/. Embryol. exp. Morph., Vol. 17, 2, pp. 405^t23, April 1967
With 2 plates
Printed in Great Britain
405
Studies on an embryonic lethal hybrid in
Drosophila
By JANICE D. KINSEY 1
From the Department of Zoology, The University of Texas, Austin
The problems of nucleocytoplasmic interactions are of central importance in
development and genetics. Studies of such interactions may help elucidate the
mechanisms of differentiation of cells receiving the same genetic complement.
One approach to a study of nucleocytoplasmic interactions is by examination of
the development of interspecific hybrids, particularly those in which either
syngamy fails to occur, or an abnormal development is produced.
These studies describe a lethal hybrid which occurs between Drosophila
montana and D. texana. In 1944, Patterson & Griffen described a genetic mechanism which acts in the hybrid females produced by crossing D. montana
females to D. texana males. In this cross, only male offspring were produced,
so that there appeared to be some incompatibility between the D. montana
ooplasm and the D. texana X-chromosome which acted to kill the female hybrid
embryos before hatching. Further studies by Patterson & Griffen indicated that
the effect of the D. texana X-chromosome was restricted to a small region, and
that the D. montana phase of the incompatibility was manifested only in eggs
produced by flies carrying the entire D. montana chromosomal complement.
The studies reported here were an attempt to determine the nature and
embryological effects of this abnormal nucleocytoplasmic interaction between
D. montana egg cytoplasm and the D. texana X-chromosome. Concurrent
studies were made of the hybrids produced by the reciprocal cross, of D. texana
9 x D. montana $.
Since the species under study are very effectively sexually isolated due to
behavioural differences and to the poor viability of D. texana sperm in the ducts
of the D. montana females, the cross under study is very infrequent; when it
does occur, only a few fertilized eggs are laid (Patterson & Griffen, 1944 a, b).
Therefore, in order to obtain enough hybrid embryos for study, it was necessary
to resort to the subterfuge of interspecific ovary transplantation, which constituted a secondary study (Kinsey, 1966).
1
Author's address: Department of Genetics, The University of Washington, Seattle,
Washington 98105, U.S.A.
406
J. D. KINSEY
MATERIALS AND METHODS
D. montana strain 1218.8d and D. texana strain 1148.9 were obtained from
the stock collection of the Genetics Foundation of the University of Texas,
where they had been maintained as standard stocks since their collection in 1941.
The stocks were reared in •§• pint milk bottles on banana-agar medium to which
a suspension of live yeast had been added. Both the stocks and the embryos
were kept at 22-23 °C.
For egg collections of normal controls of both species flies were allowed to
lay eggs on smears of banana-yeast food on strips of filter paper which were
inserted into shell vials containing 30-50 pairs of 10 to 12-day-old flies. Collections were made at 30 min intervals, so that the ages of the eggs were known
±15 min. Eggs were fixed by immersion in F.A.A. fixative (commercial formalin:
absolute alcohol: glacial acetic acid (6:16:1), plus 30 parts distilled water)
followed by puncturing of the egg membranes with fine glass needles in order to
allow the fixative to penetrate. Unlike D. melanogaster and some other species,
D. montana and D. texana do not retain fertilized eggs within the uterus, so the
egg can be considered to be just fertilized at hour 0, when it is deposited.
The fixed embryos were dehydrated through an ethanol: normal butanol
series, infiltrated and embedded in paraffin, sectioned at 4 /*, and stained with
Heidenhain's iron hematoxylin with no counterstain.
Living embryos, both normal and hybrid, were watched and photographed
during their development by means of still photographs and time-lapse cinematography. Embryos to be observed were dechorionated with fine watch-maker
forceps and hair loops; following dechorionation the embryos were put into
hanging drops of 0-5 M glycerol in deep-well depression slides, and the coverslips were sealed to the slides with petroleum jelly (Rabinowitz, 1941). When
embryos treated in this way are examined with transmitted light, the developmental processes may be seen through the transparent vitelline membrane.
Time-lapse movies of embryos treated thus were made with a Cine-Kodak
16 mm movie camera with Kodak Plus-X Reversal film. A time-lapse drive unit
allowed control of the interval between exposures. Development as shown in the
films was reviewed with an Industrialist Model SFDR modified Analyst Kodak
projector.
In order to overcome sexual isolation of the species, reciprocal interspecific
ovary transplantations were made (Kinsey, 1966). Host females of one species
carrying functional ovaries of the other species were mated to males of the same
species as the host females, so that the eggs produced by the transplanted ovaries
were fertilized to produce hybrid embryos, and eggs produced by the hosts'
own ovaries produced normal embryos.
As a control to check for the possible effect on the embryos of the development of an ovary within a female of another species, reciprocal interspecific
transplants were made, and two normal D. montana ? x D . texana $ crosses
407
Embryonic lethal hybrid
y.
oo
m.g.
n.s.
0-2 mm
Text-fig. 1. Normal embryonic development of Drosophila.
A, lsth; fertilization; fusing pronuclei in cytoplasmic island, with polar body
nuclei in peripheral cytoplasmic layer. Micropylar extension of vitelline membrane
(anterior) to right, flat side of egg dorsal, y = yolk mass.
B, 2nd and 3rdh; cleavage; syncytium of nuclei, each surrounded by cytoplasmic island. Some nuclei have entered posterior pole plasm (left).
C, 4th h; syncytial blastema, early; nuclei arranged in peripheral layer, except for
some nuclei (vitellophags) which remain in yolk mass. p.c. = pole cells.
D, 5th h; syncytial blastema, late. Nuclei elongating; nucleoli appear.
E, Gastrulation; after cell furrows separate nuclei into cells, mesoderm invaginates ventrally, while proctodeal and posterior mid-gut rudiments invaginate, with
pole cells sinking into depression thus formed. Germ band extension begins.
F, Germ band extension, m. = mesoderm; m.g. = mid-gut rudiments (endoderm).
G, Germ-band extension. Proctodeal invagination lengthens, carrying pole cells
posteriorly.
H, Germ-band extension. Stomodeal rudiment begins invaginating (s.).
I, Maximum germ-band extension. Segmentation has begun; Malpighian tubules
evaginate from proctodeum. Anterior and posterior mid-gut rudiments meet
ventrally.
J, Shortening of germ band and dorsal closure. Mid-gut closes dorsally, and mesoderm grows up laterally and closes body dorsally, along with ectoderm. Pole cells
migrate through mid-gut, and eventually settle in forming fat body (mesoderm).
Mesoderm forms visceral and body wall musculature, heart. Neuroblasts («.) form.
K, Fairly complete larva. Tracheae (tr.) invaginate, neuroblasts condense to form
brain (b.) and nervous system (n.s.). Hind-gut coils; salivaries invaginate from
stomodeum.
26
JEEM 17
408
J. D. KINSEY
were detected soon enough after mating for fertilized eggs to be obtained before
the sperm were killed by the duct secretions of the females. The embryos produced by these two normal hybridizations were dechorionated, examined in a
hanging drop, and were then fixed and sectioned. For the reciprocal control
cross, D. texana $ x D . montana <$, three crosses were obtained. The resulting
embryos were treated as described above.
OBSERVATIONS AND RESULTS
Time-lapse films were made of the development of one D. texana and four
D. montananormal embryos, beginning with freshly laid eggs. For about the
first hour following oviposition, the sperm may be seen vibrating within the
ooplasm, so that it is possible to determine whether an egg has been fertilized
by examining it in a hanging drop. Sections of normal embryos were made of
both species. For each, samples consisting of 5-7 embryos (ages known ±15
min) were fixed hourly from 0 (freshly laid) to 25 h.
The development of both D. montana and D. texana is nearly identical to that
of D. melanogaster, which has been described in detail elsewhere (Poulson, 1937,
1950; Sonnenblick, 1950). Both D. montana and D. texana take slightly longer
to develop than D. melanogaster, requiring 26-30 h for hatching, compared with
22-24 h for melanogaster. A diagrammatic representation of the early embryonic
development is shown in Text-fig. 1, based on observations on living embryos,
time-lapse cinematography, and sectioned material. Plate 1, figs. A-D, shows
photographs of whole normal embryos at early stages.
Two series of transplantations of larval ovaries were made: series M h T d
(montana host, texana donor) and series T h M d (texana host, montana donor).
The establishment of functional oviduct connexions could be determined by an
examination of the eggs laid by the operated flies; the eggs of D. montana are
slightly larger than those of D. texana, and the D. montana chorionic filaments
are considerably longer and thicker than those of D. texana. These filamental
differences are maintained in the eggs from transplanted ovaries, so that the
hybrid embryos were readily distinguishable.
Drosophila texana ? x D . montana <$'• normal cross
In order to determine whether the host had any effect on the eggs produced
by an implanted ovary of another species, it was necessary to obtain hybrids
from normal interspecific crosses. In the first test, 3 normal hybridizations of
D. texana $ x D. montana $ were obtained, giving a total of 11 viable male and
14 female flies (see Tables 1 and 2 for a summary of results). In addition, seven
eggs were found which contained larvae showing a variety of abnormalities,
no two abnormals being the same. It is probable that some eggs of this type were
overlooked, since in order to find them a very large number of unfertilized eggs
also had to be examined, and some eggs may have escaped detection in the food.
Embryonic lethal hybrid
409
The rather low number of fertilized eggs produced is probably explained by the
sperm mortality, as fertilized eggs were laid only during the first day or so after
insemination.
Drosophila texana ? x D . montana <J: hybrids from ovary implants
In the M h T d series (Table 1), 3 functional transplanted ovaries produced a
total of 30 fertilized eggs, which produced a total of 21 viable hybrids (12 males
and 9 females). There were also 9 embryos produced that showed larval development, but having various abnormalities. The hybrids that hatched were indistinguishable from those produced by the normal interspecific crosses. In both, the
larvae, pupae, and imagos closely resembled D. texana, the female parent. The
abnormal larvae in both groups showed few similarities in their abnormalities,
but all approximated being larvae, as opposed to masses of undifferentiated cells.
Many showed active muscular movements.
Table 1. Drosophila texana $ x D. montana <$ hybrids
Experimental series
Crosses
Viable
$
Non-transplant series
Transplant series
3
3
14
12
Viable
Abnormal
<$ Abnormals Fertile (%)
11
9
7
9
22
30
32
30
Table 2. Drosophila montana ? x D . texana ( 2 hybrids
Experimental series
Non-transplant series
Transplant series
PreCrosses blastema
2
10
0
34
Undetermined
sex
1
9
?
$
Fertilized Hatched
9
71
11
81
21
203
2
8
Drosophila montana ? x D . texana $: normal crosses
This is the cross in which the lethal interaction of the D. texana X-chromosome with the D. montana ooplasm kills all the female hybrids prior to hatching.
Patterson & Griffen (19446) reported that the cross is very seldom effected,
giving only 1 to 3 offspring (all male) when it does occur. They attributed this
paucity of male offspring to gamete mortality, but these studies indicate that
poor hybrid viability may also be a cause.
In these experiments, two crosses of D. montana females to D. texana males
were obtained, each of which produced only one viable male offspring. As soon
as the hatching of an egg was detected in a vial, the rest of the eggs in the vial
were dechorionated, put into hanging drops, and examined. One vial yielded 8
and the other 11 eggs which showed some development, besides the one hatched
larva in each vial. As seen in Table 2, the eggs showed two types of development
26-2
410
J. D. KINSEY
in about equal numbers; the first type of embryo was a mass of undifferentiated
cells, while the second type showed varying degrees of larval development. The
gross appearances and sections of these embryos corresponded to those of the
hybrids obtained from transplanted ovaries.
Drosophila montana $ x D . texana S: hybrids from ovary implants
The hybrid embryos from transplanted ovaries were denuded, put into
hanging drops and examined, and then fixed and sectioned. As shown in Table 2,
a total of 289 eggs were produced by transplanted ovaries, of which 203 were
found to be fertilized. Time-lapse movies were made of the development of
twenty of the hybrid embryos.
The embryos produced by the transplants showed two major types of development, corresponding to those shown in the normal hybridizations.
Male hybrids. In approximately half the embryos, development proceeded
normally until formation of the cellular blastoderm was complete (Plate 1,
fig. E), after which a wide spectrum of abnormalities could occur to produce
anything from a highly abnormal larva whose only recognizable structures were
ectodermal tubular structures, to a normal larva which hatched and continued
normal development to the imago. All the embryos that hatched were males,
and in the abnormal embryos that were allowed to develop to the hatching stage,
PLATE 1
In these and all subsequent figures, the flat (dorsal) side of the egg is toward the top of the
page. The anterior end is characterized by the micropylar projection of the vitelline membrane.
Fig. A. Normal Drosophila montana, early blastema stage. Nuclei visible at periphery.
Fig. B. Normal D. montana, late syncytial blastema stage. Beginning of nuclear elongation,
with pole cells formed at posterior end.
Fig. C. Normal D. montana, cellular blastoderm.
Fig. D. Normal D. texana. Cephalic furrow, beginning of proctodeal invagination and germ
band extension; gastrulation.
Fig. E. Normal (D. montana ? x D . texana <?) male hybrid, cellular blastoderm. Later hatched
normally.
Fig. F. Abnormal (D, montana ? x i ) . texana <?) male hybrid, defective blastoderm with
beginning of proctodeal invagination. Later formed very abnormal embryo, with some
differentiated ectodermal structures.
Fig. G. Abnormal (D. montana ? x D. texana <?) male hybrid; nearly 24 h old. Formed from
an irregular blastoderm similar to that in Fig. F.
Fig. H. Abnormal (D. montana ? x D . texana <?) male hybrid, similar to that in Fig. G.
Only recognizable structure is hind gut. Sectioned at 4 fi, stained with Heidenhain's hematoxylin.
Fig. I. Abnormal (D. montana $ x D. texana <?) male hybrid, 28 h old. Resulted from failure
of dorsal closure and head involution.
Fig. J. Abnormal (D. montana ? x D . texana <J) male hybrid. Normal posteriorly, but with
abnormal head due to failure of head involution.
J. Embryo/, exp. Morph., Vol. 17, Part 2
PLATE 1
H
0-1 mm
J. D. K1NSEY
facing p. 410
/ . Embryo/, exp. Morph., Vol. 17, Part 2
PLATE 2
M
0-1 mm
K-S
J. O. KINSEY
005 mm
T
facing p. 4J1
Embryonic lethal hybrid
411
sections showed the gonads to be about the same size as the group of larger
gonads in the controls, which are testes (Sonnenblick, 1950). From this it was
decided that the group of embryos showing any larval-type development,
abnormal or normal, were males.
From Table 2 it is seen that about one in four or five males hatched, which
indicates that the low number of offspring reported by Patterson & Griffen is
due not only to sperm mortality, but also to the embryonic death of many of the
hybrids formed. The normal embryos that did hatch in the transplant series were
indistinguishable from those produced by the normal hybridization crosses;
both groups strongly resembled the female parent, D. montana, in larval, pupal,
and imaginal stages.
The male hybrids that developed abnormally showed a wide range of defects,
about which some generalizations can be made.
(1) In embryos that became abnormal just after blastoderm formation or
germ band extension (Plate 1, fig. F) and that were allowed to develop to a stage
corresponding to hatching stage in a normal, the resulting 'larva' consisted
chiefly of gut tubules and hypodermis (Plate 1, figs. G, H). Such structures as
heart, salivary glands, fat body and, usually, discrete nervous tissue, were
undifferentiated. Generally, in embryos of this type, the abnormality was first
PLATE 2
Fig. K. (£>. montana ? x D . texana (?) male hybrid. Nearly normal larva, except for abnormal
chitinous mass in place of mouth hooks. Unable to hatch, though active hatching movements
occurred.
Fig. L. Abnormal (D. montana $ x D. texana <?) male hybrid. Nearly complete larva posteriorly;
yolk extruded anteriorly, due to a weakness in the head region. Active muscular movements.
Fig. M. Section of embryo shown in Fig. L. Nervous system, gut, musculature well developed.
Sectioned at 4 ft, stained with Heidenhain's hematoxylin.
Fig. N. Abnormal (£>. montana ? x D . texana <?) female hybrid; normal-appearing blastema,
between two large abnormal yolk contractions.
Fig. O. Same embryo as in Fig. N, 20 min later. Yolk system has contracted toward posterior,
moving most of periplasm and nuclei to anterior.
Fig. P. Same embryo as in Fig. N, 1 h 20 min later. Yolk contractions stopped; some of
nuclei are elongating.
Fig. Q. Abnormal (D. montana $ x D. texana (?) female hybrid. About 10 h old. Fairly large
mass of yolk, with proliferated, undifferentiated cells.
Fig. R. Abnormal (D. montana ?xZ). texana (?) female hybrid. About 20 h old. Yolk nearly
gone.
Fig. S. Abnormal {£>. montana $ x D. texana (?) female hybrid. Section of embryo showing
mass of cells of varying sizes, some with pycnotic regions. Darkly staining granules are yolk
spheres. Cut at 4 /*, stained with Heidenhain's hematoxylin.
Fig. T. Abnormal (D. montana $ x D. texana (?) female hybrid. Fixed about 1 h after contractions ceased. Nuclear abnormalities visible. Large polyploid nucleus (p.), some nuclei with
pycnotic regions (pyc), various sizes of nuclei. Sectioned at 4 ft, stained with Heidenhain's
hematoxylin.
412
J. D. KINSEY
seen as an irregularity of the blastoderm, resulting in abnormal germ band
extension and/or gastrulation.
(2) When the shortening of the germ band was reduced or absent, a very
abnormal embryo resulted (Plate 1, fig. I). Generally, when there was a disruption of this shortening, there were concurrent disturbances of dorsal closure
and head involution, although either of the latter could become abnormal
without a previous failure of shortening. When all three occurred, nearly every
organ became morphologically abnormal, although cytologically differentiated.
(3) In some of the embryos the only abnormality was in the head structure.
Often head involution was abnormal, either grossly or slightly, which resulted
in an otherwise apparently normal larva whose head was so abnormal that it was
unable to hatch (Plate 1, fig. J), or was unable to feed if mechanically freed from
the vitelline membrane. Sometimes head involution appeared normal, but the
larva had an abnormality in the mouth hooks (Plate 2, fig. K), rendering it
unable to tear its way out of the membrane and hatch.
(4) Rather frequently, embryos were found which consisted of a fairly normal
larva posteriorly, with the entire yolk mass extruded anteriorly (Plate 2, figs. L,
M). This was observed sometimes to result from a failure of dorsal closure of
the embryo. The resulting movement of the yolk to the anterior appeared to
interfere with the development of the head, and the gut was also abnormal.
Active muscular contractions resembling normal hatching movements occurred,
though the embryos showed varying degrees of abnormality. Sometimes a
weakness somewhere in the head region resulted in a herniation of the yolk,
which produced a larva resembling those in which dorsal closure failed to occur
properly.
Since no two embryos developed exactly the same pattern of abnormalities,
these categories of larvae are made in an attempt to show the beginnings of
the various departures from the normal developmental scheme. Subsequent
development resulted in a whole spectrum of abnormalities which, when traced
from their origins, were generally found to result from one or a combination of
the developmental errors indicated.
Female hybrids. The second major group of embryos comprised about half
the hybrids studied, and is assumed to consist of the females, which are the
embryos in which the lethal action of the D. texana Z-chromosome in the D.
montana ooplasm is manifested. The abnormalities in these embryos were more
homogeneous than in the males.
In normal controls of both D. montana and D. texana embryos, time-lapse
cinematography showed slight contractions of the yolk system beginning in the
early syncytial blastema, at the time when the pole plasm becomes lobulated in
preparation for pole cell formation. Text-fig. 2 illustrates these contractions
which were measured by means of tracings of the projected movie images. The
distances as shown in the drawings are proportional, so that measurements need
not be given. The yolk contractions in the controls were longitudinal, and slightly
Embryonic lethal hybrid
413
more pronounced in the anterior end. The opaque central yolk mass appeared
to be drawing together toward a point located two-fifths of the distance from
the posterior pole. As shown in Text-fig. 2, there were corresponding obvious
movements of the periplasm and the nuclei which it contained; these movements
were directed toward the poles, going in opposite directions from the focal point
of the yolk-mass contraction. No changes in total egg volume were apparent
-40%-
I 5 mir
c. 45 min
0-2 mm
t 10 min
Text-fig. 2. Yolk system contractions in normal Drosophila montana. Stippled area
indicates yolk mass; clear area represents peripheral cytoplasm containing blastema
nuclei. Arrows indicate directions of movement. Times given represent intervals
between the stages pictured. All figures begin with early syncytial blastema, at time
of first appearance of lobulated pole plasm.
-40%-^
8 min
12 min
84 min
15 min
5 min
0-2 mm
Text-fig. 3. Yolk-system contractions in ? hybrid of Drosophila montana ?
x D. Texana $.
414
J. D. KINSEY
during these contractions. The expansion of the yolk appeared in the film as a
relaxation, with the periplasm and nuclei simultaneously flowing back to the
positions they had occupied prior to the contractions. Individual nuclei can be
followed in the films; it appears that the nuclei are passively carried along by
the moving peripheral cytoplasm. Also, it appears that the periplasm is quite
fluid, and that its movements are also passive ones caused by changes in the
more internal yolk system. The movements of the periplasm and the adjacent
yolk mass were always in opposite directions.
In the lethal female hybrids, there were contractions of the yolk system beginning at about the same time as those in normal embryos, but they were of a
noticeably different nature. Time-lapse films were used in diagramming these
contractions in four of the female hybrid embryos, a representative one of which
is shown in Text-fig. 3. Whereas the contractions in normal embryos were fairly
consistent with respect to time of onset, duration, extent, and pattern, those of
the abnormals varied in all these respects. In one of the abnormals, there were
two contractions quite similar to those seen in the normal controls, but immediately thereafter abnormal contractions began. These abnormal contractions
were not identical in any two of the embryos firmed, but had similarities.
Except in the aforementioned case, in which two normal-appearing contractions
preceded the onset of abnormality, the contractions either were not directed
at all toward a point in the interior of the egg's yolk system, or else were
directed toward an area in the anterior half of the egg instead of the posterior.
Even in the cases in which the early contraction or contractions were internally
directed, subsequent contractions resulted in unidirectional movement of the
yolk mass toward one pole, with a corresponding movement of the periplasm
and nuclei toward the other.
The massive movements of the yolk system resulted in the abnormal distribution of the cortical cytoplasm and its nuclei (Plate 2, figs. N-P). After the
contractions ceased, many of the nuclei elongated as do normal blastoderm
nuclei, and most of the nuclei were separated into cells by the formation of cell
furrows. There followed a proliferation, which continued until all the yolk had
been utilized and all the space in the egg was occupied; various nuclear abnormalities developed in many of the embryos (Plate 2, figs. Q-T).
Thus, though the female embryos showed some variation in the pattern of
their abnormal yolk contractions, all were highly abnormal and resulted in an
* embryo' which was essentially a mass of varying amounts of yolk, undifferentiated cells, some free nuclei, and varying cytological abnormalities.
Sections of these embryos revealed normal development to the blastema stage;
in fact, these embryos were indistinguishable from the males prior to the onset
of the abnormal contractions. During the contractions no abnormalities were
visible in the nuclei. During the formation of cells and their proliferation cytological abnormalities sometimes occurred. Occasionally a cell was formed which
contained more than one nucleus. Some nuclei developed what appeared to be
Embryonic
lethal hybrid
415
polyploidy, and some contained pycnotic masses. There was no recognizable
differentiation found in any of the sections of this group of embryos.
DISCUSSION
Transplantation
The technique for transplantation of larval organs developed by Ephrussi &
Beadle (1935) has been used for testing the autonomy or heteronomy of various
organs of many different mutants in Drosophila. Some mutant effects have been
shown to be autonomous, with the organ developing as it would in situ even
after its transplantation to a normal host. Other mutant organs have been shown
to develop the normal phenotype when transplanted to normal hosts, so that
their environment in the organism controls them to some extent. There are also
cases in which an affected mutant organ develops better in a normal host than
it would in the mutant, but still is not completely normal (Hadorn, 1961).
Telfer (1965) cites several instances in many insects in which the blood proteins
have been shown to contribute to the contents of the eggs, specifically to the
yolk spheres.
In the reciprocal transplantations of ovaries between D. montana and D.
texana, it was found that the implanted ovaries developed autonomously both
with respect to morphology and to the development of the eggs which they
produced. In the case of the transplantation of D. montana ovaries to D. texana
hosts, and the fertilization of the montana eggs with the texana Z-bearing sperm,
it would seem that if the transplantation were to have any effect on the development of the hybrids thus produced, it would be to make the ooplasm of the
montana eggs more like that of texana. This should lessen the deleterious effect
of the lethal factor. Since this was not found to be so, it is concluded that the
D. montana phase of the incompatibility is restricted to the ovary itself, and is
free from any influences of the internal milieu of the animal.
This is believed to be the first case in which interspecific ovary transplantation
was used to obtain hybrids of sexually isolated species. This technique may be
useful in overcoming isolation in other species, and could be an important tool
in cytogenetic work in which hitherto unobtainable hybrids important in evolutionary studies may be produced.
Drosophila texana $ x D. montana S\ male and female hybrids
All abnormal embryos found showed larval-type development; they were all
different. It seems probable that they were caused by a disturbance of the genie
balance involved in the major morphogenetic events, probably due to the combinations of two unlike genetic complements. This will be discussed further in
the following section.
416
J. D. KINSEY
Drosophila montana ? x D . texana <J: male hybrids
Since the range of anomalies shown in this group was so large, it was impossible to do any detailed study on any one type of abnormality. Therefore
the discussion of the development of the male hybrids must be of a general
nature.
As shown in Table 2, about one-tenth of the male embryos hatched. All ten
of those that hatched completed development to the imago. This suggests that
the viability of the male hybrids was good once they attained normal larval form.
The other male hybrid embryos became abnormal at various stages of their
ontogeny. Generally, the earlier the point of departure from normal development, the more extensive the abnormalities. This is what one would expect in view
of the progressive nature of development.
In general, the abnormalities first appeared during periods of major embryonic
rearrangements. The embryos then became progressively more abnormal in
various ways.
In embryos which formed a defective blastoderm, or in which gastrulation
and/or germ band extension were abnormal, about the only distinguishable
embryonic features were some ectodermal structures. This is not surprising, as
the ectoderm of insect embryos is believed to be a self-differentiating system
(Bodenstein, 1956). Usually, no differentiated mesodermal structures were
found in these embryos. This can be explained by the failure of the ectoderm
to undergo the proper movements in relation to the underlying mesoderm which
it induces. Also, gastrulation was frequently disturbed, so that the mesoderm
may never have become a discrete layer. The sex-linked mutant X-2 (Ede, 1951a)
also shows this type of development. Generally, a weakness in the blastoderm
caused the enclosed yolk to herniate, which in itself was sufficient to cause
abnormal germ-band extension. Formation of an abnormal blastoderm generally resulted in disruption of all subsequent morphogenesis, although gastrulation and/or germ band extension could become abnormal without necessarily
being preceded by an abnormal blastoderm. Poulson (1940) described the effects
of loss of half of the Z-chromosome; in this case, an abnormal blastoderm
formed and subsequent development was arrested. In this hybrid, however, it
was quite common to find extreme spatial disorganization of the organs, but
good tissue differentiation. The failure of much of the tissue differentiation in
some of the abnormal male hybrids probably resulted from an early disruption
of spatial relationships within the embryo, so that the normal inter-tissue and
inter-organ reactions could not occur.
The type of embryo in which a fair larva formed with the yolk mass outside
could result from failure of dorsal closure to occur, or from an extrusion of the
yolk through a weak spot anywhere in the embryo. This usually interfered with
development in the area where the yolk mass came to lie, but otherwise differentiation proceeded fairly normally. Minor defects in this type of embryo prob-
Embryonic lethal hybrid
All
ably arose from the altering of normal spatial arrangement of internal organs as
a result of the lack of a mass of yolk within the mid-gut.
Many embryos became abnormal at the time when three major morphogenetic
events were occurring concurrently—shortening of the germ band, dorsal closure
of the mid-gut and body wall, and involution of the head. These processes could
become abnormal singly or in any combination.
The formation of the male hybrids between D. montana females and D. texana
males might be called, in the terminology of Hadorn (1961), a polyphasic lethal
factor showing incomplete penetrance. In inviable embryos, the first discernible
abnormality nearly always appeared during one of the periods of major embryonic rearrangement. These periods have been called 'epigenetic crises' (Waddington, 1956), in which there is a major morphogenetic process which is controlled
by a number of normally delicately balanced genetically controlled processes.
In a hybrid, the combining of the rather dissimilar chromosomal complements
of different species might cause an upset of this genie balance at various stages
in development.
The polyphasic or aphasic onset of abnormal development is not unique to
this system. Ede (19576, c), in a study of induced sex-linked lethals in D. melanogaster, found that the mutants S-9 and X-10 showed several types of action
of the lethal. It is possible that many lethal hybrids show a range of developmental abnormalities.
Drosophila montana $ x D. texana <$: female hybrids
The failure of the female embryos to survive in the D. montana $ x D. texana $
cross has been genetically analysed by Patterson & Griffen. They found that
hybrid males from crosses of texana $ $ to <£ $ of other species of the virilis
group would, when crossed to montana 9 $, give only male offspring. Since the
hybrid males thus tested carried the texana X-chromosome, the factor involved
is entirely sex-linked, and is independent of the rest of the parental genome.
By means of cross-overs, stocks were produced with several Z-chromosome
cross-over types accompanied by D. virilis autosomes. By crossing these to
D. montana females, Patterson & Griffen found that only male offspring were
produced when the Z-chromosome of the male parent carried the ec-si2 interval,
or possibly the shorter ec-cv interval, whereas all other recombinant JT-chromosomes produced offspring of both sexes.
Patterson & Griffen also found that when montana was crossed reciprocally
to other members of the virilis group, the resulting hybrid females would, when
crossed to texana, give offspring of both sexes in equal numbers. This indicates
that the full D. montana genetic complement must be present in order that the
egg cytoplasm which is formed may show the lethal incompatibility with the
texana Z-chromosomal segment. The fact that the reciprocal cross {texana $ x
montana $) produced viable hybrids in equal numbers showed that the lethality
was not simply an inability of the Z-chromosomes of the two species to interact
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J. D. KINSEY
properly. The results of the aforementioned crosses of {montana $ x virilis $)
$ x texana $ also support this observation.
The data from the ovary transplantations indicate that the formation of the
D. montana phase of the incompatibility is not affected by transplantation of the
montana ovary to a texana host. Thus, the montana ovary is autonomous with
respect to this phase of egg formation.
This lethal factor, then, results from an incompatibility between a small
chromosomal area and the ooplasm into which it is introduced. The developmental action of this incompatibility was examined by means of time-lapse
analysis of the movements involved, with corresponding light-microscope
examinations of sectioned material.
The first visible manifestation of the incompatibility is in the abnormal
behavior of the yolk system. The mechanism of normal yolk contraction is not
understood, but is an important morphogenetic process in most insects. Seidel
(1936) analysed the factors involved in the early development of the dragonfly
Platycnemis. He found that the entrance of cleavage nuclei into the posterior
pole of the egg somehow incites the 'activation center' to produce what is
presumed to be a substance of some sort. This substance diffuses away from the
activation center, producing changes in the yolk system as it goes. In Platycnemis, the changes consist of an increase in transparency and viscosity, and
there is a contraction of the yolk system which aids in the formation of the
germ band. Ede & Counce (1956) have noted this contraction involved in germband formation in D. melanogaster. The contraction begins at the ' differentiation center' which is in the area of the future prothorax. The contraction then
spreads anteriorly and posteriorly.
Some work has been done on various 'control centers', as they are called,
in many insects; however, the knowledge concerning them is still fragmentary
and has not been well correlated from one group to another. In general, the
presence and action of activation centers in most insects is not well demonstrated
except in Platycnemis, for technical reasons. About all one can say is that there
does appear to be some important function traceable to the posterior pole
during late cleavage stages in all the insects studied (Counce, 1961).
Nearly every insect studied has been shown to have a differentiation center,
which is always in the future prothoracic region. In Platycnemis (Seidel, 1934)
and Apis (Schnetter, 1934), it has been shown to be the starting-point for germband formation, gastrulation, and segmentation.
Imaizumi (1954) described a contraction of the entire egg substance which
occurs soon after the egg is laid, followed by relaxation about the time of nuclear
migration. This also occurs in unfertilized and in Nullo-X eggs. However, there
is a second slight contraction of the whole egg which occurs only in developing
eggs and accompanies the ventral germ band formation and its associated yolk
contraction.
Imaizumi did not discuss any contractions of the yolk system, as he was
Embryonic lethal hybrid
419
measuring changes only in the entire egg. The first contraction which he described was seen in this study in thefilmsof both normal and abnormal embryos,
and was found to be quite slow compared to the yolk contractions. It is not
known whether this is produced by the same mechanism as the yolk system
contractions, which are not yet understood.
The cellular proliferation without differentiation, and the appearance of
nuclear abnormalities in the lethal embryos is interpreted as being due to the
massive rearrangements of the cytoplasm by the yolk movements. Pauli (1927)
found that centrifuged insect eggs in which the cytoplasm was drastically
disturbed produced masses of undifferentiated cells. Counce & Selman (1955)
obtained similar results in ultrasonic treatment of Drosophila embryos. In
treatments of cleavage stages, they found that sonic levels which disarranged
the cytoplasm, but had no apparent effect on the nuclei themselves, produced
cytological abnormalities. They also found that the maximum lethality occurred
when embryos in the syncytial blastema stage were treated. Again, the nuclei
themselves were seldom affected directly, but the resulting disorganization of
the contents of the egg resulted in cellular proliferation without differentiation.
Whatever their mechanism, the large rearrangements of the yolk plasmodium
in the lethal hybrids may therefore have caused the failure of the proliferating
cells to differentiate, and the subsequent development of nuclear abnormalities.
The early dipteran egg, prior to the blastoderm stage, is currently thought to
be less determinate than was previously believed (Anderson, 1962). However,
it is generally agreed that it can no longer regulate at the blastema stage, at
which time the totipotent nuclei move into areas of differentiated cytoplasm.
Many instances are known in which a certain area of cytoplasm affects nuclei
entering it (Hegner, 1911; Von Borstel, 1957; Nicklas, 1959).
Therefore, it is quite possible that the failure of differentiation and the production of nuclear abnormalities in the female hybrids results from the mixing of
the peripheral cytoplasm during the syncytial blastema stage. The normal
cleavages and initial blastema formation suggest that the Z-chromosomal factor
per se does not prevent normal pairing and replication, but rather has some effect
on the yolk plasmodium whose abnormal behavior in turn disrupts the cortical
cytoplasm, causing subsequent failure of differentiation.
The D. melanogaster X-chromosome has been studied more than the autosomes with respect to effects of its mutants, rearrangements, and deficiencies on
early development. Poulson (1940) studied eggs which lacked the entire Xchromosome or either half of it. He found that when either half was missing,
the embryo either failed to form a normal blastoderm, or else it produced a
normal blastoderm but failed to form a normal germ band. When the entire
Z-chromosome was lacking, development became abnormal in late cleavage,
during which the nuclei remained in the anterior half of the egg instead of being
evenly distributed. Only the anterior peripheral cytoplasm was then nucleated
420
J. D. KINSEY
and formed into cells, which proliferated there to form a mass of undifferentiated cells, in which nuclear irregularities appeared.
It is interesting to note that those cells which first form in Nullo-X embryos
have the appearance of normal blastoderm cells, as do some of the cells which
first form in the lethal female hybrid. Poulson concluded that both halves of
the ^-chromosome carry genes important in early embryonic development.
The end-result of the Nullo-X development in D. melanogaster is quite similar
to that of the (D. montana ?xZ). texana £) hybrid female embryos, although
their basic mode of formation differs. The hybrid containing the incompatible
chromosomal area actually remains normal longer than the Nullo-X, so that
some of the -^-chromosome probably functions normally in the D. montana
ooplasm. In the Nullo-X, some part of the Z-chromosome is shown to be
involved in the proper distribution of the cleavage nuclei; when the X\s lacking,
the nuclei are not distributed to the posterior part of the egg. The subsequent
failure of yolk system contraction is probably evidence for the existence of an
activation center in the posterior region, which fails to act when the nuclei do
not reach it.
Since the mechanism of normal contraction of the yolk system is unknown,
it seems fruitless to speculate in detail on specific actions of the lethal interaction.
It appears that the ^-chromosome controls yolk-system contractions, first by
allowing proper migration of nuclei to the activation center area, and later by
some means which is disrupted in the lethal interaction under study. The hybrids
in the texana $ x montana $ cross show the normal contractions, so that the
X-chromosomal segment of either or both species together can function in the
texana ooplasm; also, the fact that the male hybrids in the montana ? x texana $
cross show the normal contractions indicates that a single montana X-chromosomal segment can function normally in montana ooplasm. Therefore, the addition of the lethal chromosomal segment of the texana X to the ooplasm of the
montana egg most likely acts either by producing an abnormal amount of some
substance, or an abnormal substance, which reacts in an atypical fashion with
the yolk system. The resulting disruption of the cytoplasmic arrangement would
then result in the lethal undifferentiated masses of cells described.
SUMMARY
1. Reciprocal interspecific ovary transplantations were made between
Drosophila montana and D. texana to determine the possible effects of such
transplantations on the development of the transplanted ovaries and of the
hybrid embryos formed from the eggs developed in these ovaries. The transplants were also a means of overcoming sexual isolation between the two species,
and obtaining hybrids.
2. Hybrid embryos of normal reciprocal crosses of D. montana and D. texana
were found to be indistinguishable from hybrids formed from eggs produced
Embryonic lethal hybrid
421
by the transplanted ovaries. The transplanted ovaries developed autonomously
both morphologically and with respect to the embryos formed from their eggs.
3. In the hybridization of D. texana $ x D. montana & offspring of both sexes
were produced in equal numbers, about a quarter of the fertilized eggs showing
abnormal development of various types. The abnormal development resulted in
embryos having recognizable larval structures, and was believed to result from
the disruption of genie balance at various ' epigenetic crises' by the combination
of rather dissimilar chromosomal complements.
4. In the hybridization of D. montana $xD. texana $, two types of offspring
were obtained in equal numbers; those that showed larval-type development
ranging from extremely abnormal to completely normal, and those that showed
cell proliferation without differentiation. Since the larvae formed were males,
the undifferentiated masses of cells were assumed to represent the female hybrids.
5. The abnormalities that developed in the male embryos showed their onsets
during periods of major morphogenetic reorganization, no two abnormal
embryos showing exactly the same patterns of development. They were assumed
to result from the same type of gene balance disruption as the hybrids of the
reciprocal cross.
6. The development of the female embryos was analysed by time-lapse
cinematography and sectioned material. It was determined that the primary
incompatibility of the texana Z-chromosome and the montana ooplasm is manifested as massive abnormal contractions of the yolk system during the syncytial
blastoderm stage. This resulted in abnormal rearrangements of the cortical
cytoplasm and the nuclei which it contained. The resulting cellular proliferation
without differentiation and eventual development of cytological abnormalities
are believed to be due to a disruption of the mosaic cortical cytoplasm.
ZUSAMMENFASSUNG
Untersuchungen ilber einen embryonal-letalen Artbastard bei Drosophila
1. Reziproke Ovarial-Transplantationen wurden ausgefiihrt zwischen Drosophila montana und D. texana, um etwaige Wirkungen solcher Transplantationen
auf die Entwicklung der transplantierten Ovarien zu studieren, und auf die der
Artbastarde, die aus den Eiern solcher transplantierten Ovarien hervorgehen.
Sie dienten auch dem Zweck, die sexuelle Isolierung zwischen diesen Arten zu
iiberwinden und so Artbastarde zu erhalten.
2. Artbastarde aus normalen reziproken Kreuzungen zwischen D. montana
und D. texana unterscheiden sich nicht von Artbastarden aus transplantierten
Ovarien. Die transplantierten Ovarien entwickeln sich autonom morphologisch
als auch hinsichtlich der Embryonen, die von diesen Eiern abstammen.
3. Aus der Kreuzung von D. texana $xZ). montana <£ gingen beide Geschlechter in etwa gleichen Zahlen hervor, wobei ungefahr \ der befruchteten
Eier verschiedenartige Abnormitaten aufwiesen. Die abnorme Entwicklung
fiihrte zu Embryonen mit erkennbaren Larvenstrukturen, und es scheint, dass sie
422
J. D. KINSEY
zuxiickzufiihren ist auf Storungen des genetischen Gleichgewichtes in verschiedenen * epigenetischen Krisen' durch die Kombination ziemlich verschiedenartiger Chromosomensatze.
4. Bei der Kreuzung von D. montana $ x D. texana <J entstanden zwei Typen
von Nachkommenschaft in ungefahr gleicher Anzahl; diese zeigten entweder
larvenartige Entwicklung von extrem abnorm zu vollig normal; oder sie zeigten
Zellvermehrung ohne Differenzierung. Da die Larven Mannchen waren, liegt
die Vermutung nahe, dass die undifferenzierten Zellmassen die weiblichen
Artbastarde darstellen.
5. Die Anomalien der mannlichen Embryonen entstehen wahrend Perioden,
in denen erhebliche morphogenetische Reorganisationen stattfinden, und kaum
zwei solcher Embryonen zeigen genau die gleichen Entwicklungsmuster.
Offenbar sind diese Anomalien das Ergebnis der gleichen Stoning des genetischen Gleichgewichtes wie bei den Bastarden der reziproken Kreuzung.
6. Die Entwicklung der weiblichen Embryonen wurde untersucht mittelst
Zeitraffer-Kinematographie und in Schnittserien. Die primare Unvertraglichkeit des texana X-Chromosoms mit dem montana Ooplasma zeigt sich in Form
von massiven abnormen Kontraktionen des Dottersystemes wahrend des
syncytialen Blastodermstadiums. Dies fiihrt zu gestorten Lagebeziehungen
zwischen dem corticalen cytoplasma und den darin enthaltenen Zellkernen.
Die darauf folgende Zellvermehrung ohne Differenzierung nebst der Entwicklung cytologischer Anomalien sind vermutlich eine Folge der Stoning des
corticalen cytoplasma-Mosaikes.
This is a portion of a dissertation submitted to the Faculty of the University of Texas in
partial fulfilment of the requirements for the degree of Doctor of Philosophy. This investigation was supported by U.S. Public Health Service Research Grants HD-00725-05 and GM
11609, and was carried out while the author was a National Science Foundation Graduate
Fellow. The author wishes to thank Dr A. G. Jacobson for his support, interest, and criticism
of the manuscript.
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