TO understand the evolution of developmental regu- served in both invertebrates and vertebrates (Murtha

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1 Copyright 2000 by the Genetics Society of America A Genetic Screen for Zygotic Embryonic Lethal Mutations Affecting Cuticular Morphology in the Wasp Nasonia vitripennis Mary Anne Pultz, Kristin K. Zimmerman, 1 Neal M. Alto, 2 Matt Kaeberlein, 3 Sarah K. Lange, 4 Jason N. Pitt, 5 Nick L. Reeves 6 and Darin L. Zehrung 7 Biology Department, Western Washington University, Bellingham, Washington Manuscript received September 14, 1999 Accepted for publication November 11, 1999 ABSTRACT We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera; Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F 1 females. We describe 74 of 100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila. TO understand the evolution of developmental regu- served in both invertebrates and vertebrates (Murtha latory gene systems, we need to identify genes of et al. 1991); in contrast, bicoid, which also plays a very interest and to examine their functions in relatives of fundamental role in Drosophila embryogenesis, dithe best-characterized genetic model organisms. A com- verges rapidly within the Diptera (Schröder and parative developmental genetic approach the isola- Sander 1993; Stauber et al. 1999). Comparisons among tion and analysis of recessive loss-of-function mutations distantly related organisms reveal the most profoundly in new model genetic organisms emphasizes the study conserved relationships, but comparative studies using of gene functions and eliminates the bias of studying more closely related organisms are also important for only homologs of previously identified genes. understanding in full detail the spectrum of evolution- Genetic pathways and their individual components ary conservation and change in developmental reguladiffer in degree of evolutionary conservation. For exam- tory genes (Raff 1996; Eizinger et al. 1999). ple, some of the genes involved in anteroposterior pat- Work in the flour beetle Tribolium castaneum first demterning in Drosophila, such as caudal, are widely con- onstrated the value of a comparative genetic approach to understanding insect development. For example, the Tribolium ortholog of the Drosophila pair-rule gene Corresponding author: Mary Anne Pultz, Biology Department, Westfushi tarazu (ftz) is expressed in a pair-rule pattern, yet ern Washington University, Bellingham, WA pultz@biol.wwu.edu this gene can be deleted without producing any pair- 1 Present address: College of Pharmacy, University of Georgia, Athens, rule segmentation defect (Stuart et al. 1991; Brown GA et al. 1994). Tribolium thus does not share with Drosoph- Present address: Department of Cellular and Developmental Biology, Oregon Health Sciences University, Portland, OR ila the unique role of ftz in patterning embryonic seg- 3 Present address: Department of Biology, Massachusetts Institute of mentation. Genetic screens in Tribolium have identified Technology, Cambridge, MA developmental mutants with gap, pair-rule, homeotic, 4 Present address: Huntsman Cancer Institute Center for Children, and other phenotypes (Denell et al. 1996; Sulston Department of Oncological Sciences, University of Utah, Salt Lake City, UT and Anderson 1996; Maderspacher et al. 1998). The 5 Present address: Division of Basic Sciences, Fred Hutchinson Cancer comparison of these genes with their Drosophila coun- Research Center, Seattle, WA terparts is placing the details of Drosophila develop- 6 Present address: Department of Biology, University of California, San mental gene functions into evolutionary perspective. Diego, La Jolla, CA Present address: Program for Appropriate Technology in Health, We have chosen the solitary parasitoid wasp Nasonia Seattle, WA vitripennis (formerly known as Mormoniella) as an ex- Genetics 154: (March 2000)

2 1214 M. A. Pultz et al. perimental organism for comparative developmental approximately the first 100 zygotic embryonic lethal genetic study of embryonic patterning. Nasonia (Hymenoptera) is more closely related to Drosophila (Diptera) this approach, we have identified both similarities and morphological mutations isolated in Nasonia. Through than is Tribolium (Coleoptera), although both the Hymenoptera and the Coleoptera diverged from the Dip- functions of Nasonia and Drosophila. differences in the zygotic embryonic patterning gene tera between 200 and 300 mya (Hennig 1981). Unlike Tribolium, Nasonia shares with Drosophila a long germ band mode of embryogenesis (Bull 1982). Therefore, MATERIALS AND METHODS the comparative study of embryonic patterning in Na- Host pupae: Nasonia stocks were maintained on Sarcophaga sonia and Drosophila will illustrate which features of bullata pupae, purchased from Carolina Biological or raised embryogenesis have diverged in two insects with mor- on site. Nasonia embryos were collected using Calliphorid phologically similar modes of development. Interestor on Sarcophaga pupae. Drosophila were raised on instant pupae, purchased as larvae from Border Bait (Porthill, ID), ingly, multiple lineages of Hymenopterans have Drosophila medium (Carolina Biological), and embryos were switched from syncytial to holoblastic embryonic cleavcollected on molasses-agar plates. age, or to polyembryonic development (Grbic et al. Genetic strains: Marker gene alleles and their linkage posi- 1996, 1998; Grbic and Strand 1997; Strand and tions are based on the 47-marker genetic map of five Nasonia Grbic 1997). This raises the question whether the genumeral to each marker allele to indicate its linkage group. linkage groups (Saul et al. 1967). We have appended a Roman neric Nasonia syncytial embryo might have novel gene The linkage map was constructed using mostly radiationfunctions or unique features of its developmental proinduced mutant alleles of marker genes. The location of gram that could facilitate such evolutionary transitions. black-424 (bk-424) on linkage group III is tentative, according Nasonia s primary advantage as a genetic organism to the original map. Although reverent originally appeared to derives from its method of sex determination. As in be closely linked to oyster, the two loci assort independently other Hymenoptera, fertilized eggs develop as diploid in our hands (data not shown). We have kept the original linkage group I designation for reverent, assuming that the females while unfertilized eggs develop as haploid (mooriginal map distance may have been reduced due to interfernoploid) males, facilitating screens for recessive zygotic ence from an internal chromosomal rearrangement that was mutations (Whiting 1967). However, unlike most other subsequently lost. Integration of these visible markers with a Hymenoptera, Nasonia does not determine sex through 100-marker, 765-cM molecular linkage map will now be possicomplementary sex determination (CSD), a system in ble (Gadau et al. 1999). Markers built into the EMS screens were purple plum -I (pu pm ), which homozygosity for a sex-determining locus specireddish-5-ii (rdh-5) and scarlet-5219-iii (st-5219). For phenofies male development (Beukeboom 1995). Instead, Na- typic descriptions of these markers, see Figure 1. For the diesonia has been hypothesized to determine sex through poxybutane (DEB) screen, we used the quadruple marker an imprinting mechanism (Dobson and Tanouye combination pu pm ; rdh-5; st-5219, bk-424. scarlet, black eyes are 1998). This is of practical significance, because the abadditional markers used for mapping embryonic lethal muta- white, and this mutant phenotype is epistatic to reddish. The sence of CSD in Nasonia facilitates the maintenance tions included reverent-i (rev), oyster-i (oy), vestigial-i, distantenof highly inbred genetic stocks. Genetic resources for napedia-ii, blue-13-iii, orange-123-iv, scarlet-318-v (st-318), and Nasonia include visible genetic markers for each of the five mickey mouse-v (mm). All of the above are allele designations chromosomes (Saul et al. 1967) and a randomly ampliand Perrot Minnot 1999). For wild-type Nasonia, we used from Saul et al. (1967), except for distantennapedia (Werren fied polymorphic DNA map of the genome (Gadau et the competent (comp ) strain, or an isogenic line of this al. 1999). In addition, a triploid stock generates fertile strain (comp B1) constructed through six generations of diploid males that can be used to construct double- sister-brother pair matings. mutant combinations of embryonic lethal mutations Screening for embryonic lethal mutations: Figure 1 outlines (Whiting 1967; M. A. Pultz, unpublished data). The the design of the genetic screen. Parental wild-type males small adult size and rapid life cycle of Nasonia (similar (comp or isogenic comp B1) were mutagenized 1 day after eclosion with freshly opened EMS (0.025 m; asinlewis and to Drosophila in both respects) contribute to ease of Bacher 1986) or DEB (0.005 m), delivered in 400 l of10% handling in the laboratory. Stock maintenance is facili- honey water wicked onto the side of a glass vial. The time of tated by maintaining mutant strains as refrigerated dia- exposure to the mutagen was varied from 3 to 12 hr for EMS pause larvae for as long as 16 months (Schneiderman and from 3 to 24 hr for DEB (see Table 1). Males were fed and Horowitz 1957). prior to mutagenesis because starved males did not mate after mutagen treatment. In our hands, the mutation frequency was To initiate a study of embryogenesis in Nasonia, we limited by difficulty in obtaining daughters after administering screened for embryonic lethal mutants that fail to hatch EMS in longer exposures or higher concentrations. However, and have defects in larval cuticular morphology. Within recent work has shown that both longer exposures and higher this group, we focused on mutations affecting anteropersonal communication). concentrations can be successfully administered (C. Trent, posterior patterning. This approach allows for direct comparison to work in Drosophila (Nüsslein-Volhard The mutagenized males were allowed to mate for 1 day with genetically marked females (Figure 1) and then discarded. and Wieschaus 1980; Jürgens et al. 1984; Nüsslein- The parental females were cultured at 18. These females Volhard et al. 1984; Wieschaus et al. 1984). Here we were cultured individually to determine whether any parental present an overview of mutant phenotypes based on female was already carrying a spontaneous embryonic lethal

3 Nasonia Embryonic Lethal Mutants 1215 mutation, in which case the mutation would be carried by half linked to the markers built into the screen, it was then crossed of her daughters. We detected such preexisting spontaneous and backcrossed to other marker loci and evaluated similarly. embryonic lethal mutations in only three isolated cases: un- By crossing to a set of 12 marker loci representing the five shaven, spontaneous-4, and one mutant that was not further linkage groups, linked markers were identified for each of characterized. the 12 lethal loci so analyzed. The F 2 male mapping data were The age and handling of the F 1 females proved critical also congruent with female data in these cases, except that for efficient screening: they must be old enough to lay eggs the reverent-i marker is not 100% penetrant. efficiently when set unmated, but young enough for successful Stock maintenance: Embryonic lethal mutations are main- pair mating after the embryonic progeny have been scored tained as diapause larvae, an overwintering stage that is maternally for mutations. Virgin females were set for egg collections 2 induced in response to short day length and cool temperfor days ( 25 ), or 3 days (22 25 ) after eclosion. When set atures (Schneiderman and Horowitz 1957). Our current unmated, females lay all haploid male progeny. They were set stock maintenance protocol is as follows. Females bearing the individually at 28 overnight. Each female was given one host dominant wild-type allele of a linked marker gene eclose in pupa, oriented in a P1000 pipet tip so that only the head of the dark at 18 and are then set unmated to identify the lethal, the host was accessible for egg laying. The next day, embryos marker /lethal, marker genotypes. Each lethal-bearing female were removed from inside the host pupal cases with a moistened is pair-mated to a male mutant for the linked marker gene. fine brush and placed onto a grid on a 1% agar plate. The matings are observed, to avoid subculturing unmated The females were held in a refrigerator (8 ) while the embryos females. Females are set individually in the dark at 16, because developed to hatching for 24 hr at 28. Most mothers laid females cultured individually lay diapause larvae more readily. from 12 to 30 eggs under these conditions. Females that did The females are subcultured, removing parasitized pupae and not lay at least 12 eggs were reset once, then discarded if they adding two to three fresh pupae, twice per week for 3 4 wk. did not lay on the second setting. Reset frequencies ranged By the third week, most females will be laying progeny that from 10 to 35% and were sensitive both to female age and will enter diapause. Approximately 8 15 individual female to host quality. lines are set every 6 months for each stock not all mated After 24 hr at 28, plates were examined to determine which lines produce daughters, and some mothers will have depleted mothers had laid 50% unhatched embryos. A probability their supply of sperm before beginning to lay diapausing prog- table was used to determine the minimum number of eggs eny (older mothers are not likely to remate). Mothers produce that should be unhatched (0.95 probability) for a given total 30 progeny per host pupa and some produce as many as number of eggs laid. For example, if an embryonic lethal 90% females, because they fertilize most of the eggs. Diapause bearing mother laid 12 eggs, at least three embryos should larvae are stored in the refrigerator (8 ). Since stocks must be unhatched. The incidence of F 1 germ-line mosaics should remain in diapause for at least 4 months and can remain in be negligible because most Nasonia embryos form only a single diapause for up to 16 months, stock maintenance twice per pole bud, with a single nucleus that divides to generate a year allows sufficient time to verify that there are daughters population of pole cells (Bull 1982). When working with (by breaking diapause) before the previous cultures are too Nasonia, there is no background of eggs that fail to develop old to use. After 4 months, stocks can be removed from diapause because they have not been fertilized. at any time by returning them to room temperature. To score for cuticular phenotypes, the unhatched embryos Using this system, about two dozen embryonic lethal stocks of interest were mounted in 90% lactic acid/10% ethanol, can be reliably maintained with 10 hr per week of continuous cleared at 56, and examined using dark-field optics. In most effort, making the economics of embryonic lethal stock maintenance cases, the unhatched embryos showed no obvious cuticular very similar to what has been described for Tribolium defects, or the defects were very subtle or highly variable. The (Berghammer et al. 1999). mothers of such embryos were discarded. In a small percentage, We established diapause lines from F 2 females for all of the the unhatched embryos had died early before elaborating 100 mutant lines with distinct heritable phenotypes, to test cuticle. These were cloudy in appearance and lacked the white the efficiency with which diapause stocks could be established gut contents of mature embryos. The mothers of these em- for a large group of mutants. Diapause was obtained for 70 bryos were also discarded. The percentage of lines in the latter 80% of the stocks on the first round and for 100% within category was difficult to determine precisely, because of a three rounds of culturing as above. However, we found that variable low background level of embryos that died early in the percentage of diapause cultures with female progeny was development. In each case where the embryos showed consis- highly variable among different stockkeepers, varying from tent cuticular defects, the mother was selected and mated to 90% to 50%. This variation probably depended largely on a genetically marked male (Figure 1). Approximately 85% how carefully the mothers were handled. Since the percentage of the selected females produced sufficient F 2 daughters for of cultures with female progeny cannot be determined until at further mutant analysis, genetic mapping, and stock mainte- least 4 months after cultures have been established, embryonic nance. After the progeny of F 2 females were examined, approximately lethal diapause stocks should be kept by an established and one-third of these lines were discarded, either because skilled stockkeeper. there was no phenotype or because the phenotype was excessively A few embryonic lethal stocks, notably headless, head only, variable. and speechless, acquired weakened phenotypes in some lines Genetic mapping of lethal mutations: Linkage analysis of after several generations of stock maintenance, as though hav- F 2 progeny is explained in Figure 1. The F 2 adult male data ing acquired a spontaneous suppressor mutation linked to were generally congruent with the female data, indicating that the mutation of interest. Such variability of phenotypic there was little or no bias of haploid male survival for the strength with genetic background does not appear to be a markers used, even in the triple-mutant marker combination. phenomenon that distinguishes Nasonia from Drosophila. For Because of the fairly low mutation frequency, we found only example, various stocks of Drosophila caudal null alleles differ two lines with spurious lethal mutations linked to marker loci. in the strengths of their mutant phenotypes in a manner Two additional lines yielded linkage results that were not that resembles the variable strengths of mutant phenotypes interpretable. Overall, the male data were highly reliable at in different lines of the single head only mutant allele (M. A. indicating probable linkage relationships for mutations of in- Pultz, unpublished observations). Using the stock maintenance terest. If a mutant of special interest was found not to be procedures described above, an embryonic lethal mu-

4 1216 M. A. Pultz et al. tant phenotype can change abruptly if a closely linked sponta- mutations with defects in cuticular morphology. EMS neous suppressor mutation is acquired as a mutation of produces mostly point mutations, identifying single interest is passaged through a single-female culture. Maintegenes that can be readily mapped meiotically. DEB often nance and phenotypic monitoring of parallel cultures allows for choice of lines with consistent phenotypes, and diapause produces small deletions (or other chromosomal re- stocks preserve access to genotypes that have been maintained arrangements). These are more likely to be null mutaduring the previous 16 months. tions and are potentially useful for molecular testing Estimate of the percentage of lethal mutations affecting of candidate gene hypotheses. In the EMS screen, we embryonic morphology: The total percentage of embryonic lethal mutations for the EMS screen was calculated from data recovered all mutations with morphological defects; in for the 4456 EMS-mutagenized genomes shown in Table 1. the DEB screen, we chose to recover a more limited This percentage was applied to the total of 6937 genomes subset of the mutations. We also describe the phenomutagenized with EMS to obtain an estimate of 600 embry- types of several spontaneous embryonic lethal mutations onic lethal mutations for the EMS screen. Approximately 100 that were recovered in the course of mutant screening EMS-induced mutations affecting embryo morphology were isolated, so the percentage is estimated as 17% of all embryonic or analysis. lethal mutations. For embryonic lethal screening, mutagenized males Recovery of spontaneous embryonic lethal morphological were mated to females homozygous for visible markers mutations: In addition to the mutations induced by EMS or on two or three of the five linkage groups, using the DEB, we have described here the phenotypes of five spontane- protocol outlined in Figure 1. Analysis of markers identious mutations that were found incidentally. Two (unshaven and spontaneous-4) were found while screening for induced fied a subset of the embryonic lethal mutations by linkmutations, as explained above. The other three (spontaneous- age group in the F 2 generation. The linkage data distin- 1, -2, and -3) were identified among the progeny of wild- guished some cases where more than one gene could type females. These were found while prescreening wild-type be mutated to produce a given mutant phenotype. (comp ) females to verify that they were not carrying lethal These data also confirmed that mutations of interest mutations when being used as controls for experiments. Fixation and antibody staining: Selected anteroposterior were zygotic and that they arose on the chromosomes patterning mutants and Drosophila giant mutant embryos of parental males. were stained with phylogenetically cross-reactive monoclonal Approximately 100 embryonic lethal mutations were antibodies: 4D9 recognizes ENGRAILED (Patel et al. 1989) isolated, of which 74 are described here. All mutants and FP6.87 recognizes both ULTRABITHORAX and ABDOMwere analyzed for cuticular defects, and selected mu- INAL-A (Kelsh et al. 1994). Fixation, mass devitellination of Nasonia embryos in cold 1:1 heptane:methanol, and antibody tants were assayed for engrailed or trunk Hox gene exstaining were carried out as described previously (Pultz et al. pression. Only recessive embryonic phenotypes were 1999). To collect embryos mutant for Drosophila giant, y 1,sc 1, analyzed we have not examined heterozygous females giant x11 /FM6 females were crossed to Canton-S males, giant- in detail for evidence of subtle dominant haploinsuffibearing females were crossed again to Canton-S males, and cient defects. The only striking haploinsufficient phenoembryonic progeny were collected. Complementation testing of embryonic lethal mutations is type that we noted is a low penetrance hunched thorax not straightforward: We did not carry out complementation defect of female pupae carrying the head only mutation. tests, despite the availability of fertile diploid Nasonia males, Mutation frequencies: Table 1 summarizes mutation because this procedure is not straightforward. Nasonia triploid frequencies that were obtained with increasing length females produce mostly aneuploid progeny. When set unof exposure to EMS and DEB. The mutation frequencies mated, their euploid progeny are approximately half haploid males and half diploid males (Whiting 1967). Because Nasonia males are normally haploid, spermatogenesis does not were higher in the mutagenized groups than in the increased with length of exposure to the mutagens and include meiosis I. Therefore, the progeny of diploid males control group. These results indicate that most of the are triploid females. It is difficult to determine whether a embryonic lethal mutations described here were induced triploid female is carrying an embryonic lethal mutation by inspection of embryonic progeny, when most embryos are by EMS or DEB. Overall, the frequency of embryonic dying of aneuploidy, and there is evidence suggesting that the lethal mutations per Nasonia genome was 9%; the maxi- chromosomes of triploid females do not segregate randomly mum frequencies were 18% for EMS and 15% for DEB. (Dobson and Tanoye 1998). Triploid females carrying two In the Drosophila saturation screens for zygotic mutamutant alleles of an embryonic lethal gene may be poorly tions affecting embryonic morphology, EMS-induced viable or fertile, precluding the necessary analysis of further generations; yet such a phenotype cannot be rigorously internome (Jürgens et al. 1984) to 82% per genome (Nüss- embryonic lethal frequencies ranged from 35% per gepreted as allelism rather than second-site noncomplementation. It may be possible to use this procedure to establish lein-volhard et al. 1984). On a per-chromosome basis, allelism in cases of exceptional interest, and we are currently mutation frequencies ranged from 8% (Wieschaus et investigating whether this approach can be used to test al. 1984) to 33% (Nüsslein-Volhard et al. 1984). whether alternate and five band are allelic. However, complementation testing of embryonic lethal mutations using diploid Wild-type Nasonia: As background to the description males is not routinely feasible in Nasonia. of mutant phenotypes, we first briefly describe cuticular features of the wild-type Nasonia first instar larva and the embryonic expression of engrailed and trunk Hox RESULTS genes. The wild-type first instar larval cuticle is shown We screened 6937 EMS-mutagenized genomes and in Figure 2, A C. A denticle belt delineates each of the 1126 DEB-mutagenized genomes for embryonic lethal 3 thoracic and 10 abdominal segments; each denticle

5 Nasonia Embryonic Lethal Mutants 1217 TABLE 1 Frequency of embryonic lethal lines after different mutagen exposures Embryonic lethal lines Genomes screened No. % Control EMS (0.025 m) 3 hr hr hr hr hr hr hr Total a DEB (0.005 m) 3hr hr hr hr Total a Figure 1. Screening for zygotic embryonic lethal mutaestimate that the mutation frequencies for the additional ge- A total of 6937 genomes were mutagenized with EMS. We tions. Parental multiply marked females were mated to mutanomes screened were approximately equivalent to the average genized parental wild-type males. F 1 females were set unmated to score haploid male progeny, and then each female bearing for those recorded here. a mutation of interest was mated to carry the mutation through the female line. We have illustrated at each generation a hypothetical example in which a newly induced mutation is closely the embryo bears a tubular anus that everts after linked to the scarlet eye-color marker. Approximately 3500 EMS-mutagenized genomes were screened using the triplehatching. mutant pu pm -I; rdh-5-ii; st-5219-iii genotype indicated here. The To follow expression of the segment polarity gene remainder of the EMS-mutagenized genomes were screened engrailed (en) in Nasonia, we used the monoclonal anti- using the double-mutant genotype rdh-5; st pu, purple body 4D9 (Patel et al. 1989). As gastrulation begins, body color; rdh, reddish eye color; st, scarlet eye color. The rdh-5; the first EN stripes appear in the antennal, mandibular, st-5219 double-mutant phenotype is distinguishable from those of the single mutants. Linkage of a zygotic lethal mutalary and maxillary stripes and then by thoracic stripes and labial segments, followed soon thereafter by interca- tion to a marker gene would first be deduced by scoring marker phenotypes of the F 2 (haploid) male progeny. These (Figure 2D). At the onset of expression, the labial EN data are designated m in Table 2. To verify that the lethal stripe is wider than the other stripes, as in bees (Flieg mutation linked to the marker was the mutation of interest, 1990), and the trunk stripes are two cells in width. As F 2 females were sorted by marker and then set unmated to score the embryonic progeny of each female. These data are the germ band extends, with both head and tail moving designated f in Table 2. For example, st-5219: m,31/161; dorsally, EN is expressed in 5 head stripes and 12 trunk f,5/24 indicates that 31 of 161 surviving F 2 males were st, stripes (Figure 2E). The head stripes take on characteris- putative crossover events and that 5 of 24 F 2 females repre- tic morphologies that have been described for a variety sented putative crossover events (including both phenotypi- of other insects, such as the dorsal fusion of the maxilcally st females that carried the embryonic lethal mutation and phenotypically st females that did not carry the lethal lary and labial stripes and the formation of intercalary mutation). These results would indicate that the embryonic spots (Rogers and Kaufman 1996). The number of lethal mutation is located 20 cm from st-5219 (see also materials and methods). aged Drosophila embryos (Dinardo et al. 1985). trunk stripes at this stage is the same as in comparably To follow the expression of trunk Hox genes, we used the monoclonal antibody FP6.87 (Kelsh et al. 1994), belt grades from finer denticles anteriorly to coarser which recognizes both ULTRABITHORAX (UBX) and denticles posteriorly. Large spiracles are located on the ABDOMINAL-A (ABD-A). Expression of UBX-ABD-A in second thoracic and first three abdominal segments. Nasonia is similar to the expression patterns for these The larval head bears a dorsolateral pair of antennal genes in Drosophila and Tribolium (Kelsh et al. 1994; papillae, and the ventral mandibles are supported by a Castelli-Gair and Akam 1995; Shippy et al. 1998). chitinized ring the anterior arch, the epistoma, is a labral derivative and the posterior arch, the tentorium, is a first thoracic derivative (Azab et al. 1967). Caudally, Weak expression extends from the posterior second through the third thoracic segments; strong expression extends from the first through the seventh abdominal

6 1218 M. A. Pultz et al. Figure 2. Wild-type Nasonia and axial mutant phenotypes. Anterior is to the left; dorsal is up in all except B, C, and G. (A F) Wild type. (A) First instar larval cuticle. Arrowheads, spiracle-bearing second thoracic and first three abdominal segments. (B) First instar ventral larval head, after Azab et al. (1967); ant, antennal sensory papillae; epi, epistoma; mn, mandibles; r, chitinized rod; tent, tentorium. (C) First instar larval tail. (D) Initiation of EN expression; ant, antennal; int, intercalary; mn, mandibular; mx, maxillary; lab, labial. For the EN panels, the arrowhead indicates the antennal stripe and the arrow indicates the labial stripe. (E) Elaboration of EN expression as the germ band is extending. The tail of the embryo has become straightened during fixation. (F) UBX-ABD-A in segmenting embryo. T2, second thoracic; A8, eighth abdominal. (G) Cuticle of head only (ho) mutant. (H) Cuticle of headless (hl) mutant. (I) Cuticle of squiggy (sq) mutant. (J) Cuticle of expanded (exp) mutant. (K) EN initiation in exp mutant embryo. The posterior abdominal EN stripes are not yet expressed. Bars, 50 m. rior gaps. The anterior gap includes thoracic, gnathal, and more anterior head segments, and the posterior gap includes the posterior three abdominal segments. squiggy (sq) mutant embryos lack anterior and posterior segments only four midtrunk segments are consistently present. ho has been hypothesized to be Nasonia caudal, and hl has been hypothesized to be Nasonia hunchback. sq has no obvious counterpart in Drosophila (Pultz et al. 1999; also see discussion). Expanded thorax: In expanded (exp) mutant embryos, thoracic segments are widened. Posterior to the thoracic region, abdominal segments are compressed; anteriorly, the tentorium is defective (Figure 2J). The tentorium has been described as a first thoracic derivative (Azab et al. 1967). exp affects EN expression (Figure 2K): as EN initiates, the EN thoracic stripes are already farther apart and the abdominal stripes more closely spaced than in wild-type embryos. In some embryos the expres- sion of the first thoracic EN stripe is also reduced. The spacing of EN stripes in the head is not expanded or moved posteriorly and may be slightly compressed ante- segments, while the eighth abdominal segment stains more weakly than the anterior abdominal segments (Figure 2F). Description of mutant phenotypes: Table 2 briefly describes mutant phenotypes for 74 embryonic lethal lines. Because such mutations are relatively easy to isolate but costly to keep (see materials and methods), only a subset of these mutations are maintained currently. The mutant isolation names are included as a part of the description of mutant phenotypes. The mutations are organized by phenotype, and data indicating probable linkage (or lack of linkage) of the embryonic lethal loci to marker loci are included. The mutant phenotypes are described below. Axial defects with large gaps: The head only, headless, and squiggy mutant phenotypes (Figure 2, G I) have been described in detail (Pultz et al. 1999). These all have large regions where several contiguous segments fail to form properly. head only (ho) lacks thoracic and abdominal segments, and caudal structures are lacking or defective. headless (hl) embryos have both anterior and poste-

7 Nasonia Embryonic Lethal Mutants 1219 TABLE 2 Summary of mutant phenotypes and linkage data Axial defects with large gaps head only a (mm-v: m,13/2006; f,0/71): b defective development of trunk segments headless a (rev-i: m,25/79; f,83/255): large anterior gap, also lacks posterior segments squiggy (rdh-5-ii: m,32/127; f,4/22): lacks head, anterior thorax, posterior abdominal segments Expanded thorax expanded (rdh-5-ii: m,5/56; f,1/12): thorax expanded, abdomen compressed, tent defective Small gaps in thorax and abdomen minus stripes a (mm-v: m,37/139; f,13/45): defects in T1, T2, region of A6-A7 Pair-rule or ectopic denticles odd-defective a (pu-i: m,7/98; f,3/43): pair-rule, odd abdominal segments defective five band-1 a (mm-v: m,1/117; f,0/28): pair-rule, even abdominal segments defective five band-2 a (mm-v: m,1/111; f,0/18) (DEB): c like five band-1 alternate a (mm-v: m,0/67; f,1/84): pair-rule, weak allele, even abdominal segments defective big hair a (oy-i: m,58/821; f,9/147): denticle lawn with naked strip posteriorly unshaven a (st-318-v: m,11/135; f,2/35): (spontaneous) denticle lawn speckled a (st-318-v: m,2/33; f,2/34) (DEB): like unshaven rambutan a (rev-i: m,24/107; f,13/61): ectopic denticles Polycomb-like mustache a (rev-i: m,26/83, f,11/62): missing or ectopic spiracles, head and dorsal closure defective Segment fusions throughout spontaneous-4 (NL: rs,139): segment fusions, denticle belts with normal polarity helter skelter (rdh-5-ii: m,3/58, f,2/15): segment fusions, denticle belts with normal polarity confused (rdh-5-ii: m,22/77; f,nd): segment fusions, denticle belts with normal polarity Y-stripes (NL: rsp,20): segment fusions, denticle belts with normal polarity Deep grooves gnarled (NL: rs,201): head sometimes separated from body, probable mouthparts in posterior spontaneous-2 (oy-i: m,7/307; f,nd): deep grooves with incompletely penetrant pair-rule register Long body wormy (NL: rs,36) (DEB): body elongated, normal segment number salamander (ND): body elongated, normal segment number Small cuticle h-small (rdh-5-ii: m,1/20; f,1/10): small cuticle dorsally another small (NL: rs,19): small cuticle dorsally new small (rdh-5-ii: m,0/30; f,nd): small cuticle dorsally classic small (ND): small cuticle dorsally scrunched (NL: rsp,19): small cuticle ventrally ventral only (NL: rs,65): small cuticle ventrally Dorsal defects fadeaway (NL: rsp,72): only ventral denticles in most segments bareback (ND): few denticles dorsally n-bareback (NL: rsp,31): few denticles dorsally sparse dorsal-2 (NL: rsp,185): few denticles dorsally disclosure (rdh-5-ii: m,1/6; f,1/10): fails in dorsal closure Anterior defects toothless a (st-5219-iii: m,31/161; f,5/24): anterior head skeleton missing, ectopic sense organs speechless (NL: rs,17): no ant, head skeleton lacking anterior structures or completely missing embryonic head-2 (NL: rs,180): anterior head skeleton missing, not like toothless flasher (rdh-5-ii: m,0/76; f,1/15): no ant, head skeleton reduced or missing, or anterior unformed extra skeleton (NL: rs,24): duplicated head skeletal structures head hole (rdh-5-ii: m,2/21; f,0/15): holes in cuticle in neck region (Continued)

8 1220 M. A. Pultz et al. TABLE 2 (Continued) Anterior defects (continued) neck (NL: rs,35): neck region elongated, with anterior holes mouth joint (NI): connection of tentorium to anterior head skeleton disrupted or branched m-flexed (st-5219-iii: m,1/12; f,0/5): tentorium not attached tent-17 (NL: rs,7): tentorium defect double chin (ND): duplicated tentorium, T1 and dorsal T2 denticle belts missing weak-h (rdh-5-ii: m,0/21; f,0/3): tentorium incomplete, ventral T1, T2 denticle belts missing m-1 (NL: rs,45): tentorium and T1 denticle belt missing, posterior denticles fade out boring (st-5219-iii: m,18/117; f,0/3): tentorium and T1 denticle belt missing t&t(nl: rsp,9): tentorium variable, T-shaped gut consistent, variable abdominal tracheae t-gut-2 (NL: rsp,138): tentorium defect and T-shaped gut Defects of every trunk segment threadbare (NL: rs,47): segment boundaries formed, few or no denticles j-threadbare (NL: rsp,28): segment boundaries formed, few or no denticles d-threadbare (ND) (DEB): segment boundaries formed, few or no denticles spontaneous-1 (ND): segment boundaries formed, few or no denticles nude (NL: rs,67): denticle rudiments visible with phase contrast but not with dark-field optics m-nude (NL: rs,12): denticles absent in whole embryo or in posterior, mandibles only striptease (NI): partially naked, irregularly spaced segment boundaries n-threadbare (NL: rs,46): partially naked, irregularly spaced segment boundaries seminude (ND): denticles mostly absent, no regular segment boundaries, tent absent dribble (NL: rsp,16): denticle belts with one denticle row if any, mouthparts disorganized spontaneous-3 (ND): single row of denticles in each segment single line (ND) (DEB): single row of denticles in each segment chipped tooth (rdh-5-ii: m,38/112; f,nd): thin denticle belts, severely reduced mouthparts thin denticles (rdh-5-ii: m,1/34; f,0/13): thin denticle belts peppery (rdh-5-ii: m,0/17; f,0/10): sparsely scattered fine denticles coarse unruly (NL: rs,182): somewhat scattered coarse denticles thin stripes (NL: rs,57): thin denticle belts, segment polarity problems, no tent band polarity (NL: rs,87): denticle belts with mirror-image polarity wrong way (NL: rs,50): denticle belt polarity defects wrinkled (NL: rsp,39): segment boundaries have thin denticle belts on both sides lots of segments (NL: rsp,37) (DEB): appears to have extra segment boundaries Posterior defects pinched (st-5219-iii: m,12/34; f,nd): posterior abdominal segments narrow, 50% expressivity skinny tail (st-5219-iii: m,2/10; f,nd): tail thin and curled upwards no posterior denticles (rdh-5-ii: m,0/25, f,1/9): ventral and posterior denticles reduced posterior scrunched (rdh-5-ii: m,3/24; f,nd): posterior of embryo compressed Summary of mutants described: 64 EMS-induced, 5 DEB-induced, and 5 spontaneous mutants. Underlined mutants are shown in figures. ant, antennal sensilla; tent, tentorium; T, thoracic; A, abdominal. a Indicates mutant strains currently maintained. b For mutants mapped with respect to multiple marker loci, the closest marker is indicated. NL, not linked. ND, not done. NI, not interpretable. r, rdh-5-ii; s, st-5219-iii; p, pu-i; For cases of potential linkage, putative crossover events per total for male data (m) and female data (f) are indicated (see Figure 1). If not linked, m and f data are combined. NL: rs,45 means that there is no apparent linkage to r or s, based on 45 individuals. c DEB-induced and spontaneous mutants are indicated; all others are EMS-induced mutants. Four spontaneous mutants were so named. riorly. The exp mutant phenotype appears to be caused by a modest expansion of the thoracic region of the embryonic fate map at the expense of both anterior and posterior embryonic regions. There is no clear counterpart of exp in Drosophila. Small thoracic and abdominal gaps: The minus stripes (ms) cuticular mutant phenotype is shown in Figure 3A. In ms mutant embryos, the first and second thoracic denticle belts are missing or defective. The tentorium is usually formed completely, but is often misshapen (not shown). Abdominal segments are disrupted in a variable region around the sixth and seventh abdominal segments. Denticle belts are missing ventrally and fused dorsally in this region. The ms mutant phenotype invites comparison to that of the gap gene giant in Drosophila. giant mutant embryos have defects of the labial segment, with transient anterior thoracic defects during embryogenesis, and these embryos also have missing or

9 Nasonia Embryonic Lethal Mutants 1221 Figure 3. Comparison of Nasonia minus stripes to Drosophila giant. Anterior, left; dorsal, up. (A and C) First instar larval cuticle and EN expression of Nasonia minus stripes mutant embryos. The arrow in A indicates the lack of a second thoracic spiracle. (B and D) First instar larval cuticle and EN expression of Drosophila giant X11 mutant embryos. giant X11 is an amorphic allele (Petschek et al. 1986). mx, maxillary; lab, labial; T1/T2 indicates fusion or partial fusion of EN in first and second thoracic segments; A6/A7 indicates fusion of sixth and seventh abdominal segments. the odd-numbered abdominal segments. The posteriormost abdominal segments are rarely affected and the second thoracic segment is also less often affected than the anterior abdominal segments, so the od mutation is probably a hypomorphic allele. The od cuticular phenotype is similar to that of Drosophila odd-skipped. The second pair-rule locus, five band (fb) is represented by one EMS-induced mutation with a strong mu- tant phenotype (Figure 4B) and one DEB-induced mutation with an identical phenotype (not shown). These mutations are both closely linked to the mickey mouse- V(mm) marker locus (Table 2) and are therefore probably allelic. fb mutant embryos have five large denticle belts with mirror-image symmetry, plus an additional posterior ventral denticle field. The first three denticle belts all have spiracles, indicating that second thoracic, first abdominal, and third abdominal identities are maintained while third thoracic and second abdominal identities are not represented. The alternate (alt) mutation is also closely linked to mm (Table 2). alt mutant embryos (Figure 4C) have a weak pair-rule phenotype. Typically, denticle belts in alternate segments are miss- ing or narrowed, lacking the more posterior rows of coarse denticles; the narrowed denticle belts are often fused posteriorly with those of adjacent segments. The second abdominal spiracle is often reduced in size or misplaced, identifying the register of the even abdominal segments as defective. The similarity in the register of pair-rule defects in alt and fb mutant embryos and the close linkage of both loci to mm suggest that alt may be allelic to fb. The fb mutant phenotype is unlike that of any Drosophila pair-rule gene loss-of-function pheno- type. The od, fb, and alt pair-rule mutant phenotypes will be described and discussed in more detail elsewhere. The first gene with a denticle lawn phenotype is big hair (bh), represented by a single EMS-induced temperature-sensitive allele linked to oyster-i (Table 2). At 28, bh mutant embryos have a denticle lawn divided defective denticle belts in a variable region around the sixth and seventh abdominal segments (Figure 3B; Perrimon et al. 1984; Petschek et al. 1986). EN expression in Nasonia ms mutant embryos (Figure 3C) was compared to EN expression in Drosophila giant mutant embryos (Figure 3D). In ms mutant embryos, the head EN stripes all form, but EN expression is defective in the anterior thorax most often, the first and second thoracic stripes fuse. In the posterior abdomen, there is fusion of EN stripes and ectopic EN expression in the region of the sixth and seventh abdominal segments. These defects appear as EN initiates (not shown). Drosophila giant mutant embryos resemble ms mutant embryos in that EN stripes from several posterior abdominal segments are also fused. However, there is no labial EN expression in the anterior defective region of giant mutant embryos (Petschek and Mahowald 1990). Nasonia ms appears to be similar to Drosophila giant in that both have anterior as well as posterior gap defects although the anterior defective regions are differently centered, the posterior defects are very similar. The functional disparity in anterior defects might be due to underlying differences in redundant or overlapping genetic functions in the two organisms, or to possible residual function of the ms allele. An ortholog for giant has been sought but not found in Tribolium, although the Krüppel and hunchback gap genes are conserved (Sommer et al. 1992; Maderspacher et al. 1998). The ms mutant phenotype is the first indication that giant may be conserved as a gap gene beyond the Diptera. Pair-rule and ectopic denticle phenotypes: Eight mutants, representing at least five genes, have pair-rule or ectopic denticle phenotypes. At least two loci have clear pairrule phenotypes and at least three loci have denticle lawn or ectopic denticle phenotypes. The first pair-rule gene, odd-defective (od), is represented by a single EMS-induced allele (Figure 4A). od mutant embryos have reduced or missing denticles in

10 1222 M. A. Pultz et al. Figure 4. Pair-rule and ectopic denticle mutant phenotypes. Anterior, left; dorsal is up in lateral views. Arrows indicate spiracles, fully or partially formed. (A) Cuticle of odd-defective (od) mutant first instar larva. The large spiracles are in the second thoracic and second abdominal segments. (B) five band (fb). (C) Detail of alternate (alt) larval cuticle. In this mutant embryo, the second abdominal spiracle is slightly misplaced but not reduced in size. (D) big hair (bh). (E) bh ventral head. epi, epistoma; mn, mandible. (F) EN initiation in bh mutant embryo. mn, mandibular; pos, posterior. (G) speckled (spe). (H) spe ventral head. tent, tentorium. (I) EN expression in unshaven (unsh) mutant embryo, early germ band elongation. A1, first abdominal. ( J and K) Range of EN expression in older unsh mutant embryos, extending germ band. Ventral (J) and ventrolateral (K) views. Arrowhead indicates ventral lack of EN expression. (L) rambutan (ram). (M) ram ventral head. DEB-induced spe mutation and the spontaneous unsh mutation are closely linked to scarlet-318-v (Table 2) and are probably allelic. spe and unsh mutant embryos have a denticle field (spe; Figure 4G). The number of large spiracles, three or four, is variable even contralaterally within individual embryos. The tentorium is formed, but detached from the more anterior head skeletal structures (spe; Figure 4H). Mandibles are missing or defective, and antennal sense organs are lacking. EN expression is identical in spe and unsh mutant embryos. By the early stages of germ band extension, many of these mutant embryos show a pair-rule periodicity in dorsolateral EN expression, defective in the register of the even abdominal segments (unsh; Figure 4I). Even as EN is initiating, some embryos show a slight pair-rule bias in the strength of trunk EN stripes and weakened expression of the premandibular EN stripes, though such early subtle defects are not always bilaterally sym- metrical (not shown). The number of cells expressing EN diminishes as germ band extension proceeds. About one-third of the mutant embryos lose EN expression equally in all segments (unsh; Figure 4J), while about two-thirds of the embryos show slight to marked pairrule periodicity in the dorsolateral loss of EN expression (unsh; Figure 4K). The latter class of mutant embryos all express EN in every segment in ventrolateral cell clusters. All of the mutant embryos fail to express EN by a posterior strip of naked cuticle (Figure 4D). The denticle lawn region bears a single incompletely formed spiracle on each side of the embryo. When mutant embryos are cultured at 18, the denticle lawn is interspersed with additional disorganized regions of naked cuticle (not shown). bh mutant embryos have antennal sense organs (not shown) and anterior head skeletal structures including mandibles, epistoma, and lateral supporting skeletal structures (Figure 4E). More posterior head skeletal structures and the tentorium do not develop in these embryos. EN fails to initiate normally in bh mutant embryos (Figure 4F). The mandibular and more anterior EN stripes are initiated anteriorly, in the same positions as in wild-type embryos, and a single EN stripe is consistently initiated at the posterior of the embryo. Once initiated, these stripes are maintained as in wild-type embryos (not shown). In Drosophila, evenskipped null mutants also fail to initiate EN in gnathal and trunk segments (Macdonald et al. 1986; Dinardo and O Farrell 1987). In contrast, Drosophila embryos lacking function of the trunk gap gene Krüppel initiate EN ectopically in the defective region of the embryo (Ingham et al. 1986). Therefore, big hair may function at the pair-rule level of gene regulation. The speckled (spe) and unshaven (unsh) mutations identify a second locus with a denticle lawn mutant phenotype, distinct from that of bh mutant embryos. Both the

11 Nasonia Embryonic Lethal Mutants 1223 in the ventralmost region of the embryo. The effects of spe/unsh on EN maintenance indicate that these mutations may identify a segment polarity gene that is not expressed equally or not needed equally in all segments, perhaps because of the regulation of segment polarity genes by the pair-rule gene system. For example, engrailed in Drosophila has a transient pair-rule pattern of expression in wild-type embryos (Dinardo et al. 1985) and a two-segment register to its cuticular phenotype (Nüsslein-Volhard and Wieschaus 1980). The third gene with an ectopic denticle mutant phenotype is rambutan (ram), identified by a single EMSinduced allele linked to reverent-i. At 28, ram mutant embryos have ectopic denticles a denticle lawn interrupted by irregular strips of naked cuticle that are most prevalent dorsolaterally and three or four large spiracles on each side of the embryo (Figure 4L). The phenotype is slightly stronger at lower temperatures, with fewer strips or no strips of naked cuticle interrupting the denticle lawn when the mutant embryos are cultured at 16 (not shown). ram mutant embryos have no tentorium, posterior head skeletal elements, or mandibles (Figure 4M). EN expression was examined only preliminarily in a collection of 40 embryos, ranging in age from the early gastrulation to gnathal lobe formation, collected from unmated ram-bearing mothers. EN was expressed in an approximately normal pattern (though with quan- titative variation) in all embryos and showed no sign of pair-rule periodicity. Function of ram function appears not to be as critical for EN expression as the functions of big hair and speckled/unshaven, but ram may also contribute to the maintenance of normal EN expression. Polycomb-group gene: One gene, mustache (mus), is similar to the Drosophila Polycomb-group genes, based on a combination of cuticular mutant phenotype and effects on Hox gene expression. Embryos with a strong mus mutant phenotype lack antennal sense organs as well as all head skeleton derivatives except for mandibles (Figure 5A). Some mus mutant embryos bear ectopic denticles above the mandibles (Figure 5B), indicating partial homeotic transformation of head to trunk identity. The number of trunk segments in mus mutant embryos is normal, but the spiracle pattern is variable. Less than 5% of mus mutant embryos bear a normal spiracle pattern, and approximately one-third have no spiracles. In most mutant embryos, spiracles are present in the second trunk segment, variably present in the third and fourth trunk segments, and lacking in the fifth and sixth trunk segments. In comparison, Drosophila Polycomb mutant embryos have ectopic denticles on the dorsal head, and the second thoracic segment is anteriorly transformed to prothorax while abdominal segments are posteriorly transformed (Denell and Frederick Figure 5. mustache (mus) mutant phenotypes. Anterior, left; dorsal, up. (A) mus first instar larval cuticle. (B) mus head. Arrow indicates ectopic denticles on dorsal head. (C) Beginning of UBX-ABD-A derepression in mus mutant em- bryo. Arrow indicates labial lobe. (D) UBX-ABD-A expression in mus mutant embryo that has completed germ band retrac- tion. Polycomb mutant embryos fail in dorsal closure of the head region. Functional similarity of mus to Drosophila Polycomb- group genes is supported by the effects of mus on trunk homeotic gene expression. At the earliest stage of UBX-ABD-A expression, mus mutant embryos were not distinguishable from their phenotypically wild-type sib- lings. However, by the time of gnathal lobe formation, ectopic UBX-ABD-A expression in a limited number of more anterior cells is evident (Figure 5C). In much later mutant embryos, after germ band retraction, UBX-ABD-A is expressed strongly throughout most of the anterior of the embryo (Figure 5D). In Drosophila Polycomb mutant embryos, UBX is initiated normally, but is increasingly derepressed during embryonic devel- opment (Wedeen et al. 1986) in a manner very similar to the UBX-ABD-A expression of mus mutant embryos. Segment fusions throughout: Four mutations caused ex- treme segmental fusions throughout the embryo (e.g., spontaneous-4; Figure 6A and Table 2), with only rudi- mentary mandibles developing in the head region. Al- 1983). The mus mutant phenotype may be caused by though segmentation was severely disrupted, the resimilar transformations. mus mutant embryos also fail to maining fragments of denticle belts maintained normal complete dorsal closure (not shown), while Drosophila anteroposterior and dorsoventral polarity (not shown).

12 1224 M. A. Pultz et al. Deep furrows: Two mutations, gnarled and spontaneous-2 (spont-2) both caused variable deep furrows, though the two mutant phenotypes differed in other respects (Table 2). In gnarled mutant embryos (not shown), the deep furrowing often resulted in a complete dissociation of the head from the rest of the body. Anteriorly, only mandibles were formed. Many gnarled mutant embryos also bore what appeared to be poorly developed mandibles at the posterior end of the embryo. spont-2 mutant embryos (Figure 6B) varied in the number of deep furrows present. Spacing of the grooves with two-segment periodicity, in register with even abdominal segments, was variably penetrant. The denticle bands had normal anteroposterior polarity. spont-2 mutant embryos had antennal sense organs, mandibles, and attached lateral head skeletal elements, but the epistoma was detached and malformed (not shown). The relationship of these genes to Drosophila genes is not clear. Long body: Two mutations, wormy (Figure 6C) and salamander (Table 2), resulted in elongation of first instar larvae as much as twofold relative to wild-type first instar larvae, although the phenotype was variable in expressivity. The elongated mutant embryos had the normal number of segments. wormy mutant embryos also appeared to be uncoordinated or paralyzed in the thoracic region, which may account for the failure of these mutants to hatch. In the Drosophila zygotic embryonic lethal saturation screens, no similar phenotype was reported, though such a mutant phenotype might have been classified as subtle and discarded. Small cuticle: Six mutations caused mutant embryos to develop a very small cuticle (Table 2), always dorsally (Figure 6D) or always ventrally (Figure 6E). The denticle bands had a reduced number of denticle rows and distinct bands of naked cuticle were present in each segment. Linkage data indicate that more than one gene can mutate to cause the dorsal small cuticle mutant phenotype. Comparison of these mutant phenotypes to Figure 6. Diverse mutant cuticular phenotypes. Anterior, left; dorsal is up on lateral views. (A) spontaneous-4, ventral the spectrum of mutant phenotypes from the Drosoph- view. (B) spontaneous-2, lateral view. (C) wormy, ventral view. ila embryonic lethal saturation screens suggests that the (D) classic small, lateral view. (E) scrunched, lateral view. Bars, dorsal small cuticle phenotypes might be counterparts 100 m. of neurogenic phenotypes in Drosophila, while the ventral small cuticle phenotypes may be comparable to those of genes such as canoe (Jürgens et al. 1984; Nüss- Examination of EN expression in helter skelter mutant lein-volhard et al. 1984; Wieschaus et al. 1984). embryos revealed that EN stripes were disorganized Dorsal defects: Four mutations reduced or eliminated from the time of EN initiation (not shown). Linkage dorsal denticles on the mutant embryos (Table 2). fadedata indicate that more than one gene can mutate to away mutant embryos developed only the ventralmost cause segment fusions throughout the embryo. In Dro- denticles in most segments. Three other mutations also sophila, variable segment fusions appear to be caused resulted in a lack of denticles dorsally, though the dentimore frequently by loss of maternal gene functions (e.g., cles developed ventrally and laterally. One additional Schüpbach and Wieschaus 1988) than by loss of zy- mutant appeared to fail only in dorsal closure, showing gotic functions, although some of such mutant pheno- no other obvious cuticular defects (Table 2). Lack of types might have been discarded as severely defective in screens for Drosophila zygotic embryonic lethal genes (see below). dorsal denticles is a phenotype without counterpart in Drosophila, since wild-type Drosophila embryos bear denticles only ventrolaterally. Numerous genes are in-

13 Nasonia Embryonic Lethal Mutants 1225 volved in the process of dorsal closure in Drosophila (Noselli 1998). Anterior defects: Fifteen mutations caused specifically or primarily anterior defects (Table 2). These included defects of the head and the anterior thorax. toothless mutant embryos lack mandibles and more anterior head skeletal structures (Figure 7A). Anteriorly, the normal antennal sense organs are present, accompanied by what appears to be an ectopic pair of antennal sense organs on the labrum (Figure 7, B and C). This apparent homeotic transformation has no known counterpart in Drosophila. speechless (spls) mutant embryos (not shown) had no antennal sense organs and no head skeletal structures when the mutation was first isolated. After spls had been maintained for several generations, partial posterior head skeletons, not including mandibles or more anterior structures, began to appear in the mutant embryos. Antennal sense organs were always missing. We examined EN expression in a small collection of spls mutant embryos and found that EN stripes were present but disorganized in the heads of spls mutant embryos (not shown). Missing or duplicated head structures were caused by three additional mutations, and another two mutations caused characteristic defects at the boundary between head and thorax. Eight of the mutations described here caused defects primarily or specifically in the tentorium region, and several additional mutations that were isolated but not characterized (see below) also had tentorium defects. Three mutations caused tentorium defects coupled with defects of first thoracic denticle belt, and two mutations caused tentorium defects associated with a characteristic T-shaped gut mutant phenotype (Table 2). The Nasonia tentorium has no direct counterpart in Drosophila. Defects of every trunk segment: Twenty-one of the mutations described here caused defects that were repeated in every trunk segment, although many of these also caused head defects (Table 2). Many of the mutations Figure 7. toothless mutant cuticular phenotypes. Anterior, left. (A) Ventral view, showing that only posterior head skeletal structures are formed. (B) Lateral view, dorsal up. Arrow indicates ectopic sense organ on labrum. (C) Dorsal view of head. Asterisks indicate antennal sense organs; arrows indicate ec- topic sense organs. that were isolated but not characterized (see below) are also in this category. The majority of these mutant phenotypes were characterized by the lack of a full complement of denticles in each segment and included embryos with normal segment boundaries but few or no denticles, embryos with irregularly spaced segment boundaries and few denticles, embryos with a single row of denticles in most segments, embryos with thin denticle belts, and embryos with a disorganized denticle pattern. In addition, two mutations caused disorganized denticle belts that lacked the normal anterior-posterior gradation of fine to coarse denticles, two mutations caused mirror-image patterning of denticle rows in each segment, and one mutation caused what appeared to be an extra segment boundary in each segment. Some of these mutations may affect components of a segment polarity system homologous to that of Drosophila. Posterior defects: Four mutations caused characteristic posterior defects. In pinched mutant embryos, as many as six of the most posterior abdominal segments were narrowed, as though transformed to more posterior identities. In skinny tail mutant embryos, the tails were narrow and curled upward. In no posterior denticles mutant embryos, both ventral and posterior denticles were reduced, and in posteriorly scrunched mutant embryos, the posterior of the embryo appeared compressed. These mutations probably caused additional internal defects that accounted for the failure of the mutant embryos to hatch. Mutants not characterized: Thirty additional mutant lines were tested in the F 2 generation and found to have consistent defects, but were not characterized. Most of these had defects judged to be least likely to indicate specific patterning defects, including sparse or slightly disorganized denticles, tentorium defects, and severely defective cuticular phenotypes. Also included in this group were pleiotropic mutants that were difficult to characterize because some defects were consistent while others were variable. Do the mutations identify zygotic functions? Most of the mutations described above are zygotic rather than leaky dominant maternal-effect mutations, for the fol- lowing reasons: in 20 lines, data for lethal-bearing females indicated linkage of an embryonic lethal muta- tion to a visible adult genetic marker, and with only one exception, differential recovery of the marker in haploid male progeny was as expected if the linked embryonic lethal mutation were zygotic (see materials and meth- ods). In the one exceptional line (h-small), there were originally two mutations first, a zygotic mutation with a small cuticle phenotype linked to reddish-5 and second, a leaky dominant maternal-effect mutation with a prothoracic denticle belt defect linked to scarlet Although most of the recovered mutations with consistent phenotypes were zygotic, an unknown proportion of discarded lines with excessively variable phenotypes may have had leaky dominant maternal-effect mutations. In