Cell Differentiation, 6 (1977) 319--330 © Elsevier/North-Holland Scientific Publishers Ltd.

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ASPECTS O F D E V E L O P M E N T O F IMAGINAL DISCS IN A NON-PUPARIATING LETHAL MUTANT IN D R O S O P H I L A M E L A N O G A S T E R

Collin MURPHY* and James W. FRISTROM

Department o f Genetics, University o f California, Berkeley, CA 94 720, U.S.A.

Allen SHEARN

Department o f Biology, Johns Hopkins Uniuersity, Baltimore, MD 21218, U.S.A.

Accepted 4 August, 1977

Imaginal discs from a non-pupariating mutant, lethal-1 a, were cultured in vitro. Re- sponses of the discs to B~cdysone are abnormal, including failure to maintain an in- creased level of RNA synthesis, depression of protein synthesis and cell death. When lethal-I a discs are transplanted into wild-type larvae for metamorphosis, almost all bristle-forming regions fail to differentiate, whereas non-bristle-forming regions do so normally. Mutant ~arvae apparently secrete molting hormone, and can synthesize cuticle within unevaginated discs. The in vitro experiments suggest that abnormal responses of mutant tissue to ~cdysone in situ result in non-pupariation , failure to disc evagination and death.

Metamorphosis in Drosophila melanogaster is under the control of the hormone, ecdysone, which is produced by the ring gland in the head of the larva. High ti tres of ecdysone prior to metamorphosis induce the larval epidermis to pupariate and the imaginal discs to undergo morphogenesis. Imaginal discs respond to ecdysone first by an increase in macromolecular synthesis (Ralkow and Fristrom, 1971; Fristrom et al., 1974; Logan et al., 1975) and then undergo evagination to take on t h e / o r m of the adult struc- tures (Fristrom and Fristrom, 1975). Cell death does not seem to play a significant part in the early stages of disc metamorphosis (Sprey, 1971; Fristrom and Fristrom, 1975).

In the hope of genetically dissecting the process of disc evagination we have been studying lethal mut~tnts affecting imaginal discs (Sheam et al., 1971; Stewart et al., 1972; Murphy, 1974). One of these mutants , lethal (1) discs normal-1 a (hereafter called 1-1a), a non-pupariating lethal, was singled ou t for extensive s tudy because of its developmental defects and is the subject of the present report . Specifically, 1-1 a is characterized by ex- tensive degenerat ion of the imaginal discs at the stage when puparium form- ation would normally occur, and by incomplete evagination in vitro. In ring-X elimination tests for au tonomy, larval-pupal gynanders are formed

* Current address: Department of Biology, San Francisco State University, San Francisco, Ca 94132, U.S.A.

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with the portion of the body hemizygous for the lethal genotypes failing to pupariate (Kiss et al., 1976).

Several gynanders were produced in which the anterior half (lethal geno- type), containing the ring gland, failed to pupariate while the posterior part (wild-type) pupariated normally. This indicates that the mutant ring gland is able to induce metamorphosis in normal tissue (i.e. produce adequate titres o f /~cdysone ) , but that the mutant tissue is not able to respond normally to the hormone. The extensive degeneration observed in mutant discs in the late third instar suggested that cell death is the ultimate response of the imaginal disc cells of 1-1 a to metamorphic concentrations of ecdysone. In order to test this possibility we have taken discs from early third instar larvae (when the in vivo titre of ecdysone is still low) and studied the effect of #3-ecdysone in vitro on macromolecular synthesis and cell death.

We have also investigated the action of the 1-1 a mutant gene in terms of the capa,~ty of the mutant discs to differentiate when transplanted to wild-type hosts. Clearly, there can be no differentiation in situ or even in mutant/wild-type gynanders since the failure to pvpariate results in death of the entire organism. 'However, earlier studies, where mature lethal leg discs were transplanted to wild-type larvae, showed that the mutant discs had a limited capacity to differentiate, i.e. lethality was only partially autonomous (Stewart et al., 1972). These studies have now been extended by transplanting young mutant discs into Imval and adult wild-type hosts.

MATERIALS AND METHODS

Stocks

For descriptions of genetic stocks see Lindsley and Grell (1967). The lethal is balanced over Binsn and is carried on a chromosome marked with y, w and sn 3 . Two types of male larvae are present, non-lethal Binsn larvae and y, l-1 a, w,sn 3 larvae. The latter are recognizable because of the ye l low larval mouth- parts. An Ore-R stock was also used.

Cell death s tudies

Larvae were grown at 25°C. on standard Drosophila medium. Develop- mental age was timed from egg deposition (+/- 4 h). Twenty.four hours prior to dissection, male lethal larvae were separated and placed on fresh medium. At 96 h after egg deposition, wing imaginal discs were dissected in ice-cold Ringer's solution. (Larvae at this age have a low titre of ~;-E (Borst et al., 1974)). Discs were subsequently transferred with a micropipet to Robb's culture medium (Robb, 1969), containing ~-E (0.1 ug/ml or 1.0/~g/ ml) or no hormone (control) and incubated at 25°C. for 4 h. Following incubation, discs were rinsed for 10 min in 0.1 M Na cacodylate buffer (pH 7.5), fixed for I h in 1% glutaraldehyde and 1% OsO4 in 0.05 M cacody-

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late buffer at 0°C., dehydrated in ethanol and then embedded in Epon 812. Discs from the same age larvae which had not been incubated in Robb's medium were also fixed as were discs from lethal larvae in age from 120 to 232 h after egg deposition. Sections (1/~m) for light microscopy were cu~: with glass knives and stained with 1% toluidine blue. Thin sections were cut with a diamond knife and stained with uranyl acetate and Reynold's leaA citrate.

To determine the amount of cell death present, sections were examined using light microscopy at approx. 1000× as follows:

Approximately every fourth section of each wing disc was examined; since disc cells are approx. 1 ~m across, seeing the same cells more than once is avoided. When disc cells die, they fragment into several pieces which are later removed by phagocytosis or exocytosis (Fristrom, 1969; Sprey, 1971). These fragments, as well as intact but dead or dying cells stain deeply with toluidine blue. Each darkly staining cell or fragment was counted as a 'dead fragment'; no attempt was made to distinguish dead cells and cell fragments. Live cells were also counted. Sections from the outermost regions of the specimens were excluded since they contained very few cells. Despite close- ness of chronological age, the wing imaginal discs varied greatly in size. To standardize comparisons between discs, a ratio was calculated by dividing the number of dead fragments in all sections scored from a single disc by the number of live cells present.

Incorporation studies

Eggs were collected over 12-h periods and larvae were grown at 25°C and recovered from the medium 4 days after hatching. The imvae were rinsed with 1 M perchloric acid and then exhaustively washed with wa~;er. Wing discs were dissected from non-lethal (Binsn) and lethal (1.1 a) male larvae in Robb's medium, rinsed three times and then incubated in 0.1 ml of Robb's medium in a microtiter plate at 25°C with and without ~-E (1 ~g/ ml). For studies on incorporation into RNA, the discs were incubated singly in standard Robb's medium. After 1, 3 and 5 h of incubation, [3H] uridine (5 Ci/mmol) was added to a final concentration of 0.5 mCi/ml and incuba- tions were continued for an additional 30 rain. For studies on incorpora- tion into protein, groups of 4 discs were incubated in 0.05 ml of leucine-free Robb's medium With and without ~-E (1 /~g/ml) for 3.5 h. [aH]leucine (5 Ci/mmol) was added to a final concentratiorr of 2.5 mCi/ml and incuba- tions were continued for an additional 30 min. Incubations were terminated in both sets of experiments by removing the culture medium and adding 0.1--0.2 ml of 70% ethanol. After standing overnight the discs were then washed 8 times in 0.1 ml vols. of 0.5 M perchloric acid and 2 times in 95% ethanol. Single discs were transferred with stainless steel needles to scintilla- tion vials to which 0.5 ml of Soluene (Pack'~rd Corporation) was then added. Vials sat overnight at room temperature after which 20 ~l of H20 and 5 ml

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of a to luene based scinti l lation cocktai l were added. Incorporated radio- activity was t h e n determined using an LKB scinti l lat ion counter. Using these condit ions and methods , incorporat ion o f [3H] uridine and [ 3 H ] l e u c i n e into acid insoluble material was f o u n d to be linear with t ime for up to 1 h.

Transplantation studies

Discs from early and Iv.re third instar 1-1 a larvae were transplanted to both larval and adult hosts using standard transplantation techniques. The sizes of discs were estimated by the product of their length and width measured with an ocular micrometer. Mutant discs which had undergone metamorphosis along with wild-type hosts were recovered and mounted on slides for observa- tion.

RESULTS

Cell death in lethal 1-1 a and non-lethal wing discs

Male larvae hemizygous for the 1-1 a trait develol~ slowly. At 96 h a ~ e r egg deposition, mutant larvae and imaginal discs are about the same size as

TABLE I

Cell death in l e t h a l - 1 a and non-lethal wing imaginal discs a

Incubat ion l e t h a l - I a discs condit ions

Non-lethal discs

d.f. l.c. d.f.]l.c, d.f. l.c. d.f.]l.c.

No 448 ± 2767 -~ 0.19 *- 32 -+ 4944 ± 0.013 -+ Incl~bation 213 1880 0.10 21 4675 0.018

(n = 6) (n ffi 4)

No 886 +- 1684 ± 0.54 ± 339 ± 5946 ± 0.057 ± hormone 351 778 0.11 148 796 0.023

(n = 9) (n = 3)

+0.1 #g/ml 2776 ± 2971 ± 0.95 ± ~-ecdysone 558 529 0.19

(n = 8)

+1.0 #g/ml 3049 ± 2527 ± 1.34 ± -ecdysone 950 660 0.8

(n ffi 3)

239 ~ 4518 ± 0 .053 ± 148 1108 0.019

(n ffi 3) b

a All numbel's are means and standard deviations, d.f. = dead fragments; I.e. = liver ceils; d.f./l.c. = ratio o f dead fragments to live cells. Large standard deviations in some categories reflect the great variability and small numbers o f discs scored.

b All ecdyson ,~. treated non-lethal discs are t reated together.

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early third instar, 72-h-old non-lethal larvae, although there is much variabil- ity. The amount of cell death in discs from lethal and non-lethal larvae at the time of explantation and after incubation with and without fl-E is summarized in Table I.

As can be seen from Table I, I - 1 a discs show substantially more degenera- tion than their control counterparts and greater degeneration in the presence of hormone than in its absence. The effect of ecdysone on cell death in the lethal discs is dramatic with about half the tissue present being degenerate. It should be pointed out that the ratio of dead to live cells in lethal discs incubated without hormone may be artificially high, because the discs in this category were by chance much smaller at the beginning of the incubation than the hormone-treated discs or the non-incubated discs. This may increase the ratio of dead to live cells in two ways: (1) A given amount of cell death in a small disc (with fewer live cells) will result in a much higher ratio than the same amount of cell death in a larger disc. (2) Since the discs were un- usually small at the beginning of the incubation, it is possible that there were more dead cells than normal at the outset. The effects of culturing 1-1 a discs in ecdysone is perhaps more evident in .the photographs of representative sections (Fig. 1). As can be seen, lethal discs cultured with ecdysone show far more cellular degeneration (as judged by the number of darkly staining bodies) than nondethal discs or lethal discs cultured without hormone. Examination of histological sections revealed only one area where degenera- tion appeared to be localized. In the wing pouch which gives rise to the wing blade, wing margin and wing hinges, there is particularly extensive cell death in cultured and uncultured 1 - I a discs at all stages examined.

Incorporation into RNA

Previous results (Raikow and Fristrom, 1971) have demonstrated that immediately upon introduction into culture the rate of incorporation of [3H] uridine into RNA in discs is high, but then decreases to reach a steady state level after about 3 h of incubation. When 5-E is added at the start of the incubation the final steady state level of [3H] uridine incorporation into RNA is 2--4 times greater than that of control discs. In the present experi- ments ~-E was present or absent at the start of the incubations. In the absence of ~-E, non-lethal discs had an initial elevated level of [3H] uridine incorporation which then declined to a steady state level (Fig. 2A). In the presence of ~-E, non-lethal discs had elevated levels of incorporation after 1, 3 and 5 h of incubation (Fig. 2A, B). Lethal discs cultured in the absence of ~-E had an elevated level of incorporation into RNA at the start of the incubation which declined to a steady state value similar to that of the non-lethal discs cultured without ~-E (Fig. 2A, B). In the presence of/~-E, an elevated rate of incorporation of [3H] uridine into RNA is found in the I-I a discs after I h of incubation. However, after 5 h of incubation, incorporation

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Fig. 1. A--D: Wing imaginal discs from larvae 96 h after egg deposition, (25°C), cultured for 4 h in vitro. A: Non-lethal, without ~-E; B: non-lethal, with 0.1 ~g/ml S-E; C: lethal-1 a, without ~-E; D: lethal-1 a, with 0.1 ~g/ml ~-E. Note extensive degeneration. E--F: Discs from lethal-1 a larvae 205 h after egg deposition (25°C). E: Note cuticle on luminal sur- face of disc and dead cellsin the lumen; F: electron micrograph of cuticle on the luminal surface, d.c. = dead cells; p.c. = pupal cuticle.

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into RNA is lower than that found in control discs. These results indicate that 1-1 a discs are capable of responding to/3-E with an increased level of RNA synthesis, but in contrast to non-lethal discs, do not maintain the increased level.

Incorporation into protein

Imaginal discs respond to ~-E with an increased level of protein synthesis

TABLE II

Incorporation of [3H]leucine into protein in l e t h a l - I a and non-lethal discs at 3.5 h after addition of ~-ecdysone (means and standard deviations).

Treatment L e t h a l - 1 a discs (cpm/disc/30 rain)

Non-lethal discs (cpm/disc/30min)

No hormone 8,300 -+ 613 15,550 -+ 1,229 (n = 4) (n = 4)

With hormone 4,950 +- 1,568 2.4,900 +- 2 ,881 (n = 7) (n = 6)

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TABLE III

Effect of culturing in wild-type hosts on size of wing discs from l e t h a l - I a

Treatment or age of discs at 22°C Size of discs in mm 2

Mutant n Normal n

Early third instar, no culture Three days in larval host Seven days in adult host Late third instar, no culture

0.020 +- 0.(J08 (66) 0.026 -+ 0.007 (27) 0.115 ± 0.066 (12) 0.161 ± 0.121 (38) 0.031 ± 0.007 (50) 0.091 ± 0.038 ( 9 ) 0.072 ± 0.049 (37) 0.179 ± 0.007 ( 4 )

with a maximum response being achieved 4--5 h after the start of incubation (Fristrom et al., 1974). In the current experiments incorporation of [3H]- leucine into protein was monitored after a 3.5-h incubation with ~-E. The non-lethal discs responded to incubation with the hormone with an increased level of incorporation into protein (Table II). However, no such response is seen with 1.1 a discs. Indeed the level of incorporation into proetin is depressed in the presence of ~-E (Table II).

T r a n s p l a n t a t i o n s t u d i e s

As indicated in the introductory section, discs from mature 1-1 a l a rvae are

often smaller than their wild-type counterparts and leg discs are capable of only limited differentiation when transplanted to wild-type hosts (Stewart et al., 1972). We have here repeated the experiments with mature leg discs, as well as with wing and eye-antennal disc. We have also investigated whether transplantation of young 1-1 a wing discs to young wild-type larvae or to adult females can influence the ability of the discs to grow anO differentiate. The results of these experiments are summarized in Table III. Young discs transplanted to young wild-type larvae become substantially larger than those left in 1- I a larvae (0.115 ram: vs. 0.072 mm 2 ). However, early third instar 1-1 a discs cultured in adult female abdomens for 7 days only reach a size of 0.031 mm 2 .

Following in vivo culture for 3 days in wild-type larvae or for 7 days in adult female abdomens, or following direct transplantation of mature ,aiscs to metamorphosing wild-type hosts, 1.1 a wing discs differentiate as follows (Fig. 3): In all implants recovered, there is normal differentiation of cer-

Fig. 3. Implants derived from wing imaginal discs injected into wild-type metamorphosing larval hosts. A--C: Norm~J disc from late third instar larva; D,E: mutant disc from young third instar larva cultured 3 days in larval host, then reinjected into metamorphosing host; F,G: mutant disc from late third instar larva injected into metamorphosing host. Abbre- viations used: Alu, alula; Cos, costa; DoT, dorsal mesothorax; Teg, tegula; TR, triple row; WBT, wing blade trichomes. Note absence of bristles on wing margin of mutant implants, D and F × 134.4.

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rain parts of the dorsal thorax (including 2--7 of the normal 11 macrochae- tae, 0--2 of the normal 100 microchaetae), ventral thorax and wing blade. The parts of the wing disc which differentiate do so completely normally. However, missing from the recovered implants are the bristles along the entire wing margin, the alula, costa and tegula, and most of the macro- and micro~haetae of the dorsal thorax. Elimination of primordia arising in the pouch region of the wing such as wing margin is not surprising given the localized degeneration found in sectioned discs, as mentioned above. Al- though certain re~ions of the presumptive thorax may also be d.estroyed by localized degeneration, no consistent cell death pattern was noted in sections of this relatively small region of the wing disc.

Similarly, transplanted leg and eye-antennal discs differentiate as follows: Leg discs differentiate only 2--3 poorly formed chaetae, whereas control legs contain hundreds of them (cf. Stewart et al., 1972). Eye-antennal discs differentiate eye facets, prefrons and ptilinum, but no cephalic chaetae or antennal parts. Thus, the new transplantation results indicate that the lethal gene preferentially affects the bristle primordia suggesting that the wild-type allele of 1-1 a plays a vital role in the developmental program of these cells.

Observat ions o f 1-1 a discs in over-age larvae

Mutant larvae continue to live long past the normal time for puparium formation (120 h from egg deposition). At 186 h a~out 50% of mutant lar- vae are still alive, but by 220 h almost all of the larvae are dead. During this p£-otracted larval stage, the larvae do not grow beyond the normal third in- star size, but the wing discs continue to grow, taking on complex folding and containing either fibrous or amorphous material in the lumen. Cell death is variable, with some sections having few dead cells and others having extensive degeneration, often in the wing pouch.

The most striking change in appearance is the presence of pupal cuticle in these non-evaginated wing discs. Cuticle deposition is variable, ranging from the formation of only the cuticulin layer up to layers 3 ~m thick which display the typical sculptured appearance resulting from the helicoidal struc- ture of cuticle (Fig. 1E,F). However, there are no signs of apolysis of the pupal cuticle, formation of an adu l t cuticle or of bristle differentiation. Pupal cuticle deposition has occurred in these discs in the absence of evagi- nation. Evidently evagination is not a prerequisite for cuticle formation, even though in normal discs cuticle deposition occurs following evegination.

DISCUSSION

The results and conclusions of the various experimental approaches used here to elucidate the action of lethal 1.1 a can be summarized as follows:

1) Early third instar lethal discs degenerate when incubated in the pres- ence of ecdysone as indicated by the large number of degenerating cell fragments seen in toluidine blue stained sections. Cell death represents an

329

abnormal response of the le|ihal disc cells to the presence of the hormone which normally induces disc cells to proceed with morphogenesis and differentiation of adult structures.

2) Lethal 1.1 a disc cells, like normal disc cells, respond ip~ially to the presence of the hormone by an increase in RNA synthesis. Tb;s indicates that the mutant cells contain ecdysone receptors which are able to bind to chromatin and trigger the first phase of the metamorphic sequence of gene action. However, the low level of RNA and protein synthesis in mutant discs after a 5-h exposure to hormone suggests a failure to proceed with the developmental program. A general reduction of synthetic activities can be regarded as coinciding with the process of cell death.

3) The transplantation results indicate that the lethal effect of 1-1 a is not necessarily expressed in all cells of the disc. Rather, it is bristle precur- sor cells which are most adversely affected by the mutant gene and which generally fail to differentiate in transplanted discs. Thus there seem to be two classes of cells in the mutant discs; those which are able to differentiate under appropriate conditions, i.e. in which the action of the gene 1-1 a is not lethal and those in which the action of 1-1a is lethal.

4) The presence of cuticle in mutant discs which have been allowed to develop in situ is an important observation since these discs have n o t evag -

i n a t e d . This provides an example of the uncoupling of two developmental events both of which are dependent on ecdysone. Since evagination and cuticle deposition are distinctly different biological processes, the existence of separate developmental pathways is reasonable. However, since pre- mature deposition of cuticle would enclose the disc in an inflexible shell and prevent evagination, it also seems reasonable to assume that precocious cuticle deposition is prevented in wild type discs. The hormonal signals for cuticle deposition and evagination are probably somewhat different (Logan et al., in preparation), and would normally prevent premature cuticle depo- sition. Thus, cuticle deposition in discs is not triggered by the intrinsic morphogenetic stage of the disc, but is independently triggered by the external hormonal milieu which apparently is normal in 1 . I a larvae (Kiss et al., 1976).

It is also important to note that the cells of the mutant discs which differentiate in transplants may well do so without evagination of the disc as a whole. This is almost certainly true of those discs which are trans- planted directly to host larvae about to pupariate since such discs are unable to ev~inate in vitro or in situ.

In conclusion then, although we are a long way from understanding the molecular basis of the action of 1- I a we have been able to define some developmental events in which the action of the allele appears to be crucial. These include puparium formation, some early event in the process of bristle differentiation and evagir, ation. If we can discover what these diverse developmental processes have in common it should bring us a good deal closer to an understanding of the action of the gene as well as of the processes themselves.

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ACKNOWLEDGEMENTS

Some of the work reported in this paper was performed while James W. Fristrom was a research guest in the Genetics Institute, Biological Research Cen ter, Hungarian Academy of Sciences, Szeged. We are indebted to Drs. Janos Szabad and Janos Gausz for assisting in some of the experiments.

We thank Evelyn Keigler for her skillful technical assistance. This work was supported by a grant from ERDA EO4334PA186, to JWF, and by a grzmt from NSF GB40737, to AS.

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178,285--302 (1975). Robb, J.A.: J. Ce!! Biol. 41,876--884 (1969). Shearn A., A. Garen and W. Gehring: Proc. Natl. Acad. Sci. USA 68, 2594--2598 (1971). Sprey, T." Neth. J. Zool. 21,221--264 (1971). Stewart, M., C. Murphy and J.W. Fristrom: Dev. Biol. 27, 71--83 (1972).