Developmental genetics of Drosophila.

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Ann. Rev. Genet. 1976. 1O:Z09-5Z Copyright © 1976 by Annual Reviews Inc. All rights reserved

DEVELOPMENTAL GENETICS

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Annu. Rev. Genet. 1976.10:209-252. Downloaded from www.annualreviews.org by ILLINOIS STATE UNIVERSITY on 11/21/12. For personal use only.

OF DROSOPHILA Walter J. Gehring Department of Cell Biology, Biozentrum, University of Basel, Basel, Switzerland

CONTENTS INTRODUCfION OOGENESIS

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Female-Sterile Mutants ............. ..... . . . . .. ........ .................................... . ............ Ribosomal RNA Genes in Oogenesis ... . . ...... .. ..................................................... SPERMATOGENESIS . . . . .. . . .. . .. ..... . . . ... . .. . .. .. . .. . .. .. .... . . .. .. . . . . . Function and Structure of the Y Chromosome .............. .............. ...... ...................... X-Linked and Autosomal Mutants Affecting Male Fertility .................................. In Vitro Culture of Testis and Isolated Cysts ........................ ... . ........ ... ....... .... . .... EMBRYOGENESIS . . .. ...... ...... . . ...... . . . . . . ... . ..... . .. . ....... . ........... . .. . .. . .. . . . Nuclear Transplantation Experiments ........................... . .................. ...................... Germ CeJJ Determination ...... .... ........................... ................. ............... ....... ..... ........ Gynandromorph Mapping .... . .. . . . ..... . ............................. .. .. . . .. .. . ... . . . . . .. . Cell Determination at Early Embryonic Stages ....... . .. ............ .. . ..... ...... . .. . . . Molecular Aspects of Embryogenesis . . . . . . .. . . . .. . . .

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POSTEMBRYONIC DEVELOPMENT OF IMAGINAL DISCS .. . . .. .. . . . . . . . . . Determination During Larval Stages .......... . . . . .. ................ .................... ................ Compartmentalization and the Genetic Control of Disc Development .. . ........ . .. . ... .

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Regeneration . . . ..................................................... ........................................... Transdetermination .. . . ... . . .. ........................................ ...... .. . ......................... .. ..... Metamorphosis .. .. . .. . ................. . ........ ............. .. ...... .................... ... .................... CONCLUSIONS ... . . .. .. . . . .. ..... . . ... . .... ........ .... . . . ... .... ... ......... ....... ..... ........ .. .. . . .....

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209 210 210 216 217 217 220 220 221 221 222 224 227 230 231 231 233 236 237 238 240

INTRODUCTION

This article covers the literature on the genetics of Drosophila development from 1973 to 1975; the older literature has been reviewed by Fristrom ( 1 ) and Postleth wait & Schneiderman (2). There is a vast literature relating directly or indirectly to Drosophila development which makes it necessary for any reviewer to concentrate on certain aspects. I have confined this review to gametogenesis, embryogenesis, and determination in imaginal discs, although I realize that such a choi ce i s rather 209

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subjective. A more comprehensive coverage of the topic will be given in The Genetics and Biology 0/Drosophila (3), which is being written by a large number of contribu tors; it will, however, be some time before it is completed. OOGENESIS


Female-Sterile Mutants

Since the publication of King's monograph (4) on ovarian development, consider able progress has been made in the genetic analysis of oogenesis (5, 6). Several laboratories have begun a systematic study of female-sterile mutants on both the X chromosome (7, 8) and the autosomes (9, to). Female-sterile mutants can be subdivided into two major groups: those that do not lay eggs and those that produce defective eggs. Most of the mutants that do not deposit eggs are blocked in ovarian development. The defect can primarily concern the oocyte, nurse cells (sister cells of the oocyte connected to it by cytoplasmic canals), mesodermal components (follicle cells and ovarian sheath), or extraovarian tissues (e.g. the fat body). The effect of extraovarian tissues on oogenesis can be tested by ovarian trans plants. The question of whether a mutation affects the germ line directly or whether the follicle cells are primarily affected can be analyzed by transplanting mutant pole cells into wild-type embryos and vice versa ( 11). Only few of the newly isolated mutants have been analyzed experimentally, but the morphological descriptions sometimes provide indications about the nature of the defect. In some mutants the ovaries are very small, and appear to be blocked at an early stage of development before any egg follicles are formed (7, 10). The cystocyte divisions leading to 15 nurse cells and one oocyte may be abnormal (7), or vitellogenesis (the deposition of yolk in the eggs) may be blocked (7, 10). The major yolk protein, vitellogenin, is synthesized in the fat body, secreted into the hemolymph, and taken up by the oocyte (12, 13). In the mutant ft(3)Al7, no vitellogenin has been found in the hemolymph and it appears to be blocked in vitellogenin synthesis (13, 10). Four other mutant strains produce vitellogenin, but the oocyte is unable to take it up and incorporate it into yolk spheres. Topical application of a juvenile �ormone analog partially restores the uptake of vitellogenin in some of the mutants (13, 14). New mutants that retain mature stage 14 oocytes in the ovary have also been isolated (7, 10) and it has been suggested that they might be defective in the mechanism that releases the eggs from the ovary, i.e., in ovulation. A substance stimulating oviposition has been found in the male paragonial glands (15, 16) which is transferred to the female during mating. Partial purification of this substance from Drosophila melanogaster has been reported previously (17-19). Virgin females re ceiving transplants of male paragonia show both an increase in oviposition rate and a switch-off in sexual receptivity to males (20, 2 1). Recently two substances PS-l and PS-2 have been isolated from paragonial glands in Drosophila/unebris (22-24). PS- l , when injected into virgin females, reduces their receptivity to males, whereas


DEVELOPMENTAL GENETICS OF DROSOPHILA

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PS-2 stimulates oviposition. PS-I is a peptide consisting of 27 amino acid residues, the sequence of which has been determined (25). PS-2 is a low molecular weight substance containing glycine, ammonia, and carbohydrate (23). For the mutant Hairy-wing 49c, which retains the eggs in the oviduct, it was shown by ovarian transplantation that the sterility of homozygous mutant females is the result of a malfunctioning oviduct derived from the genital imaginal disc rather than the ovary anlage (26, 27). Among female-sterile mutants that produce eggs and also deposit them, three classes will be distinguished: (a) mutants with abnormal egg structure, (b) mutants affecting meiosis, and (c) maternal-effect mutants. Mutants with abnormal egg structure are relatively common among female-sterile mutants. In many cases the eggs lack the usual turgor and possess fragile and malformed membranes. Normal eggs have an elaborate eggshell consisting of three layers, the vitellin membrane, the endochorion, and the exochorion, which are sequentially secreted by the follicular epithelium. Whereas normal eggs are highly impermeable to vital dyes, eggs of the mutant js(1)180 (7) readily absorb neutral red from the medium, indicating an alteration in permeability. An ultrastructural analysis of mutant js(2)A9 showed that the vitellin membrane of this mutant is unusually thin and that the chorion is incomplete (28). The chorion proteins of the eggshell are now under intensive study (29, 30). The problem of limiting amounts of material has in part been overcome by the development of a method for mass isolation of follicles (30). With this technique the egg shell proteins have been studied in mature (stage 14) oocytes (29). The chorion contains 5 major and 16 minor polypeptides, varying in molecular weight from 8,000 to 80,000. The amino acid composition of the total chorion proteins is unusual: proline is 11 molar percent, alanine 15%, and glycine 16%. The proteins of the vitelline membrane consist of 18% proline, 29% alanine, and 10% glycine. The sequential synthesis of these proteins from stage 11 to 13 of oogenesis have been studied by labeling with 3H-proline. These proteins will certainly provide an interesting system for the study of the genetic control of sequential protein synthesis. Mutants affecting meiosis are reviewed separately in this volume and therefore, we give only a brief summary here. The spectrum of meiotic mutants found in Drosophila melanogaster now involves some 40 loci, most of which affect the female (31-33). They influence the rate of recombination, the distribution of exchanges along the chromosome, the regularity of centromere separation at anaphase I or anaphase II, movement of chromosomes to the anaphase poles, and the behavior of chromosomes in the gametes and immediately upon fertilization. Previously, no mutants affecting a function shared by both meiosis and mitosis had been recovered since such mutants are likely to be celi lethais. However, it should be possible to isolate conditional mutants of this type, and a first mutant belonging to this cate gory, (l)TW-6CS, has been described (34). It is a cold-sensitive zygotic lethal which in both homozygous and heterozygous females induces nondisjunction of all four chromosomes at both high and low temperatures; it also causes mitotic irregularities in zygotes at both temperatures.


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MATERNAL-EFFECT MDTANTS In this class of mutants, the homozygous female produces abnormal eggs giving rise to abnormal or lethal progeny. For some of the maternal-effect lethals it has been found that they are rescuable when fertilized by a wild-type sperm, indicating that these genes are not active exclusively during oogenesis, but also act in the zygote. A classical example is deep orange (dar): homozygous females lay defective eggs which, when fertilized by dar sperm, become disorganized at the gastrula stage and die. However, when they are fertilized by dor+-bearing sperm, some of the embryos develop normally and become adults. Progeny from heterozygous females are viable irrespective of the genotype of the sperm (dar or dor+). The defect in the egg cytoplasm has been demonstrated directly by injection experiments (35). Cytoplasm from normal, but not from dar/dar females, can repair the developmental defect in a large fraction of the dar embryos. At present, there is no information about the identity of the defective component. In the case of rudimentary (r), which shows a similar maternal effect, the analysis has proceeded further. N�rby (36) has shown that r mutants have a nutritional requirement for pyrimidines, and Bahn (37) demonstrated that the female fertility can be restored if they are grown on medium enriched with cytidi.ne. Eggs of homozygous females do not develop into viable adults unless fertilized by a r+ sperm. Furthermore, the lethal maternal-effect can be repaired by injection of r+ ooplasm or pyrimidines into the eggs (38). The female sterility phenotype has been elegantly used for the analysis of the genetic fine structure of the r locus (39) which was interpreted according to a model of a single cistron consisting of 7 complemen tation units. However, a recent biochemical analysis indicates that the r locus is multifunctional (40-42) and affects the first three enzymes of pyrimidine biosynthe sis. Mutants belonging to complementation units I, III, and I-IV lack aspartate transcarbamylase activity (40-42), one mutant classified in complementation unit V-VI lacks carbamyl phosphatesynthetase activity (41), and one mutant from com plementation unit VII lacks the third enzyme of the pyrimidine pathway-dihy droorotase (42). Two mutants that do not complement with any other of the tested r alleles lack at least two, and probably all three, enzymes (41, 42), whereas the fourth enzyme, dihydroorotate dehydrogenase, is present. It will be interesting to study the molecular nature of the defect in those mutants which show a complex pattern of complementation. Homozygous alm ondex (am x) females produce defective eggs which become abnormal after germ band extension when fertilized with an amx- sperm. A small fraction of the defective eggs can be rescued by amx+ sperm. Survival of these zygotes to the adult stage is increased at higher temperature and with increasing maternal age (43, 44). Another mutant, cinnamon (cin), has been found on the X chromosome which shows a similar maternal effect (45). The locus controls a process which is essential for embryogenesis. The cin+ product can be supplied to the zygote either via the egg cytoplasm or by the expression of a cin+ locus in the zygote's genome during development. The cin locus also controls pteridin pigments of the eyes. The rare cin/cin and cin /Y progeny recovered from homozygous cin females have a reddish-



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brown (cinnamon-like) eye color, whereas the cin progeny of heterozygous females have wild-type eye color. Apparently, the eggs from heterozygous females contain enough of a cin+ substance to ensure normal eye pigmentation in the absence of cin+ allele in the zygote. cin has also been characterized biochemically and it was shown that the mutant lacks xanthine dehydrogenase (XDH), accumulates its pu rine and pteridine substrates (hypoxanthine, xanthine, inosin, guanosin, and 2amino-4-hydroxy pteridine), and lacks its products (no isoxanthopterin and reduced levels of drosopterins). In this respect, it is very similar to the mutants rosy (ry) and maroonlike (mal) (see 46). mal also shows a maternal effect with respect to eye pigments, but both ry and mal are viable although they lack XDH. ry is a structural gene for XDH. The roles of mal and cin are not clear. The recent success in the purification and characterization of XDH (47-50) should help to clarify this problem. Gans et al (7) have isolated 12 sex-linked maternal-effect lethals which are rescua ble by wild-type sperm. The effective lethal phase of these mutants varies: one mutant continues to the gastrula stage, four die late during embryogenesis, one reaches the larval stage, one the pupal stage, and one produces abnormal adults. All the rescuable mutants so far described are sex-linked; only a few mutants of this type have tentatively been identified on the third chromosome (9). The fact that these maternal-effect mutants are rescuable by the paternal wild-type allele indicates that their expression is not limited to oogenesis but also may occur in the zygote. However, in many cases the paternal gene may become active at too late a stage of development and therefore the defective egg cannot be rescued. This may be the case in the pts (ponte thermosensible) mutant which is temperature-sensitive and maps to the third chromosome (51). When homozygous pts females are shifted to high temperature, oogenesis is blocked at stage 7 prior to vitellogenesis. Eggs that have gone through this stage before the temperature shift fail to develop. However, a small fraction (7-8%) of the eggs hatch if they are fertilized by a pts+ sperm (Table 2 in reference 51). The temperature-sensitive period (TSP) is not restricted to oogenesis, but it includes also the first 10 hours of embryogenesis and early larval stages. These TSPs and the partial rescue by pts+ sperm strongly suggest that the paternal gene is active in the zygote, but in most cases it fails to rescue the defective egg. Furthermore, it is to be expected that a large fraction of those mutants now classified as zygotic, also are expressed during oogenesis and show a maternal effect, since it has been found for many enzymes that in heterozygotes the early embryonic stages contain the maternal isozyme, whereas the paternal isozyme appears later in development (52). A substantial number of maternal-effect mutants have been identified in which the eggs laid by homozygous females cannot be rescued by a paternal wild-type allele. A highly interesting class of such mutants is blocked in the formation of pole cells (primordial germ cells) owing to a maternal effect. In these mutants, the homozy gous female produces defective eggs which give rise to sterile adults lacking germ cells in their gonads, irrespective of the paternal genotype. Since these homozygous females produce children but no grandchildren, the first mutant of this type, found in Drosophila subobscura. was designated as grandchildless (gs) (53). The mainte-


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nance of this mutant stock was rather difficult (54) but more recently we succeeded in establishing a balanced stock with almost complete penetrance (55). Mutants of similar type have been isolated on the X chromosome of Drosophila melanogaster (7, 56-58). In these mutants, the homozygous females show reduced fertility and a fraction of their offspring lack germ cells. Grandchildless 87 maps close to the ct locus and is temperature-sensitive. Homozygous females produce normal offspring at 16°C, but when they are bred at 29°C, with either mutant or wild-type males, they produce few offspring and more than half of the surviving adults lack germ cells. The original stock contained a modifier gene which increases the percentage of agametic offspring from 12 to 60%. The temperature-sensitive period lasts from stage 7 to 14 of oogenesis (58) and the examination of blastoderms collected at the restrictive temperature showed, in many cases, that pole cells were absent, which explains the production of agametic offspring. Two other mutants 1(1)1103 and fs(l)1122, map close to the tip of the X chromosome and do not complement (7, and D. Thierry-Mieg, personal communi cation). At 16°C the fertility of the homozygous females is normal but approxi mately 20% of the offspring lack germ cells. At 23°C, all their descendants are agametic and show morphological abnormalities of the abdominal tergites, wings, eyes, or antennae. At 29°C, the fecundity of homozygous females is low but some eggs develop to the blastoderm stage and apparently show waves of mitoses propa gating towards the anterior end (59). fs(l)1122 has been mapped genetically to map position 1-1.4 ± 0 1 and cytologi cally to band 3B3 (D. Thierry-Mieg, personal communication). Females heterozy gous for fs(l)1122 and a deletion including 3B3 express the grandchildless phenotype. However, fs(l)1122 complements with (l)zw12 (60) which maps in this band. The temperature-sensitive period (TSP) of fs(l)1122 begins very early in embryogenesis of the homozygous mother, when pole cells are formed, and extends to the early stages of oogenesis prior to yolk deposition. The early embryonic TSP is surprising since the effect appears only in the following generation. It remains to be seen whether these grandchildless mutants affect the germ cell determinant (see below) or whether they block some other step in germ cell development. An electron microscopic examination of developing eggs produced by homozy gous gs females in Drosophila subobscura showed that these eggs contain polar granules which appear morphologically normal, although they may be reduced in number (55). The cleavage nuclei do not migrate into the polar cytoplasm and no pole cells are formed, even though the polar granules are incorporated, after some delay, into blastoderm cells at the posterior pole. Polar granules have also been found in grandchildless 87 eggs (58). Bakken ( 1 0) has described 12 maternal-effect lethals, most of which die at early embryonic stages prior to blastoderm formation. Three of them do not seem to contain any stainable chromatin and may not be fertilized. The others can be fertilized, but most are arrested during cleavage divisions, presumably because of abnormal mitoses. One mutant shows abnormal gastrulation and one seems to reach later embryonic stages, but never hatches as a larva. Rice & Garen (9, 61) have isolated maternal-effect mutants on the third chromo some, one of which [mat(3)1] forms only pole cells but no blastoderm cells. The .



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cleavage nuclei migrate into the cortex but cell membranes are formed only around the pole cells at the posterior end. Even though no blastoderm is formed, the pole cells migrate anteriorly, as they do normally during extension of the germ band; apparently this migration does not depend on cellularization. Another maternal effect lethal [mat(3)6] forms a partial blastoderm only; cellularization is confined to the anterior and posterior ends. In a small number of in vivo cultures, only imaginal structures of the head and abdomen were formed; (that is, there were no thoracic structures). Also in this mutant, the pole cells migrate and even invaginate with the posterior midgut anlage. mat(3)2 and mat(3)4 are blocked during gastrulation and do not form the ventral furrow; in one of them, but not in the other the posterior midgut invagination occurs. Finally, mat (3)3 is a temperature-sensitive mutant with a temperature-sensitive period during the last 10 to 12 hours of oogenesis. At 29°C it is an embryonic lethal. At 20°C it is viable but some 20% of the surviving progeny from homozygous females show adult defects, mostly missing halteres and third legs. In comparison, only 0.3% of the progeny from heterozygous females lack adult parts. Dissection of the mutant larvae produced by homozygous females shows that in some of the larvae the respective imaginal discs are missing. The defect can even be traced back to the blastoderm stage, where a fraction of the cells is not formed. There is a good correlation between the gynandromorph fate map and the structures that are missing in the adult (A. Garen, personal communication). A similar mutant, [s(l)J456, has been found in the X chromosome (7, 59). At 16°C it appears normal, at 29°C it behaves as a partial zygotic lethal. If homozygous females are raised at 16°C and then transferred to 29°C so that oogenesis takes place at 29°C, the eggs are arrested prior to gastrulation, even when fertilized with a wild-type sperm and about half of the eggs contain haploid nuclei. At an intermedi ate temperature (23°C) the adult progeny display morphological anomalies, such as missing halteres and legs, similar to those of mat(3)3. However, these defects do not depend exclusively on the maternal genotype. The frequency of adult defects amounts to 26% in the progeny of homozygous females, and 12% in the progeny of heterozygous females. A third mutant, (l)ERts, with similar properties mapping close to [s(l)J456, has been characterized (62). The mutant expression is biphasic, producing a temper ature-sensitive maternal product essential for early embryogenesis and exhibiting a second temperature-sensitive period during the first three days of larval life. There fore, the gene is expressed both during oogenesis and in the zygote even though a wild-type allele introduced by the sperm cannot rescue the defective egg. The defective eggs have been examined by light and electron microscopy, and it was found that there are disturbances in the distribution of nuclei, cytoplasm, and yolk. In addition, there were abnormal configurations of the rough endoplasmic reticu lum. Among the 95 female-sterile mutants isolated by Gans et al (7) on the X chromo some, 32 mutants belonging to 27 different complementation groups showed a maternal effect which was not repaired by a wild-type sperm. These mutants were analyzed by light and electron microscopy (59). They can be subdivided into five phenotypically distinct groups. Mutant eggs in group one showed essentially no development, most remaining unfertilized or arresting prior to syngamy. In the


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second group, at least some nuclear divisions were observed, but the "cleavages" soon stopped and the nuclei became polyploid and degenerated. The third group produced eggs that developed to the cellular blastoderm stage or beyond, but the development was abnormal and the cells proved to be haploid. It is not known whether the haploidy is a consequence of parthenogenesis or a failure of syngamy with independent development of one of the pronuclei, but in one specimen a Y chromosome was found. In the fourth group, abnormal blastoderms were formed with excess or reduced numbers of nuclei, spindle abnormalities, or failure of cellularization. In the last case, irregular cleavage furrows are observed. The last group comprises mutants forming abnormal gastrulae. The effective lethal phase of all these mutants is early in embryogenesis and only a fraction of the offspring of the leaky mutants develop to later stages of development. Maternal-effect mutants are of particular importance with respect to the identifi cation of cytoplasmic determinants in the egg. Unfortunately, this aspect has not yet been developed very successfully. Only very few mutants which prevent the formation of a specific cell-type have been found. The best case is still grandchildless which prevents the formation of pole cells. But even in this case there is no evidence that the germ cell determinant is directly affected. So far, no maternal-effect mutants have been found which, for example, prevent the formation of a specific imaginal disc. Mutants such as mat(3)3 and possibly fs(J)1456, form incomplete blas toderms with a variable number of cells missing. According to the cell lineage relationships, the respective adult structures also are missing, since there is no or at least insufficient compensation of the defect. If the mutation would affect a specific determinant, for example a determinant for the haltere disc, then one would expect both halteres only to be missing, but no other structures. However, in mat(3)3 the defects are variable and often asymmetric, and not only may the haltere be missing but also the third leg or abdominal segments. Therefore, it seems unlikely that this mutant affects a disc-specific determinant. This issue is discussed further in the sections on embryogenesis and imaginal discs. Ribosomal RNA Genes in Oogenesis

Amplification of the genes coding for ribosomal RNA has been found in the oocytes of amphibians, some insects, and several other animal species (reviewed in 63). Drosophila has ovaries of the meroistic type, containing egg chambers in which one oocyte is connected to 15 nurse cells and surrounded by follicle cells. The polyploid nurse cells produce large amounts of RNA which seems to be transferred into the oocyte via intercellular canals (4). Since the nurse cells are highly polyploid (up to 100 n), the question arises whether amplification of the ribosomal RNA genes also occurs in this case. Saturation hybridization studies indicate that the ribosomal genes in D. hydei are underreplicated in adult ovaries (64). The rDNA content of nurse cell nuclei is 40% of that of normal diploid cells; for follicle cell nuclei a value of 63% was found. Underreplication of rDNA was also found in pupal ovaries of D. virilis (65) which contain nurse cell nuclei with low levels of polyploidy. The mutation abnormal oocyte (abo) may be involved in the regulation of the genes for ribosomal RNA. It is a recessive autosomal mutation and shows a marked



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maternal effect on the sex ratio of the offspring. When mated to normal males carrying an attached X Y chromosome, homozygous abo females produce a large deficit of XO males relative to XX Y female progeny (66). Zygotes developing from eggs produced by homozygous abo females have a reduced survival relative to genetically identical zygotes from eggs from heterozygous females: XO male off spring survive only 6% as often as in the control, while the XX Y female offspring survive 72% as often as in the control; it is this that leads to the distorted sex ratio. The reduced survival can, however, be partially compensated for by an additional Y chromosome or by the basal heterochromatic region of the X chromosome either in the mother or introduced by the sperm. Since the basal heterochromatin of the X chromosome contains hardly any genes other than the ribosomal RNA genes, Sandler proposed that abo+ regulates the ribosomal genes (66-68). Mapping of the region of the basal heterochromatin (Xh"iKJ) responsible for rescuing eggs from the abo-induced lethality was accomplished by crossing homozygous abo females to males carrying, in addition to the attached XY chromosome, any one of a series of X -chromosome duplications (69). A clear separation of bobbed (i.e., the ribosomal RNA genes) and Xh"iKJ, with Xh"iJo occupying a more distal position, was found. In homozygous abo stocks, a gradual loss of the abo phenotype is observed (70). When abo chromosomes which no longer produced the abnormal sex ratio were returned to the heterozygous condition, a gradual return of the capacity to express the abo phenotype was observed. This instability is formally similar to the magnifi cation phenomenon of the ribosomal genes (71) and may well be a property of the X chromosome and not the gene abo itself. Indeed, the multiplicity of the genes for ribosomal RNA was determined in saturation hybridization experiments using DNA from whole flies (70) and was found to be changed in these experiments. Homozygous abo females which no longer expressed the abo phenotype had nearly seven times more rDNA than the controls, whereas abo/abo females expressing the abo phenotype had only twice as much rDNA. This correlation between the mutli plicity of the ribosomal RNA genes and the expression of abo further suggests a functional interaction between these genes, but it is not clear at what level this interaction takes place. SPERMATOGENESIS

The genetic control of morphogenesis, the relationship between structure and func tion of a chromosome, and the problem of activation and inactivation of chromo somes can be particularly well studied in spermatogenesis. The genetic control of sperm morphogenesis has been reviewed extensively by Kiefer (72). Several authors have summarized their studies on the structure and function of the Y chromosome (73-78), Romrell has compiled a list of mutants affecting male fertility (79), and Geer has reviewed certain aspects of metabolism during spermatogenesis (80--82). Function and Structure of the Y Chromosome

With the possible exception of the nucleolus organizer region, the Y chromosome appears to be heterochromatic and inactive in all tissues except the male germ line.


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Males lacking a Y chromosome are morphologically and behaviorally normal, but they are sterile because of a failure during spermatogenesis. The activity of the Y chromosome appears to be limited to the primary spermatocyte stage, when it forms characteristic loops typical of lampbrush chromosomes. Two Drosophila species have been analyzed particularly: D. hydei, which shows the most prominent lamp brush structures, and D. melanogaster. which has obvious advantages for genetic analysis. The two species are basically similar but they differ in some respects. For example, in D. hydei. spermatogenesis in XO males is generally arrested before meiotic metaphase, whereas in D. melanogaster males lacking a Y chromosome, spermatogenesis proceeds to the spermatid stage (83). Using both X-ray and ethyl methanesulfonate treatment, mutants belonging to seven complementation groups, also designated as fertility factors, have been iso lated (84-86). This is a very small number relative to the size of the Y chromosome which contains roughly 10% of the total nuclear DNA. It seems that the rest of the DNA is either silent, with the exception of the nucleolus organizer, or contains repetitive genes for which it is difficult to recover mutants. The fact that tempera ture-sensitive (ts) alleles have been recovered for at least five of the seven com plementation groups suggests that the fertility factors represent unique genes, for the reason that ts mutants are generally missense mutants owing to single-base changes (87). Alternatively, the yrs mutants could be dominant mutations within repetitive segments of the chromosome (see also below). The Y chromosome of D. hydei has not yet been analyzed genetically to the same extent as the melanogaster Y, but cytologically the number of lampbrush loops it contains is similar to the number of fertility factors that have been found in D. melanogaster (83). Each pair of loops has a characteristic morphology when visual ized by phase-contrast microscopy. The evidence that these structures are formed by the Y chromosome comes from the observation that they are absent in XO spermatocYtes and duplicated in nuclei of X YY spermatocytes. Furthermore, the absence of individual loops correlates with the deletion of specific Y segments. The absence of anyone of these loop pairs, owing to a deletion on the Y, results in male sterility. Transcriptional activity of the Y chromosome has been demonstrated by au toradiography (88) and by RNA/DNA hybridization (89). Use of the Miller tech nique (90) for spreading the chromosomes, transcriptional activity of the nucleolus (91), the Y chromosome loops (73, 78), and the other chromosomes (92) has been visualized in spermatocytes of D. hydei by means of electron microscopy. The length measurements of the putative ribosomal DNA matrix, 2.5 /-Lm in D. hydei and 2.6 /-Lm in D. melanogaster, correspond rather well to the molecular weight determi nants of the precursor of rRNA (2.6 and 2.85 X 106 daltons respectively), whereas the length of the RNP fibrils is foreshortened by a factor of approximately 5 to 10 (91,93,94). Transcripts found on the Y chromosome loops are as >11uch as 50 times longer, which would correspond to molecular weights of 107 to 108 (73,78). Similar transcript lengths were measured in lampbrush chromosomes of amphibian oocytes (90)



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Under normal metabolic conditions, no evidence for the occurrence of several initiation points within a single loop of the Y chromosome was obtained (78). However, after X-irradiation leading to a partial inhibition of RNA synthesis, a maximum oftive transcriptional units were observed within a single loop (73). While this possibly indicates the existence of repeats, the point obviously needs to be clarified. In XO spermatocytes, the transcription of the autosomes and of the X chromo some can be visualized (92). Besides the putative rRNA cistrons, transcribing regions are found in which the RNP fibrils were not as closely spaced and were considerably longer (10-100 fLm assuming a foreshortening of the RNP fibrils by a factor of 10). The molecular weight estimates for such transcripts also range from 107 to 108• For transcripts in embryonic Drosophila cells, a minimum estimate of 1.75 X 106 daltons was obtained (93). It is possible that the transcripts in XO spermatocytes were not only derived from autosomes but also from the X chromo some, since they were also found close to the nucleolus, where the X chromosomal material is preferentially located (73, 92). RNA/DNA hybridization studies indicate that testes of D. hydei contain RNA complementary to repetitive DNA sequences of the Y chromosome (89). These studies have been extended to males carrying Y chromosome fragments, yLd and yLpS, which are complementary (73). Competition hybridization experiments indi cate that the repetitive sequences in the Y chromosome transcribed during sper matogenesis are not restricted to either of two complementary fragments. Autoradiographic evidence and inhibitor studies indicate that RNA synthesis is restricted to the spermatocyte stage (88, 95, 96) and that no significant incorporation of 3H-uridine is observed in spermatids. However, protein synthesis can be detected in both pre- and postmeiotic stages (i.e. spermatocytes and spermatids) indicating that the mRNA is relatively long-lived (96, 97). The temperature-sensitive period (TSP) of the majority of the mutants in D. melanogaster coincides with the spermatocyte stage (86). However, one heat-sensi tive (A12), and one cold-sensitive (k15), mutant (98) have their TSP during the spermatid stage. The existence of cold-sensitive mutants among the fertility factors of the Y chromosome may indicate that these genes are involved in some assembly process, because a class of cold-sensitive mutants in E. coli is known to affect the in vivo assembly of ribosomes (99), while the assembly of microtubules in different organisms is also cold-sensitive (100). Little is known about the biochemistry of fertility factors. Hennig et al (73) have analyzed protein patterns in testes from males of D. hydei with different genetic constitution by electrophoresis on SDS-acrylamide gels. A particular protein band was present in X Y and absent in XO testes. This band appears only in the postmei otic stages of development. Since it is present in males carrying the Y fragment, yLpS, and absent in males carrying yLd, it may be coded for, or regulated by, a gene on yLpS. This protein has a different electrophoretic mobility in the sibling species, D. neohydei. Since fertile hybrids between these two species can be obtained, it should be possible to map the gene coding for this protein.

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With respect to morphogenesis, the studies on the ultrastructure of sperm in males carrying the various Y mutations have been somewhat disappointing. It has not been possible to relate the presence of a structural or developmental defect to the absence of a specific Y locus (72). The differences between the various mutants are more quantitative than qualitative and in no case are all spermatids affected to the same extent. It seems very difficult to draw any conclusion about the nature of the defect in the various mutants from these morphological studies.


X-Linked and Autosomal Mutants Afftcting Male Fertility

A considerable number of male-sterile mutants either map to the X chromosome or the autosomes, or are the result of X-autosome translocations (72, 79). Lifschytz . (101) has isolated an X chromosomal mutant, st(l)XL2, in D. hyde/: which blocks the normal unfolding of all of the Y chromosome loops. In mutant spermatocytes, the loops begin to unfold but they appear to be arrested at an early stage. At low temperature, loop development proceeds somewhat further. While it is possible that st(l)XL2 controls transcription of the Y chromosome or that it is directly responsi ble for the unfolding ofthe Y-DNA, more recent evidence indicates that it, and two other mutants of this type, exert a pleiotropic effect on somatic tissues indicating that they are not Y chromosome specific (1OIa). On the basis of genetic and cytological evidence, Lifschytz & Lindsley (102, 103) have proposed that the X chromosome is inactivated during a critical stage of spermatogenesis. In a large sample of reciprocal X-autosome translocations, it was found that such translocations are male sterile except when one breakpoint is in the proximal heterochromatin of the X or when the chromosome tips are interchanged. The sterility is dominant; the addition of a duplication for the segments of the X in which the breakpoint is located does not restore fertility. This is in contrast to Y autosome or X- Y translocations, where fertility can be restored by the addition of a normal Y chromosome. Since the X chromosome in many heterogametic organisms condenses prema turely during the first meiotic prophase, it is assumed that the dominant sterility associated with X -autosome translocations is due to interference with the normal inactivation process. This hypothesis needs to be tested more directly. Two X autosome translocations have been examined by electron microscopy. Both have rather specific morphological effects: T( 1;2)H is defective in the differentiation of the sperm head, and morphogenesis of the flagellum appears normal (104); T(l;3) 10;939 is defective in flagellar differentiation as evidenced by 50-80% of the ax onemes lacking the central pair of microtubules (72). The high degree of specific ity of these two translocations cannot easily be explained on the basis of the X inactivation hypothesis; obviously the problem has to be examined further. In Vitro Culture of Testis and Isolated Cysts

Considerable progress has been made in the development of techniques for studying spermatogenesis in vitro. Testes of third instar larvae or 1-2 day pupae can be cultured in commercial Grace's medium supplemented with 9% fetal calf serum. Under these conditions, primary spermatocytes will complete meiosis and the result-


22 1

ing spermatids will begin to elongate (96). By 24 h in vitro, the most advanced spermatids reach about 20% of their final lengths of 1.8 mm. Excellent results were obtained with Mandaron's medium in which single cysts of primary spermatocytes underwent meiosis and gave rise to elongated spermatids within 30 h ( lOS). With these techniques, spermatogenesis can be analyzed cytologically in living cells and the conditions for radioactive labeling or other manipulations can be better con trolled than in vivo.


EMBRYOGENESIS Nuclear Transplantation Experiments

What is the relative importance of the nucleus and the cytoplasm in cell differentia tion? Do nuclei become programmed for a certain developmental pathway? Are the regulatory controls imposed on the nucleus reversible or is there an irreversible restriction to a certain developmental pathway? These are some of the basic prob lems which can be studied by nuclear transplantation. A substantial body of evi dence relating to these questions has been accumulated in amphibia (see 106). The experiments in Drosophila, which confirm and extend these results considerably, have recently been summarized by Illmensee (107). Early Drosophila development differs from amphibian embryogenesis signifi cantly in that the cleavage nuclei are not immediately segregated into different cells, but rather the egg remains a syncytium in which the nuclei divide synchronously. After the eighth division, the nuclei migrate to the periphery of the egg cytoplasm and a small group of cells is formed at the posterior pole (pole cells); at this time, there are about 512 nuclei. The other nuclei form a monolayer in the cortical cytoplasm (syncytial blastoderm). After 3 (108, 109) or 4 additional synchronous divisions, cell membranes are formed (M. Zalokar, personal communication). At this stage, designated as blastoderm, the embryo essentially consists of a monolayer of cells, a group of pole cells, and some nuclei which remain in the central yolk. The blastoderm stage is followed by gastrulation which leads to the formation of the three germ layers by invagination and complicated morphogenetic movements. Nuclei from all these early embryonic stages have been tested by transplanting them into unfertilized or fertilized eggs carrying different genetic markers. In earlier studies, nuclei from cleavage and syncytial blastoderm stages were injected into unfertilized eggs, which developed up to embryonic or larval stages. By in' vivo culturing ( lID) of the imaginal discs from the defective embryos or larvae, it was possible to demonstrate that the transplanted nuclei can give rise to virtually all cuticular structures of the adult (111-113). This indicates that up to the blastoderm stage the nuclei are not irreversibly restricted in their potential for forming imaginal structures. Evidence for the totipotency of preblastoderm nuclei was also obtained by transplantations into fertilized eggs (113-116). Nuclei withdrawn from the an terior end of genetically marked preblastoderm donors and injected into the poste rior pole of fertilized recipients can give rise to chimeric adults. Several of these chimeras produced progeny of the donor genotype providing evidence that under these conditions the preblastoderm nuclei are totipotent.


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Since cell determination can be demonstrated as early as the blastoderm stage (117), it was important to test nuclei from blastoderm and gastrula stages in order to see whether cell determination involves restriction of the developmental potential of the nucleus. Nuclei were taken from five different regions of gastrula embryos and injected into unfertilized eggs (118). For technical reasons, most recipients die as embryos or larvae. However, the potential of their nuclei can nevertheless be tested by either transplanting the gonads from the defective larvae into healthy recipients or by transplanting pole cells (i.e. primordial germ cells) from the defective blas toderm embryo into normal blastoderms. In both cases, chimeric adults were ob tained which produced fertile offspring of the donor genotype indicating that the gastrula nuclei are totipotent. By this procedure, clones of individuals derived from a single gastrula nucleus can be obtained. Recently, fertile adults were obtained directly by transplanting nuclei from blastoderm cells into unfertilized eggs without having to go through a second transplantation of the germ cells (107). Thus, the nuclear transplantation experiments demonstrate the totipotency of nuclei from determined cells and fail to show any irreversible restrictions imposed on them at least up to the gastrula stage. Therefore, the determinative factors involved in cell differentiation are presumed to reside in the egg cytoplasm. Some direct evidence on this point comes from the cytoplasmic transplantation experi

ments, which are discussed below. Nuclear transplantation can also be used in genetic experiments: interspecific chimeras have been produced in this way between four species of the melanagaster subgroup (D. simulans. D. mauritiana. D. teissieri. and D. yakuba) and D. melana gaster (119). The cleavage nuclei of these four species became integrated into the recipient embryo and gave rise to adult chimeras in which the morphological differentiation of host and donor cells was autonomous. No evidence for the pres ence of donor cells in the germ line was found. Nuclei from cell lines established in vitro (Schneider's line I, 2, and 3; Echalier's line K and 52-84) were transplanted successfully into the posterior region of genetically marked cleavage embryos (107). In the resulting chimeras. the donor nuclei were capable of forming a large variety of tissues indicating that cells that have been cultured in vitro for almost five years have retained a fairly normal genotype. So far, no germ-line chimeras have been found. The integration of nuclei from cultured cells into the germ line would provide a means for mapping and characterizing mutants induced in vitro in cultured cells. Germ Cell Determination

In Drosophila. the germ line is separated very early in development from the somatic cells. The potential for forming germ cells is restricted to the pole cells, as shown by UV irradiation experiments (120-124), by the analysis of grandchildless mutants (see section on maternal effect mutants), and by pole cell transplantation experiments (118). UV irradiation experiments suggest that the pole cells may also give rise to the cuprophilic cells of the midgut (121). but the evidence on this point is controversial (for discussion, see 125). Cuprophilic cells have been found in grandchildless flies lacking germ cells (54). However, in recent pole cell transplanta-



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tion experiments, donor cells were also recovered in the midgut (107). It remains to be seen whether these are cuprophilic cells or germ cells which did not reach the gonad. It has long been proposed that the polar cytoplasm contains special substances, designated as germ cell determinants, which interact with the entering nuclei. This interaction is thought to lead to germ cell determination (126). The polar plasm contains a specific type of granule, the polar granule, which consists of a fibrillar core and becomes associated with polysomes (127). Polar granules are localized in a peripheral layer of the cytoplasm. There is some cytochemical evidence that they contain both RNA and protein, but no DNA (128). Pole cell nuclei are characterized by dense structures called nuclear bodies (130). The interaction of nuclei and polar cytoplasm has been studied by cytoplasmic injection experiments. Pole cell formation has been restored in UV-irradiated em bryos by the injection of polar cytoplasm from untreated embryos (126, 129). However, these experiments do not conclusively demonstrate the presence of a germ cell determinant, because it is not possible to distinguish between a specific inactiva tion of a determinant and a more general lesion of the polar cytoplasm which can be compensated for by the injection of normal polar plasm. The most conclusive experiment, involving heterotopic rather than homotopic transplantation of cytoplasm, was performed by Illmensee & Mahowald (130). The polar cytoplasm from early cleavage stage embryos was injected into the anterior end of recipient embryos of the same stage. The nuclei entering this area of the cortical cytoplasm formed cells showing the characteristics of pole cells (polar granules and nuclear bodies). Subsequent transplantation of the induced pole cells into genetically marked host blastoderm established that they are functional and can give rise to progeny of the donor genotype. Anterior blastoderm cells from unin jected controls did not produce germ-line chimeras (see below). Subsequently, it was shown that polar plasm injected into the midventral region of the cleavage embryo also induces the formation of functional pole cells (131). Normal pole cells, when transferred into the midventral region of genetically marked embryos can reach the gonad and contribute to the germ line (131). These experiments provide conclusive evidence for determinative factors, local ized in a specific region of the egg cytoplasm, capable of inducing the formation of a certain type of cell. Cleavage nuclei from various regions of the egg can interact with these determinants and participate in germ-cell formation which indicates that they are equivalent and confirms our interpretation of the nuclear transplantation experiments. Little is known about the nature of the germ-cell determinants. Since there is a good correlation between the occurrence of polar granules and the forma tion of germ cells in both insects and amphibia (128, 133-136), and since polysomes are associated with the granules, it has been postulated that the polar granules themselves represent the germ-cell determinants and that they contain information in the form of maternal messenger RNA (137). The question of whether the occurrence of active polar plasm correlates with the appearance of polar granules during oogenesis has been studied by transplanting


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cytoplasm from the posterior pole of unfertilized eggs and oocytes into the anterior pole of cleavage embryos ( 1 32). Functional pole cells can be induced by injection of polar plasm from unfertilized eggs and late oocytes (stage 1 3 and 14), whereas cytoplasm from earlier stages of oogenesis (stages 10 to 12) was inactive. Since the polar granules can be observed as early as stage 10 (128), there is not an absolute correlation between the presence of polar granules and the inductive capacity of the polar cytoplasm. However, one may argue that the granules require some matura tion step before they become active. As mentioned in the section on maternal effect mutants, polar granules are also present in the grandchildless mutant in D. subobscura (55), but they may either be inactive because of the mutation or because they become incorporated into blas toderm cells with some delay. It remains to be seen whether the polar granules really represent the germ-cell determinants. However, the available evidence strongly suggests that the germ-cell determinants consist of at least two components ( 1 38), one involved in pole cell formation and one concerned with the determination of these cells to become germ cells, since only a fraction of the pole cells become germ cells. Furthermore, I present evidence in the section on cell determination at early embryonic stages that the germ-cell determinants differ from the cytoplasmic com ponents that specify the anterior-posterior polarity of the embryo. Gynandromorph Mapping

Gynandromorph mapping has become a powerful tool in the analysis of develop ment and behavior. The lucid article by Hotta & Benzer ( 1 39 , 140) gives an excellent outline of this technique and some of its applications. Gynandromorphs, or similar mosaics, can be produced by various genetic techniques (14 1). The best method for generating XX-XO gynandromorphs involves the use of the unstable ring-X chromosomes, In (1) wvC (142) and R (1)5A (143). These chromosomes, when intro duced through the father or the mother, are frequently lost during one of the earliest cleavage divisions of the embryo giving rise to XX-XO mosaics. Alternatively, mutants leading to chromosome loss during cleavage mitoses can be used. The first mutant of this kind, described by Sturtevant ( 144) was claret in D. simulans. The corresponding mutant in D. melanogaster is claret-nondisjunctional (cand, 3 - 1 00. 7) ( 145 , 146). Nondisjunction and chromosome loss occur during gamete formation and in the cleavage embryos from homozygous mothers giving rise to exceptional progeny including gynandromorphs, haplo-4 mosaics, and haplo-diplo mosaics having lost the maternal chromosomes ( 141). In contrast to cand, the mutant paternal loss (pal, 2-37.5) can cause the loss of any paternal chromosome during early cleavage in the progeny of homozygous males (147). A third mutant, mitotic loss inducer (mit, 1 -57±), leads to the loss of both maternal and paternal chromosomes during cleavage in the progeny of homozygous mothers (148). mit-induced loss seems to occur mostly during the third and fourth mitotic division, whereas cand and pal lead to chromosome loss during the first or second division (148). Using these three mutants, mosaics for the X. Y. and fourth chromosomes can be generated. Aneuploidy for the second and third chromosomes appears to be lethal.



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With these mosaics, the location of the primordial cells for the various larval and adult structures in the early embryo can be deduced ( 144). Chromosome loss during the first mitotic division leads to gynandromorphs consisting of approximately 50% male (XO) and 50% female (XX) cells, which are not freely intermingled but occupy rather large continuous areas consisting exclusively of cells of one genotype. The orientation of the genotypic boundary between the XX and the XO cells, which can be recognized by marker genes, is largely random. The probability of this boundary passing between two cells at the blastoderm stage, when the embryo essentially consists of a monolayer of cells, is proportionlj.1 to the arc distance between them ( 144, 149). Because the location of a cell within the blastoderm largely decides its fate, the frequency of the genotypic boundary passing between two adult (or larval) cells can be taken as a measure for the arc distance between their primordial cells in the blastoderm (139, 140). A frequency of 1% of the genotypic boundary passing between two structures is defined as one sturt (139, 140). For mosaics generated by chromosome loss in a later mitotic division, Gelbart has introduced the Sturtoid in order to correct for the smaller fraction of male cells. The fate map distance in Sturtoids equals n (a9bd) + n (adb9) / n (ad) + n (b9) X 100, where n is the total number of gynandromorphs in a given class, a and b are any two structures on the fly, and the subscripts 9 and d' indicate the sex of a given structure. By calculating such distances and then triangulating, fate maps for the cuticular markers have been constructed using ca (149), the unstable ring X chromosome, In (1) w·c (139, 140, 150), the mit mutant ( 148), and the pal mutant (147) for generating the mosaics. In all cases, quite similar maps were obtained. Various enzyme mutants have been used for mapping internal structures. The enzyme aldehyde oxidase proved to be particularly useful in this respect because sex-linked mutants (maroon-like, mal) lacking this enzyme activity are available and a simple cytochemical procedure can be used to demonstrate enzyme activity (151-157). The structures of the alimentary tract, the Malpighian tubules, and the internal genitalia have been mapped in the adult (153, 150) and in the larva ( 1 54, and W. Janning, personal communication). The enzymes glucose-6-phosphate dehy drogenase and 6-phosphogluconate dehydrogenase have also been used for mapping internal structures (158). Sex-linked, enzyme-negative mutants for these two dehy drogenases are available, but the cytochemistry is somewhat more difficult, because the enzyme or one of the reaction products is diffusible unless the tissue sections are covered with gelatin. In addition to structures of the alimentary tract, the oenocytes can be mapped by means of these dehydrogenases. For mapping the nervous system, a translocation of the acid phophatase locus from the third to the X chromosome was constructed and gynandromorphs were generated with the pal system (158). In general, the various fate maps agree fairly well with Poulson's embryonic fate map derived from histological data (159), but there are some discrepancies. For example. the anterior structures of the adult gut have been mapped to anterior ventral regions of the blastoderm using the aldehyde oxidase marker (150) roughly corresponding to the anterior midgut rudiment in Poulson's fate map. However, with the dehydrogenase and acid phosphatase mark ers, a more dorsal position was obtained (158). Also it appears that the brain anlage


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reaches more to the dorsal side in the embryological map than in the gynan dromorph map (158). We obviously need some direct tests for the mapping proce dures. A good candidate for such a test is the primordial germ cells, since they form a morphologically distinct group of cells at the posterior pole of the blastoderm. Gynandromorph mapping of the gonad gave conflicting results: Falk et al ( 1 60) obtained a median and dorsal position for the larval gonad, whereas Hotta & Benzer (139), using closer landmarks as reference points, mapped it to the posterior pole. A posterior and ventral location wa� obtained for the adult gonad ( ISO). However, in each of these cases the gonad was mapped as a whole and no distinction was made between the somatic tissues of the gonad, which are of mesodermal origin, and the germ cells. With both normal and transformed gynandromorphs, the primordial germ cells and the gonadal mesoderm were mapped separately ( 161). Transformed gynandromorphs (162, 163) are homozygous for the recessive autosomal mutation transformer (tra), which transforms females into sterile males (164). Such gynan dromorphs possess only male genitalia and male gonads, which facilitates the analy sis. Similarly, male (XY-XO) and female (XXY-XXO) mosaics can also be generated with the pal system (147). The mapping of the pole cells and the gonadal mesoderm ( 1 6 1) was based upon the assumption that pole cells are incapable of undergoing sex reversal and fail to differentiate when associated with mesoderm of the opposite sex. Direct evidence for this assumption has been obtained by pole cell transplantation (E. Van Deusen and W. J. Gehring, in preparation; K. IlImensee, personal communication). Relative to the adult cuticular markers, the germ cells map as the posterior-most structure coinciding with their known location in the blastoderm embryo. These data strongly support the hypothesis that the gynandromorph map reflects the real position of the primordial cells in the embryo, and that it refers to the blastoderin stage since the pole cells migrate anteriorly in subsequent stages of development. The gonadal mesoderm maps along the ventral midline anterior to the pole cells and the primor dial cells for the genital disc. The calculated map positions for the various primor dial cells can also be tested directly by homotopic transplantation of marked single cells from one blastoderm to another (107; see below). The mapping procedure is not restricted to morphological structures; a fly that is mosaic for any physiological, behavioral, or biochemical character can be scored as mutant or normal, and the focus of the character can be mapped (139). For example, the mutant hyperkinetic ( Hk �, which causes shaking of the legs of flies under ether anesthesia, was analyzed and separate mutant foci were found for each of the legs; the foci were ventral to the primordial cells for the leg imaginal discs ( 139). This position coincides with the position of the thoracic ganglia (158), which is consistent with electrophysiological experiments which indicate that mutant flies have defective motor neurons in the thoracic ganglia ( 165, 166). The mutant focus may be more complex than in Hk : There may be several interacting foci, domineering and submissive foci, or, if the mutant character affects all the cells, no focus at all ( 139). A case of bilateral submissive foci is provided by the drop-dead mutant ( 1 39). The mutant foci map in an area of the blastoderm to



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which the ganglia of the head have been localized (158), consistent with the observa tion that the brain degenerates in mutant flies. The map locations of these mutant foci confirm the data obtained with morphological characters, but more impor tantly, they indicate the primary site of action of the mutant gene which often is difficult to find by other means. The mutant foci were also analyzed for a sample of recessive lethals (167) and the eye color mutant vermilion which lacks tryptophane pyrrolase activity (168). In the latter case, two mutant foci for the production of kynurenin, the product of tryptophan pyrrolase, were found which presumably correspond to the larval fat body and the Malpighian tubules. The number and location of the primordial cells for the imaginal discs can be deduced from gynandromorph data. Such data clearly indicate that the imaginal discs are not clonal in origin but arise from several primordial cells. The estimates range from about 2 to 40 cells for the various discs (see 169). Similar values are obtained if the estimates are based on mosaics obtained by induced mitotic recombi nation at the blastoderm stage (180). The location of the primordial cells on the map roughly corresponds to the location of the discs in the larva, with some exceptions which presumably are due to morphogenetic movements. Detailed gynandromorph maps were generated for the wing (170) and the wing and the three leg discs (171, 172). For these thoracic discs, it was found that their primordia map close together and probably occupy adjacent areas of the blastoderm, but the centers of the primordia occupy distinct sites (171, 172). Map distances measured within the leg discs are larger than expected, especially when measured between leg segments. This map expansion is assumed to be due to some indeterminacy in cell lineage (139, 172). The mapping procedure is based on the assumption that the lineage of a given cell is largely determined by its position on the blastoderm, but the cell lineage is not absolutely fixed and varies somewhat from fly to fly (173-175). Cell Determination at Early Embryonic Stages

The genetic control of cell determination in the embryo has been reviewed recently (55). The most decisive test for cell determination is the isolation and combination experiment, in which the cells to be tested are dissociated, combined with cells of different types, and allowed to differentiate. If, under these conditions, a cell differen tiates autonomously, it is considered to be determined. With this criterion, blas toderm cells have been shown to be determined, at least with respect to forming anterior versus posterior imaginal structures (117). Recently, these results have been confirmed by some heterotopic transplantations of isolated blastoderm cells (107, 130). For example, cells taken from the anterior end of a blastoderm and injected into the posterior end of a genetically marked recipient blastoderm differentiate autonomously into imaginal structures of the head region and do not become integrated into the recipient tissues. After homotopic transplantation, blastoderm cells are capable of forming integrated mosaic structures. The determination of blastoderm embryos has also been tested by defect experi ments. In this case, it is assumed that the blastoderin as a whole or the individual blastoderm cells are determined if injury or removal of cells results in specific


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damage or losses in the developing animal. However, defect experiments can give at most indirect evidence for determination, since their outcome largely depends upon the regulative or regenerative capacities of the surrounding cells. Microcautery with a hot needle leads to localized defects which are correlated with the site of the blastoderm that has been damaged ( 1 76, 177). Although the resolving power of this kind of defect experiment is limited, the damage observed in the surviving adult flies does roughly correspond to the position of the respective primordia as determined by gynandromorph mapping. Similar results are obtained after pricking . with a needle which leads to the extrusion of some cells from the blastoderm ( 1 78). Never theless, after both microcautery and pricking, a large fraction of the surviving adults appeared to be normal; from this it was concluded that some form of regulation or regeneration was taking place. However, the possibility that some of the damage remained undetected, especially in embryonic and larval tissues, cannot be ruled out. Localized UV irradiation of blastoderm stages leads to embryonic defects which are also correlated with the site of irradiation ( 1 79). The experiments mentioned so far indicate that the blastoderm cells are deter mined, but they give relatively little information on the specificity of this determina tion. One approach to this problem is to study the fate of single blastoderm cells marked by mitotic recombination. Mitotic recombination can be induced by X irradiation as early as the blastoderm stage (180). In fact, the blastoderm stage is relatively resistant to irradiation; most of the clones induced at the blastoderm stage are confined to adult structures derived from one imaginal disc. However, some ofthe clones in the thoracic region, where the disc primordia map close to each other (see section on gynandromorph mapping), overlap structures derived from both the wing and the second leg disc. From this result we can conclude that at least some ofthe primordial disc cells are not yet disc-specifically determined at the blastoderm stage. The observation that no clones were found to overlap discs from different body segments might suggest that determination is segment specific at the blastoderm stage. No overlapping clones were found in the thoracic region after irradiation at gastrula (7h) and later stages. Recently, these results have been confirmed by using the Minute technique (see section on compartmentalization and the genetic control of disc development) for generating unusually large clones ( 1 81, 182). Clones overlapping discs within the thoracic segments were found but none which crossed the segmental boundaries. Further experiments testing the potential, rather than the fate, of individual blastoderm cells are needed before any definite conclusions about the specificity of determination can be drawn. It is, however, clear that the blastoderm cells are determined at least with respect to general (anterior versus posterior) properties and possibly with respect to segments, but the cells retain some developmental flexibility as indicated by their potential to contribute to two different imaginal discs within the same segment. Some flexibility is also suggested by transplantation experiments of single blastoderm cells from the ventral region of the embryo into the posterior region of genetically marked recipient blastoderms ( 1 07). In this case, the ventral cells became integrated into the host tissues, Malpighian tubules, and fat body. Since



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the Malpighian tubules are located near the posterior end of the gynandromorph fate map (139, 150, 153), this result possibly indicates that the ventral cells may differentiate nonautonomously according to their new location. Because the cleavage nuclei are totipotent and determination occurs as soon as cells are formed at the blastoderm stage, it seems necessary to assume that the egg cytoplasm contains some localized developmental information. The presence of germ cell determinants localized in the cytoplasm at the posterior pole has been convincingly demonstrated (see section on germ cell determination). There is also some evidence for determinative factors for somatic cells localized in the egg cyto plasm. Defect experiments, aimed at removing or destroying specific regions of the cortical cytoplasm during cleavage stages. gave relatively little information. Local ized microcautery (176) and pricking (1 1 2) yielded some adult defects correlated with the site of damage in the cortical egg cytoplasm, but many of the surviving adults appeared normal. UV irradiation (179) resulted in embryonic defects roughly corresponding to the site of irradiation, but the "double abdomen" syndrome was not observed. Embryos with this syndrome can be induced by UV irradiation of the anterior pole of the egg in various chironomic midges (183, 184). They consist of two abdom:na in mirror image symmetry: the head, the thorax, and the anterior abdominal segments are replaced by a second set of posterior abdominal segments, but pole cells are present at the original posterior pole only (185). The UV-sensitive targets are located in the anterior one eighth of the egg cytoplasm and not in the cleavage nuclei (186). Double abdomens can also be induced by irradiating anterior halves of embryos, which indicates that the transformation does not depend upon an interaction between the primary posterior end and the anterior end (186). Action spectra and photoreversion studies suggest that the target is a nucleic acid-protein complex (187, 188) but mitochondria do not seem to be involved (189). By UV irradiation of the posterior end of the egg, "double heads" can be induced (183). In Drosophila, the same syndrome is produced by the bicaudal mutant which shows a maternal-effect indicating some defect in the egg cytoplasm (190). Therefore, we have postulated that there are factors present in the egg cytoplasm that determine the anterior-posterior polarity of the embryo (55). Since the pole cells are only formed at the original posterior pole, even in bicaudal (190; C. Niisslein and W. J. Gehring, unpublished), these determinative factors must be different from the germ cell determinants. Since UV irradiation affects only the surface layer of the egg, the targets must be localized in the cortical cytoplasm or the plasma membrane. Egg polarity has also been studied by fragmentation experiments (191, 192). Drosophila eggs can be fragmented by pressing down onto the egg with a blunt razor blade; this separates the egg contents without severing the vitelline membrane. If embryos are fragmented at preblastoderm stages, the developing larvae lack one or two of the intermediate body segments and the respective imaginal discs, whereas the terminal segments are formed. Fragmentation at the blastoderm yields half larvae. which, when added together, contain all body segments. With increasing age of the embryo, the gap of missing segments becomes increasingly smaller, until it

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disappears at the time of blastoderm formation. These observations may be ex plained by assuming that the intermediate segments are determined by the interac tion of two polarity gradients ( 193).


Molecular Aspects of Embryogenesis

The biochemistry of Drosophila embryogenesis is still very embryonic, but during the last few years important technical problems have been solved and some major molecular components have been isolated and characterized. Methods for the large scale production of unfertilized eggs and synchronized embryos (194) are available. Procedures for the permeabilization of the vitelline membrane have been worked out (195, 196); these are prerequisites for radioactive labeling or treatment with various drugs and inhibitors. Since early embryonic development is extremely rapid, one would expect that some of the organelles and molecular components are preformed during oogenesis and stored in the unfertilized egg. This prediction is fulfilled for at least two of the components involved in the rapid nuclear divisions during cleavage; DNA poly merase activity is highest in unfertilized eggs and declines during the second half of embryogenesis (197). Also, the pool of cytoplasmic microtubule proteins involved in the formation of mitotic spindles remains constant throughout early development at least up to the gastrula stage ( 198). Furthermore, studies on bobbed mutants lacking the genes for ribosomal RNA indicate that the egg contains a pool of ribosomes sufficient to support development up to early larval stages in the absence of new ribosomal synthesis (199). The dramatic decrease in the ratio of mitochon drial to nuclear DNA in the course of embryogenesis also suggests that mito chondria are stored in the unfertilized egg (200). Unfertilized eggs contain roughly equal amounts of mitochondrial DNA and DNA of unknown function with a density that differs from that of main band nuclear DNA (201, 202). The histones of the embryo have been characterized, and as expected all five major classes were present (203). They were similar to histones from other species with respect to amino acid composition, with the exception of histone F I which contains larger amounts of both hydrophilic and hydrophobic amino acids than does mam malian F l . The amino acid composition of the C-terminal half of the molecule is lysine- and alanine-rich as in vertebrates, but it contains considerably less proline. Histone Fz., is very similar to, or identical with, the calf protein. Several groups have studied RNA polymerases in embryos (204, 205), larvae (205-207), imaginal discs, and tissue culture cells (208). The embryo contains two major forms ofthe enzyme: RNA polymerase I which is resistant to a-amanitin and presumably responsible for rRNA synthesis, and form II which is a-amanitin sensitive and involved in the transcription of mRNA. If the enzymes are purified by phosphocellulose chromatography followed by fractionation on DEAE-cellulose, both of the major forms can be resolved into two distinct peaks of activity (204). RNA polymerase II (or B), which is similar or identical in embryos and larvae, has been isolated in essentially pure form from third-instar larvae and characterized extensively (207). It is similar in its enzymatic and structural properties to the respective enzyme in other eucaryotes. It preferentially transcribes DNA containing


DEVELOPMENTAL GENETlCS OF DROSOPHILA

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single-stranded regions. Its subunit structure is complex and at present it is not known how many polypeptides constitute the core enzyme. An antiserum directed against the Drosophila enzyme also inhibits the activity of calf and yeast RNA polymerase II. However, only the Drosophila enzyme gave a precipitin reaction. If preparations of giant chromosomes are treated with fluorescent antibodies against Drosophila RNA polymerase II, specific fluorescence is observed in the puffed regions of the chromosome (U.F. Plagens and E. K. Bautz, personal communica tion). Protein synthesis has been investigated in cell-free preparations of mature ovarian oocytes (stage 14), unfertilized and fertilized eggs, and early embryos (209). This cell-free system supports a linear incorporation of labeled amino acids into acid precipitable material for at least 1 5 min. The specific activity of amino acid incorpo ration in postmitochondrial supernatants with endogenous RNA templates was found to increase five- to sixfold in oviposited unfertilized eggs as compared to ovarian oocytes. Fertilized eggs show only a slight increase as compared to unfertil ized eggs. It is known that the mature ovarian oocytes are arrested in the first meiotic division and that meiosis is completed in deposited unfertilized eggs (210, 210a). Also some ultrastructural changes are observed in both fertilized and unfertilized eggs after deposition (A. P. Mahowald, personal communication), indicating that both are activated. These observations agree with the finding that protein synthesis is activated in both fertilized and unfertilized deposited eggs. It has been suggested that this activation is under hormonal control (209); alternatively, the activation may be mechanical as in the ichneumonid wasp Pimpla. The Pimpla egg is activated by mechanical pressure when passing through the narrow opening of the ovipositor (212). No polyribosomes are detected in ovarian oocytes, whereas 35% of the ribosomes from both unfertilized and fertilized eggs sediment in the polyribosome region of sucrose density gradients (209), confirming the interpretation of the amino acid-incorporation data. The specific activity in protein synthesis decreases in celJ free extracts from blastoderm stages and returns during gastrulation to the same level as in fertilized eggs. Few data are available on the cytoplasmic localization of specific macromolecules. A first step in this direction has been made in a study of soluble antigens in various fragments of the egg (211). Two antigens were shown by immunoelectrophoresis to be localized in the posterior part, whereas one antigen was limited to the anterior 30% of the egg. The functional importance of this localization and the mechanism of localized deposition of these antigens remain to be elucidated.

POSTEMBRYONIC DEVELOPMENT OF IMAGINAL DISCS Determination During Larval Stages

During embryogenesis, groups of cells forming the imaginal discs are segregated from the larval cells (see 2 1 3). As shown by transplantation experiments, each disc forms a specific area of the adult epidermis and in some cases also mesodermal


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structures. By cutting the disc into defined fragments, the anlagen for the various adult structures can be precisely localized in a fate map. The list of such fate maps (213) has been supplemented with maps of the haltere disc (214) and the wing disc (215). The detailed map of the wing disc of Drosophila is in good agreement with that of two other flies, Calliphora (216) and Zaprionus (21 7). With respect to the female genital disc, Drosophila differs from Musca, which forms three genital discs instead of a single median one (218). The fact that isolated disc fragments, when transplanted into a host larva of the same age as the donor, develop strictly according to their fates, indicates that at least by the third larval instar the important events of pattern formation have already occurred. Bryant assumes that by this time the disc cells have had their positional information specified, but considers the changes involved in this specification as transitory and nonheritable (219). He reserves the term determination for heritable events (or states) as they presumably occur in disc-specific determination. However, long-term culture experiments do not allow a clear distinction between specification and determination (see section on transdetermination). Therefore we have used the term determination also for events that program the various anlagen within a disc (213). A third concept, that of compartmentalization, is discussed in the following section. There is accumulating evidence that determination is a process of stepwise restric tion of the developmental potential of cells (220, 221). To my knowledge, the best example is the eye-antenna! disc. Dissociation-reaggregation experiments (117) indi cate that anterior blastoderm cells are determined to form anterior structures and cannot form any abdominal or genital structures, even when combined with poste rior cells. In the course of development, the primordial cells of the eye-antennal disc can still give rise to both eye and antennal structures, since clones of genetically marked cells induced at the blastoderm stage by X irradiation frequently overlap both the eye and antennal region ( 180). However, in late-third instar larva, the determination of eye versus antennal cells has occurred (222). Therefore, at least two determinative steps can be demonstrated. The distinction between eye- and antennal cells in the discs of late-third instar larvae is in fact reflected at the biochemical level; cells in the antennal region of the disc show aldehyde oxidase activity as demonstrated by histochemical staining, whereas cells ofthe eye region lack this enzyme activity (152). The specificity of the histochemical reaction is shown by the observation that cells of the antenna! region of the disc do not stain if the substrate is omitted; a similar result obtains in the mal and aldox"i mutants which lack the enzyme activity. Differential permeability to the reagents is also ruled out, because the same staining pattern is observed in frozen sections and in whole mounts ( 1 57). Therefore, these results provide evidence that the eye and antennal disc cells are not only programmed, but that they begin to express part of their program as shown by their different biochemical properties. However, the largest part of their program is not expressed until metamorphosis when morphological differentiation sets in (see section on metamorphosis). The time course of this biochemical differentiation was studied by histochemical analysis of eye-antennal discs from larvae of different stages (221). No aldehyde



233

oxidase activity was detected in first-instar discs which consist of very few cells, although staining was observed in other tissues. The first aldehyde oxidase positive cells were found in the early second instar, and by the middle second instar the staining reaction was quite strong. It is interesting to note that in the third instar disc there is a sharp borderline between the positive cells of the antennal region and the cells of the eye region which lack the enzyme activity. It remains to be seen whether the time of appearance of the differential enzyme activity coincides with the time of determination. Studies on genetic mosaics have shown that cell lineage is not absolutely fixed, but varies to some extent from fly to fly. Recent studies on cell lineage in the eye disc cast some doubt on the dogma of ommatidial differentiation as stated in various textbooks. The extreme regularity and the number of eight receptor cells per om matidium favor the idea that these cells arise by a defined number of differential cell divisions from a single primordial cell, as suggested by some morphological studies (see 223). However, the analysis of genetic mosaics indicates that a single om matidium can contain a mixture of receptor cells of different genotypes and there fore, cannot be derived from a single ancestral cell (224). The interpretation of these mosaics is based upOn the assumption that the marker gene, white in this case, is cell autonomous which is difficult to prove rigorously. However, the pattern of mosaicism as indicated by Benzer can hardly be explained by partial nonautonomy. A long-standing problem concerns the origin of the mesodermal cells in the imaginal discs. Interspecific transplantation experiments between Drosophila and Zaprionus, which have different electrophoretic variants of arginine kinase, indicate clearly that the precursors ofthe muscle cells are present in the leg discs of late third instar larvae (225). Ultrastructural studies indicate that the adepithelial cells (225a) are the precursors of the muscle cells (225b), but it is not known whether they originate from the disc itself or migrate into the disc during early development. For the genital disc, recent histological studies support the idea that some mesodermal cells from the hemolymph attach themselves specifically to the ectodermal disc epithelium and give rise to mesodermal structures in the genitalia (22Sc) . Compartmentalization and the Genetic Control of Disc Development

The clonal analysis of imaginal disc development by X ray-induced mitotic recom bination has led to the discovery that clones initiated after a given time in develop ment do not cross certain boundaries which delimit what has been called a compartment (226-228). The occurrence of compartments may be a general feature of insect development (229). A compartment is thought to comprise all of the descendants of a small group of founder cells and therefore has been designated as a polyclone (230). The boundary between compartments is precisely defined and, in the case of the anterior-posterior compartments of the wing disc, it follows a straight line between the third and the fourth wing vein (226). The edges of the clones are somewhat irregular except where they follow the compartment boundary. A boundary is observed by using cuticular markers in wild-type wings, but its detection is greatly facilitated by the use of the Min ute technique (231). Heterozy gous Minute larvae (M/M+) show a reduced rate of cell division in their imaginal -


·

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discs. Mitotic recombination in such discs leads to M/M cells which are lethal M+/M+ cells which overgrow the heterozygous cells and form unusually large clones. Furthermore. the Minute genotype is characterized by an increased fre quency of induced mitotic recombination (232). Using this technique, compartmen talization has been studied in detail in the wing disc (22�228) and in the leg disc (181). The earliest clones (0-24 h) induced in the wing already define the anterior posterior demarcation line subdividing the disc into two compartments. As develop ment and cell division proceeds, these large compartments become split into pairs of smaller ones. For the leg disc, it has been shown that the anterior-posterior compartment boundary is already established at the blastoderm stage ( 1 81). A few clones were found to overlap the anterior and posterior compartments, which strongly suggests that the founder cells for the two compartments lie close to each other in the blastoderm. Preliminary gynandromorph mapping data (233) leads to the same conclusion, but there are limitations to this technique owing to map expansion when applied to very short distances (172). The mechanism of compartment formation is not understood. The most likely mechanism seems to involve "geographical partition" within a patch of epithelial cells separating the founder cells of one subcompartment from those of another (230). But it is difficult to understand how a straight demarcation line is formed between cells which appear morphologically identical. It has been proposed that negative cell affinities between cells of different compartments might lead to the formation of such demarcation lines (227). Straight demarcation lines are also found in gynandromorphs. In many cases, the boundary between male and female cells follows exactly the longitudinal midline and the segmental borders, which is a consequence of the fact that the adult fly is "constructed" from segmentally ar ranged pairs of imaginal discs. The sharp demarcation line between left and right disc for example, is an artifact, since at early embryonic stages the disc primordia are located far apart and an irregular genotypic boundary may run across cells that separate the disc primordia but do not contribute to the adult cuticle. This raises the question of whether the compartment boundaries result from the way the cuticular elements are "constructed" and whether they really represent the clonal boundary. In these studies, the assumption is usually made that all the cells of a clone or a polyclone are visible on the adult cuticle, but the possibility that a fraction of the cells die during morphogenesis (programmed cell death) or form some inter nal structures has not been rigorously ruled out. At least some of the compartment boundaries may be explained by the folding pattern of the disc during metamorpho sis and possibly also by localized cell death (215). The existence of defined compart ment boundaries does not necessarily prove that clonal or polycionaJ restrictions exist. If they do exist, they are certainly confined to normal development, since both in the wing (215) and in the leg disc (18 1 , 234) regeneration across compartment borders does occur. An important piece of evidence for the biological significance of compartments is provided by homeotic mutants. It turns out that a number of such mutants affect certain compartments. It has been known for a long time that various bithorax (bx) alleles transform either the anterior or posterior half of a segment (235).



235

Recently it has been shown that bx3, for example, specifically transforms the an terior compartment of the haltere disc into an anterior wing compartment (236). The mutant engrailed (en) leads to the transformation of the posterior compartment into the anterior compartment of the wing disc, and similar anterior-anterior dupli cations of all the thoracic discs (227, 237). In a series of elegant experiments, it was shown that the en+ allele is needed for maintenance of the anterior-posterior com partment border in the wing (238). Homozygous en/en clones induced in the anterior wing compartment of en/en+ flies "respect" the compartment boundary, whereas clones in the posterior compartment may cross it and extend into the anterior compartment. Furthermore, marked clones in en/en wings can extend across the compartment boundary. These expe:'iments suggest that the activity of the en+ gene is restricted to the posterior compartment where it "labels" the cells so that they do not mix with the anterior cells (238). It is interesting to nc,te that in en/en flies, the two wing discs often fuse incompletely leaving a cleft in the scutellum, indicating that the mutation affects both interdisc and compartment boundaries, which suggests that both may arise by a similar mechanism (W. 1. Gehring, unpublished observations). Numerous laboratories have been involved in the isolation and characterization of zygotic mutants affecting disc development. Because there are no selective proce dures for isolating such mutants, pupal lethals that are enriched for mutants affect ing disc development, or cell lethals that block the development of disc cells are isolated. However, other classes of mutants controlling disc development are either missed or recovered by accident. A relatively large number of mutants have been mapped, tested for complementa tion, and examined for their effects on disc development by transplantation experi ments (239-243), by gynandromorph tests (167 242, 244, 247), by mitotic recombination (244-247), or studied by light and electron microscopy (239, 240, 242, 248). It has been estimated that about one fifth of all complementation groups in the genome are essential for the development of all imaginal discs and not for the development of the larva (240). These mutants can be classified in various ways and, because their mode of action is not known, several fancy models and nomenclatures have been proposed. However, a priori we can assume that there are different genetic programs for different discs and different cell types, and that at least two classes of genes exist: one controlling the type of program performed and one involved in the actual performance. These correspond to the regulatory and structural genes de scribed in procaryotes. The fundamental problem in cell differentiation concerns the problem of how the structural genes are regulated. Good candidates for regulatory genes are the ho meotic genes. The work on the homeotic mutants, which shift cells into a different developmental pathway, has been reviewed recently (249) and only some of the most important points are summarized here. The specific effect of homeotic mutants on compartments has been mentioned above. Mitotic recombination studies show that the action of these mutants is cell autonomous, indicating that the wild-type gene products are not diffusible (235-237, 250-252). Indirect evidence from studies in which clones of homozygous mutant cells were induced at different times in develop,


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ment and the analysis of temperature-sensitive mutants suggest that various hom eotic genes may become active at different times in development, the bithorax genes as early as the blastoderm stage (227), the Antennapedia and aristapedia genes apparently only in the early third-instar larva (253, 254). ' The continued expression of the genes seems to be required for the maintenance of the determined state (see 227, 228). The aristapedia mutation continues to be expressed during proliferation, when antennal or eye discs are cultured in vivo (255). Antennae regenerated from the eye disc of a temperature-sensitive aristapedia allele (Ss>40a) differentiate according to the temperature at which regeneration takes place independently of the temperature at which the donor larva was raised (256). For several mutants it has been found that homozygous mutant clones induced some 3-4 divisions prior to metamorphosis, differentiate nonautonomously into wild-type structures (236, 237, 25 I, 252). This has been explained by assuming that the wild-type gene product has a relatively long half-life (perdurance, 257). This inter pretation is supported by the observation that perdurance of bithorax is lost after in vivo culture (A. Garcia-Bellido, cited in 258). These studies also indicate that different developmental pathways are mutually exclusive. Unfortunately, we still do not know anything about the molecular basis of homeotic mutants. Since reversions of the dominant mutant Nasobemia can be induced by X or 'Y irradiation and are associated with deletions, the hypothesis has been advanced that this mutation is either neomorphic, producing an effect not produced to an appreciable extent by the normal gene, or a duplication (259, 260); however, the biochemical nature of the gene product remains unknown. Regeneration

Regeneration and duplication in imaginal discs have been summarized recently by Bryant (26 1 ). If defined fragments of a disc are transplanted into host larvae which have the same age as the donor, they give rise to a defined set of cuticular structures which can be predicted from the fate map of the disc (see section on determination during larval stages). However, if the fragment is cultured for a short time, thus allowing the cells to proliferate, these structures will either be duplicated, or struc tures that are normally derived from other areas of the disc are regenerated (222, 234, 262). Regeneration is also observed after bisecting a disc in situ (263). Recently, the wing (2 1 5, 26 1 , 264, 265) and the haltere disc (266) have been examined in detail. If all the data are considered together, an interesting fact emerges: when a particular fragment shows duplication, the complementary piece shows regeneration. This finding has been interpreted in terms of a simple gradient model (2 1 9, 263); the position of a cell in the gradient specifies its developmental capacity. Cells at any position in the gradient would have the developmental capac ity to replace all the lower levels but not higher levels in the gradient. A large number of fragmentation experiments have been performed with one or two cuts either in the longitudinal, transverse, or diagonal direction (2 1 5). The results are consistent with the model, assuming that the high point of the gradient is roughly in the center of the disc. However, when central pieces were i!>olated by four cuts, contrary to expectation, they showed a stronger tendency to duplicate than to regenerate. Studies on pattern formation after irradiating first-instar larvae with 'Y



237

rays to kill a fraction of the disc cells gave similar results which can be interpreted in the same way (265). Surprisingly, the data from haltere discs differ significantly from those of the wing disc (266). In this case, the anterior (proximal) fragments can regenerate whereas the posterior pieces tend to duplicate. This difference, which has been confirmed by irradiation experiments (265), is unexpected because the two discs are homologous from an evolutionary standpoint and they have very similar fate maps. The most recent experiments (264) indicate that intercalary regeneration occurs in the wing disc. The crucial experiments consist of intermixing genetically marked fragments from distant regions of the disc so that an aggregate of cells is formed. If cells from the proximal and distal tip of the disc are intermixed, they become capable of extensive regeneration, whereas cells from distal or proximal fragments alone duplicate but do not regenerate. If this result is confirmed, it provides the first evidence for an altered determination of imaginal disc cells as a result of cellular interactions. Transdetermination

One of the most fascinating phenomena in developmental biology is transdetermina tion (2 1 3 , 267, 268). Imaginal discs, disc fragments, or dissociated and reaggregated disc cells can be cultured in vivo in the abdomen of adult female hosts. In the hemolymph of the host fly, which has a low titer of ecdysone (269), the implant proliferates but does not differentiate morphologically. Morphological differentia tion requires a high titer of ecdysone which can be provided by transplanting the tissue into metamorphosing larvae. Long-term cultures of imaginal discs can be established by serial transfer from one adult generation to the next, and in general the cells retain their capacity for differentiation and a normal karyotype. A given state of determination can be reproduced over a large number of transfer generations. For example, anal plates were formed by some of the subcultures for more than 70 transfer generations or approximately one thousand replication cycles (267). It is important to note that other structures derived from the genital disc, for example claspers, were not formed in these subcultures. Other subcultures produced claspers but no analplates. Therefore, intradisc determination is heritable and can not be distinguished from disc-specific determination on the basis of this criterion, as proposed by Bryant (2 1 9). In addition to the autotypic structures which are normally derived from the cultured disc, allotypic structures, which in normal development arise from other discs, are formed. Hadorn has called this phenomenon transdetermination. Cell proliferation seems to be a prerequisite for transdetermination. By inducing clones of genetically marked cells, it has been shown that both autotypic and allotypic cells can occur in the same clone and that different types of allotypic structures appear successively in subsequent transfer generations (270). Allotypic structures appear in a certain sequence and with a certain probability, which presumably reflects the circuits of the controlling genes. A model based upon this assumption has been proposed (27 1 , 272). It assumes that determination is carried by entities possessing only two stable alternate states, for example "on" and "off." Several of these independent bistable circuits could


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constitute an epigenetic code word standing for a certain state of determination. The model allows certain predictions which can be tested experimentally. The homeotic mutants resemble these bistable circuits in that they presumably select a certain developmental pathway, repress an alternative pathway, and maintain their state of activity by feedback control (227). For most sequences of transdetermination the corresponding homeotic mutants are known (see 249). However, there are exceptions which are not simply a consequence of the fact that the respective mutant has not yet been isolated. For example, no transdetermination of wing into haltere has been found although the respective homeotic mutants are known. The frequency of transdetermination increases with increasing age of the donor larva and drops off at the prepupal stage which may be a result of changes in ecdysone titer (273, 274). The analysis of clones of genetically marked cells provides strong evidence that transdetermination occurs in groups of cells rather than in single cells (270, 275, 276). Similarly, the homeotic transformation of the antenna into a leg in Anten napedia is not clonal but occurs in a group of cells. This observation suggests that cellular interactions are involved in these changes. However, cell mixing experi ments and studies on genetic mosaics failed to demonstrate such interactions, which would lead to nonautonomous differentiation (2 1 3). Alternatively, it can be assumed that a group of cells may proliferate in synchrony and undergo the same changes leading to transdetermination at the same time. It remains to be shown whether a group of cells, after having acquired a new state of determination, transmits this state to its descendants as a polyclone or whether the apparent cell heredity is because there is continued cellular interaction. In several long-term cultures of imaginal discs, sublines have been isolated which no longer differentiate normally or fail to differentiate completely. The changes leading to such lines are associated with a marked increase in the rate of prolifera tion and characteristic morphological changes of the disc epithelium (277). These changes resemble those observed in neoplasms which arise spontaneously or can be readily obtained from discs of certain mutants (278, 279). Metamorphosis

During larval development, the imaginal discs gradually acquire competence to metamorphose. This can be shown by transplanting discs from young larvae into hosts that are ready to pupate. For example, eye-antennal discs from first-instar larvae are unable to differentiate under these conditions; discs from second-instar larvae are capable of synthesizing ommochromes, but the competence to form a full complement of cuticular structures is only acquired during the third-larval instar (280, 28 1). This competence seems to depend on a minimum number of mitoses (280). Both in the leg disc (282) and the eye-antennal disc (28 1 , 283) different cells acquire the competence to metamorphose at different times as shown by light and electron microscopy. Autoradiographic evidence indicates that during the third larval instar the eye anlage is traversed by two consecutive mitotic waves, producing different categories of receptor cells (283).



239

The hormonal control of disc metamorphosis is most clearly demonstrated by in vitro studies. In his pioneering experiments, Mandaron obtained complete metamor phosis of isolated leg, and wing discs in vitro upon addition of ecdysone in a chemically defined medium (284). This remarkable morphogenesis from a disc to a moving leg has also been documented by time-lapse cinematography. There has been some controversy about whether a- or /3-ecdysone is the active compound. Mandaron finds that complete metamorphosis is obtained only with a-ecdysone (285), whereas Fristrom and collaborators were able to induce differentiation with /3-ecdysone using different media (285a). This difference may in part be due to impure hormone preparations. Milner & Sang (286) found both compounds to be active in promoting metamorphosis, although the /3-hormone seemed more active. However, one would like to see photographs in order to evaluate the degree of differentiation obtained. The mechanism of disc evagination, which is the first step in metamorphosis, has been investigated in vitro (284, 287-292). Evagination can be subdivided into two phases: during the first phase, the disc is unfolded and the peripodial membrane ruptures; in the second phase, there is a marked change in cell shape from columnar to cuboidal and the epithelium becomes thinner and elongates. These changes also occur in disc fragments and seem to be mediated by direct action of the hormone on the cells. Recent ultrastructural studies indicate that both in vivo (292) and in vitro (293) the intercellular junctions do not change during this process. Several inhibitors and enzymes, including concanavalin A, cytochalasin B, trypsine, and neuraminidase, have been tested for their effect on evagination (292, 288-290a) and a mechanism has been proposed (291). Fristrom and co-workers have examined the effect of /3-ecdysone on mass prepa rations of imaginal discs from late-third instar larvae. /3-Ecdysone increases overall RNA synthesis, predominantly rRNA (290), which is paralleled by an increase in RNA polymerase I (208a). Similarly, ecdysone increases the rate of incorporation of 3H-Ieucine into protein (294) with the strongest effect on the ribosomal protein fraction (295). Both a- and /3-ecdysone were also found to stimulate the rate of 3H-thymidine incorporation into disc DNA (296). This stimulation is blocked by juvenile hormone and it has been proposed that these two hormones might regu late DNA synthesis in a balanced fashion. Recent studies on the uptake and bind ing of /3-ecdysone demonstrate specific hormone binding sites in the imaginal discs (297). The in vitro culturing system will obviously be a powerful tool in the analysis of hormone action in cell differentiation and morphogenesis. No disc cell lines pro liferating in vitro are presently available, but a step in this direction has been made (298-300). In cultures of cells from 6-8 h old embryos, vesicles of imaginal disc epithelium have been obtained in which the cells divide. Addition of a- or /3ecdysone to the culture medium leads to the differentiation of cuticular structures in these vesicles. Recognizable patterns are not formed though, unless these vesicles are cultured in vivo. It remains to be seen whether such vesicles can be cultured permanently as isolated cells and if so, whether they retain their capacity for differentiation under these conditions.

240

GEHRING


CONCLUSIONS

It is obvious that we are still far from an understanding of the mechanisms of development. The availability of elaborate genetic techniques, giant chromosomes, imaginal discs, and numerous other advantages make Drosophila one of the organ isms most frequently used in the study of development. We are just now entering a phase of research in which molecular techniques can be applied to the problems of developmental genetics; in this context, the new techniques of genetic engineering have to be mentioned (301-303). These involve the insertion of DNA sequences from Drosophila and other eucaryotic organisms into bacterial plasmids or phages, and the cloning of such sequences. These techniques provide a general method for gene isolation which is a prerequisite for studying gene regulation as it occurs during development in higher organisms. Most relevant, although not directly related to developmental problems, are recent studies on heat-inducible proteins (304--306). It has been found that heat treatment of salivary glands induces a small number of specific puffs and a corre sponding number of newly synthesized polypeptides; concomitantly, the synthesis of other proteins is interrupted. Because these heat-shock proteins can also be induced in other tissues including cell cultures, it has been possible to isolate a fraction of mRNA which presumably codes for these proteins. In this system it seems possible to correlate a particular puff with a gene, its mRNA, and its protein products. Furthermore, it allows us to study the coordinate control of a group of genes. With these new techniques and systems it may become possible to elucidate some of the mechanisms of gene regulation in eucaryotes, which is of major impor tance for the understanding of development. Literature Cited I . Fristrom, J. W. 1 970. The developmen 2.

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1 3. KambyseJlis, M . P., Gelti-Douka, H. 1974. Vitellogenesis in Drosophila: Ge netic and hormonal controls. Genetics 77(I):Suppl.s33 (Abstr.) 14. Postlethwait, J. H., Weiser, K. 1973. Vitellogenesis induced in the female sterile mutant apterous-four of Droso phila. Nature New Bioi. 244:284-85 1 5 . Fox, A. S., Mead, C. G., Munyon, I. L. 1959. Sex peptide of Drosophila melano gaster. Science 1 2 3 : 1489 16. Chen, P. S., Diem, C. 1961. A sex specific ninhydrin-positive substance found in the paragonia of adult males of Drosophila melanogaster. 1. Insect PhysioL 7:289-98 17. Fox, A. S., Munyon, 1. L., Singh, !. P., Sweeney, E. A. 1962. Genetic determi nation, amino acid composition, and synthesis of the sex-peptide of males in Drosophila melanogaster. Genetics 47: 953 18. Leahy, M., Lowe, M. L. 1967. Purifica tion of the male factor increasing egg deposition in Drosophila melanogaster. Life Sci. 6: 1 5 1-56 19. Chen, P. S., BUhler, R. 1970. Paragonial substance (sex peptide) and other free ninhydrin-positive components in male and female adults of Drosophila melano gaster. 1. Insect Physiol. 16:61 5-27 20. Garcia-Bellido, A. 1 964. Das Sekret der Paragonien als Stimulus der Fekunditat bei Weibchen von Drosophila melano gaster. Z. Naturforsch. 19b:491-95 2 1 . Burnet, 8., Connolly, K., Kearney, M., Cook, R. 1973. Effects of male paragonial gland secretion on sexual receptivity and courtship behavior of female Drosophila melanogaster. J. In sect Physiol. 19:242 1-3 1 22. Baumann, H., Chen, P. S. 1973. Ges chlechtsspezifische ninhydrin positive Substanzen in Adultmannchen von

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80:685-90 23. Baumann, H. 1974. The isolation, par tial characterization and biosynthesis of the paragonial substances, PS-I and PS-2, of D. funebris. J. Insect Physiol. 20:2 1 8 1-94 24. Baumann, H. 1974. The biological effects of the paragonial substances, PS- I and PS-2, in female of Drosophila funebris. 1. Insect PhysioL 20:2347-62 25. Baumann, H., Wilson, K. J., Chen, P. S., Humbel, R. E. 1975. The amino acid sequence of peptide (PS-I) from Drosophila funebris: A paragonial pep tide from males whi

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