/ . Embryol. exp. Morph., Vol. 17, 2, pp. 375-384, April 1967 3 7 5With 1 plate

Printed in Great Britain

Developmental genetics of the mutantgrandchildless of Drosophila subobscura

By C.J.FIELDING1

From the Department of Zoology and Comparative Anatomy,University College London

A study of the embryology of female-sterile mutants can provide informationon the course of normal development, particularly when several mutants affectthe same organ system. A favourable situation for an investigation of this kindarises with the formation and migration of the pole cells in Drosophila species.A number of genetic characters are known whose expression in the embryologyof the offspring of affected mutant females involves the complete loss of polecells or a change in their distribution after gastrulation. Thus the sex-linkedfemale sterile mutant fsna3A (Counce & Ede, 1957), and the sex-linked lethalsLfll (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 mutantgrandchildless (gs) of Drosophila subobscura (Spurway, 1948). The homozygousmutant female lays eggs which develop into adults with rudimentary gonads,which are otherwise phenotypically normal. There is no phenotypic effect in theoffspring of homozygous males by normal females, although the gene is ex-pressed in the offspring of homozygous females whatever the genotype of themale parent.

MATERIALS AND METHODS

The gene grandchildless, which is autosomal and recessive, is maintained bysibmating within cultures of the first cousins of gonadless individuals. Themaintenance of this stock has been described in detail by Suley (1953).

Freshly eclosed cousins of gonadless individuals were put into shell vials, amale 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 transferredevery 4 days to a new food medium, until the female genotype could be estab-lished from the phenotype of the offspring. Because of the number of culturesinvolved only the adults could be examined for the presence of gonads, about20 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

376 C. J. FIELDING

childless females and sufficient of their sisters, taken at random from thepopulation, provided a supply of eggs for analysis; only eggs from activelylaying females were used.

In this paper the term 'grandchildless egg' refers to an egg laid by a homo-zygous mutant female, and 'normal egg' to one laid by her phenotypicallynormal sister; and similarly with larvae and adult offspring.

In some experiments whole ovaries and isolated mature oocytes (stage 14 ofKing, Rubinson & Smith, 1956) were frozen in liquid nitrogen, sectioned at 5 fiin a cryostat, thawed and fixed in formol-calcium (Baker, 1944), and stainedwith 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 werecut at 5 fi and stained with iron haematoxylin.

Freshly excised larval midguts were suspended in aqueous sodium diethyldi-thiocarbamate 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 theovary rudiment of the newly eclosed female offspring of the homozygousgrandchildless female there were no egg chambers or oogonia. The organ con-tained about a dozen columnar masses of small cells, within a substantialmatrix of connective tissue (Plate 1, fig. 1). At its anterior end each cell massnarrowed to a thread of a single thickness of cells. The position of the rudimentin the abdominal cavity, and its association with a group of tracheoles whosecomponents passed between the columnar masses, were as in the normal ovary.Within a few days of eclosion the individual cells of the masses coalesced, andstained darkly with haematoxylin.

In the adult male offspring of the homozygous grandchildless female thetestis rudiment was a minute structure attached to the vas deferens. It wascomposed throughout its length of the orange pigmented tissue characteristic ofthe vas deferens wall, and tunica externa of the normal testis. Whilst the rudi-ment usually consisted of a spherical knob attached by a thin thread of thesame orange tissue, in a few individuals (the proportion varying with the residualgenotype) it lay unattached in the body cavity, or was completely absent. Inoccasional cultures which contain gonadless individuals there are a few flieswith one or both gonads, which contain maturing sperm or eggs, and these arefully fertile.

/ . Embryol. exp. Morph., Vol. 17, Part 2 PLATE 1

Fig. 1. Ovary rudiment of newly eclosed gonadless female offspring of a homozygous grand-child/ess mother. Iron haematoxylin. x 275.Fig. 2. Grandchild/ess ovum, early cleavage, showing the chains of vacuoles. Iron haemato-xylin. 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

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 abdominalsegment on each side, an organ of a few large cells within a single-layeredenvelope of small cuboidal cells. This is the larval gonad. In the second andthird instars, when the differentiation of the gonad had proceeded farther, therelationship of the gonad to the segmental fat body was the same as thatdescribed for Drosophila melanogaster by Kerkis (1933): in the female the fatbody tissue appeared to surround the gonad, whereas in the male the gonad ismerely apposed to it by the external face. Out of twelve complete series ofsections 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 inseven, surrounded by the segmental fat body. In the remaining five no organwas found, and the fat body tissue, which was normally developed, was con-tinuous across the expected position of the gonad.

Embryology of normal and grandchildless ova

Yolk granules and large vacuoles are distributed rather evenly through thevolume of the freshly laid normal egg, except that at each pole there is an area ofclear cytoplasm, devoid of yolk spheres, which extends also as a narrow cortexabout 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 theegg pronucleus. After the second maturation division has been completed there,this moves inwards to meet the sperm head. The polar bodies remain behindat the surface. In Drosophila subobscura as in D. melanogaster the maturationdivision spindles have no centrioles or asters (Huettner, 1923).

In the grandchildless eggs of the same stock there were far fewer yolk spheresand an increased number of large vacuoles. Further, these vacuoles were notdistributed 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 maturationdivision, fixed at up to 8 h after laying (25 out of 97 series, 26 %), show avariety of spindle disorders, including half-spindles, spindles of very large sizecontaining diffuse masses of chromatin, and closely apposed triplets of completespindles, in eggs containing other normal nuclei. In some cases the distortedspindles are peripheral and are presumably derived from the first or secondmaturation divisions, in some cases by further division of the polar bodies. Inothers it appears to be early cleavage nuclei in the central ooplasm which areinvolved.

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 of1773 hatched (71-1 %). The observed frequency of eggs not developing beyond

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378 C. J. FIELDING

maturation or early cleavage could therefore account for the whole of the in-creased mortality found in the mutant series, and in practice dying embryos oflater stages are not encountered.

In the normal embryos at 3 h after laying, the cleavage nuclei had moved outto the circumference of the egg into the cortex. Since the cleavage divisionsinvolved not only a multiplication but a distribution of nuclei in the ooplasm,the whole cortex was nucleated at approximately the same time, although thezygote was formed in the anterior third. As nuclei reached the poles of theegg, they bulged out but remained spherical. Elsewhere they became flattenedagainst the vitelline membrane. At about 3-£ h after laying the first pole cellswere cut off from the clear posterior pole plasm. At the anterior pole the bulgingnuclei were withdrawn.

A few nuclei remained behind in the yolky ooplasm after the formation of theblastoderm, 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 and21 pole cells were cut off. Other nuclei in the polar region pushed out but weredrawn back without severance of the cytoplasmic connexion. At this time theblastoderm has not yet been completed posteriorly, and these nuclei migrateanteriorly in the ooplasm, because in sections of embryos at a slightly laterstage, when the blastoderm wall has been completed, nuclei identical in appear-ance to those of the pole cells, with a prominent nucleolus, appear just anteriorto 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 receivednuclei 3 h after laying. Not until 4 -4 | h do nuclei reach the anterior edge of thepole plasm. Pole cells have not been found in any grandchildless embryo;before the arrival of the first cleavage nuclei the posterior pole plasm isobserved to be crumbling and vacuolated (Plate 1, fig. 3). The extent and size ofthe damaged area is the same as that of the normal pole plasm, defined as thearea of clear cytoplasm found at the posterior pole in the mature egg. By 5 hafter laying, however, the blastoderm is completed posteriorly but the poleplasm has been undercut and is excluded from the embryo.

Certain of the cleavage nuclei during the outwards migration aggregatedanterior to the degenerating pole plasm, and these were seen to remain theretogether as the blastula wall extended back around them (Plate 1, fig. 3). Theprimary vitellophags were present as usual, distributed singly in the ooplasm.The multiplication of nuclei in the blastoderm, and the development of cleavagefurrows between them, proceeded normally in the mutant embryos.

In the normal embryo, 5 h after laying, the blastula wall nuclei are placedperipherally, next to the vitelline membrane. In embryos fixed slightly later itwas seen that at the postero-dorsal surface the nuclei were drawn inwards and asmall depression had appeared over them. Into it passed the pole cells which hadremained together posteriorly, outside the completed blastula wall. In sections

Genetics of mutant grandchildless 379of later embryos it was seen that the depression moved anteriorly and at thesame time curved backwards inside the embryo so that at its maximum exten-sion, at about 7 h after laying, the opening was in the anterior third, and thediverticulum stretched back almost to the posterior blastoderm wall. This organis clearly homologous with the midgut diverticulum of D. melanogaster andother Diptera (Sonnenblick, 1950).

In embryos older than 7 h the pole cells in the diverticulum have becomefewer and by 9 h have disappeared entirely from the lumen.

In the grandchildless embryos, where no pole cells are cut off, the midgutdiverticulum was found to be normally developed. The mass of nuclei which hadaggregated anterior to the degenerated pole plasm became associated with thediverticulum 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 ofthe pole cells. The number of calycocytes in the midguts of normal and grand-childless larvae was counted, using preparations stained with sodium diethyldi-thiocarbamate solution to display copper (Waterhouse, 1945). Normal larvaecontain an average of 69 calycocytes (69 examples, range 57-86); grandchildlesslarvae contain an average of 75 (33 examples, range 57-84). A complete absenceof pole cells is therefore not associated with any reduction in the number ofcalycocytes of the mutant larva.

Oogenesis in homozygous grandchildless females and their normal sistersOogenesis in homozygous grandchildless females shows the following unusual

features. Firstly, in all mutant ovaries there is a high proportion of egg chambersat stage 10 of King et al. (1956), that is, with the oocyte occupying about halfthe volume of the chamber, and the nurse cell chromosomes at their maximumsize. It is at this stage in both normal and mutant oogenesis that the amountof neutral lipid in the egg chamber rapidly increases, particularly in the nursecells, 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 ispresent as a few large globules (Plate 1, fig. 4), rather than as the mass of finedroplets 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 specieshas been reported previously, not as the result of the action of a single gene, asin the present case, but in two other circumstances. In a number of inter-specificcrosses [Drosophila aztecaxD. athebasca (Sturtevant & Dobzhansky, 1936),

380 C. J. FIELDING

D. melanogaster xD. simulans (Bonnier, 1924; Sturtevant, 1929; Kerkis, 1933;Pontecorvo, 1942), D. mirandaxD. pseudoobscura (Dobzhansky, 1938)] thetestes of the male offspring are minute and not functional. In the first two casesthe ovary is also reduced, but in the third the female hybrid offspring have adelayed but superficially normal oogenesis, but of the eggs produced, nonedevelop beyond the maturation division or early cleavage stages and show avariety 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 havebeen irradiated with ultraviolet light (Geigy, 1931; Aboim, 1945; Poulson &Waterhouse, 1960). In adult females from irradiated eggs the ovary is rudi-mentary and contains columnar masses of flattened cells. These cells havebeen shown to develop from the envelope of the larval gonad, which wouldnormally provide the follicle cells of the egg chambers (Geigy, 1931). Germ cellsand 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 mentionedis known only for D. melanogaster x D. simulans, where Pontecorvo (1942) hasshown that certain masses of round cells (not present in the grandchildlessrudiment) represent germ cells which have undergone multiple mitotic divisions.

The close resemblance between the structures of the ovaries of offspring ofirradiated and homozygous mutant females, together with the presence of onlythe small cuboidal cells in the position of the mutant larval gonad in thoselarvae possessing the organ, suggests that the mutant rudimentary ovarycontains no germ cells. The observation that the individuals bearing theseorgans develop from embryos without pole cells gives confirmation to previousresearch showing that the two cell types have the same embryological origin.

In adult males developing from eggs irradiated at the posterior pole the testisis represented by connective tissue and tunic only (Geigy, 1931). In the maleoffspring of homozygous grandchildless females it consists entirely of the pig-mented 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 bodythe gonad rudiments which are found in grandchildless larvae correspond tothose of the normal female, it is likely that those larvae showing no rudiment aregenetically 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 likelythat in the intervening generations a loss of the inner mass of loose tissue hasoccurred, and that it was this component which was present in the male grand-childless larva in the gonad transplantation studies of Suley (1953).

There is no sign in the testis rudiment of irradiated males or those from thegrandchildless stock of a cell type equivalent to the follicle cells of the ovary.

Genetics of mutant grandchildless 381This supports the conclusion reached by Bodenstein (1950) from a study ofthe normal embryology of D. melanogaster.

In D. subobscura, as in Diptera generally, there is a very early separation ofthe germ line from the remainder of the embryo by the formation of pole cellsafter nucleation of the posterior pole plasm. Not all the pole cells contributeto the germ line (Rabinowitz, 1941a; Sonnenblick, 1941; Poulson, 1947). Thesecells migrate by two distinct and alternative routes: by passage between the cellsof the posterior blastoderm wall, and later by migration into the growing hollowof the midgut diverticulum. Counce (1961), who termed these migrations phase Iand phase II respectively, has suggested that the migration is continuous ratherthan diphasic.

Hathaway & Selman (1961) used an ultraviolet microbeam to irradiate theposterior pole of embryos at three early stages of development: prior to theformation of any pole cells, after completion of pole cell formation, and afterphase I migration but before the migration of the remaining pole cells into themidgut diverticulum. The number of pole cells found in the embryonic gonadwas counted, and it was found that the same significant reduction in the numberof gonadal pole cells was found after irradiation of all the pole cells or of theremaining phase II cells only. Thus a clear demonstration was obtained that thepole cells migrating at phase II give rise to the germ line.

Embryos from homozygous grandchildless females show no phase II migra-tion, 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 cannotbe equipotent, because otherwise a deficiency of phase II pole cells caused byultraviolet treatment would presumably be compensated for by a contributionof phase I cells to the gonad. However, these authors' results are not able toprovide information on the equipotency of the pole cells before the phase Imigration. As Rabinowitz (1941 a) has shown, the phase I nuclei fuse with thecytoplasm of the blastoderm, whereas the phase II nuclei retain the cytoplasmwith which they were associated at the time of their formation. Thus a differentia-tion into germ-line and calycocyte-line pole cells at the time of budding from thepole plasm, during the period outside the embryo, or during the phase I migra-tion, 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 anypole cells, but irradiation at both stages would show the same reduction.

This distinction is of considerable importance because of two observationsthat do not fit readily into the simple scheme that phase I gives rise to the caly-cocytes and phase II to the germ line. In grandchildless embryos no nuclei enterthe pole plasm because of its early degeneration, and therefore the aggregationof nuclei anterior to it derive their cytoplasm from the blastoderm directly.However, grandchildless larvae developed from these contain the normal

382 C. J. FIELDING

complement of calycocytes. Since in normal development the calycocytes arederived from pole cells which have been budded off from the main body of theembryo, it is reasonable to conclude that the irreversible differentiation of germline from calycocytes required by the observations of Hathaway & Selmanoccurs after the migration of the phase I nuclei, which fuse with the blastoderm.

Secondly, Counce & Ede (1957) found that in the mutant/snasA there was nophase II migration; nevertheless gonads were formed in some cases. If, as seemslikely from plate 1, fig. C of these authors, the pole cells migrating back throughthe blastoderm do not readily fuse with it, but retain their original cytoplasm,then the determination of the presumptive pole cell nuclei in this mutant iscompatible with that in normal and irradiated embryos, and of grandchildlessembryos. In this case, the determination of the germ line would require theinteraction of presumptive pole cell nuclei and pole plasm at the onset of gastrula-tion, after the migration of the phase I nuclei, which, rejoined with the generalblastoderm, would there complete their differentiation to calycocytes.

SUMMARY

1. The development of the gonad rudiment of the offspring of femaleDrosophila subobscura homozygous for the gene grandchildless, in the embryoand larva, has been investigated.

2. An absence of germ cells in the larval and adult gonads has been correlatedwith the absence of pole cells in the midgut diverticulum of the embryo.

3. Early in the development of the mutant embryo the posterior pole plasmdegenerates. The calycocytes of the larval midgut, which are derived from polecells, are present in full number.

4. In the grandchildless embryo the presumptive pole cell nuclei stop shortof the pole plasm, and aggregate anterior to it. These nuclei are thought toenter subsequently the midgut.

5. Observations on the fate of the presumptive pole cell nuclei in mutantembryos confirm previous research on the origin of the germ-line pole cells. Theimplications of the development of calycocytes in these embryos are discussed.

RESUME

Genetique du developpement du mutant 'grandchildless' de Drosophilapseudoobscura

1. Le developpement de l'ebauche gonadique de la progeniture de femelles deDrosophila subobscura homozygotes pour le gene 'grandchildless' ('absence depetits-enfants') a ete etudie chez Pembryon et la larve.

2. L'absence de gonocytes dans les gonades larvaires et adultes est correlativede l'absence de cellules polaires dans le diverticule de l'intestin moyen del'embryon.

Genetics of mutant grandchildless 3833. Au debut du developpement de l'embryon mutant, le plasme polaire

posterieur degenere. Les calyocytes de l'intestin moyen de la larve, qui deriventdes cellules polaires, sont presents en totalite.

4. Chez l'embryon 'grandchildless', les noyaux presomptifs des cellulespolaires s'arretent en avant du plasme polaire et s'agregent anterieurement alui. On pense que ces noyaux entrent par la suite dans l'intestin anterieur.

5. Des observations sur le sort des noyaux presomptifs des cellules polaireschez les embryons mutants confirment les recherches precedentes sur l'originedes cellules polaires de la lignee germinale. On discute les implications dudeveloppement des calyocytes chez ces embryons.

This research is part of that presented to the University of London for the award of aDoctorate of Philosophy, and was supported by a Postgraduate Studentship of the Agricul-tural Research Council. I am indebted to Professor John Maynard Smith for his supervisionof this investigation.

REFERENCES

ABOIM, A. N. (1945). Developpement embryonnaire et post-embryonnaire des gonadesnormales et agametiques de Drosophila melanogaster. Revue suisse Zool. 52, 53-154.

BAKER, J. R. (1944). The structure and chemical composition of the Golgi element. Q. Jl.microsc. Sci. 85, 1-71.

BODENSTEIN, D. (1950). The postembryonic development of Drosophila. In Biology ofDrosophila, ed. M. Demerec. New York: Wiley and Sons.

BONNIER, G. (1924). Contributions to the knowledge of intra- and inter-specific relationshipsin Drosophila. Ada. zool., Stockh. 5, 1-122.

COUNCE, S. J. (1961). The analysis of insect embryogenesis. Ann. Rev. Entomol. 6, 295-312.COUNCE, S. J. & EDE, D. A. (1957). The effect in embryogenesis of a sex-linked female-

sterility factor in Drosophila melanogaster. J. Embryol. exp. Morph. 5, 404—21.DOBZHANSKY, T. (1937). Further data on Drosophila miranda and its hybrids with Drosophila

pseudoobscura. J. Genet. 34, 134-52.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-linkedlethal stocks, and an analysis of the mutant Lffll. Wilhelm Roux Arch. EntwMech. 148,416-36.

EDE, D. A. (19566). Ibid. II. An analysis of the mutant X2. Wilhelm Roux Arch. EntwMech.148,437-51.

EDE, D. A. (1956C). Ibid. III. An analysis of the mutant X27. Wilhelm Roux Arch. Entw.-Mech. 149, 88-100.

EDE, D. A. (1956d). Ibid. V. An analysis of the mutant X10. Wilhelm Roux Arch. Entw.-Mech. 149, 247-58.

GEIGY, R. (1931). Action de l'ultra-violet sur le pole germinal dans l'oeuf de Drosophilamelanogaster. Revue suisse Zool. 38, 187-288.

HATHAWAY, D. S. & SELMAN, G. G. (1961). Certain aspects of cell lineage and morpho-genesis studied in Drosophila melanogaster with an ultraviolet microbeam. J. Embryol. exp.Morph. 9, 310-25.

HuETTNER, A. F. (1923). The origin of the germ cells in Drosophila melanogaster. J. Morph.37, 385-423.

KAUFFMAN, B. P. (1940). The nature of hybrid sterility—abnormal development in eggs ofhybrids between Drosophila miranda and Drosophila pseudoobscura. J. Morph. 66,197-213.

KERKIS, J. (1933). Development of gonads in hybrids between Drosophila melanogaster andDrosophila simulans. J. exp. Zool. 66, 477-509.

384 C. J. FIELDING

KING, R. C, RUBINSON, A. C. & SMITH, R. F. (1956). Oogenesis in adult Drosophila melano-gaster. Growth 20, 121-57.

LILLIE, R. D. (1944). Various oil soluble dyes as fat stains in the supersaturated isopropanoltechnic. Stain Technol. 19, 55-8.

PARKS, H. B. (1936). Cleavage patterns in Drosophila and mosaic formation. Ann. ent. Soc.Am. 29, 350-92.

PONTECORVO, G. (1942). Hybrid sterility in artificially produced recombinants betweenDrosophila melanogaster and Drosophila simulans. Proc. R. Soc. Edinb. 61, 385-97.

POULSON, D. F. (1947). The pole cells of Diptera, their fate and significance. Proc. natn. Acad.Sci. U.S.A. 6, 182-4.

POULSON, D. F. (1950). Histogenesis, organogenesis, and differentiation in the embryo ofDrosophila melanogaster (Meigen). In Biology of Drosophila, ed. M. Demerec. New York:Wiley and Sons.

POULSON, D. F. & WATERHOUSE, D. F. (1960). Experimental studies on pole cells and midgutdifferentiation in Diptera. Aust. J. biol. Sci. 13, 541-66.

RABINOWTTZ, M. (1941a). Studies on the cytology and early embryology of the egg ofDrosophila melanogaster. J. Morph. 69, 1-49.

RABINOWTTZ, M. (19416). Yolk nuclei in the egg of Drosophila melanogaster. Anat. Rec. 81(Suppl. 2), 80-1.

SMITH, S. G. (1940). A new embedding schedule for insect cytology. Stain Technol. 15, 175-6.SONNENBLICK, B. P. (1941). Germ cell movements and sex differentiation of the gonads in

the Drosophila embryo. Proc. natn. Acad. Sci. U.S.A. 27, 484-9.SONNENBLICK, B. P. (1950). The early embryology of Drosophila melanogaster. In Biology of

Drosophila, ed. M. Demerec. New York: Wiley and Sons.SPURWAY, H. (1948). Genetics and cytology of Drosophila subobscura. IV. An extreme

example of delay in gene action, causing sterility. J. Genet. 49, 126-40.STERN, C. & HADORN, E. (1939). The relation between the colour of testis and vas deferens in

Drosophila. Genetics, Princeton 24, 162-9.STURTEVANT, A. H. (1929). The genetics of Drosophila. Publs Carnegie Instn 399, 1-62.STURTEVANT, A. H. & DOBZHANSKY, T. (1936). Observations on the species related to Droso-

phila affinis, with descriptions of seven new forms. Am. Nat. 70, 574-84.SULEY, A. C. E. (1953). Genetics of Drosophila subobscura. VIII. Studies on the mutant

grandchildless. J. Genet. 51, 375-405.WATERHOUSE, D. F. (1945). Studies on the physiology and toxicology of blowflies. 10. A

histochemical examination of the distribution of copper in Lucilia cuprina. Bull. Coun.scient. ind. Res., Melb. 191, 5-20.

{Manuscript received 18 October 1966)