A Genetic Screen for Zygotic Embryonic Lethal Mutations Affecting Cuticular Morphology in the Wasp Nasonia vitripennis

Corresponding author: Mary Anne Pultz, Biology Department, Western Washington University, Bellingham, WA 98225-9160., pultz{at}biol.wwu.edu (E-mail)

Communicating editor: K. ANDERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 F₁ 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 regulatory gene systems, we need to identify genes of interest and to examine their functions in relatives of the best-characterized genetic model organisms. A comparative developmental genetic approach—the isolation and analysis of recessive loss-of-function mutations in new model genetic organisms—emphasizes the study of gene functions and eliminates the bias of studying only homologs of previously identified genes.

Genetic pathways and their individual components differ in degree of evolutionary conservation. For example, some of the genes involved in anteroposterior patterning in Drosophila, such as caudal, are widely conserved in both invertebrates and vertebrates (MURTHA et al. 1991 ); in contrast, bicoid, which also plays a very fundamental role in Drosophila embryogenesis, diverges rapidly within the Diptera (SCHRODER and SANDER 1993 ; STAUBER et al. 1999 ). Comparisons among distantly related organisms reveal the most profoundly conserved relationships, but comparative studies using more closely related organisms are also important for understanding in full detail the spectrum of evolutionary conservation and change in developmental regulatory genes (RAFF 1996 ; EIZINGER et al. 1999 ).

Work in the flour beetle Tribolium castaneum first demonstrated the value of a comparative genetic approach to understanding insect development. For example, the Tribolium ortholog of the Drosophila pair-rule gene fushi tarazu (ftz) is expressed in a pair-rule pattern, yet this gene can be deleted without producing any pair-rule segmentation defect (STUART et al. 1991 ; BROWN et al. 1994 ). Tribolium thus does not share with Drosophila the unique role of ftz in patterning embryonic segmentation. Genetic screens in Tribolium have identified developmental mutants with gap, pair-rule, homeotic, and other phenotypes (DENELL et al. 1996 ; SULSTON and ANDERSON 1996 ; MADERSPACHER et al. 1998 ). The comparison of these genes with their Drosophila counterparts is placing the details of Drosophila developmental gene functions into evolutionary perspective.

We have chosen the solitary parasitoid wasp Nasonia vitripennis (formerly known as Mormoniella) as an experimental organism for comparative developmental genetic study of embryonic patterning. Nasonia (Hymenoptera) is more closely related to Drosophila (Diptera) than is Tribolium (Coleoptera), although both the Hymenoptera and the Coleoptera diverged from the Diptera between 200 and 300 mya (HENNIG 1981 ). Unlike Tribolium, Nasonia shares with Drosophila a long germ band mode of embryogenesis (BULL 1982 ). Therefore, the comparative study of embryonic patterning in Nasonia and Drosophila will illustrate which features of embryogenesis have diverged in two insects with morphologically similar modes of development. Interestingly, multiple lineages of Hymenopterans have switched from syncytial to holoblastic embryonic cleavage, or to polyembryonic development (GRBIC et al. 1996 , GRBIC et al. 1998 ; GRBIC and STRAND 1997 ; STRAND and GRBIC 1997 ). This raises the question whether the generic Nasonia syncytial embryo might have novel gene functions or unique features of its developmental program that could facilitate such evolutionary transitions.

Nasonia's primary advantage as a genetic organism derives from its method of sex determination. As in other Hymenoptera, fertilized eggs develop as diploid females while unfertilized eggs develop as haploid (monoploid) males, facilitating screens for recessive zygotic mutations (WHITING 1967 ). However, unlike most other Hymenoptera, Nasonia does not determine sex through complementary sex determination (CSD), a system in which homozygosity for a sex-determining locus specifies male development (BEUKEBOOM 1995 ). Instead, Nasonia has been hypothesized to determine sex through an imprinting mechanism (DOBSON and TANOUYE 1998 ). This is of practical significance, because the absence of CSD in Nasonia facilitates the maintenance of highly inbred genetic stocks. Genetic resources for Nasonia include visible genetic markers for each of the five chromosomes (SAUL et al. 1967 ) and a randomly amplified polymorphic DNA map of the genome (GADAU et al. 1999 ). In addition, a triploid stock generates fertile diploid males that can be used to construct double-mutant combinations of embryonic lethal mutations (WHITING 1967 ; M. A. PULTZ, unpublished data). The small adult size and rapid life cycle of Nasonia (similar to Drosophila in both respects) contribute to ease of handling in the laboratory. Stock maintenance is facilitated by maintaining mutant strains as refrigerated diapause larvae for as long as 16 months (SCHNEIDERMAN and HOROWITZ 1957 ).

To initiate a study of embryogenesis in Nasonia, we screened for embryonic lethal mutants that fail to hatch and have defects in larval cuticular morphology. Within this group, we focused on mutations affecting anteroposterior patterning. This approach allows for direct comparison to work in Drosophila (NUSSLEIN-VOLHARD and WIESCHAUS 1980 ; JURGENS et al. 1984 ; NUSSLEIN-VOLHARD et al. 1984 ; WIESCHAUS et al. 1984 ). Here we present an overview of mutant phenotypes based on approximately the first 100 zygotic embryonic lethal morphological mutations isolated in Nasonia. Through this approach, we have identified both similarities and differences in the zygotic embryonic patterning gene functions of Nasonia and Drosophila.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Host pupae:
Nasonia stocks were maintained on Sarcophaga bullata pupae, purchased from Carolina Biological or raised on site. Nasonia embryos were collected using Calliphorid pupae, purchased as larvae from Border Bait (Porthill, ID), or on Sarcophaga pupae. Drosophila were raised on instant Drosophila medium (Carolina Biological), and embryos were collected on molasses-agar plates.

Genetic strains:
Marker gene alleles and their linkage positions are based on the 47-marker genetic map of five Nasonia linkage groups (SAUL et al. 1967 ). We have appended a Roman numeral to each marker allele to indicate its linkage group. The linkage map was constructed using mostly radiationinduced mutant alleles of marker genes. The location of black-424 (bk-424) on linkage group III is tentative, according to the original map. Although reverent originally appeared to be closely linked to oyster, the two loci assort independently in our hands (data not shown). We have kept the original linkage group I designation for reverent, assuming that the original map distance may have been reduced due to interference from an internal chromosomal rearrangement that was subsequently lost. Integration of these visible markers with a 100-marker, 765-cM molecular linkage map will now be possible (GADAU et al. 1999 ).

Markers built into the EMS screens were purple^plum-I (pu^pm), reddish-5-II (rdh-5) and scarlet-5219-III (st-5219). For phenotypic descriptions of these markers, see Fig 1. For the diepoxybutane (DEB) screen, we used the quadruple marker combination pu^pm; rdh-5; st-5219, bk-424. scarlet, black eyes are white, and this mutant phenotype is epistatic to reddish. The additional markers used for mapping embryonic lethal mutations included reverent-I (rev), oyster-I (oy), vestigial-I, distantennapedia-II, blue-13-III, orange-123-IV, scarlet-318-V (st-318), and mickey mouse-V (mm). All of the above are allele designations from SAUL et al. 1967 , except for distantennapedia (WERREN and PERROT MINNOT 1999 ). For wild-type Nasonia, we used the competent+ (comp+) strain, or an isogenic line of this strain (comp+ B1) constructed through six generations of sister-brother pair matings.

View larger version (27K): In this window In a new window Download PPT slide   Figure 1. Screening for zygotic embryonic lethal mutations. Parental multiply marked females were mated to mutagenized parental wild-type males. F1 females were set unmated to score haploid male progeny, and then each female bearing 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 linked to the scarlet eye-color marker. Approximately 3500 EMS-mutagenized genomes were screened using the triple-mutant pupm-I; rdh-5-II; st-5219-III genotype indicated here. The remainder of the EMS-mutagenized genomes were screened using the double-mutant genotype rdh-5; st-5219. pu, purple body color; rdh, reddish eye color; st, scarlet eye color. The rdh-5; st-5219 double-mutant phenotype is distinguishable from those of the single mutants. Linkage of a zygotic lethal mutation to a marker gene would first be deduced by scoring marker phenotypes of the F2 (haploid) male progeny. These data are designated "m" in Table 2. To verify that the lethal mutation linked to the marker was the mutation of interest, F2 females were sorted by marker and then set unmated to score the embryonic progeny of each female. These data are designated "f" in Table 2. For example, st-5219: m,31/161; f,5/24 indicates that 31 of 161 surviving F2 males were st+, putative crossover events and that 5 of 24 F2 females represented putative crossover events (including both phenotypically st females that carried the embryonic lethal mutation and phenotypically st+ females that did not carry the lethal mutation). These results would indicate that the embryonic lethal mutation is located ~20 cM from st-5219 (see also MATERIALS AND METHODS).

Screening for embryonic lethal mutations:
Fig 1 outlines the design of the genetic screen. Parental wild-type males (comp+ or isogenic comp+ B1) were mutagenized 1 day after eclosion with freshly opened EMS (0.025 M; as in LEWIS and BACHER 1986 ) or DEB (0.005 M), delivered in 400 µl of 10% honey water wicked onto the side of a glass vial. The time of exposure to the mutagen was varied from 3 to 12 hr for EMS and from 3 to 24 hr for DEB (see Table 1). Males were fed prior to mutagenesis because starved males did not mate after mutagen treatment. In our hands, the mutation frequency was limited by difficulty in obtaining daughters after administering EMS in longer exposures or higher concentrations. However, recent work has shown that both longer exposures and higher concentrations can be successfully administered (C. TRENT, personal communication).

The mutagenized males were allowed to mate for 1 day with genetically marked females (Fig 1) and then discarded. The parental females were cultured at 18°. These females were cultured individually to determine whether any parental female was already carrying a spontaneous embryonic lethal mutation, in which case the mutation would be carried by half of her daughters. We detected such preexisting spontaneous embryonic lethal mutations in only three isolated cases: unshaven, spontaneous-4, and one mutant that was not further characterized.

The age and handling of the F₁ females proved critical for efficient screening: they must be old enough to lay eggs efficiently when set unmated, but young enough for successful pair mating after the embryonic progeny have been scored for mutations. Virgin females were set for egg collections 2 days (25°), or 3 days (22°–25°) after eclosion. When set unmated, females lay all haploid male progeny. They were set individually at 28° overnight. Each female was given one host pupa, oriented in a P1000 pipet tip so that only the head of the host was accessible for egg laying. The next day, embryos were removed from inside the host pupal cases with a moistened fine brush and placed onto a grid on a 1% agar plate. The females were held in a refrigerator (8°) while the embryos developed to hatching for 24 hr at 28°. Most mothers laid from 12 to 30 eggs under these conditions. Females that did not lay at least 12 eggs were reset once, then discarded if they did not lay on the second setting. Reset frequencies ranged from <10 to ~35% and were sensitive both to female age and to host quality.

After 24 hr at 28°, plates were examined to determine which mothers had laid ~50% unhatched embryos. A probability table was used to determine the minimum number of eggs that should be unhatched (0.95 probability) for a given total number of eggs laid. For example, if an embryonic lethal bearing mother laid 12 eggs, at least three embryos should be unhatched. The incidence of F₁ germ-line mosaics should be negligible because most Nasonia embryos form only a single pole bud, with a single nucleus that divides to generate a population of pole cells (BULL 1982 ). When working with Nasonia, there is no background of eggs that fail to develop because they have not been fertilized.

To score for cuticular phenotypes, the unhatched embryos of interest were mounted in 90% lactic acid/10% ethanol, cleared at 56°, and examined using dark-field optics. In most cases, the unhatched embryos showed no obvious cuticular defects, or the defects were very subtle or highly variable. The mothers of such embryos were discarded. In a small percentage, the unhatched embryos had died early before elaborating cuticle. These were cloudy in appearance and lacked the white gut contents of mature embryos. The mothers of these embryos were also discarded. The percentage of lines in the latter category was difficult to determine precisely, because of a variable low background level of embryos that died early in development. In each case where the embryos showed consistent cuticular defects, the mother was selected and mated to a genetically marked male (Fig 1). Approximately 85% of the selected females produced sufficient F₂ daughters for further mutant analysis, genetic mapping, and stock maintenance. After the progeny of F₂ females were examined, approximately one-third of these lines were discarded, either because there was no phenotype or because the phenotype was excessively variable.

Genetic mapping of lethal mutations:
Linkage analysis of F₂ progeny is explained in Fig 1. The F₂ adult male data were generally congruent with the female data, indicating that there was little or no bias of haploid male survival for the markers used, even in the triple-mutant marker combination. Because of the fairly low mutation frequency, we found only two lines with spurious lethal mutations linked to marker loci. Two additional lines yielded linkage results that were not interpretable. Overall, the male data were highly reliable at indicating probable linkage relationships for mutations of interest. If a mutant of special interest was found not to be linked to the markers built into the screen, it was then crossed and backcrossed to other marker loci and evaluated similarly. By crossing to a set of 12 marker loci representing the five linkage groups, linked markers were identified for each of the 12 lethal loci so analyzed. The F₂ male mapping data were also congruent with female data in these cases, except that the reverent-I marker is not 100% penetrant.

Stock maintenance:
Embryonic lethal mutations are maintained as diapause larvae, an overwintering stage that is maternally induced in response to short day length and cool temperatures (SCHNEIDERMAN and HOROWITZ 1957 ). Our current stock maintenance protocol is as follows. Females bearing the dominant wild-type allele of a linked marker gene eclose in the dark at 18° and are then set unmated to identify the lethal, marker⁺/lethal⁺, marker genotypes. Each lethal-bearing female is pair-mated to a male mutant for the linked marker gene. The matings are observed, to avoid subculturing unmated females. Females are set individually in the dark at 16°, because females cultured individually lay diapause larvae more readily. The females are subcultured, removing parasitized pupae and adding two to three fresh pupae, twice per week for 3–4 wk. By the third week, most females will be laying progeny that will enter diapause. Approximately 8–15 individual female lines are set every 6 months for each stock—not all mated lines produce daughters, and some mothers will have depleted their supply of sperm before beginning to lay diapausing progeny (older mothers are not likely to remate). Mothers produce ~30 progeny per host pupa and some produce as many as 90% females, because they fertilize most of the eggs. Diapause larvae are stored in the refrigerator (8°). Since stocks must remain in diapause for at least 4 months and can remain in diapause for up to 16 months, stock maintenance twice per year allows sufficient time to verify that there are daughters (by breaking diapause) before the previous cultures are too old to use. After 4 months, stocks can be removed from diapause at any time by returning them to room temperature. Using this system, about two dozen embryonic lethal stocks can be reliably maintained with 10 hr per week of continuous effort, making the economics of embryonic lethal stock maintenance very similar to what has been described for Tribolium (BERGHAMMER et al. 1999 ).

We established diapause lines from F₂ females for all of the >100 mutant lines with distinct heritable phenotypes, to test the efficiency with which diapause stocks could be established for a large group of mutants. Diapause was obtained for ~70–80% of the stocks on the first round and for 100% within three rounds of culturing as above. However, we found that the percentage of diapause cultures with female progeny was highly variable among different stockkeepers, varying from >90% to <50%. This variation probably depended largely on how carefully the mothers were handled. Since the percentage of cultures with female progeny cannot be determined until at least 4 months after cultures have been established, embryonic lethal diapause stocks should be kept by an established and skilled stockkeeper.

A few embryonic lethal stocks, notably headless, head only, and speechless, acquired weakened phenotypes in some lines after several generations of stock maintenance, as though having acquired a spontaneous suppressor mutation linked to the mutation of interest. Such variability of phenotypic strength with genetic background does not appear to be a phenomenon that distinguishes Nasonia from Drosophila. For example, various stocks of Drosophila caudal null alleles differ in the strengths of their mutant phenotypes in a manner that resembles the variable strengths of mutant phenotypes in different lines of the single head only mutant allele (M. A. PULTZ, unpublished observations). Using the stock maintenance procedures described above, an embryonic lethal mutant phenotype can change abruptly if a closely linked spontaneous suppressor mutation is acquired as a mutation of interest is passaged through a single-female culture. Maintenance and phenotypic monitoring of parallel cultures allows for choice of lines with consistent phenotypes, and diapause stocks preserve access to genotypes that have been maintained during the previous 16 months.

Estimate of the percentage of lethal mutations affecting embryonic morphology:
The total percentage of embryonic lethal mutations for the EMS screen was calculated from data for the 4456 EMS-mutagenized genomes shown in Table 1. This percentage was applied to the total of 6937 genomes mutagenized with EMS to obtain an estimate of ~600 embryonic lethal mutations for the EMS screen. Approximately 100 EMS-induced mutations affecting embryo morphology were isolated, so the percentage is estimated as 17% of all embryonic lethal mutations.

Recovery of spontaneous embryonic lethal morphological mutations:
In addition to the mutations induced by EMS or DEB, we have described here the phenotypes of five spontaneous mutations that were found incidentally. Two (unshaven and spontaneous-4) were found while screening for induced mutations, as explained above. The other three (spontaneous-1, -2, and -3) were identified among the progeny of wild-type females. These were found while prescreening wild-type (comp+) females to verify that they were not carrying lethal mutations when being used as controls for experiments.

Fixation and antibody staining:
Selected anteroposterior patterning mutants and Drosophila giant mutant embryos were stained with phylogenetically cross-reactive monoclonal antibodies: 4D9 recognizes ENGRAILED (PATEL et al. 1989 ) and FP6.87 recognizes both ULTRABITHORAX and ABDOMINAL-A (KELSH et al. 1994 ). Fixation, mass devitellination of Nasonia embryos in cold 1:1 heptane:methanol, and antibody staining were carried out as described previously (PULTZ et al. 1999 ). To collect embryos mutant for Drosophila giant, y¹,sc¹, giant^x11/FM6 females were crossed to Canton-S males, giant-bearing females were crossed again to Canton-S males, and embryonic progeny were collected.

Complementation testing of embryonic lethal mutations is not straightforward:
We did not carry out complementation tests, despite the availability of fertile diploid Nasonia males, because this procedure is not straightforward. Nasonia triploid females produce mostly aneuploid progeny. When set unmated, their euploid progeny are approximately half haploid males and half diploid males (WHITING 1967 ). Because Nasonia males are normally haploid, spermatogenesis does not include meiosis I. Therefore, the progeny of diploid males are triploid females. It is difficult to determine whether a triploid female is carrying an embryonic lethal mutation by inspection of embryonic progeny, when most embryos are dying of aneuploidy, and there is evidence suggesting that the chromosomes of triploid females do not segregate randomly (DOBSON and TANOYE 1998). Triploid females carrying two mutant alleles of an embryonic lethal gene may be poorly viable or fertile, precluding the necessary analysis of further generations; yet such a phenotype cannot be rigorously interpreted as allelism rather than second-site noncomplementation. It may be possible to use this procedure to establish allelism in cases of exceptional interest, and we are currently investigating whether this approach can be used to test whether alternate and five band are allelic. However, complementation testing of embryonic lethal mutations using diploid males is not routinely feasible in Nasonia.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We screened 6937 EMS-mutagenized genomes and 1126 DEB-mutagenized genomes for embryonic lethal mutations with defects in cuticular morphology. EMS produces mostly point mutations, identifying single genes that can be readily mapped meiotically. DEB often produces small deletions (or other chromosomal rearrangements). These are more likely to be null mutations and are potentially useful for molecular testing of candidate gene hypotheses. In the EMS screen, we recovered all mutations with morphological defects; in the DEB screen, we chose to recover a more limited subset of the mutations. We also describe the phenotypes of several spontaneous embryonic lethal mutations that were recovered in the course of mutant screening or analysis.

For embryonic lethal screening, mutagenized males were mated to females homozygous for visible markers on two or three of the five linkage groups, using the protocol outlined in Fig 1. Analysis of markers identified a subset of the embryonic lethal mutations by linkage group in the F₂ generation. The linkage data distinguished some cases where more than one gene could be mutated to produce a given mutant phenotype. These data also confirmed that mutations of interest were zygotic and that they arose on the chromosomes of parental males.

Approximately 100 embryonic lethal mutations were isolated, of which 74 are described here. All mutants were analyzed for cuticular defects, and selected mutants were assayed for engrailed or trunk Hox gene expression. Only recessive embryonic phenotypes were analyzed—we have not examined heterozygous females in detail for evidence of subtle dominant haploinsufficient defects. The only striking haploinsufficient phenotype that we noted is a low penetrance hunched thorax defect of female pupae carrying the head only mutation.

Mutation frequencies:
Table 1 summarizes mutation frequencies that were obtained with increasing length of exposure to EMS and DEB. The mutation frequencies increased with length of exposure to the mutagens and were higher in the mutagenized groups than in the control group. These results indicate that most of the embryonic lethal mutations described here were induced by EMS or DEB. Overall, the frequency of embryonic lethal mutations per Nasonia genome was 9%; the maximum frequencies were 18% for EMS and 15% for DEB. In the Drosophila saturation screens for zygotic mutations affecting embryonic morphology, EMS-induced embryonic lethal frequencies ranged from 35% per genome (JURGENS et al. 1984 ) to 82% per genome (NUSSLEIN-VOLHARD et al. 1984 ). On a per-chromosome basis, mutation frequencies ranged from 8% (WIESCHAUS et al. 1984 ) to 33% (NUSSLEIN-VOLHARD et al. 1984 ).

Wild-type Nasonia:
As background to the description 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 genes. The wild-type first instar larval cuticle is shown in Fig 2A&NDASH;C. A denticle belt delineates each of the 3 thoracic and 10 abdominal segments; each denticle belt grades from finer denticles anteriorly to coarser denticles posteriorly. Large spiracles are located on the second thoracic and first three abdominal segments. The larval head bears a dorsolateral pair of antennal papillae, and the ventral mandibles are supported by a 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, the embryo bears a tubular anus that everts after hatching.

View larger version (142K): In this window In a new window Download PPT slide   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 µm.

To follow expression of the segment polarity gene engrailed (en) in Nasonia, we used the monoclonal antibody 4D9 (PATEL et al. 1989 ). As gastrulation begins, the first EN stripes appear in the antennal, mandibular, and labial segments, followed soon thereafter by intercalary and maxillary stripes and then by thoracic stripes (Fig 2D). At the onset of expression, the labial EN stripe is wider than the other stripes, as in bees (FLIEG 1990 ), and the trunk stripes are two cells in width. As the germ band extends, with both head and tail moving dorsally, EN is expressed in 5 head stripes and 12 trunk stripes (Fig 2E). The head stripes take on characteristic morphologies that have been described for a variety of other insects, such as the dorsal fusion of the maxillary and labial stripes and the formation of intercalary spots (ROGERS and KAUFMAN 1996 ). The number of trunk stripes at this stage is the same as in comparably aged Drosophila embryos (DINARDO et al. 1985 ).

To follow the expression of trunk Hox genes, we used the monoclonal antibody FP6.87 (KELSH et al. 1994 ), which recognizes both ULTRABITHORAX (UBX) and ABDOMINAL-A (ABD-A). Expression of UBX-ABD-A in Nasonia is similar to the expression patterns for these genes in Drosophila and Tribolium (KELSH et al. 1994 ; CASTELLI-GAIR and AKAM 1995 ; SHIPPY et al. 1998 ). Weak expression extends from the posterior second through the third thoracic segments; strong expression extends from the first through the seventh abdominal segments, while the eighth abdominal segment stains more weakly than the anterior abdominal segments (Fig 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 (Fig 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 posterior 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 (Fig 2J). The tentorium has been described as a first thoracic derivative (AZAB et al. 1967 ). exp affects EN expression (Fig 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 expression 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 anteriorly. 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 Fig 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 defective denticle belts in a variable region around the sixth and seventh abdominal segments (Fig 3B; PERRIMON et al. 1984 ; PETSCHEK et al. 1986 ). EN expression in Nasonia ms mutant embryos (Fig 3C) was compared to EN expression in Drosophila giant mutant embryos (Fig 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.

View larger version (72K): In this window In a new window Download PPT slide   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 giantX11 mutant embryos. giantX11 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.

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 pair-rule 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 (Fig 4A). od mutant embryos have reduced or missing denticles in 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.

View larger version (131K): In this window In a new window Download PPT slide   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.

The second pair-rule locus, five band (fb) is represented by one EMS-induced mutation with a strong mutant phenotype (Fig 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 (Fig 4C) have a weak pair-rule phenotype. Typically, denticle belts in alternate segments are missing 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 phenotype. 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 by a posterior strip of naked cuticle (Fig 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 (Fig 4E). More posterior head skeletal structures and the tentorium do not develop in these embryos. EN fails to initiate normally in bh mutant embryos (Fig 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, even-skipped 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 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; Fig 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; Fig 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; Fig 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 symmetrical (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; Fig 4J), while about two-thirds of the embryos show slight to marked pair-rule periodicity in the dorsolateral loss of EN expression (unsh; Fig 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 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 (NUSSLEIN-VOLHARD and WIESCHAUS 1980 ).

The third gene with an ectopic denticle mutant phenotype is rambutan (ram), identified by a single EMS-induced 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 (Fig 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 (Fig 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 quantitative 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 (Fig 5A). Some mus mutant embryos bear ectopic denticles above the mandibles (Fig 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 1983 ). The mus mutant phenotype may be caused by similar transformations. mus mutant embryos also fail to complete dorsal closure (not shown), while Drosophila Polycomb mutant embryos fail in dorsal closure of the head region.

View larger version (117K): In this window In a new window Download PPT slide   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 embryo. Arrow indicates labial lobe. (D) UBX-ABD-A expression in mus mutant embryo that has completed germ band retraction.

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 siblings. However, by the time of gnathal lobe formation, ectopic UBX-ABD-A expression in a limited number of more anterior cells is evident (Fig 5C). In much later mutant embryos, after germ band retraction, UBX-ABD-A is expressed strongly throughout most of the anterior of the embryo (Fig 5D). In Drosophila Polycomb mutant embryos, UBX is initiated normally, but is increasingly derepressed during embryonic development (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 extreme segmental fusions throughout the embryo (e.g., spontaneous-4; Fig 6A and Table 2), with only rudimentary mandibles developing in the head region. Although segmentation was severely disrupted, the remaining fragments of denticle belts maintained normal anteroposterior and dorsoventral polarity (not shown). Examination of EN expression in helter skelter mutant embryos revealed that EN stripes were disorganized from the time of EN initiation (not shown). Linkage data indicate that more than one gene can mutate to cause segment fusions throughout the embryo. In Drosophila, variable segment fusions appear to be caused more frequently by loss of maternal gene functions (e.g., SCHUPBACH and WIESCHAUS 1988 ) than by loss of zygotic functions, although some of such mutant phenotypes might have been discarded as severely defective in screens for Drosophila zygotic embryonic lethal genes (see below).

View larger version (63K): In this window In a new window Download PPT slide   Figure 6. Diverse mutant cuticular phenotypes. Anterior, left; dorsal is up on lateral views. (A) spontaneous-4, ventral view. (B) spontaneous-2, lateral view. (C) wormy, ventral view. (D) classic small, lateral view. (E) scrunched, lateral view. Bars, 100 µm.

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 (Fig 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 (Fig 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 (Fig 6D) or always ventrally (Fig 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 the spectrum of mutant phenotypes from the Drosophila embryonic lethal saturation screens suggests that the dorsal small cuticle phenotypes might be counterparts of neurogenic phenotypes in Drosophila, while the ventral small cuticle phenotypes may be comparable to those of genes such as canoe (JURGENS et al. 1984 ; NUSSLEIN-VOLHARD et al. 1984 ; WIESCHAUS et al. 1984 ).

Dorsal defects: Four mutations reduced or eliminated dorsal denticles on the mutant embryos (Table 2). fadeaway mutant embryos developed only the ventralmost denticles in most segments. Three other mutations also resulted in a lack of denticles dorsally, though the denticles developed ventrally and laterally. One additional mutant appeared to fail only in dorsal closure, showing no other obvious cuticular defects (Table 2). Lack of dorsal denticles is a phenotype without counterpart in Drosophila, since wild-type Drosophila embryos bear denticles only ventrolaterally. Numerous genes are involved 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 (Fig 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 (Fig 7B and Fig C). This apparent homeotic transformation has no known counterpart in Drosophila.

View larger version (83K): In this window In a new window Download PPT slide   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 ectopic sense organs.

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 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 F₂ 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 following reasons: in >20 lines, data for lethal-bearing females indicated linkage of an embryonic lethal mutation 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 METHODS). 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-5219. 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 the screen of the Drosophila second chromosome for zygotic embryonic lethal mutations, ~2% of the mutant lines had leaky dominant maternal-effect mutations (NUSSLEIN-VOLHARD et al. 1984 ).

The percentage of morphological mutants:
After discarding mutants that were judged as having very subtle defects or being very poorly differentiated, we found that ~17% of the EMS-induced Nasonia embryonic lethal lines showed heritable cuticular defects (including head defects; see MATERIALS AND METHODS). In the Drosophila saturation screens for mutations causing larval cuticular defects, ~14% of the second-chromosome embryonic lethal lines were classified as having abnormal morphology, including head defects, and 14% were classified as poorly differentiated; 11% of third-chromosome lines were classified as having abnormal morphology, excluding head defects, and 24% were classified as having subtle defects (including poor differentiation and head defects); 17% of X chromosome and fourth chromosome lines were classified as having zygotic visible phenotypes (JURGENS et al. 1984 ; NUSSLEIN-VOLHARD et al. 1984 ; WIESCHAUS et al. 1984 ). Head defects were classified separately from other morphological defects in two of the Drosophila screens because the failure of head involution is a common and often nonspecific class of developmental defect in this organism. In Nasonia, although the embryo undergoes stomodeal involution and elaborates an internal head skeleton (BULL 1982 ), we did not find such a common class of nonspecific head defects—rather, the head defects of individual mutant lines were each distinctive. In contrast, defects of the tentorium—a prothoracic derivative (AZAB et al. 1967 )—were frequently associated with failure of embryos to hatch. Overall, the percentages of embryonic lethal lines with abnormal morphology indicate that a roughly comparable proportion of the zygotic embryonically essential genome is needed for normal morphology of the first instar larva in Nasonia and Drosophila.

The percentage of genes identified:
We can estimate the percentage of Nasonia embryonic lethal genes identified in this screen, although the mutations cannot be sorted by complementation and most were not mapped genetically. First, focusing on the mutations that are currently maintained, which have been mapped, there are 9 genes represented by single alleles, one putative complementation group with 2 alleles, and one with 3 alleles—about 1.3 alleles per gene—suggesting that about one-quarter to one-third of the patterning genes of interest have been identified in the combined EMS and DEB screens. Second, if we estimate the minimum total number of genes identified with distinct morphological phenotypes (or distinctive linkage to markers) in the EMS screen, we count >50 genes. In the Drosophila embryonic lethal saturation screens, 144 genes were identified (and the number would be ~160 had head defects been uniformly included). Though numbers of morphological mutants are not straightforward to compare, these numbers are consistent with the first estimate that about one-quarter to one-third of the genes have been identified. Both measures indicate that fewer than half of the genes of interest have been identified.

Homeotic phenotypes:
Of the mutants described here, only mustache and toothless were identified as having homeotic phenotypes. In the Drosophila saturation screens at least eight loci with homeotic phenotypes were identified, including Polycomb-group genes, trunk Hox genes, Sex combs reduced, homothorax, and extradenticle. The single Polycomb-group gene identified here appears to be consistent with our estimate that about one-fourth to one-third of the Nasonia genes of interest have been identified. Nasonia mutant phenotypes comparable to those of most of these Drosophila genes could in principle have been identified through altered spiracle patterns—in most cases, the absence of spiracles on a specific segment. Several Nasonia embryonic lethal lines with aberrant spiracle patterns—in some cases including ectopic spiracles—were discarded because the mutant phenotypes were too variable to maintain effectively by selection. Some of these might have had partial loss-of-function mutations in genes with homeotic phenotypes. The similar appearance of all denticle bands makes Nasonia less favorable for easily recognizing homeotic phenotypes than other insects with greater larval segmental variation, such as Drosophila or Tribolium. Mutations in Nasonia ANT-C and BX-C genes could be sought most efficiently if the locations of these genes were first determined molecularly, and then appropriate linked genetic markers were used to identify candidate embryonic lethal mutations during the course of a genetic screen.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The broadly defined goal of this work has been to test the potential of the haplo-diploid wasp N. vitripennis as a genetic experimental organism for understanding the evolution of developmental regulatory gene networks. We have begun to survey genetically which embryonic anteroposterior patterning functions have changed and which have been conserved since the divergence of the Hymenoptera from the Diptera, by isolating recessive mutations affecting larval cuticular morphology in Nasonia and comparing the mutant phenotypes to those of Drosophila genes. Both Nasonia and Drosophila have syncytial long germ band embryos, with developmental programs separated by >200 million years of evolution.

This initial collection of mutant phenotypes has begun to outline some of the variations on the themes of Drosophila developmental pathways that are likely to emerge from further study of embryonic patterning in Nasonia. In brief, we have isolated Nasonia zygotic mutations with axial, gap, pair-rule, segment polarity, homeotic, and Polycomb-group functions, indicating that each of these classes of genes plays a role in patterning the Nasonia embryo. Some of the Nasonia mutant phenotypes closely resemble those of Drosophila genes; for example, Nasonia odd-defective resembles Drosophila odd-skipped, suggesting that the functions of some segmentation genes are well conserved. In contrast, specific axial, pair-rule, and homeotic mutant phenotypes are unlike those of Drosophila genes. Nasonia homologs of Drosophila genes may control a different spectrum of patterning functions, or patterning functions may be carried out in Nasonia by genes that do not function as embryonic patterning genes in Drosophila. Specific comparisons of the Nasonia genes to Drosophila genes are detailed in RESULTS.

The Nasonia axial patterning mutant phenotypes have pointed toward a lesser role of maternal contributions to embryonic patterning in Nasonia than in Drosophila (PULTZ et al. 1999 ). The zygotic head only mutant phenotype is most similar to that of Drosophila embryos lacking both maternal and zygotic caudal function, while the zygotic headless mutant phenotype is most similar to that of Drosophila embryos lacking both maternal and zygotic hunchback function (MACDONALD and STRUHL 1986 ; LEHMANN and NUSSLEIN-VOLHARD 1987 ; SIMPSON-BROSE et al. 1994 ; PULTZ et al. 1999 ). We are testing the hypotheses that head only is Nasonia caudal and that headless is Nasonia hunchback, through linkage analysis, then through sequencing of the candidate genes from mutant chromosomes. We reason that the zygotic products of these genes in Nasonia may be responsible for functions that are covered jointly by maternal and zygotic gene products in Drosophila. A high degree of reliance on zygotic gene products to control axial patterning may allow for the evolution of holoblastic cleavage and polyembryonic development in Hymenopteran lineages (STRAND and GRBIC 1997 ).

While some mutant phenotypes such as those of headless and head only suggest testable candidate gene hypotheses, novel mutant phenotypes—such as those of the axial patterning genes squiggy and expanded—will depend on further technical advances before they can be characterized at a molecular level. Such molecular characterization of novel phenotypes will require developing a system for transposon mutagenesis or a system of molecular genetic markers to support positional gene cloning in Nasonia (e.g., GADAU et al. 1999 ).

Isolating a more complete set of mutations identifying Nasonia patterning genes is feasible. The screening described here (including handling of F₂ individuals and initial diapause stock establishment; see MATERIALS AND METHODS) was carried out largely by undergraduates and required the equivalent of ~12–15 months of full-time work by one investigator. The efficiency of screening can be enhanced by increasing the mutation frequency and by selecting only a focused group of mutations affecting a specific developmental process.

A complementary approach to achieving a thorough understanding of embryonic patterning is through use of RNA interference (RNAi; HUNTER 1999 ; SHARP 1999 ). Recently, RNAi has been shown to be effective at producing phenocopies of loss-of-function mutant phenotypes in Tribolium as well as in Drosophila (KENNERDELL and CARTHEW 1998 ; BROWN et al. 1999 ). If effective also in Nasonia, RNAi will be useful for probing functions of cloned regulatory genes and for testing whether candidate gene hypotheses are plausible.

An important consideration for comparative genetic investigations is the cost of genetic stock maintenance. In Nasonia, embryonic lethal lines can be maintained for >1 year as diapause larvae; however, the cost of embryonic lethal stock maintenance for Nasonia (see MATERIALS AND METHODS) is currently similar to that reported for Tribolium (BERGHAMMER et al. 1999 ). This has made it necessary to choose a limited number of mutant lines to maintain from this pilot study, and we have focused on those for which candidate gene hypotheses may be useful in relating our understanding of Nasonia development to that of Drosophila. If mutations of interest are relatively easy to isolate in Nasonia but relatively costly to maintain, what approaches should be taken for further study of this organism? First, by developing an array of molecular probes to assay expression and linkage for Nasonia orthologs of key developmental regulatory genes, Nasonia mutations affecting a given developmental process of interest can be identified relatively rapidly in the course of genetic screening. Second, development of biological cryopreservation capabilities for stock maintenance would greatly enhance the potential of Nasonia as a genetic experimental organism. Techniques for cryopreservation of Drosophila embryos have been developed (MAZUR et al. 1992 ; STEPONKUS and CALDWELL 1993 ), but judged not to be cost effective because Drosophila balancer chromosomes offer a powerful alternative. The Drosophila cryopreservation techniques may be adaptable and cost effective for Nasonia.

In conclusion, screening for recessive loss-of-function mutations in Nasonia is feasible for the comparative analysis of genetic regulatory systems, and further technical advances will enhance the power of this approach. The mutations isolated here have begun to illustrate that some aspects of Drosophila patterning are conserved in Nasonia while others display evolutionary flexibility in these long germ band insects.

	FOOTNOTES

¹ Present address: College of Pharmacy, Uni