DEVELOPMENTAL GENETICS OF DROSOPHILA

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DEVELOPMENTAL GENETICS O F DROSOPHILA1

HEINRICH URSPRUNG

Laboratory for Developmental Biology, Swiss Federal Institute of Technology,

Zurich, Switzerland

HE fruitfly, once the organism of choice in genetic studies of many investi- Tgators, has lost much of its popularity in the last decade. Its biological com- plexity and slow breeding presented difficulties of resolving power for genetic fine structure analyses. Its small size, it was argued, posed problems for sophisticated biochemical work on proteins and nucleic acids, thus rendering the vast collec- tion of data on morphological mutants of little exploitable use. Only a few mutants were known that affected known metabolic steps, and it appeared hard indeed to generate more such mutants experimentally. As a consequence fine structure analysis by saturation genetics and biochemically meaningful complementation studies seemed out of reach. Meaningful studies on cell differentiation appeared difficult, again because of the small size of the organism, and more importantly, the difficulty of isolating and biochemically characterizing tissues and cells. The lack of convenient cell and tissue culture methods for insect material, largely caused by the difficulties encountered in reproducing subtle changes in hormone concentration, represented another handicap for such studies. Cell fusion and nuclear transplantation, methods successfully used in vertebrate embryology, were unknown in Drosophila and appeared not to be feasible.

This situation has markedly changed over the last few years, largely because of the hard work of a relative small group of scientists who stubbornly kept believing in the potential of their organism.

Genetic and cytogenetic fine structure analyses

CHOVNICK, SCHALET, KERNAGHAN and TALSMA (1962) in a pioneering study used closely linked lethals flanking the rosy cistron in such a way as to permit only the (rare) crossovers to survive. By this procedure, recombination tests representing the order of

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offspring were carried out, and it was possible to calculate intracistronic distances as well as to estimate the entire length of the rosy cistron. When these map distances were converted to nucleotide pairs, using the estimates of RUDKIN (see

RUDKIN

1972 for references), a total length of 11.8 X

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nucleotide pairs was calculated for the rosy cistron, which would permit a code f o r a protein with a molecular weight of about 390,000. These calcula- tions were made with many assumptions, one of which was that the entire cistron

Original work reported in this review was in part supported by a grant from the Swiss National Science Foundation, project 3.8640.72.

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3 74 H . URSPRUNG

be reflected in the protein product. Our present knowledge of the transcriptional unit in eukaryotes (GEORGIEV 1972) would lead to a substantially lower estimate of the molecular weight of this product.

In a more recent, very elegant study, JUDD has presented a genetic fine struc- ture analysis at the cytogenetic level (

JUDD,

SHEN and KAUFMAN 1972). These authors have saturated the cytogenetic region 3A2-3C2 by inducing mutations with X-rays, ethyl methanesulfonate and N-methyl-N’-nitro-N-nitroso-guani-

dine. They then analysed the treated chromosomes by crosses with known de- ficiencies of that chromosomal region, which comprises 15 bands. The mutants were furthermore subjected to complementation analysis and arranged into 16 complementation groups. The data permit the conclusion that each chromomere

(i.e., a band, an interband, or a band and its interband) represents one functional group. Expressed in terms of DNA content per haploid equivalent, this means that functional units could range from about 5,000 to some 50,000 nucleotide pairs. An average chromomere then would leave room for some 30 genes each a 1,000 nucleotides long.

Much additional work is obviously needed for reconciling this finding with the results obtained from formal-genetic analysis of, e.g., the rosy cistron. What is clearly needed for such studies is precise molecular information on enzymes with a known genetic and/or cytogenetic basis. Amylase (DOANE 1970), alcohol dehydrogenase ( SOFER and URSPRUNG 1968; JACOBSON, MURPHY and HARTMAN 1970; JACOBSON and PFUDERER 1970; L. GERACE, M. SCHWARTZ and

W.

SOFER, in preparation). This enzyme is of particular interest, because ADH-negative strains have recently been produced using a new selective principle. Mutagenized flies were exposed to the vapors of pentenol. Because of their ADH content wild type flies oxidize this compound into a toxic ketone. Only those flies survive that lack the enzyme. Using this method, SOFER and HATKOFF (1972) have isolated 11 new Adhneg strains. The enzyme pair xanthine dehydrogenase and aldehyde oxidase also appears well suited f o r this purpose.

Genetics and Biochemistry of Xanthine Dehydrogenase ( X D H ) and Aldehyde Oxidase ( A D X )

Loci of enzymes for which electrophoretic variants or null alleles are available can be mapped without much difficulty. In Drosophila, this has been done for some 30 enzymes as of today (see Fox, ABACHERLI and

URSPRUNG

1972; MAD-

HAVAN and URSPRUNG 1973, for additional enzymes). This gene-enzyme map

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Much of the earlier work on this interesting gene-enzyme system has been accomplished and reviewed by GLASSMAN (1965). Largely from his work emerged two models that could explain this peculiar trans-control of several enzymes by one distant locus. One of these assumes that the enzymes be multi- nieric molecules composed of at least two different subunits, each under the control of one of the loci involved. To test this model requires pure enzymes. For

X D H ,

purification to homogeneity an acrylamide gels and

in

isoelectric focussing

gels has recently been occomplished by SEYBOLD (1973), who used a combination of ammonium sulfate fractionation, heat precipitation (70"), and chromatog- raphy on DEAE-cellulose, hydroxyl apatite, and Sephadex G-200 to obtain XDH of a molecular weight of about 300,000. Upon SDS electrophoresis, this molecule splits up into subunits of about one half this size. The precise size of the subunits is not known at the moment, nor is it known with certainty whether they are of identical size. Aldehyde oxidase is currently being purified by R. ANDRES (per- sonal communication), and it is interesting to note that it co-purifies with XDH to a large extent. Particularly noteworthy is the fact that it tolerates the rather drastic heat step. This enzyme too has a molecular weight of about 300,000. What is being tested now is whether the ry+, lao+ and ma-l+ loci each code for one of the two subunits of an enzyme. It would seem to make sense that XDH and AOX contained a common subunit, coded by the ma-Z+ locus, and a specific subunit, coded by ry+ or Zao+, respectively. These latter subunits then would be responsible for the substrate specificities of the two multimers.

The second model assumed that the ma-1 locus is not responsible for a shared subunit, but rather for the production or attachment of a necessary cofactor. Through analogy with vertebrate XDH and AOX, molybdenum, FAD, and iron offered themselves for this role. We have examined our pure XDH preparations for their content of two of these cofactors, with the following preliminary results (W. LUTZ and

W.

D. SEYBOLD, in preparation). Using flameless atomic absorp- tion photometry, a value of 1 Fe/NIW = 160,000 was found (cf. 8 FeJMW = 300,000 for chicken liver XDH) . Using the same method, no molybdenum was found under conditions that would have detected one MO atom per 3 X

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MW. These findings cast doubt on the homology of Drosophila and vertebrate XDH. Perhaps other cofactors will have to be found before the cofactor hypothesis can be tested.

Stage and tissue specificity of enzymes

My student

M.

CONSCIENCE-EGLI (1973) has screened larval fat body, intestine, Malpighian tubules, and salivary glands for the content of over 20 enzymes. She has furthermore assayed 12 enzymes in adult fat body, intestine, muscle, and eggs. As was to be expected from similar studies in vertebrates (KNOX

19 72) marked tissue and stage-specific diflerences were found. By histochemical methods, striking demonstrations of cell specificity were possible. Direct chemical assay has permitted a quantitative determination of such differences (URSPRUNG, SOFER and BURROUGHS 1970).

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376 H. URSPRUNG

ticular enzyme, but also whether it synthesizes that enzyme by itself. AlcohoI dehydrogenase may serve as a n example. This enzyme is not detectable in imaginal disks-not by biochemical assays of disks mass-isolated by the

FRISTROM

technique (FRISTROM 1972), nor histochemically. It is present in substantial quantities, however, in the adult derivatives of, for example, the genital disk. When this undifferentiated larval epithelium differentiates during metamorphosis into various cell types of its adult product organ, do its cells actively synthesize ADH, or do they merely sequester the enzyme from the surrounding hemo- lymph or from other cells? These questions were answered by transplanting genital disks of one electrophoretic

ADH

variety into host larvae of the opposite ADH genotype (URSPRUNG, SOFER and BURROUGHS 1970). The disks metamorphosed in their host from which fully differentiated implants could be recovered and subjected to electrophoretic analysis. Their ADH was of the donor’s genotype, indicating that the cells of the implant had synthesized the e n z y m e autonomously. The same conclusion was reached in a similar experiment, in which wild-type genital disks were transplanted into ADH-negative ( GRELL, JACOBSON and MURPHY 1968) hosts. After metamorphosis, the implants contained ADH, which they must have synthesized themselves, for their hosts were deficient in the respective genetic information.

This same kind of reasoning is not only useful for solving questions of cell- autonomous enzyme synthesis, but also for cell-lineage analyses. Using this method, a long-standing controversy on holometabolous insect myogenesis has recently been solved-namely, the question of whether the musculature of appendages is formed from cells contained in imaginal disks or from cells that populate the appendages during metamorphosis. The strategy of the experiment was to raise leg imaginal disks of one electrophoretic variant of arginine kinase in hosts of a different variant of this muscle enzyme. Although an extensive survey of Drosophila stocks and species showed no such variants, the related Drosophilid Zaprionus uittiger turned out to have a n arginine kinase that can readily be resolved from the Drosophila enzyme by electrophoresis on starch gels (WALLIMANN and EPPENBERGER 1973). When Zaprionus leg disks were grown in Drosophila hosts, and the metamorphosed legs were recovered from the hosts after metamorphosis and electrophoresed, they were seen to contain Zapnonus- type arginine kinase (URSPRUNG et al. 1972). The mere fact that these implants contain musculature is no proof that the respective cells stem from the leg disks; they could have invaded the disks during the metamorphosis. But the fact that the arginine kinase is of the donor t y p e clearly shows that the cells producing it were already present in the disk at the time of transplantation. Using recent electron microscope information on leg disks (POODRY and SCHNEIDERMAN 1970),

we can speculate that the so-called adepithelial cells are myoblasts. Direct proof of this assumption requires histochemical demonstration of arginine kinase or other muscle-specific proteins in these cells as they differentiate; this has not been done.

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far been successfully used to trace the lineage, or establish cell-autonomous gene expression, of individual cells. For the solution of the latter type of problem, the induction of genetic mosaics coupled with histochemical staining has been used successfully by W. JANNING (unpublished). Using DICKINSON’S (1971 ) findings on the tissue specificity of the enzyme aldehyde oxidase, and the known fact that maroon-like genotypes lack this enzyme (see above), he produced ring/rod-X chromosome heterozygotes carrying ma-1 on the rod-X. The ring-X chromosome is lost during early cleavage, leading to a genetic mosaicism with cells lacking aldehyde oxidase (ma-l/O) o r possessing it ( m a d / + ) , respectively.

He then visualized aldehyde oxidase activity in freshly dissected tissues by histo- chemistry. In Malpighian tubules, individual cells can be distinguished by this procedure. As the rod-X used also carried the marker white, and since all white cells in the Malpighian tubules of gynandromorphs lacked aldehyde oxidase, while wild-type cells contain the enzyme, the conclusion is safe that aldehyde oxidase is expressed cell autonomously. “Clonal dissection” of organ primordia, finally, is also feasible with such technology, as JANNING (unpublished) demon- strated recently. I n genetic mosaics of the kind described above, he discovered mosaic antennal disks composed of cells that do or do not contain aldehyde oxidase.

Cell culture and cell fusion

I n vertebrates, cell cultures and cell fusion have been used successfully in numerous attempts to study the regulation of established cell specific enzyme syntheses (see EPHRUSSI 1972 f o r a recent review). I n Drosophila, such studies are just now beginning to become feasible. Although Drosophila cells have been kept in culture for several years now (ECHALIER and OHANESSIAN 1970; DOLFINI

1971), only recently have these cell lines been described‘in enzymatic terms (DEBEC, in press). DEBEC assayed 18 cell lines or clones for their content of thirteen enzymes. The lines were all derived from Drosophila embryos. Aliquots of these lines were subcultured in suspension culture or on glass surfaces in a medium supplemented with

5%

fetal calf serum. The specific activities of enzymes were determined in crude homogenates of such cultures. Quantitative and in some instances qualitatiue diferences in enzyme content of the uarious lines were obserued. When these findings were compared with the known tissue- specificity ( CONSCIENCE-EGLI 1973) some tentative conclusions regarding the origin and/or fate of these embryo-derived cells were drawn: one line, roundish cells, was suspected of being an “imaginal disk” line; another line, cells inter- woven into networks, was suspected of being a “neuronal line.”

The fact that the lines are different from one another in their enzyme content is interesting from the point of view o i determination. CHAN and GEHRING

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3 78 H. URSPRUNG

by the blastoderm stage of development. As the donor embryos used in DEBEC’S analyses were considerably older than the blastoderm stage (ECHALIER and OHANESSIAN 1970). determination no doubt had proceeded to a considerable extent in these cells.

I

find the prospects for cloning cells of known enzymatic content coupled with in vivo culture techniques that might permit differentiation of such cells into larval or adult structures most fascinating f o r a n eventual under- standing of the chemical basis of determination. This will be all the more feasible since new cell lines of known karyotype are also being reported (MOSNA and DOLFINI 1972).

For the study of gene regulation, the method of fusing like or unlike cells to one another has become a n important tool in vertebrates. I n Drosophila, fusion has been reported for embryonic cell lines by BECKER ( 1973)

,

who used Concana- vallin-A to induce fusion. Virus-induced fusion has been reported between human and mosquito cells by ZEPP et al. (1971). These recent findings give us new hope that an experiment we proposed five years ago (URSPRUNG et al. 1968) will become feasible. The experiment is designed to answer the question of whether gene function is regulated by positive control mechanisms. Let me illustrate the experiment with an example. Salivary gland cells of Drosophila larvae are known not to synthesize alcohol dehydrogenase (ADH)

,

whereas this enzyme occurs in many other cell types, e.g., the larval fat body. The cognate locus, whose position on the cytogenetic map is known with reasonable accuracy through the work of GRELL, JACOBSON and MURPHY (1965)

,

may be under positive control by cytoplasmic signals. If we succeed in fusing a salivary cell to a cell synthesizing ADH, mechanically, chemically, or by means of a virus, we might expect derepression of the Adh locus in the salivary genome.

If

we choose cells with a different electrophoretic phenotype of ADH as partners in this fusion experiment, we would expect expression of the salivary enzyme in the heterokaryon. By 3H-uridine autoradiography of the Adh region of the giant chromosomes we could then ascer- tain whether the bands known to contain the Adh locus are caused to puff or synthesize RNA. I believe this experiment will make a contribution to our under- standing of the immediacy of the control of gene function and of nucleo-cytoplasmic interactions that a n organism without giant chromosomes could not make.

Nuclear transplantation and related techniques

In vertebrates, nucleo-cytoplasmic interactions have been successfully studied by nuclear transplantation. Such technology has now also been developed and utilized for Drosophila in several laboratories (ILLMENSEE 1968, 1973; SCHU-

BIGER and SCHNEIDERMAN 1971; ZALOKAR 1971). I n his recent publication,

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Through two different manipulations,

ILLMENSEE

managed to obtain fertile off spring from such transplantation embryos. One involved the transplantation of pole cells of transplant embryos into blastoderm hosts. This led to gonadal mosaics; and it was then possible, by appropriate breeding, to produce off spring with chromosomes derived from the original transplant nucleus. The other involved transplantation of gonadal primordia from transplant embryos that developed into larval stages. The primordia were implanted into genetically labelled host larvae, where they become attached to the external genitalia in competition with the host’s own gonadal primordia. The resulting gametes could unambigu- ously be attributed to the original donor nucleus or the foster parent’s genotype. There is no doubt from these elegant studies that at least u p to gastrulation, Dro- sophila cell nuclei are totipotent. The technique of nuclear transplantation in Drosophila is no more difficult than in Amphibian eggs. Quite obviously, the wealth of genetic information of the fruitfly can now be exploited with entirely new perspectives not only for the solution of yet another question of changes in totipotency, but more importantly, for regulatory studies.

It is appropriate in this connection to mention experiments by GAREN and GEHRING (1972), who used microinjection procedures similar to those used in nuclear transplantation for correcting the consequences of the genetic constitu- tion deep orange ( d o r )

. By injecting wild-type egg extracts into

dor eggs, they were able substantially to rectify the developmental abnormalities that these eggs normally show.

This technique will doubtless be exploited to develop functional assays for the elucidation of the biological significance of egg inclusions with a suspected developmental role. I am thinking primarily of the pole plasm, and its characteristic inclusions, the polar granules, so beautifully documented by MAHOWALD’S (1972) electron micrographs. Much classical information attributes a significant role to this region of the ooplasm in the determination of germ cells. Injection experiments involving cytoplasm of known egg regions, and fractions of known chemical composition obtained from cytoplasm may soon solve this important problem of cytoplasmic localization.

Concluding remarks

This brief review covers but a small segment of recent progress in Drosophila developmental genetics. I excluded from it chapters that I know were covered elsewhere in this Genetics Congress, for example, the recent work on the “genetic dissection” of the nervous system (BENZER 1971). I largely left out the cell and molecular biology of giant chromosomes which has been summarized recently in

a volume devoted entirely to this interesting system of analysis (BEERMAN 1972). For a review of the state of transdetermination see GEHRING (1972). Other topics pertinent to this discussion would have been the problem of pattern formation

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380 H. URSPRUNG

activity (FRISTROM 1972;

HANLY

and STEWART 1972). Time and space allotted at this symposium did not allow me to include a discussion of these topics, nor of the potentially very useful temperature-sensitive mutants (Suzuxr 1970).

There is no doubt however that Drosophila is having a n impressive comeback as an organism of great promise for the solution of many fundamental problems of eukaryote developmental genetics, as most of the obstacles mentioned in the introduction have been overcome already.

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