Developmental genetics of the mutant grandchildless of Drosophila subobscura

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1 /. Embryol. exp. Morph., Vol. 17, 2, pp , April With 1 plate Printed in Great Britain Developmental genetics of the mutant grandchildless of Drosophila subobscura By C.J.FIELDING 1 From the Department of Zoology and Comparative Anatomy, University College London A study of the embryology of female-sterile mutants can provide information on the course of normal development, particularly when several mutants affect the same organ system. A favourable situation for an investigation of this kind arises with the formation and migration of the pole cells in Drosophila species. A number of genetic characters are known whose expression in the embryology of the offspring of affected mutant females involves the complete loss of pole cells or a change in their distribution after gastrulation. Thus the sex-linked female sterile mutant fs na3a (Counce & Ede, 1957), and the sex-linked lethals Lfll (Ede, 1956a), X2 (Ede, 19566), X27 (Ede, 1956c) and X10 (Ede, 1956c?) all show disturbances of the pole cell complement. This paper presents an account of the embryological effects of the mutant grandchildless (gs) of Drosophila subobscura (Spurway, 1948). The homozygous mutant female lays eggs which develop into adults with rudimentary gonads, which are otherwise phenotypically normal. There is no phenotypic effect in the offspring of homozygous males by normal females, although the gene is expressed in the offspring of homozygous females whatever the genotype of the male parent. MATERIALS AND METHODS The gene grandchildless, which is autosomal and recessive, is maintained by sibmating within cultures of the first cousins of gonadless individuals. The maintenance of this stock has been described in detail by Suley (1953). Freshly eclosed cousins of gonadless individuals were put into shell vials, a male and a female to each. The vials contained a mixture of dried yeast, agar, molasses and corn meal, seeded with live yeast. The pairs were transferred every 4 days to a new food medium, until the female genotype could be established from the phenotype of the offspring. Because of the number of cultures involved only the adults could be examined for the presence of gonads, about 20 days after the eclosion of the parents. Thereafter the homozygous grand- 1 Author's address: Department of Biochemistry, University of Oxford, Oxford, U.K. 24 JEEM 17

2 376 C. J. FIELDING childless females and sufficient of their sisters, taken at random from the population, provided a supply of eggs for analysis; only eggs from actively laying females were used. In this paper the term 'grandchildless egg' refers to an egg laid by a homozygous mutant female, and 'normal egg' to one laid by her phenotypically normal sister; and similarly with larvae and adult offspring. In some experiments whole ovaries and isolated mature oocytes (stage 14 of King, Rubinson & Smith, 1956) were frozen in liquid nitrogen, sectioned at 5 fi in a cryostat, thawed and fixed in formol-calcium (Baker, 1944), and stained with Oil Red O (Lillie, 1944) to display neutral glyceride. Developing eggs were fixed with formol-alcohol-acetic acid (Smith, 1940) and after impregnation with paraffin wax (melting point 58 C) sections were cut at 5 fi and stained with iron haematoxylin. Freshly excised larval midguts were suspended in aqueous sodium diethyldithiocarbamate to show the presence of copper (Waterhouse, 1945). RESULTS Structure of the gonad rudiment of adult offspring of grandchildless females The ovary of the newly eclosed female contains about a dozen ovarioles, containing egg chambers at stages 1-5 of ICing, Rubinson & Smith (1956). In the ovary rudiment of the newly eclosed female offspring of the homozygous grandchildless female there were no egg chambers or oogonia. The organ contained about a dozen columnar masses of small cells, within a substantial matrix of connective tissue (Plate 1, fig. 1). At its anterior end each cell mass narrowed to a thread of a single thickness of cells. The position of the rudiment in the abdominal cavity, and its association with a group of tracheoles whose components passed between the columnar masses, were as in the normal ovary. Within a few days of eclosion the individual cells of the masses coalesced, and stained darkly with haematoxylin. In the adult male offspring of the homozygous grandchildless female the testis rudiment was a minute structure attached to the vas deferens. It was composed throughout its length of the orange pigmented tissue characteristic of the vas deferens wall, and tunica externa of the normal testis. Whilst the rudiment usually consisted of a spherical knob attached by a thin thread of the same orange tissue, in a few individuals (the proportion varying with the residual genotype) it lay unattached in the body cavity, or was completely absent. In occasional cultures which contain gonadless individuals there are a few flies with one or both gonads, which contain maturing sperm or eggs, and these are fully fertile.

3 /. Embryol. exp. Morph., Vol. 17, Part 2 PLATE 1 Fig. 1. Ovary rudiment of newly eclosed gonadless female offspring of a homozygous grandchild/ess mother. Iron haematoxylin. x 275. Fig. 2. Grandchild/ess ovum, early cleavage, showing the chains of vacuoles. Iron haematoxylin. x520. Fig. 3. Grandchildless embryo, blastula showing degenerated pole plasm. Iron haematoxylin. x 520. Fig. 4. Egg chamber of homozygous grandchildless female. Oil Red O. x 275. Fig. 5. Egg chamber of normal female of the same stock. Oil Red O. x 275. C. J. FIELDING facing p. 376

4 Genetics of mutant grandchildless 377 Structure of the gonad rudiment in larvae of grandchildless females In the newly hatched normal larva there was found, in the fifth abdominal segment on each side, an organ of a few large cells within a single-layered envelope of small cuboidal cells. This is the larval gonad. In the second and third instars, when the differentiation of the gonad had proceeded farther, the relationship of the gonad to the segmental fat body was the same as that described for Drosophila melanogaster by Kerkis (1933): in the female the fat body tissue appeared to surround the gonad, whereas in the male the gonad is merely apposed to it by the external face. Out of twelve complete series of sections of second and third instar larvae of homozygous grandchildless females, a ball of cuboidal cells in the position of the normal larval gonad was found in seven, surrounded by the segmental fat body. In the remaining five no organ was found, and the fat body tissue, which was normally developed, was continuous across the expected position of the gonad. Embryology of normal and grandchildless ova Yolk granules and large vacuoles are distributed rather evenly through the volume of the freshly laid normal egg, except that at each pole there is an area of clear cytoplasm, devoid of yolk spheres, which extends also as a narrow cortex about the entire periphery. At a point below the base of the chorionic filament, and close to the surface of the egg, the clear layer is expanded to include the egg pronucleus. After the second maturation division has been completed there, this moves inwards to meet the sperm head. The polar bodies remain behind at the surface. In Drosophila subobscura as in D. melanogaster the maturation division spindles have no centrioles or asters (Huettner, 1923). In the grandchildless eggs of the same stock there were far fewer yolk spheres and an increased number of large vacuoles. Further, these vacuoles were not distributed singly through the cytoplasm but were associated together in chains (Plate 1, fig. 2). Although the fertility of a few of these malformed eggs cannot be decided, approximately one-quarter of the eggs which have passed the second maturation division, fixed at up to 8 h after laying (25 out of 97 series, 26 %), show a variety of spindle disorders, including half-spindles, spindles of very large size containing diffuse masses of chromatin, and closely apposed triplets of complete spindles, in eggs containing other normal nuclei. In some cases the distorted spindles are peripheral and are presumably derived from the first or second maturation divisions, in some cases by further division of the polar bodies. In others it appears to be early cleavage nuclei in the central ooplasm which are involved. In the entire series of experiments 8616 out of 9370 normal eggs hatched (92-0%). Of the eggs laid by homozygous grandchildless females 1259 out of 1773 hatched (71-1 %). The observed frequency of eggs not developing beyond 24-2

5 378 C. J. FIELDING maturation or early cleavage could therefore account for the whole of the increased mortality found in the mutant series, and in practice dying embryos of later stages are not encountered. In the normal embryos at 3 h after laying, the cleavage nuclei had moved out to the circumference of the egg into the cortex. Since the cleavage divisions involved not only a multiplication but a distribution of nuclei in the ooplasm, the whole cortex was nucleated at approximately the same time, although the zygote was formed in the anterior third. As nuclei reached the poles of the egg, they bulged out but remained spherical. Elsewhere they became flattened against the vitelline membrane. At about 3- h after laying the first pole cells were cut off from the clear posterior pole plasm. At the anterior pole the bulging nuclei were withdrawn. A few nuclei remained behind in the yolky ooplasm after the formation of the blastoderm, and these are the equivalent of the primary vitellophags of D. melanogaster as defined by Rabinowitz (1941Z?). In 16 examples of normal early embryos of D. subobscura between 12 and 21 pole cells were cut off. Other nuclei in the polar region pushed out but were drawn back without severance of the cytoplasmic connexion. At this time the blastoderm has not yet been completed posteriorly, and these nuclei migrate anteriorly in the ooplasm, because in sections of embryos at a slightly later stage, when the blastoderm wall has been completed, nuclei identical in appearance to those of the pole cells, with a prominent nucleolus, appear just anterior to it. No evidence was found that pole cells divide while outside the embryo. In grandchildless embryos the posterior third of the egg has not yet received nuclei 3 h after laying. Not until 4-4 h do nuclei reach the anterior edge of the pole plasm. Pole cells have not been found in any grandchildless embryo; before the arrival of the first cleavage nuclei the posterior pole plasm is observed to be crumbling and vacuolated (Plate 1, fig. 3). The extent and size of the damaged area is the same as that of the normal pole plasm, defined as the area of clear cytoplasm found at the posterior pole in the mature egg. By 5 h after laying, however, the blastoderm is completed posteriorly but the pole plasm has been undercut and is excluded from the embryo. Certain of the cleavage nuclei during the outwards migration aggregated anterior to the degenerating pole plasm, and these were seen to remain there together as the blastula wall extended back around them (Plate 1, fig. 3). The primary vitellophags were present as usual, distributed singly in the ooplasm. The multiplication of nuclei in the blastoderm, and the development of cleavage furrows between them, proceeded normally in the mutant embryos. In the normal embryo, 5 h after laying, the blastula wall nuclei are placed peripherally, next to the vitelline membrane. In embryos fixed slightly later it was seen that at the postero-dorsal surface the nuclei were drawn inwards and a small depression had appeared over them. Into it passed the pole cells which had remained together posteriorly, outside the completed blastula wall. In sections

6 Genetics of mutant grandchildless 379 of later embryos it was seen that the depression moved anteriorly and at the same time curved backwards inside the embryo so that at its maximum extension, at about 7 h after laying, the opening was in the anterior third, and the diverticulum stretched back almost to the posterior blastoderm wall. This organ is clearly homologous with the midgut diverticulum of D. melanogaster and other Diptera (Sonnenblick, 1950). In embryos older than 7 h the pole cells in the diverticulum have become fewer and by 9 h have disappeared entirely from the lumen. In the grandchildless embryos, where no pole cells are cut off, the midgut diverticulum was found to be normally developed. The mass of nuclei which had aggregated anterior to the degenerated pole plasm became associated with the diverticulum but could not be traced further. The cuprophilic cells of the larval midgut Poulson (1947, 1950) has shown that in D. melanogaster the cuprophilic cells (calycocytes) of a region of the larval midgut are derived from a proportion of the pole cells. The number of calycocytes in the midguts of normal and grandchildless larvae was counted, using preparations stained with sodium diethyldithiocarbamate solution to display copper (Waterhouse, 1945). Normal larvae contain an average of 69 calycocytes (69 examples, range 57-86); grandchildless larvae contain an average of 75 (33 examples, range 57-84). A complete absence of pole cells is therefore not associated with any reduction in the number of calycocytes of the mutant larva. Oogenesis in homozygous grandchildless females and their normal sisters Oogenesis in homozygous grandchildless females shows the following unusual features. Firstly, in all mutant ovaries there is a high proportion of egg chambers at stage 10 of King et al. (1956), that is, with the oocyte occupying about half the volume of the chamber, and the nurse cell chromosomes at their maximum size. It is at this stage in both normal and mutant oogenesis that the amount of neutral lipid in the egg chamber rapidly increases, particularly in the nurse cells, and droplets can be seen in passage from the nurse cells to the oocyte. Secondly, in both nurse cells and oocytes of mutant ovaries the neutral lipid is present as a few large globules (Plate 1, fig. 4), rather than as the mass of fine droplets characteristic of this stage of oogenesis in the normal ovary (Plate 1, fig. 5). DISCUSSION The occurrence of rudimentary gonads in the adults of Drosophila species has been reported previously, not as the result of the action of a single gene, as in the present case, but in two other circumstances. In a number of inter-specific crosses [Drosophila aztecaxd. athebasca (Sturtevant & Dobzhansky, 1936),

7 380 C. J. FIELDING D. melanogaster xd. simulans (Bonnier, 1924; Sturtevant, 1929; Kerkis, 1933; Pontecorvo, 1942), D. mirandaxd. pseudoobscura (Dobzhansky, 1938)] the testes of the male offspring are minute and not functional. In the first two cases the ovary is also reduced, but in the third the female hybrid offspring have a delayed but superficially normal oogenesis, but of the eggs produced, none develop beyond the maturation division or early cleavage stages and show a variety of disorders of the division figures (Kauffman, 1940). Rudimentary gonads are also developed in normal eggs of D. melanogaster (and presumably other species) when the pole plasm or separate pole cells have been irradiated with ultraviolet light (Geigy, 1931; Aboim, 1945; Poulson & Waterhouse, 1960). In adult females from irradiated eggs the ovary is rudimentary and contains columnar masses of flattened cells. These cells have been shown to develop from the envelope of the larval gonad, which would normally provide the follicle cells of the egg chambers (Geigy, 1931). Germ cells and their derivatives, which are produced from the pole cells of the embryo (Huettner, 1923; Rabinowitz, 1941a; Sonnenblick, 1941), are completely absent. The embryological origin of the gonad rudiment tissue in the crosses mentioned is known only for D. melanogaster x D. simulans, where Pontecorvo (1942) has shown that certain masses of round cells (not present in the grandchildless rudiment) represent germ cells which have undergone multiple mitotic divisions. The close resemblance between the structures of the ovaries of offspring of irradiated and homozygous mutant females, together with the presence of only the small cuboidal cells in the position of the mutant larval gonad in those larvae possessing the organ, suggests that the mutant rudimentary ovary contains no germ cells. The observation that the individuals bearing these organs develop from embryos without pole cells gives confirmation to previous research showing that the two cell types have the same embryological origin. In adult males developing from eggs irradiated at the posterior pole the testis is represented by connective tissue and tunic only (Geigy, 1931). In the male offspring of homozygous grandchildless females it consists entirely of the pigmented tissue that in the normal testis contributes to the tunica externa only, and which late in pupal life spreads from the gonad over the vas deferens (Stern & Hadorn, 1939). Since in their structure and their position in relation to the segmental fat body the gonad rudiments which are found in grandchildless larvae correspond to those of the normal female, it is likely that those larvae showing no rudiment are genetically male. The size of the adult male rudiment reported by Spurway (1948) is much greater than that found in the present investigation, and it seems likely that in the intervening generations a loss of the inner mass of loose tissue has occurred, and that it was this component which was present in the male grandchildless larva in the gonad transplantation studies of Suley (1953). There is no sign in the testis rudiment of irradiated males or those from the grandchildless stock of a cell type equivalent to the follicle cells of the ovary.

8 Genetics of mutant grandchildless 381 This supports the conclusion reached by Bodenstein (1950) from a study of the normal embryology of D. melanogaster. In D. subobscura, as in Diptera generally, there is a very early separation of the germ line from the remainder of the embryo by the formation of pole cells after nucleation of the posterior pole plasm. Not all the pole cells contribute to the germ line (Rabinowitz, 1941a; Sonnenblick, 1941; Poulson, 1947). These cells migrate by two distinct and alternative routes: by passage between the cells of the posterior blastoderm wall, and later by migration into the growing hollow of the midgut diverticulum. Counce (1961), who termed these migrations phase I and phase II respectively, has suggested that the migration is continuous rather than diphasic. Hathaway & Selman (1961) used an ultraviolet microbeam to irradiate the posterior pole of embryos at three early stages of development: prior to the formation of any pole cells, after completion of pole cell formation, and after phase I migration but before the migration of the remaining pole cells into the midgut diverticulum. The number of pole cells found in the embryonic gonad was counted, and it was found that the same significant reduction in the number of gonadal pole cells was found after irradiation of all the pole cells or of the remaining phase II cells only. Thus a clear demonstration was obtained that the pole cells migrating at phase II give rise to the germ line. Embryos from homozygous grandchildless females show no phase II migration, and no pole cells in the gonads, but the calycocytes are present as usual. These results are therefore completely in accord with those of Hathaway & Selman (1961). These authors also conclude that the migrating pole cells cannot be equipotent, because otherwise a deficiency of phase II pole cells caused by ultraviolet treatment would presumably be compensated for by a contribution of phase I cells to the gonad. However, these authors' results are not able to provide information on the equipotency of the pole cells before the phase I migration. As Rabinowitz (1941 a) has shown, the phase I nuclei fuse with the cytoplasm of the blastoderm, whereas the phase II nuclei retain the cytoplasm with which they were associated at the time of their formation. Thus a differentiation into germ-line and calycocyte-line pole cells at the time of budding from the pole plasm, during the period outside the embryo, or during the phase I migration, would give the same results in terms of reduction of gonadal pole cells, namely that irradiation of all the pole cells, or of the remaining phase II only, would give significantly fewer in the gonad than before the formation of any pole cells, but irradiation at both stages would show the same reduction. This distinction is of considerable importance because of two observations that do not fit readily into the simple scheme that phase I gives rise to the calycocytes and phase II to the germ line. In grandchildless embryos no nuclei enter the pole plasm because of its early degeneration, and therefore the aggregation of nuclei anterior to it derive their cytoplasm from the blastoderm directly. However, grandchildless larvae developed from these contain the normal

9 382 C. J. FIELDING complement of calycocytes. Since in normal development the calycocytes are derived from pole cells which have been budded off from the main body of the embryo, it is reasonable to conclude that the irreversible differentiation of germ line from calycocytes required by the observations of Hathaway & Selman occurs after the migration of the phase I nuclei, which fuse with the blastoderm. Secondly, Counce & Ede (1957) found that in the mutant/s nasa there was no phase II migration; nevertheless gonads were formed in some cases. If, as seems likely from plate 1, fig. C of these authors, the pole cells migrating back through the blastoderm do not readily fuse with it, but retain their original cytoplasm, then the determination of the presumptive pole cell nuclei in this mutant is compatible with that in normal and irradiated embryos, and of grandchildless embryos. In this case, the determination of the germ line would require the interaction of presumptive pole cell nuclei and pole plasm at the onset of gastrulation, after the migration of the phase I nuclei, which, rejoined with the general blastoderm, would there complete their differentiation to calycocytes. SUMMARY 1. The development of the gonad rudiment of the offspring of female Drosophila subobscura homozygous for the gene grandchildless, in the embryo and larva, has been investigated. 2. An absence of germ cells in the larval and adult gonads has been correlated with the absence of pole cells in the midgut diverticulum of the embryo. 3. Early in the development of the mutant embryo the posterior pole plasm degenerates. The calycocytes of the larval midgut, which are derived from pole cells, are present in full number. 4. In the grandchildless embryo the presumptive pole cell nuclei stop short of the pole plasm, and aggregate anterior to it. These nuclei are thought to enter subsequently the midgut. 5. Observations on the fate of the presumptive pole cell nuclei in mutant embryos confirm previous research on the origin of the germ-line pole cells. The implications of the development of calycocytes in these embryos are discussed. RESUME Genetique du developpement du mutant 'grandchildless' de Drosophila pseudoobscura 1. Le developpement de l'ebauche gonadique de la progeniture de femelles de Drosophila subobscura homozygotes pour le gene 'grandchildless' ('absence de petits-enfants') a ete etudie chez Pembryon et la larve. 2. L'absence de gonocytes dans les gonades larvaires et adultes est correlative de l'absence de cellules polaires dans le diverticule de l'intestin moyen de l'embryon.

10 Genetics of mutant grandchildless Au debut du developpement de l'embryon mutant, le plasme polaire posterieur degenere. Les calyocytes de l'intestin moyen de la larve, qui derivent des cellules polaires, sont presents en totalite. 4. Chez l'embryon 'grandchildless', les noyaux presomptifs des cellules polaires s'arretent en avant du plasme polaire et s'agregent anterieurement a lui. On pense que ces noyaux entrent par la suite dans l'intestin anterieur. 5. Des observations sur le sort des noyaux presomptifs des cellules polaires chez les embryons mutants confirment les recherches precedentes sur l'origine des cellules polaires de la lignee germinale. On discute les implications du developpement des calyocytes chez ces embryons. This research is part of that presented to the University of London for the award of a Doctorate of Philosophy, and was supported by a Postgraduate Studentship of the Agricultural Research Council. I am indebted to Professor John Maynard Smith for his supervision of this investigation. REFERENCES ABOIM, A. N. (1945). Developpement embryonnaire et post-embryonnaire des gonades normales et agametiques de Drosophila melanogaster. Revue suisse Zool. 52, BAKER, J. R. (1944). The structure and chemical composition of the Golgi element. Q. Jl. microsc. Sci. 85, BODENSTEIN, D. (1950). The postembryonic development of Drosophila. In Biology of Drosophila, ed. M. Demerec. New York: Wiley and Sons. BONNIER, G. (1924). Contributions to the knowledge of intra- and inter-specific relationships in Drosophila. Ada. zool., Stockh. 5, COUNCE, S. J. (1961). The analysis of insect embryogenesis. Ann. Rev. Entomol. 6, COUNCE, S. J. & EDE, D. A. (1957). The effect in embryogenesis of a sex-linked femalesterility factor in Drosophila melanogaster. J. Embryol. exp. Morph. 5, DOBZHANSKY, T. (1937). Further data on Drosophila miranda and its hybrids with Drosophila pseudoobscura. J. Genet. 34, EDE, D. A. (1956a). Studies on the effects of some genetic lethal factors on the embryonic development of Drosophila melanogaster. I. A preliminary survey of some sex-linked lethal stocks, and an analysis of the mutant Lffll. Wilhelm Roux Arch. EntwMech. 148, EDE, D. A. (19566). Ibid. II. An analysis of the mutant X2. Wilhelm Roux Arch. EntwMech. 148, EDE, D. A. (1956C). Ibid. III. An analysis of the mutant X27. Wilhelm Roux Arch. Entw.- Mech. 149, EDE, D. A. (1956d). Ibid. V. An analysis of the mutant X10. Wilhelm Roux Arch. Entw.- Mech. 149, GEIGY, R. (1931). Action de l'ultra-violet sur le pole germinal dans l'oeuf de Drosophila melanogaster. Revue suisse Zool. 38, HATHAWAY, D. S. & SELMAN, G. G. (1961). Certain aspects of cell lineage and morphogenesis studied in Drosophila melanogaster with an ultraviolet microbeam. J. Embryol. exp. Morph. 9, HuETTNER, A. F. (1923). The origin of the germ cells in Drosophila melanogaster. J. Morph. 37, KAUFFMAN, B. P. (1940). The nature of hybrid sterility abnormal development in eggs of hybrids between Drosophila miranda and Drosophila pseudoobscura. J. Morph. 66, KERKIS, J. (1933). Development of gonads in hybrids between Drosophila melanogaster and Drosophila simulans. J. exp. Zool. 66,

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