Drosophila embryos homozygous for strong mutations in each of the segment-polarity genes wingless (wg), engrailed (en), naked (nkd) and patched (ptc) form a larval cuticle in which there is a deletion in every segment. The mutant embryos normally fail to hatch but by in vivo culture we were able to show which could produce adult structures. Cultured wg embryos did not produce any adult structures. Cultured en embryos produced eye-antennal derivatives and rarely produced partial thoracic structures. nkd and ptc embryos produced eye-antennal and thoracic derivatives. The nkd and ptc thoracic imaginal discs developed with an abnormal morphology and abnormal pattern of en-expression. Our findings are consistent with the idea that the thoracic imaginal discs derive from two adjacent groups of cells that express wg and en respectively in the embryo.

Each hemi-segment of the adult head and thorax comprises two polyclones called the anterior and posterior compartments (e.g. Garcia-Bellido et al. 1973; Steiner, 1976; Wieschaus and Gehring, 1976; Morata and Lawrence, 1978; Struhl, 1981). These polyclones are determined early in development, as restriction to one compartment occurs during embryogenesis in all head and thoracic segments, except the eye-antenna in which the restriction occurs during larval life (ibid), engrailed (en) expression is characteristic of posterior hypodermis cells throughout development. In embryos en transcription is restricted to a band of cells that lies just posterior to the parasegmental groove ( DiNardo et al. 1985; Fjose et al. 1985; Kornberg et al. 1985; Ingham et al. 1985,Ingham et al. 1988) and in larvae, en is transcribed in the posterior compartment of the imaginal discs (Kornberg et al. 1985). wingless (wg) is transcribed by embryonic cells just anterior to those expressing en in what is likely to be the anterior compartment (Baker, 1987). However, wg-expression is not clonally inherited by anterior cells throughout development as its transcription is not limited to the anterior compartment in all imaginal discs (Baker, 1988a). Loss of the activity of either of these genes results in a deletion of part of the larval cuticle in every segment (Nüsslein-Volhard and Wieschaus, 1980) including the Keilin’s organ, a structure thought to straddle the anterior-posterior boundary (Keilin, 1915; Struhl, 1984). If the anterior and posterior adult primordia derive from embryonic cells expressing wg and en, a failure of either of these genes may also cause a loss of, or defects in, imaginal discs. We tested this by examining discs derived from wg and en mutant embryos.

wg and en are expressed in the segment-polarity mutants naked (nkd) and patched (ptc) but with an altered pattern (Martinez-Arias et al. 1988; DiNardo et al. 1988). The larval cuticles of these two mutants retain the Keilin’s organ and have deletions in the denticle rows (Nüsslein-Volhard and Wieschaus, 1980; Jurgens et al. 1984; Martinez Arias et al. 1988). If imaginal discs are derived from embryonic cells expressing wg and en, imaginal primordia should be present in both mutants, but may be altered and develop into abnormal discs. We tested this by examining discs derived from nkd and ptc mutant embryos.

Homozygotes of wg, en, nkd and ptc null alleles die as unhatched first instar larvae but by in vivo culture in adults (Hadorn et al. 1968) we have extended their development to an equivalent of the third larval instar, when imaginal discs, if present, are mature and competent to differentiate in a larval host. Thus, we have shown which of these four mutants produce imaginal discs and describe their morphology.

Stocks

Canton S females and larvae were used as hosts for transplantation. Embryos from the following stocks were implanted into hosts (for some transplants embryos were collected from a cross of two different alleles). Wildtype; Canton S; mwh red e/mwh red e and mwh red e/TM3,Sb Ser e. wingless; dp wgL4b en bw/Gla; wgCX4b pr/CyO. engrailed; stw pwn en10/ CyO, stw pwn enIK/ CyO, stwpwn en10/ CyO, en10/ CyO, en7/ CyO and stw pwn enX49/ CyO. naked; ‘ru cu ca’ nkd 7ES9 1 TMg gb e and ‘ru cu ca7HI6I TM3,Sb e. patched; stw ptcG12/ CyO, stw ptcINIO8I CyO and ptc82/ CyO. Typical mutant embryos are shown in Fig. 1 and the phenotypes were similar for each allelic combination used with one exception; the combination enX49/ en10, unlike the en” embryo shown in Fig. 1 E and F, did not exhibit a pair rule phenotype and had about one Keilin’s sensory hair per embryo. Stocks are described in Lindsley and Grell (1968), Tearle and Nüsslein-Volhard (1987), Nakano et al (in press) and Phillips et al. (submitted). The en lac-Z/CyO stock was provided by C. Hama and T. Kornberg.

Fig. 1.

Cuticle preparations of the mutants. (A) wild-type embryo (dark-field); (B) wild-type embryo, detail of the third thoracic and first abdominal segments (phase contrast); (C) wg embryo, wgCX4/wgL4, (dark-field); (D) wg embryo, detail of the thorax and first abdominal segment (phase contrast); (E) en embryo, en10/en7 (dark field); (F) en embryo, detail of the thorax and first abdominal segment (phase contrast); (G) nkd embryo, nkd7E89/nkd7E89, (dark field); (H) nkd embryo, detail of second and third thoracic segments and first abdominal segment (phase contrast); (I) ptc embryo, ptcS2/ptcIN108 (dark field) and (J) ptc embryo, detail of the third thoracic and first abdominal segments (phase contrast). Each Fig. shows a ventral aspect; arrowhead, Keilin’s organ; thorax, t and abdomen, a.

Fig. 1.

Cuticle preparations of the mutants. (A) wild-type embryo (dark-field); (B) wild-type embryo, detail of the third thoracic and first abdominal segments (phase contrast); (C) wg embryo, wgCX4/wgL4, (dark-field); (D) wg embryo, detail of the thorax and first abdominal segment (phase contrast); (E) en embryo, en10/en7 (dark field); (F) en embryo, detail of the thorax and first abdominal segment (phase contrast); (G) nkd embryo, nkd7E89/nkd7E89, (dark field); (H) nkd embryo, detail of second and third thoracic segments and first abdominal segment (phase contrast); (I) ptc embryo, ptcS2/ptcIN108 (dark field) and (J) ptc embryo, detail of the third thoracic and first abdominal segments (phase contrast). Each Fig. shows a ventral aspect; arrowhead, Keilin’s organ; thorax, t and abdomen, a.

Culture of embryos in female hosts

Embryos that were about 20 h old (25 °C) were dechorionated on double-sided tape, transferred to Ringer’s solution and examined under a dissecting scope on a white background. Live embryos with the desired cuticle phenotype were sliced posteriorly with tungsten needles and inverted. Each operated embryo was transplanted into the abdominal cavity of a young etherised Canton-S adult female. Injected hosts were mated and maintained for 10 days at 25 °C before the implanted tissue was recovered.

Culture of implants in larval hosts

Adult female hosts were dissected in Ringer’s solution and tissue derived from the cultured embryos was injected into late third instar larvae. Larval hosts were kept at 25°C. The implants were recovered 1 day after the host eclosed and mounted in Faure’s medium for examination with a compound microscope.

Histological stain for engrailed expression

en lac-Z/CyO, ptcIN108en lac-Z/ptcG12 and en lac-Z/ +; nkd7E89/ nkd7E89 embryos were implanted into adult hosts as described and cultured for ten days. The implants were recovered and stained for β-galactosidase activity as described in O’Kane and Gehring (1987). Discs were mounted in Ringer’s solution on slides for examination.

Keilin’s organs in the mutants

The various segments of the Drosophila embryo are characterized by their complement of sense organs, hairs and setae (Lohs-Schardin et al. 1979). The Keilin’s organ, a trihair sensory structure, is characteristic of the thorax and thought to straddle the A-P compartment boundary (Struhl, 1984). Wild-type embryos have a pair of Keilin’s organs on the ventral surface of each thoracic segment (Fig. 1B). wg and en embryos lack all Keilin’s organs (Fig. 1D and F). Both nkd and ptc embryos have Keilin’s organs but these often have four or more sensory hairs rather than the normal three (Fig. 1 H and J). nkd embryos occasionally have extra, discrete organs, so that three organs appear in one segment and this is usually accompanied by the loss of an organ from an adjacent segment (Fig. 1H). We show below that embryos with Keilin’s organs produced thoracic imaginal discs and further that changes in the morphology of the Keilin’s organ accompanied changes in the morphology of the discs.

In vivo culture of embryos

We chose in vivo culture, as opposed to clonal analysis or nuclear transplantation, as in this procedure the animals were wholly mutant and the imaginal primordia were not mosaic or chimaeric with wild-type tissue and thus any effects of perdurance or local nonautonomy of wild-type gene products was avoided (Morata and Lawrence, 1977).

Wild type

Wild-type embryos (Fig. 1A) transplanted into adult hosts increased in mass and occupied a large part of the host’s abdomen after the culture period. The tissue derived from the embryos included larval gut, Malpighian tubule, fat body, salivary gland, brain and imaginal discs. Examples of tissues from cultured wildtype embryos are shown in Fig. 2A, C and E. Imaginal disc tissue recovered from the implants was transplanted into larval hosts for metamorphosis. Differentiated adult cuticle was recovered from 54 embryos and cases of eye-antenna, leg and wing discs were recorded (Table 1 and Fig. 3 A, D and G). The differentiated implants from each embryo were only classified as to type not number thus, for example, an embryo that produced two wing discs both of which differentiated is scored as one entry in the ‘wing’ column of Table 1. We report only cases of eye-antenna, leg and wing implants because these come from large discs and were recovered at high frequency. Rice et al. (1979) cultured younger wild-type embryos and recovered a similar frequency of disc derivatives.

Fig. 2.

Larval tissues derived from cultured embryos. (A) wild-type gut, fat body and Malpighian tubule; (B) wg gut, fat body, Malpighian tubules, larval hypoderm and salivary gland; (C) wildtype brain and eye-antenna imaginal discs; (D) en brain and eye-antenna imaginal discs; (E) wild-type larval hypoderm, salivary gland and imaginal discs; (F) nkd larval hypoderm and imaginal discs; (G) ptc larval hypoderm and imaginal discs; (H) imaginal discs from (G) removed from larval tissue, brain, b; imaginal disc, d; fat body, fb; gut, g; larval hypoderm, h; Malpighian tubule, mt; salivary gland, sg.

Fig. 2.

Larval tissues derived from cultured embryos. (A) wild-type gut, fat body and Malpighian tubule; (B) wg gut, fat body, Malpighian tubules, larval hypoderm and salivary gland; (C) wildtype brain and eye-antenna imaginal discs; (D) en brain and eye-antenna imaginal discs; (E) wild-type larval hypoderm, salivary gland and imaginal discs; (F) nkd larval hypoderm and imaginal discs; (G) ptc larval hypoderm and imaginal discs; (H) imaginal discs from (G) removed from larval tissue, brain, b; imaginal disc, d; fat body, fb; gut, g; larval hypoderm, h; Malpighian tubule, mt; salivary gland, sg.

Table 1.

Identity of adult structures in differentiated implants

Identity of adult structures in differentiated implants
Identity of adult structures in differentiated implants
Fig. 3.

Adult structures derived from cultured embryos. (A) Wild-type head capsule with frons, F. (from eye-antenna disc); (B) en antenna showing posterior saw tooth bristles (arrow); (C) en wing, arrow marks posterior margin characteristic of an en transformation; (D) wild-type leg; (E) nkd leg; (F) ptc leg; (G) wild-type wing; (H) nkd wing, bristles on wing blade are due to h1 mutation present on the nkd chromosome (I) ptc wing.

Fig. 3.

Adult structures derived from cultured embryos. (A) Wild-type head capsule with frons, F. (from eye-antenna disc); (B) en antenna showing posterior saw tooth bristles (arrow); (C) en wing, arrow marks posterior margin characteristic of an en transformation; (D) wild-type leg; (E) nkd leg; (F) ptc leg; (G) wild-type wing; (H) nkd wing, bristles on wing blade are due to h1 mutation present on the nkd chromosome (I) ptc wing.

wingless

Homozygous wg embryos were recognized by the lawn of denticles and abnormal head skeleton (Nüsslein-Volhard and Wieschaus, 1980; Fig. 1C). On dissection from the vitelline membrane, the gut extruded from the anterior end. If the gut showed muscular contractions, the embryonic tissues were considered viable and implanted into hosts. wg embryos grew during culture but produced rather small implants. These had larval gut, salivary gland, fat body and rarely Malpighian tubules but lacked imaginal disc material (Fig. 2B). Because disc tissue may have been present in small amount but hidden in the mass of larval tissue, any tissue that was not clearly gut was transplanted to larval hosts. Metamorphosed implants derived from 30 embryos were recovered (Table 1). All of these implants were necrotic and none had recognisable adult cuticular structures.

engrailed

Homozygous en embryos were recognised by the reduced number of denticle belts and abnormal head skeleton (Nüsslein-Volhard and Wieschaus, 1980; Fig. 1E). en embryos increased in tissue mass during culture and the tissue masses recovered had imaginal discs and all larval tissues. This disc material was often associated with the brain suggesting it was eye-antenna (Fig. 2D). Metamorphosed implants derived from 51 embryos were recovered (Table 1). 50 of these embryos gave rise to implants characteristic of the eye-antenna disc (Fig. 3B). 5 implants characteristic of thoracic discs were recovered (Fig. 3C). Four different alleles of en were used in these experiments and the distribution of number and type of implants from each combination is shown in Table 2. The cuticle markers stw and pwn confirmed that the tissue was derived from homozygous en embryos in all combinations except en10/ en7, and this unmarked set served to show that the presence of pwn, a near-lethal cuticle marker, was not selectively eliminating thoracic implants (G=0.22, P>50%).

Table 2.

Distribution of adult structures derived from engrailed embryos of different allelic combinations

Distribution of adult structures derived from engrailed embryos of different allelic combinations
Distribution of adult structures derived from engrailed embryos of different allelic combinations

98% of cultured en individuals produced eyeantenna implants compared with 67 % of cultured wild types. This does not mean that en embryos had more eye-antenna discs than wild type (we observed a maximum of 2 in cultured embryos of both genotypes) but rather reflects that, to be represented in Table 1, an embryo must have produced at least one differentiated implant. In the case of en embryos, which only produced thoracic discs at 10% of the wild-type frequency, this was most likely to be an eye-antennal implant.

naked

Homozygous nkd embryos were recognised by their cuticles which were devoid of all but a few denticles (Jürgens et al. 1984; Fig. 1G). Transplanted nkd embryos produced all larval tissues and imaginal discs. Unlike those derived from cultured wild-type embryos, the nkd discs tended to grow as a merged sheet of material rather than as discrete discs (Fig. 2F). Following metamorphosis, tissue from 14 nkd embryos produced adult cuticular structures including derivatives of the eye-antennal, leg and wing discs (Table 1 and Fig. 3E and H). The absence of the cuticle marker Sb, present on the balancer chromosome, confirmed that the tissue was derived from homozygous nkd embryos.

patched

Homozygous ptc embryos were recognised by the abnormal head skeleton, mirror-image denticle belts and absence of posterior spiracles (Nüsslein-Volhard and Wieschaus, 1980; Fig. 1L). Transplanted ptc embryos produced the same inventory of larval and imaginal tissue as wild type. The imaginal discs often grew as a mass of merged tissue (Fig. 2G and H) resembling the organization observed in disc tissue from cultured nkd embryos. Following metamorphosis, tissue from 17 ptc embryos produced adult cuticular structures. The implants included structures from the eye-antenna, leg and wing discs (Table 2 and Fig. 3F and J). The cuticle marker stw confirmed that the tissue was derived from homozygous ptc embryos in the genotype stw ptcIN108I stw ptcG12 (4 embryos).

Morphology of naked and patched imaginal discs en-expression is a useful marker for posterior hypodermis cells throughout development and can be easily monitored by visualising the β-galactosidase activity associated with a strain that contains a P-transposon, carrying the en promoter fused to a prokaryotic lac-Z gene, integrated at the en locus (C. Hama and T. Kornberg, unpublished). Cultured en-lac-Z /CyO thoracic discs (n=33) gave a relatively normal pattern of en-expression with posterior staining (Fig. 4A). We examined en-lac-Z expression in cultured nkd and ptc discs in an attempt to correlate the abnormal morphology (Fig. 2H) with altered posterior domains in the discs. Cultured nkd and ptc discs showed abnormal patterns of en-expression which were often difficult to analyse because, unlike the control wild-type discs, the discs were folded and grew close together so that the limits of a single disc were difficult to discern (Fig. 4B). Four cases of discrete, mature, nkd discs were analysed and one is shown in Fig. 4C. This disc and the other three had a similar organisation with a central domain of en-expression flanked by two regions of nonexpressing cells. In contrast, discrete, mature ptc discs (n=7) had a central domain of nonexpressing cells flanked (or surrounded) by en-expressing cells (Fig. 4D).

Fig. 4.

engrailed expression in naked and patched discs. (A) cultured wild-type leg and wing discs; (B) mass of cultured nkd discs; (C) cultured nkd disc; (D) cultured ptc consistent with the results for the disc; (E) cultured wild-type eye-antenna disc with antennal and ocellar ezi-domains; (F) cultured nkd eye-antenal disc with antenna en-domain. en-exprcssion corresponds to the blue β-gal positive regions in the discs.

Fig. 4.

engrailed expression in naked and patched discs. (A) cultured wild-type leg and wing discs; (B) mass of cultured nkd discs; (C) cultured nkd disc; (D) cultured ptc consistent with the results for the disc; (E) cultured wild-type eye-antenna disc with antennal and ocellar ezi-domains; (F) cultured nkd eye-antenal disc with antenna en-domain. en-exprcssion corresponds to the blue β-gal positive regions in the discs.

Wild-type eye-antennal discs (n=22), which grow attached to the brain and mouth hooks, were more distorted but showed an antennal patch of expression and a spot of expression that corresponds to the ocellus (Brower, 1986; Fig. 4E). Cultured nkd and ptc eyeantennal discs were relatively normal but had more variable en-expression (Fig. 4F).

Morphology of engrailed adult cuticle

Three of the five thoracic implants derived from en embryos (Table 1) were sufficiently well developed to be analysed in detail and were classified as structures of anterior provenance. One implant formed a wing blade with mirror-image anterior structures (Fig. 3C), resembling the adult wings formed in situ by enr homozygotes (Garcia-Bellido and Santamaria, 1972) and two leg implants had mirror symmetric sets of duplicated sternopleural bristles. We cannot judge whether this mirror-imaging arises from a double anterior disc primordium or from subsequent epimorphic regulation in response to the formation of an anterior compartment ‘half disc’. Antennal structures from en embryos apparently included those of both compartments (Morata and Lawrence, 1978). Although there is no unequivocal marker of the antennal posterior compartment, the saw-tooth bristles are posterior except for one that sometimes lies in the anterior compartment (Morata and Lawrence, 1978). Normal numbers of saw-tooth bristles were observed in en implants (Fig. 3B) and, as these are never duplicated in en mutant clones (Morata et al. 1983), it is likely that the posterior compartment is intact.

Imaginal discs of the thoracic region

No thoracic discs were recovered from cultured wg embryos, and cultured en embryos produced thoracic discs at 10% of the wild-type frequency. By the extended germ band stage, an embryo of either genotype is functionally null for both wg and en, as continued transcription of each gene is dependent upon the transcription of the other (Martinez-Arias et al. 1988; DiNardo et al. 1988). The different results from the embryos of the two genotypes probably reflect the different requirement for the genes in the metamere (Ingham, 1988). The embryos have different cuticle phenotypes, wg embryos have more elements of the pattern deleted than en embryos (Fig. 1 D and F), and thus cultured wg embryos may fail to produce any adult thoracic structures because of the deletion of a large part of the metamere. We suggest that the rare en embryos that produced thoracic discs were individuals in which wg activity persisted sufficiently long to permit the formation of a thoracic disc primordium which, because it lacked en function, metamorphosed into a purely anterior structure. An alternative interpretation of the recovery of some thoracic discs from en embryos is that the en alleles used were not nulls and had some residual activity. However, there were no differences between the number of disc implants recovered from the various allelic combinations used (Table 2), and two alleles, en7 and en10, encode truncated proteins that lack the homeodomain and thus presumably have no function (Poole and Kornberg, 1988; T. Kornberg, personal communication).

Cultured nkd and ptc embryos gave rise to thoracic imaginal discs. Wild-type discs grew discretely, much as they do in a normal larva, whereas the cultured nkd and ptc discs were often folded and fused. In nkd and ptc embryos, ectopic borders between wg-and en-expressing cells arise in each metamere; there is a broader domain of en-expression flanked by two wg stripes in nkd embryos, whilst in ptc embryos there is a broader wg domain flanked by two en stripes (Martinez-Arias et al. 1988; Di Nardo et al. 1988). Discrete nkd and ptc discs had a disposition of engrailed domains compatible with their respective embryonic patterns.

The results suggest that thoracic imaginal primordia may arise within two adjacent bands of embryonic cells expressing wg and en respectively. Mutants in which these cells no longer express the genes either do not produce thoracic discs (wg- embryos) or produce only rare partial structures (en embryos). Mutant embryos in which ectopic cells express wg and en (nkd and ptc) have thoracic discs whose morphology is consistent with development from altered primordia incorporating these cells.

Imaginal discs from the head region

Three of the four segment-polarity mutants we cultured produced eye-antennal discs; nkd, ptc and en. wg embryos produced no discs and this may be explained by the possible loss of the cells in the large region of the antennal segment where wg is normally expressed (Baker, 1988b). That nkd and ptc embryos produced eye-antennal derivatives was consistent with the results for the thorax. Neither of these two genes appear to be essential for the production of imaginal primordia but are required for the normal morphology of thoracic discs as judged by en expression, en expression was relatively more normal in eye-antennal discs from cultured nkd and ptc embryo.

The finding that most en embryos produced eye-antennal structures was not expected based on the results for the thoracic discs. Most en embryos did not produce thoracic structures, possibly because early wg expression terminates prematurely, but most en embryos produced anterior antenna and eye structures. There is also evidence that the posterior antenna derived from en”implants was normal. A role for en in the posterior antenna was proposed because en2, unlike most en alleles, causes antennal duplications (Morata et al. 1983; Lawrence and Struhl, 1982). This allele is a rearrangement and the phenotype may be due to the disruption of another function. However, aristapedia clones show that the antennal compartments are serially homologous to A and P compartments in other segments (Morata and Lawrence, 1978, 1979). The results presented here suggest that en does not have an identical role in all body segments (see also Lawrence and Struhl, 1982; Eberlein and Russell, 1983; Gubb, 1985) and apparently no influence on the development of the eye-antenna.

We thank the Bowling Green stock centre and Dr T. Kornberg for stocks and Drs N. Tripoulas and R. Phillips for commenting on the manuscript. A. A. S., E. H. and A. S. acknowledge research support from the National Institutes of Health, J. R.S.W. and M.C.G. from the Science and Engineering Research Council of the UK and A. A. S. and J. R. S. W. from the Nuffield Foundation. I. J. H. R. is the recipient of an SERC studentship. J. R. S. W. is grateful to the Wellcome Trust and the Royal Society for jointly funding his visit to The Johns Hopkins University.

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