be either absent or minimally expressed in non-Rex chromosomes. Combined with its maternal action, this implies that Rex is active during oogenesis and puts something

402 L. G. Robbins and S. Pimpinelli

y Rex y+ Target Chromosome X

y 4 Y

y+ Target Chromosome y Rex or y

A: 'Spiral' Exchange B: 'Hairpin' Exchange

1 GI' Reversed order

FIGURE 1 .-Rex-induced exchange events. Repmediated exchange takes place in early embryos whose mothers are Rex and whose fathers contribute a target chromosome that bears two rDNA arrays. Normally, a single rDNA array of about 200 copies forms the nucleolus organizer (NO) in the heterochromatin of the X chromosome and another array is in the short arm of the Y chromosome ( RITOSW et nl. 1966). Suitable two- array targets. however, include some attached-XY chromosomes, chromosomes constructed by recombination between overlapping inversions, and chromosomes derived from these by Rex-induced exchange (SWANSOS 1987; RLWI.Y and RORRINS 1991). Reflecting the mixed orientation of rDNA re- peats, two types of exchanges occur in target chromosomes: "spiral" exchanges that delete the material between the two arrays. and 'hairpin" events that invert the entire segment between the NOS (RORRINS and SWANSON 1988). In the example shown, and in the crosses reported here, the target chromosome is an attached-XY chromosome. Heterochromatic seg- ments are drawn as heavy lines with the locations of the rDNA and fertility factor complexes (KS and KL) indicated. In this example, the spiral exchange nuclei are J+ because they have a J X chromosome and a ?+ Y chromosome. Most of these events occur very earlv. before Sphase of the first embryonic division, producing, from an XXY zygote, a male that is entirely J+ but carries no other paternal markers. A minority of events occur after first S and yield gynandromorphs in which the male tissue has one maternallv derived chromosome and the y* Y chromosome (RORRINS 1981). Hairpin events are more difficult to detect, because they do not alter gene content. To re- cover them, all of the daughters' X chromosomes must be tested for the classical inversion phenotype of crossover sup pression.

into the egg. Since the recombination events occur well before translation is detectable in embryos, the Rex product is probably, although not necessarily, a protein.

Rex's maternal action, its neomorphy, its presence in multiple copies, and the production of deletions at the sites of exchange suggest that Rex may be an inserted element that produces an endonuclease during oogenesis. Breaks in the rDNA in early embryos could then be repaired to give both types of inter-array exchange as

well as intra-array magnification, with multiple hits in one array yielding the deletions at the exchange site (RASOOLY and R O I ~ I S S 1991). One possibility (HAWIXY and MARCUS 1989) is that RPX is an active version of one of the retrotransposon-like insertion sequences (1%) found in the 28Scoding segment5 of many rDNA re-

Expression of an rDNA IS from Bombyx mon' in Es- cho-ichin coli yields reverse-transcriptase and rDNA endonuclease activity (XION(; and EICKRUSH 1988). The Drosophila and Rombyx ISs are structurally quite similar UAKUR(:~'K 41 af. 1990). and the E. coli-expressed product of a Drosophila element, although not yet as well characterized as the Bombyx protein, also has endonuclease activity (T. EI(:KRUSII, personal communication).

Of course, RPX might be an entirely novel element. If, however, it does encode an endonuclease. it is possible that only a minority of breaks are successfully repaired as crossovers or magnified arrays. This raises the possibility that we might see, cytologically, what Rex does. We report here the results of 4,Miamidino-2-phenylindole (DAPI) staining experiments that reveal a high incidence of chromosome damage and a striking developmental-arrest phenotype when that damage is not repaired.

peats (DAMID and ROBERTS 1981 ;JAKUBC7AK d d. 1990).

MATERIALS AND METHODS

Matings, egg collection and lethal phase: Matings were made at 2.5" on cornmeal medium. I n one experiment, 0.025% tetracycline was added in the generation preceding egg collection. For egg collections, mas matings were done in normal culture bottles 2 days before egg collecting began. After 1 day, the flies were transferred to empty bottles capped by yeast- pastecontaining Petri dishes for the second day. Eggs were then collected at room temperature (18-22") on acetic-acid- spiked medium. For early embryos, after discarding the eggs laid in a I-hr interval, collection plates were changed every I5 min and the eggs were immediately dechorionated and fixed. Samples from some of the 15min collections were aged 3.5 hr at 25" for cytological examination of developmental progress, and I-hr long collections from all of the matings were also examined after aging 24 hr. Larvae from the 24-hr samples were allowed to continue development to score lethal phase and adult phenotypes.

Cytology: Eggs were dechorionated in hypochlorite, rinsed briefly w i t h water, and fixed 0.5 hr to overnight in 1:3:4 glacial acetic acickabsolute ethanokchloroform. We have seen no dif- ference between eggs stored in fixative at 4" for extended pe- riods, eggs immediately processed and stained, or eggs stored at 4" after staining. Eggs were prepared for DAF'I staining by successively rinsing once with 9.5% ethanol, twice with phosphate-buffered saline (PRS = 0.01 M sodium phosphate, 0.15 M NaCI. pH 7.3) containing 0.5% Triton X-100, and once with PRS alone. Staining with 1 pg/ml DAPl in PBS for 10 min to 3 hr gave equivalent results. Eggs were rinsed with PBS, which was replaced with PBS + 20% glycerol for storage and mounting. Neither higher glycerol concentrations. nor addition of 1,4-phenylenediamine seemed to significantly improve resistance to fading, and the latter has the disadvantage of yellowing with time. Observation and photography were done with either a Nikon or Olympus epi-fluorescence microscope and Ektachrome 400 film. Occasionally, the first slide made

Rex-Induced Chromosome Damage 403

revealed poor staining, rapid fading or high background, but these problems were often eliminated when the remainder of the sample was re-processed through the fixation/rinsing/ staining sequence.

To stage such early embryos, it is essential to see all of the nuclei as well as the state of the meiotic products and polar bodies. For example, because the first mitosis is nearly com- plete before fusion of the male and female pronuclei, there are two swollen nuclei with diffuse chromatin at prophase of both the first and second divisions. The polar bodies are distinctly different at those times, however, being rotund at the first prophase and in an arrested, metaphase-like state at the second. Vigorously squashed eggs gave the best resolution of individual chromosomes, but proved impossible to stage with assurance. Mitotic figures were difficult to see clearly in un- squashed whole mounts. A compromise of mounting in PBS/ glycerol and flattening by slowly wicking liquid from under the coverslip allowed us to be quite confident of the age of the embryo, at the cost of some loss of resolution. Since repeated staining was not planned, we tried to get more permanent mounting than the usual sealing with rubber cement. Contact cement gave some improvement, but we have had the best results running a bead of quick-setting epoxy around the coverslip. Epoxy displaces the aqueous medium under the edges of the coverslip, often, but not always, giving an airtight seal that lasts for months.

We should note that, after having examined the early divisions of hundreds of normal as well as abnormal embryos in the course of this project, SONNENBLICK'S (1950) description of early mitoses remains a remarkably accurate guide to the pro- gression of events. Current techniques are more convenient, and the photographs may be prettier, but the standard for careful observation was set a long time ago.

Crosses and genotypes: Rex's rDNA-recombination phenotype is semidominant, and eggs produced by both Rex homozygotes and Rex heterozygotes were examined. A variety of controls were also done. These included non-Rex females and two controls to test whether each observed phenotype is produced by Rex or by some extraneous element in the Rex- bearing chromosome.2 For some phenotypes, such as rDNA recombination where spontaneous events are quite rare, any dominance is readily detected. For these phenotypes, comparison of Rex heterozygotes with suppressed-Rex females provides a functional test for whether the phenotype is caused by Rex. Other phenotypes, such as mortality, some of which occurs in any case, are essentially recessive. For these phenotypes, placing the Rex-bearing chromosome over a deficiency chromosome that exposes the rDNA but covers the euchromatin provides a test of location. Since the Rex-bearing nucleolus organizer (NO) is itself strongly bb, controls were also done for possible effects of bb. In addition, a cross was done to generate

gene being studied was forcefully brought home in some other work with Rex. ' The need to test whether each phenotype is actually associated with the

Those observations are reported in this footnote so that others who may work with Rex will not be similarly misled. In the course of dissecting ovaries from Rex/Rex females for in situ hybridization and RNase protection experiments, we noticed that many Rex females lacked one or even both ovaries (or had oocyte-less remnants). This gonadal dysgenesis is a very easily scored pheno-

able element active during oogenesis. The assumption that Rex causes the type, and certainly a phenotype that could reasonably be caused by a transpos-

ovarian dysgenesis, however, proved to be incorrect. The first hint came from crosses of Rex/Rex females that also carried a Y chromosome. The maternal Y chromosome nearly completely suppressed rDNA recombination in the offspring, but the frequency of missing ovaries in these Rex/Rex/Y females was even higher than in Rex/Rex females. While we thought about various expla- nations, such as production of embryos with so much damage that hardly any sulvived as rDNA exchanges, a student, MARK THOMPSON, proceeded to roughly map the dysgenesis-to between w and cv, about as far away from Rex as it is possible to be. They Rex chromosome used for the Rex/Df(lJXI cross reported in this study is a recombinant that does not produce gonadal dysgenesis,

unfertilized eggs. These eggs provided a reference for recog- nizing unfertilized eggs in the other matings, and a test for how nuclear morphology progresses when chromosomes are not damaged but division is blocked. Lastly, Rex/Rex females gen- erated following the tetracycline treatment protocol of O'NEILL and KARR 1990 were crossed to check for the possibility of commensal parasites. These crosses, and the purposes of each, are outlined in Table 1.

RESULTS

Cytology of early embryos: Many of the nuclear figures of the first three divisions of embryos produced by Rex homozygotes are clearly aberrant. They reveal bridges, fragments or excluded elements, and, even by the second division, there is often marked asynchrony of the nuclei. Examples of the chromosome damage found are shown in Figure 2, with the results enumerated in Table 2 and summarized in Figure 3. Figure 2D repre- sents the most extreme situation seen, but such embryos were not frequent. In most cases, as seen in the other panels of Figure 2, only a single element behaved out of concert with the remaining chromatin.

Because the Rex-bearing NO is bb, half of the zygotes in the first cross are bb. Any zygotic effects of the bb phenotype are, however, ruled out by the Rex/Rex X OreR/Y cross which produces only bb' embryos yet gives a frequency of abnormal figures that is not significantly different (contingency x' = 1.71, 1 d.f., P - 0.19). The Rex/Rex females are themselves also bb, and maternal rDNA insufficiency must also be ruled out if this damage is to be ascribed to Rex. Three crosses bear on this. First, Rex is semidominant in terms of the production of inter- array crossovers (ROBBINS 1981), but Rex heterozygotes are not bb. Rex is also semidominant for the cytological phenotype; the heterozygotes produce fewer cytologically aberrant embryos, but the spectrum of damage is the same. Second, we directly tested for effects of bb by examining embryos from crosses of bb2/bb- females to both attached-XY/O and X/Y males. In the first of these crosses, either maternal or zygotic effects of bb would have been evident, in the second only maternal effects would have been seen, but both crosses gave very few embryos with early damage. The few early-division abnormalities seen in these embryos, two in each cross within the first three divisions, were also quite different from most of those seen in the Rex crosses. In these embryos two sister nuclei, or the two pro-nuclei, were one mitotic stage apart, but there were no chromatin abnormalities.

We must also be sure that the cytological damage is an effect of Rex and not of some other gene that happens to be on the Rex-bearing chromosome. We did two tests of this. In one, we examined eggs produced by females heterozygous for Rex and Df(l)Xl, a deficiency that un- covers the basal heterochromatin. These hemizygous females yield a higher frequency of damage than do heterozygotes, mapping the cause of the cytological


TABLE 1

Crosses

Female Male Purpose

y e71 u f Rex/y cu u f Rex

y CU u f Rex/y cu u f Rex

y Rex /Df( l )XI

y cu u f Rex/y

y cu u f Rex/FM7, &(Rex)

y/FM7, Su(Rex)

bb2/In(l)sc4LscsR, bb-

bb2/Zn(l)sc4Lsc8R bb

Y /Y

y Rex/y Rex (tetracycline treated)

YSX.YI,, I n ( l ) E N , y u f B .y+/O

OreR/Y

YSX. YL, In( l )EN, y u f B.y+/Y

YSX.YL, In( l )EN, y u f B .y+/O

YSX.YL, In( l )EN, y u f B.y+/O YSX. YL, In( l )EN, y u f B.y+/O

OreR/Y OreR/O

YSX. YL, In( I )EN, y u f B.y+/ Y

Female is Rex/Rex, male provides a target chromosome that yields a y+ Y chromosome following spiral exchange

Maternal effect of Rex with exclusion of any possible zygotic bb effect because all embryos have a wildtype X or Y chromosome

Control for effects of X-linked genes located outside of the basal heterochromatin. D f ( 1 ) X l is deleted for most of the basal heterochromatin, including all of the rDNA and for two euchromatic postembryonic lethals. Attached-XY/Y males were used to avoid possible embryonic lethality of rDNAdeficient D f ( l ) X l / O zygotes

Effect of heterozygous Rex, and control for maternal bb phenotype of Rex/Rex females

Suppressed-Rex control; FA47 bears a strong, although not absolute, Su(Rex)

Non-Rex control

Direct control for possible maternal or zygotic effects of rDNA insufficiency on early mitotic divisions. I n ( l ) ~ c ~ ~ ~ s c ~ ~ is deleted for the entire NO

Test for maternal effects of rDNA insufficiency

Sterile mating used to generate unfertilized eggs. X 0 males were produced by mating OreR wildtype females to YSX.YI,, Zn(l)EN, y u f B . ~ + / o males

Tetracycline treated to eliminate parasites. F M 7 / D f ( l ) X I females were crossed to y Rex/Y males on medium containing 0.025% tetracycline. "Cured" y Rex/FM7 daughters were then crossed to y Rex/Y males to generate the y Rex/y Rex test females

Descriptions of markers and chromosomes may be found in LINDSLEV and ZIMM (1992).

phenotype to the basal heterochromatin. However, the cytological phenotype, like the rDNA-exchange phenotype, is semidominant, and the measured frequency of damage depends on the distribution of mitotic stages in the sample (see below). Thus, for this phenotype, deficiency mapping is a weak test at best. The availability of Su{Rex)s, much as they are a nuisance in other con- texts, provides an alternative, and more convincing, test. Thus, a suppressed-Rex control was done as well as the non-Rex control. Although Rex/Su {Rex) females remain heterozygous for Rex, not only is the production of rDNA recombinants suppressed, but visible damage is nearly absent as well (a contingency test comparison of the Rex heterozygotes and Rex/Su{Rex) yields 9 = 6.01, 1 d.f., P - 0.014).

The aggregate frequency of visible damage in the first three divisions of embryos from Rex homozygotes is 26%, but damage is not equally detectable at every point in mitosis. Observation, confirmed by statistical analysis, indicates that damage is least readily detected in interphase and prophase, and most obvious at anaphase and telophase. The extremely large and diffuse first cycle prophase nuclei are particularly difficult to score. Pool- ing the numbers for all three divisions for both Rex/Rex crosses in Table 2, we find 14% (7/49) aberrant at interphase, 6% (3/49) at prophase, 23% (14/62) at metaphase, 72% (21/29) at anaphase and 100% (6/6) at

telophase. Similarly, for the Rex/Df{l)Xl data, there were 3% aberrant interphases (1/31), 4% aberrant prophases (2/68), 8% aberrant metaphases (4/48), 53% aberrant anaphases (9/17) and 100% aberrant te- lophases (5/5), and Rex heterozygotes gave no (0/38) aberrant interphase embryos, 4% (3/85) aberrant prophases, 1% (1/96) aberrant metaphases, 35% (9/ 26) aberrant anaphases and 40% (4/10) aberrant telo- phases. Thus, the damage that is visible in these prepa- rations must underestimate the true frequency. Moreover, the measured frequency is quite sensitive to the proportion of eggs at different stages in the samples. The frequency of aberrant metaphase, anaphase and telophase figures, although most likely still an underestimate, should more closely approximate the true frequency: 67% for Rex/Rex X attached-XY/O, 34% for Rex/Rex X OreR/Y, 27% for Rex/Df{l)Xl X attached- XY/Y, and 11% for Rex/y X attached-XY/O.

There is a statistically significant increase in the frequency of aberrant embryos with successive divisions- 33, 57 and 70% among metaphase through telophase nuclei of the first three divisions of embryos from Rex homozygotes. There are, however, two sets of chromosomes per embryo throughout the gonomeric first division and during most of the second division, four from second division anaphase through metaphase of the third, and eight in third anaphase and telophase. The

Rex-Induced Chromosome Damage 405

FIGURE 2.-Examples of Rm-induced chromosome damage. (A) anaphase of first mitosis w& lagging elements (from Rex/y X XY/O cross); (B) telophase of first mitosis with excluded - Y-containing elements (from Rex/y X XY/O cross); (C) prophase of second mitosis with two excluded elements (from Rex/Rex X XY/O cross) ; (D) metaphase of second mitosis plusmi- cronucleus (from Rex/Rex X XY/O cross)-note asynchrony; (E) anaphase of second mitosis (from Rex/Rex X OreR/Y cross)-one plate has a bridge, the other an excluded element; (F) anaphase ofthird mitotic division (from Rex/y X XY/O cross)-these two sister plates have bridging elements of Y, or attached-XY, origin. The other two plates in the embryo were apparently normal.

frequency of damaged complements, therefore, remains roughly constant. Either all of the damage occurs very early, and damaged complements continue to divide, or a steady-state balance of damage and repair is quickly established. The observation that adjacent second division or later anaphase plates often evince remarkably similar aberrations (e.&, Figure 2F) supports the former.

Viability: With so much damage, why do we see only a few percent recombinants? The answer is that most of the progeny of R e x homozygotes die, and they die as embryos. These results are shown in Figure 4. Rex homozygotes produce upwards of 60% embryonic lethality, while so few eggs from R e x heterozygotes die as embryos that this phenotype is effectively recessive. Thus, the embryonic lethality seen when a portion of the basal heterochromatin including the rDNA is uncovered by Df(1)XI rules out any maternaleffect lethals elsewhere. The absence of rDNA in Df(1)Xl sons, and any embryonic lethality that might be caused by being bb-, was covered by inclusion of a Y chromosome in the male parents, but Df(1)Xl is also deleted for two essential

euchromatic genes, resulting in the absence of Df(l)XI/Y sons (Table 3) as postembryonic lethals (Figure 4).

Survival of the progeny of Rex heterozygotes is higher than that of the Su(Rex)/Rex and y/Su(Rex) controls, but most of the death in those controls is postembryonic. Moreover, the scoring of surviving adults shown in Table 3 indicates that the depressed viability in the Su(Rm) crosses was caused by the Su(Rex} chromosome which was the multiply inverted FM7balancer. The data for surviving adults also indicate that much of the inviability in the bb2/ bb- X attached-XYcross is ascribable to the lethality of the bb-/O segregant, but the lethality seen in the b/?/bb- X OreR/Y cross does suggest that maternal rDNA insufficiency also causes some zygotic lethality.

The embryonic lethal phenotype: The reason for the death of these embryos was apparent when eggs from the R e x / R e x X attached-XY cross were examined after 3.5 hr of development. These results are listed in Table 4, and examples of the embryos are in Figure 5. At this stage, normal embryos are mature gastrulae.


TABLE 2

The spectrum of ReDinduced chromosome damage

Parents First division Second division Third division Later

Female Male U pre I P M A T I P M A T I P M A T divisions

Rex/Rex

Rex/Rex

Rex /Df ( l )X l

R e d y

Rex/Su(Rex)

y/Su (Rex)

bb2/bb-

bb2/bb-

- X Y / O

OreR/Y

XY/Y

X Y / O

X Y / O

X Y / O

X Y / O

-

-

-

-

-

OreR/Y

Total: Abnormal:

Total: Abnormal:

Total: Abnormal:

Total: Abnormal:

Total: Abnormal:

Total: Abnormal:

Total: Abnormal:

Total: Abnormal:

28

27

37

19

10

6

18

19

9

10

18

4

5

11

29

10

15 2a

16 0

17 0

15 0

16 0

22 0

12 l C

7 0

12 0

31 I n

49 2

47 0

17 0

36 0

35 l C

23 0

9 7 2 2 0 4 1 1 2 0 0 0 0 4 3 7 2 0 0 3 0 1 0 0 0 0 0 3

3 3 1 2 1 1 2 5 1 0 6 1 2 1 6 3 1 18 1 7 1 4 2 3 5 1 1 0 4 2 1 14

3 4 1 3 4 1 3 1 8 1 1 3 0 1 1 3 1 1 7 2 7 4 0 1 0 2 0 1 0 2 0 1 6

66 19 6 1 8 3 1 27 5 3 5 7 3 2 1 25 0 7 2 0 0 0 1 1 0 3 ~ 1 1 1 4

30 9 7 1 3 1 0 8 3 1 1 3 1 0 0 13 1 0 0 0 0 0 0 0 0 0 0 0 0 2

28 7 1 9 7 1 4 2 2 2 3 5 0 0 32 0 0 0 0 0 0 0 0 0 0 0 0 0 1

2 0 1 0 5 5 2 6 3 2 1 4 2 2 1 32 0 0 0 0 0 0 0 0 0 0 0 0 0 3

1 5 1 3 4 3 4 8 4 0 1 4 1 0 0 2 25 0 0 0 0 0 0 l b 0 0 0 l b 0 0 1

Embryos of 0-15 min of age from the crosses indicated, as well as from a y / y X X 0 mating that yields only unfertilized eggs, were classified as follows:

U = Unfertilized; only one pronucleus and three polar bodies are present. The pronucleus of an unfertilized egg can develop as far as the first division interphase state described below, and polar body nuclei may continue to develop to the early rosette stage usually seen in the third division of fertilized eggs.

pre = Pre-mitotic; two pronuclei and three polar bodies are present. All five nuclei are tightly condensed, with the male pronucleus migrating toward the interior and the female pronucleus moving away from the polar bodies.

First division: I = Interphase; pronuclei and polar bodies are engorged, spherical and of equal size. Pronuclei have moved well away from the cortex but are

- ,

not yet tightly apposed. P = Prophase; pronuclei, which now lie side by side, become swollen and elongated and chromatin staining becomes quite diffuse. The

pronuclei are much larger than the polar bodies. M = Metaphase; chromatin of both pronuclei and polar bodies condenses and the nuclear envelopes disappear. The pronuclear complements

are tightly apposed, but do not form a single plate. A = Anaphase; depending on the angle of observation, the parallel anaphase figures of the two complements are more or less apparent. The polar bodies remain arrested in a metaphase-like state.

fused at this stage. T = Telophase; nuclear envelopes re-form. Polar body chromatin remains condensed, but the two inner members of the tetrad often appear

Second and later divisions: Interphase nuclei can appear quite similar to first division interphase, but are spherical and never get as large. The appearance of the zygotic nuclei throughout mitosis is conventional, but the polar bodies become polytene (or polyploid) and begin to assume a rosette configuration during the third division that becomes larger in later divisions. The polar bodies may all fuse, but two rosettes are often quite prominent until much later cycles.

a Polar bodies at more advanced stage than pronuclei, but otherwise normal appearing. Asynchronous, but otherwise normal appearing. Male pronucleus in metaphase, but otherwise normal appearing.

Some of the eggs produced by Rex homozygotes were abnormal gastrulae, appeared to have developed nor- normal (Figure 5, panel A), but 58 out of 11 1 eggs (52%) mally. For this phenotype, embryos from Rex/Df(l)Xl had strikingly abnormal phenotypes. Fifty-six of the 58 females provide strong assurance that the cause resides had fewer than 100 of the “exploded” nuclei seen in in the base of the X; those embryos evince the same Figure 5, panels B through D, and most of these had spectrum of nuclear damage as those from Rex/Rex fe- eight or fewer such nuclei. These nuclei are either males, although the damage is somewhat less, in accord polyploid or are aggregates of nuclei, and the chromatin with the different frequencies of chromosome aberra- is fragmented. Two of the embryos made it to the stage tions seen in the 15-min samples. seen in panel E, with many hundreds of similar, mas- Embryos from the two bb2/bb- matings were also ex- sively disrupted, nuclei. One of those had a small patch amined at 3.5 hr. Although a few of the embryos from of cortically arrayed nuclei, and an additional four em- the bb2/bb- X attached-XY mating were also arrested bryos had large areas of cortical nuclei but prominent early and had disrupted nuclei, the vast majority of these holes in the array. Although there has been much DNA embryos proceed quite nicely to gastrula. replication in these embryos, the nuclei are disrupted Do the embryos that look normal at 3.5 hr die later, and few of them are ever regularly organized as in the or are they the ones that hatch? One-hour long egg col- normal blastoderm shown in panel F. The remaining 49 lections were made from all of the matings and then embryos, with the exception of a few morphologically held 24 hr. Those data are also in Table 4. By 24 hr, any

Klx-Induced Chromosome Damage

1 -7

140 167 255 119 138 110 98 r RexV7ex Rex'X1 Rex:y Rex'Su Y/sU bb2,bb. bb7 bW

X X X X X X X

OreRW XYW XY/O XY/O XY/O XY/O OreRW

FIGI:RE 4.--I~thality of embryos produced by experimental and control crosses. .V = total number of eggs collected; X / = Df(/)X/; St1 = Su(Krx)-l)c*aring FM7chromosome; bb- = In(I)sr".srXH.

N 790 436 2466 698 2253 1545 1439 495 RexRcx Rex Rex Rrx X 1 Rex y R w Su y S u bb'bb bh'bb'

X X X X X X X X

xY/O OreRN xYW xYi0 xY/O mi0 xY/O OreRW

sunking embryos will have hatched into larvae. If some of the KPx-damaged eggs continued to develop only to die later, there would he some more-mature dead eggs in this sample, That was not the case for the embryos produced by RPX homozygotes. Degeneration of the chromatin continues, as indicated by the increased proportion of eggs with little or no staining. Nevertheless, the eggs from the RPX/RPX and RPX/J crosses that were unhatched at 24 hr and had any distinctive areas of chromatin looked very much like the abnormal 3.5-hr-old embryos, and those that were gastrulae at 3.5 hr had gone on to hatch. Thus, all of the RPx-induced lethalip is quite early, and the damaged embryos must die soon after the initial damage. This is rather different from the lethal pattern procluced by FA47 (the Su(RPx) used) or a zygotic rDNA deficit (from the bb'/hb- X xY/O cross),

where many embryos arrest well after gastrulation. Once again, embryos from Rpx/Df(l)XI females are arrested in the same fashion as embryos from RPX/RPX females, providing reasonable confidence that this is a RPX- associated phenotype.

The origin of nuclear fragmentation: Within the first three divisions, RPx-induced damage is usuallyevident in only one chromosome or a pair of chromosomes, hut the "exploded" nuclei seen later suggest widespread fragmentation. Is this a direct effect of KPX, or is this the fate that awaits any nucleus whose development is arrested? To ask whether or not the nuclei of embryos arrested in early development by some other means also develop in this way, normal females were crossed to sterile, XO, males, and the unfertilized eggs produced were examined at 3.5 and 24 hr of age. The results of that


TABLE 3

Sumiving adults

Parents Regular females Regular males Nondisjunction Rex exchange

Gynandro Lost or Female Male Class 1 class 2 Class 1 Class 2 Females Males Males morphs stuck'

Rex/Rex X Y / O 39 69 0 1 44

0 OreR/Y

2 45 Rex/Rex - 0 2

Rex/DJ(l)Xl &Y/Y 319 187 Lethal X Y / O 195

0 2 6 Rex/y - 192 200 237 0 1 1

1

354 Rex/Su(Rex) &Y/O 324 408 93 3 5 0 22

X Y / O 278 247 385 1 0 244

y /Su (Rex) 55 2 1 0 bb2/bb- X Y / O 434 285

0 210

bbz/bb- Lethal 0 17 0

172 80 0 50

OreR/Y 74 NA NA NA NA 10

Each cross produces a different spectrum of phenotypically distinct offspring. Where the two maternal X chromosomes can be distinguished, class 1 refers to the first-listed of the mother's X chromosomes. NA indicates classes that can not be scored or are not produced; nondisjunctional progeny in one cross and the products of Rex-induced exchange in the two crosses that lack a target chromosome. Adults stuck to the food, except in the Rex/Df ( l )X l cross where they were not scored, and the few escapers, were included as survivors for lethal phase calculations (Figure 4), but their phenotypes were not scored.

- 3

NA NA 36

' Flies stuck to food except for a few (1-3) lost during transfers. The marker fwas used to follow the two maternal X chromosomes, but is -9 map units from Rex.

TABLE 4

Embryonic fate

Parents Fragmented nuclei Blastulae Gastrulae

17- Post- Female Male DAFT' 1-2 3-8 9-16 -100 Hundreds Abnormal Normal Abnormal Normal gastrula

~~

3.5-hr-oid embryos: - ~~

Rex/Rex X Y / O 3 5 32 8 11 2 6 4 3 38 2 Rex/Df ( l jX l XY/Y 5 21 26 16 9 3 6 13 0 89 1 bb2/bb- X Y / O 0 0 4 6 0 0 1 0 2 76 7 bb2/bb- OreR/Y 13 0 0 0 0 0 2 1 3 70 7

Rex/Rex XY/Y 0 1 6 12 6 2 0 0 1 21 0 Y /Y - OreR/O 1 20 1

(tetracycline treated)

24hr-old embryos: - Rex/Rex X Y / O 37 13 25 7 7 14 4 1 0 0 4 Rex/Rex - OreR/Y 45 1 16 4 1 3 0 0 0 0 9 Rex/Df ( l )X l XY/Y 36 29 38 12 6 7 6 0 1 0 5 R d y X Y / O 5 2 3 0 0 0 0 0 5 0 5 Rex/Su(Rex) - X Y / O 1 0 0 0 0 1 0 0 0 0 23 y/Su(Rexi - X Y / O 7 1 3 0 0 0 0 0 0 0 21 bb2/bb- X Y / O 26 4 0 0 0 0 2 0 9 0 49 bb2/bb- OreR/Y 50 1 0 0 0 2 5 0 5 0 2 Y /Y OreR/O 27 21 2 Embryos collected in a 15min interval and aged for 3.5 hr, and embryos collected for 1 hr and aged a further 24 hr, were DAPI stained and

scored for chromatin morphology and developmental stage. The cross of y / y females to OreR/O (XO) males yields only non-fertilized eggs. The numbers of nuclei indicated for 24hr-old embryos are necessarily rough estimates because of the necrosis that occurs by this time; there is diffuse, globular background staining, and in those eggs with substantial chromatin much of it is in large aggregates.

a Most of these embryos had no DAPI-staining material or a few tiny fluorescent flecks. A few had one or more distinguishable polar bodies, but no fluorescent material in the remainder of the egg.

mating are also presented in Table 4, and two examples are shown in Figure 6. Although there are fewer nuclei in these eggs, their condition is much like that seen in eggs killed because their mothers bore Rex. These nuclei start out haploid, and they do not get as large as the nuclei of Rexdamaged eggs, but the chromatin morphology is quite similar; over-replicated and disrupted. Eventually, even these blotches of chromatin are further degraded, as in the Rex crosses, since the 24hr sample of unfertilized eggs also included many with no DAF'I signal.

LIN and WOLFNER (1991) report that aberrant staining

found in some embryos produced by the maternaleffect lethal f s ( l ) Yu is probably staining of a commensal para- site (O'NEILL and KARR 1990) rather than staining of Drosophila DNA. It is unlikely that this is the case for Rex given the variety of different stocks used to generate the test females and the bright DAF'I staining and somewhat different morphology seen, but we also tested this by

rearing the mothers of Rex/Rex females on tetracyclinecontaining medium as outlined in Table 1. The results (Table 4) are not notably different from those of the other Rex/Rex crosses.


disproportionately involved might reflect observer bias more than Rex sensitivity.

One might also argue that the widespread fragmentation of the chromatin seen a few hours after the initial damage implies injury outside the rDNA, but the “exploded” appearance of these nuclei is unlikely to be caused by Rex itself. Nuclei that have never been ex- posed to Rex, but that are arrested because the egg is not fertilized, also develop in this fashion. Once mitosis is disrupted, whether because of Rex-induced damage or not, the nuclear cycle proceeds along a programmed path toward this disrupted fate. Although one can not properly call this apoptosis, as there are no individual cells involved, the nuclear result is similar to what is seen in mammalian cells following damage by a wide variety of agents (KERR and HARMON 1991) and in a variety of situations of developmentally programmed cell death (LOCKSHIN and ZAKERI 1991).

Cytologically visible Rex-induced damage occurs far more frequently than rDNA recombination. Most of the damaged embryos die, and rDNA crossovers are only a small fraction of the sunivors. Thus, even though Rex- induced recombination is rDNA specific, these observations raise the possibility that the breakage may not be. There might not be a Rex target sequence in the rDNA; rather, the rDNA might have a higher probability of suc- cessful repair. For understanding Rex, the critical ques- tion now is whether all of the Rex-induced damage is in the rDNA.

Breaking a few chromosomes has a devastating effect on the biology of the embryo. Most often, development does not proceed much beyond the first few cycles. W h y does chromosome damage have this catastrophic effect? Comparing the damaged embryos, particularly the ones that are furthest developed, with the normal blastoderm in Figure 5 , panel F, suggests a reason. In the normal blastoderm there are not only regularly arrayed cortical nuclei, but the so-called yolk nuclei of the interior (FOE and ALBERTS 1983) that contain polyploid, fragmented complements. SULLIVAN et al. (1990) have suggested that the interior is a repository for misbehaving nuclei. In the case of the dal embryos that SULLIVAN studied, the problems occur after migration to the cortex and the rogue nuclei are actually shuttled back down into the interior. Being unable to repair all damage during the rush of the early syncytial divisions, Drosophila has a system of nuclear exclusion, and a program of nuclear death and degeneration, that removes damaged nuclei from the part of the egg that will actually form the embryo. With Rex acting so early, there are few if any un- damaged nuclei to migrate to the surface in any case, and the embryo aborts. The furthest-developed of our damaged embryos look very much like the dal embryos, except that Rexdamaged embryos, unlike those from dal, rarely show any cortical development at all.

Despite the fact that nuclear division ceases early in most of these embryos, replication obviously proceeds

for a while, albeit in a rather deranged fashion. Surely this supports the notion that check points at which damaged nuclei are held for repair are lacking during these extremely rapid divisions (RAFF and GLOVER 1988; HARTWELL 1991). But it also implies that there must be some mitotic controls still acting; in Rex-damaged embryos replication proceeds, but division usually does cease. It would be interesting to see what components of the cell’s machinery continue to function, and what elements of the cytoskeleton remain intact, when chromosome damage causes mitotic arrest.

These observations also must be seen as a caution flag for those interested in using mutants, such as f s (1 )Ya (LIN and WOLFNER 1991) and pZu (SHAMANSKI and ORR- WEAVER 1991), to dissect mitotic regulation in early embryos. Rex also causes early developmental arrest, but it is not a loss-of-function mitotic mutant. It is a repeated element whose positive action causes chromosome breakage that may well be restricted to the rDNA. It is the chromosome damage that has a profound effect on mitotic behavior. Itwill not be easy, and it will take great care and some intellectual discipline, to separate mutant effect from cellular response when faced with the nearly unbridled progress of the syncytial mitoses.

This work was supported by National Science Foundation grants MCB 8919178 and MCB 9305846 to L.G.R, and Italian National Re- search Council grant CR. No. 91.00012PF99 of Progetto finalizzato “Ingegneria Genetica” to S.P., and was largely done while L.G.R. was a National Institutes of Health Fogarty Center Senior International Fellow (1 F 0 6 TWO1657-01) at the Istituto di Genetica, Universiti di Ban. We appreciate critiques of a draft provided by PETER CRAWLEY, MAURIZIO GATTI, ELLEN SWANSON and MARK THOMPSON.

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Communicating editor: R. E. DENELL

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