/ . Embryol. exp. Morph., Vol. 17, 2, pp. 375-384, April 1967
With 1 plate
Printed in Great Britain
375
Developmental genetics of the mutant
grandchildless 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 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 fsna3A (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 grand1
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 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 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 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.
/. 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
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
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
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),
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.
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
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 n a s A 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.
Genetics of mutant grandchildless
383
3. 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.
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