/ . Embryol. exp. Morph., Vol. 15, 3, pp. 271-279, June 1966
With 2 plates
Printed in Great Britain
271
Histology of larval eye-antennal disks and cephalic
ganglia of Drosophila cultured in vitro
BylMOGENE SCHNEIDER1
From the Department of Biology, Yale University and the
Department of Entomology, Walter Reed Army Institute of Research
INTRODUCTION
In the past decade, a relatively large number of reports have been published
on the culture in vitro of organs of Drosophila melanogaster. The majority of
these reports have been concerned with the development of the eye-antennal
complex explanted, with or without the attached cephalic ganglia, from the
prepupal stage (Denial, 1956) or from late second and third larval instar stages
(Kuroda & Yamaguchi, 1956; Gottschewski, 1958, 1960, 1962; Gottschewski &
Querner, 1961; Fugio, 1962; Schneider, 1964). With the exception of the work
of Demal (1956), the above reports have been confined to descriptions of the
developing explants solely in morphological terms and, as such, are not wholly
adequate for comparisons to be made between development in vivo and in vitro.
If, however, such descriptions are supplemented with histological studies, a
more valid appreciation can be gained of the potentialities as well as the limitations of such explants under conditions in vitro.
In a recent paper (Schneider, 1964) the day-by-day morphological development of late third instar eye-antennal disks of D. melanogaster cultured in vitro
for varying lengths of time was described. The present report deals with the
extent of histological differentiation attained in these explants.
MATERIALS AND METHODS
Since the majority of explants in the previous study had been allowed to
remain in culture for as long as 2-3 weeks, a second series were placed in culture
in order to obtain explants that had been under in vitro conditions for relatively
limited periods, i.e. 24-96 h. The method of culturing was identical to that
described previously, with the exception that the culture medium was slightly
revised as follows: the concentrations of KH 2 PO 4 and NaHCO 3 were lowered to
45 and 40 mg/100 ml respectively and Na 2 HPO 4 was added in a concentration
of 70 mg/100 ml. The individual amino acids were used instead of lactalbumin
1
Author's address: Department of Entomology, Walter Reed Army Institute of Research,
Washington, B.C., U.S.A.
272
I. SCHNEIDER
hydrolysate; and TC yeastolate in a concentration of 200 mg/100 ml was employed instead of the individual B vitamins.
In addition, eye-antennal disks with cephalic ganglia were removed from intact
larvae, prepupae and pupae of D. melanogaster (raised under aseptic conditions
at 25° C and timed to ± 2 h after emergence) and fixed immediately to serve as
controls.
The above were fixed in warm FAA (6:16:1 + 30 parts water), embedded
using the Peterfi methyl benzoate-celloidin method, sectioned at 8 /i, and stained
with Harris's hemotoxylin.
Although explants from both D. melanogaster and D. virilis were used in the
previous study, this report deals only with explants from the former.
RESULTS
With the exception of orange and red pigment deposition, differentiation of
the eye-antennal disks and the brain lobes reached its peak between the 7th and
8th day in vitro. Thereafter the explants markedly deteriorated even though
further pigment deposition took place.
In general, the antennal disks were least adversely affected by being placed
in culture whereas the opposite held true for the ventral ganglion. The former
showed relatively few signs of degeneration even after 14 days in culture, whereas
in the latter blebs and vacuoles as well as increasing opaqueness became evident
after 24-48 h. The effects of culturing on the eye disks and paired brain lobes
were intermediate between these two extremes. Consequently, the histology of
the developing antennal disks most closely approached that of the controls.
The antennal disks were the only structures which consistently differentiated to
the point of approximating the adult organ. With few exceptions, both the eye
disks and the brain differentiated to an extent comparable to mid-pupal or late
pupal controls.
Contrary to an earlier assumption, based solely on the morphological
appearance of the explants, the eye-antennal disks did not evaginate but shifted
only to a position normally attained in the late prepupal stage.
Histology of the developing eye disks
After one day in vitro, longitudinal sections through the eye disk show a
series of adjacent 'goblet-like' units (terminology of Steinberg, 1943) with each
unit having a two-layered appearance. The upper portion of these units shows
some signs of organization within the presumptive cone area whereas the lower
layer is a mass of unorganized cells. This effect is further heightened by the fact
that the upper layer invariably stains less intensely than does the lower layer
(Plate 1, fig. A). In cross-section the four-celled 'clusters' of the presumptive
ommatidia are very prominent in the outer layer of the eye disks. On the third
day the pre-ommatidia are more elongate and further development takes place
Histology of cultured explants
273
in the upper layer. The pseudocone, corneagenous cells and cone cells can be
identified although they are often somewhat displaced in comparison with the
pupal controls. Beneath the cone cells are a number of oval-shaped cells, at
least four in number and possibly more. In a few instances an axial rod can be
seen extending from the pseudocone throughout the length of the retinula to
the optic nerve. There is still little or no organization of the cells in the lower
layer.
During the fifth and sixth days, the first indication of the cornea appears in the
form of a darkened layer at the upper border of the ommatidia. Between the
seventh and eighth days (see Plate 1,figs.B, C) the facets are delineated, although
occasionally one facet appears to overlap two or more ommatidia. The setiform
corneal hairs are also apparent and, in contrast to the normal controls in which
one seta is found at the intersection of three adjacent facets, as many as seven
hairs may project upward from the border of a single facet. Although the upper
area of the ommatidia (i.e. from the cone cells to the cornea) attains a reasonably
normal appearance, the retinulae are very much foreshortened and often are
situated at an oblique angle to the upper portion. In many instances the retinulae
simply taper off into an unrecognizable mass with the lower layer of cells, which
never attains any degree of organization.
In addition to the eye proper, the ocelli as well as brown and black head cuticle
and bristles differentiate from certain sectors within the eye disk. No attempt
was made to follow the course of this development in vitro other than to note
that both bristles and cuticle are present by the seventh day. The ocelli, if present,
were not detected.
No further differentiation is apparent in explants 10-15 days old. On the contrary, the structures are much less definitive in appearance and position due to
the advanced state of degeneration in the explants.
Histology of developing antennal disks
During the first 2 days in vitro the concentric folds within the antennal disk
become increasingly thickened, especially the fold destined to become the third
antennal segment. This third segment is the first to protrude from the antennal
'center' and is followed shortly thereafter by the second segment or pedicel and
the small basal scape. In the next few days the second and third segments of the
antenna increase both in length and width.
Whereas the cells in the basal segment of a 2-day-old explant are fairly uniform
in size, those within the second and third segments show a gradation, being very
large at the border and becoming progressively smaller toward the interior.
These cells appear to be partially oriented into adjacent wedge-shaped rows,
wide at the periphery, accommodating the larger cells, and tapering to a narrow
shaft as the central lumen is reached.
Indication of a lumen is present in all three segments as early as the first day.
In the basal segment the lumen is quite small, occupying perhaps a fifth of the
274
I. SCHNEIDER
total area. In the second and third segments the lumina are larger, in the former
covering as much as a third of the total area. By the fifth day (Plate 1, fig. D) the
lumina have fused into a common duct serving all three segments.
Spanning the width of the lumen in the second segment is a diagonal cell
'bridge' (occasionally two), a few cell layers thick, 1-5 or more cells in width
and from 8 to over 20 cells in length. These bridges are present by the first day
and become increasingly larger as well as more complex in structure on successive days. This increase in cell number within the bridges may be due to division,
but it seems more likely that at least part of the increase is due to a migration of
cells from certain sectors on the periphery of the lumen. As development continues, it is apparent that this internal organization leads to the formation of
Johnston's organ.
The base of the arista (segments 4 and 5 of Ferris, 1950) becomes evident
between the fifth and eighth day in vitro. The arista itself is much foreshortened
and does not appear in all of the explants (Plate 2, fig. E). The antennal nerve is
traceable from its origin in the deutocerebrum to its termination point in the
third segment by the sixth day. Formation of antennal muscles and bristles, as
well as sclerotization of the first and second segments, begins on the fifth day,
with the organ essentially adult in appearance by the seventh day (Plate 2, fig. F).
Histology of the developing brain lobes
After 1 day in vitro, the brain lobes lose their more or less spherical appearance and elongate laterally. The fusion of the two lobes mediodorsally is also
nearly completed. Aside from these two developments the brain lobes are still
essentially late larval in character. The external optic glomerulus (Plate 2, fig. G)
is closely apposed to the outer lateral edge of the optic lobe, where it is united
EXPLANATION OF PLATES
Abbreviations. A \-A 6, antennal segments 1-6; AR, axial rod of ommatidium; BM,
basement membrane; E, eye disk; EEC, external optic glomerulus; F, facet; H, corneal hair;
TEG, inner optic glomerulus; JO, Johnston's organ; MEG, middle optic glomerulus; LOR,
lower ommatidial region; O, ommatidia; UOR, upper ommatidial region. The horizontal
line under each figure presents 10 ji.
PLATE 1
Fig. A. Section of eye disk from 2-day explant. The more lightly staining upper half shows a
series of adjacent 'goblet-like' units, each composed of four cells. Little or no organization
is apparent in the darker staining lower half.
Fig. B. Ommatidia of 7-day explant. Failure of head to evaginate is probably responsible for
crowding of ommatidia and overlapping of facets. Axial rod can be traced in ommatidium
to right of the corneal hair.
Fig. C. Ommatidia of 8-day explant. Note difference in height of facets in this explant as
compared with flattened facets in fig. B. Ommatidia have attained their greatest length, less
than two-thirds of that found in late pupal controls. Inverted facet at left may or may not
be an artifact.
Fig. D. Antenna of 5-day explant. The individual lumina have fused into a common duct
serving all three segments.
/. Embryol. exp. Morph., Vol. 15, Part 3
PLATE 1
AR
I. SCHNEIDER
facing p. 274
J. Embryol. exp. Morph., Vol. 15, Part 3
PLATE 2
Fig. E. Antenna of 7-day explant. Segments 4, 5 and 6 protrude from third segment. The
arista (segment 6) is not seen in its entirety.
Fig. F. Fully formed antenna in 8-day explant. Johnston's organ is at top. Note the pronounced sclerotization of second segment and the numerous bristles bordering entire organ.
Fig. G. Brain lobe of 2-day explant. External optic glomerulus is surrounded by outer
imaginal epithelium.
Fig. H. Nine-day explant. External optic glomerulus is now fan-shaped but does not lie
completely outside the rest of the brain. Facets border upper area of the unevaginated eye.
I. SCHNEIDER
facing p. 275
Histology of cultured explants
275
with the fibrous optic nerve. The former has differentiated into an inner and
outer mass of cells divided by a lightly staining fibrous band. The middle
glomerulus as well as the anterior and posterior portions of the inner glomerulus
are well defined. The cells surrounding these optic glomeruli are very small as
contrasted with the much larger cells in the central portion of the brain.
In the next 24-48 h, fusion of the brain lobes is completed. The external optic
glomerulus, previously within the brain lobe proper, now protrudes outward to
a distance corresponding to approximately a third of its length. The middle
glomerulus increases both in length and width, in contrast to the inner glomerulus which remains essentially the same.
Between the fifth and eighth day in vitro, the external optic glomerulus
assumes a somewhat fan-shaped appearance and is in close apposition to the
ommatidia of the unevaginated eye (Plate 2, fig. H). This structure never attains
the normal position of lying outside of the brain; it is always bounded on its
anterior and outer lateral sides by a thin cellular layer of the outer imaginal
epithelium.
By the fifth day in vitro, all of the numerous components of central portion
of the adult brain as described by Miller (1950) can readily be identified in the
explants. Nonetheless, there are a number of characteristics by which the explants differ from the controls. In addition to the essentially mid-pupal position
of the external optic glomerulus, the entire brain appears flattened so that in
frontal sections the deutocerebrum does not assume its normally elevated
position but is instead strongly compressed against the protocerebrum. Finally,
although the nervous tissue (excluding the ventral ganglion) shows few signs
of degeneration, the periphery of the brain lobes, especially those portions lying
directly beneath the middle and posterior inner glomeruli, becomes increasingly
vacuolated after the third or fourth day in culture.
A summary of the results together with an in vivo timetable is given in Table 1.
DISCUSSION
The foregoing description was based on a histological study of approximately
140 explants. The rate and the extent of differentiation within these explants was
by no means uniform and consequently the description given is a more or less
generalized one.
As a rule, if a developmental sequence takes place within 24 h after puparium
formation this same sequence will occur in vitro without a serious time-lag. If
such a sequence normally takes place after this period, the time-lag not only
increases considerably but there is also much greater diversity among the
explants with respect to their degree of development (see Table 1). Undoubtedly,
an explant when first placed in culture has sufficient nutritional and hormonal
reserves to continue normal or near-normal development for a limited time. Once
such reserves have been greatly diminished or exhausted, the explant must rely
276
I. SCHNEIDER
on an artificial milieu inferior in many, if not all, respects to its normal environment. Consequently, developmental sequences taking place early in metamorphosis are much more likely to proceed normally in vitro than those which occur
later.
Table 1. Time required for differentiation of eye-antennal disks and
cephalic ganglia 0/Drosophila melanogaster: in vitro vs. in vivo
Eye disk
Definitive ommatidia
Pseudocone, corneagenous and cone cells
Cornea, facets, corneal hairs
Head cuticle, bristles
Brown pigment3
Red pigment3
Antennal disk
Protrusion of distinct segments
Formation of Johnston's organ
Formation of arista, muscles, bristles;
sclerotization of segments
Brain lobes
Fusion of lobes
Partial protrusion of external optic glomerulus
Fan-shaped external optic glomerulus
Adult form of proto- and deutocerebrum
Ventral ganglion
Metamorphosis into imaginal ganglion
In vitro1
5-8
3
5-8
6-7
5-8
In vivo2
2-3
l±-2
2-2*
7-12
2-3
2*
3
2
3-5
1
2
5-7
2-3
1-3
2-3
1
1
5-8
1*
3-5
1-2
—
2-3
1
Days after explantation.
Days after puparium formation: in aseptic cultures the puparium is formed 98-100 h
post hatching.
3
Times for in vitro deposition of pigment taken from an earlier study (Schneider, 1964).
2
The failure of the head to evaginate precludes the normal positioning of the
eyes, antennae, and external optic glomeruli. Robertson (1936) suggested that
the evagination of the head is the result of increased internal pressure brought
about by the contraction of the abdominal muscles within the prepupa. Obviously this cannot occur in a system consisting solely of organs from the
cephalic region. Similarly, Shatoury (1956) has stated that the ultimate protrusion of the external optic glomerulus is the result of the contraction of the optic
nerve during the early pupal stage. Even if such a contraction does take place
in vitro, the spatial relationships of the unevaginated eye, the optic nerve and the
glomerulus are such as to negate any profound change in the position of the
latter structure.
The fact that some organs in culture differentiate more fully than others can
probably be attributed to three main factors: (1) the relative extent of injury
during dissection, (2) vulnerability to adverse conditions in an artificial environment, and (3) inherent capacity for autonomous development. The ventral
ganglion is obviously subjected to more trauma during dissection (through the
Histology of cultured explants
277
necessity of severing the multitude of nervous fibers emanating along its entire
length) than are the eye disks and brain lobes, which are virtually untouched, or
the antennal disks, which are severed only at their apex. Furthermore, the ventral
ganglion is completely vulnerable to any adverse environmental conditions in
contrast to the rest of the brain, which is 'protected' to some extent by an outer
epithelium, or to the antenna, which is completely surrounded by other organs.
This factor may also account for the difference in the extent of differentiation
found in the upper and lower areas of the ommatidia. Finally, the culture in
vitro of isolated eye-antennal disks has shown that they display a considerable
degree of autonomous development if explanted at the end of the third larval
stage. It is evident that the ventral ganglion either lacks this degree of autonomy
or that it cannot be expressed under the conditions imposed by the environment
in vitro.
The results reported here as well as those of an earlier study (Schneider, 1964)
are at variance in a number of respects with those of Gottschewski (1960). In
the latter study eye-antennal disks with cephalic ganglia were explanted at the
end of the third larval instar (exact age and temperature not given), and differentiation of the eye disks, including the evagination movement, was complete
by 96 h, a time-lag of not more than 12 h compared with in vivo controls.
Possible explanations for the great disparity in the rate of differentiation observed in the two studies are: (1) difference in age of the explants cultured,
(2) difference in the volume of culture medium employed, and (3) differences
between the media.
Larvae grown under aseptic conditions tend to mature at a somewhat slower
pace than normal and therefore an explant may be slightly younger developmentally than the chronological age of the donor would indicate. In order to
eliminate this factor, explants from larvae grown on standard food were also
cultured (the concentration of antibiotics suppressed but did not eliminate
contamination from bacteria and/or yeast). Little improvement was noted in
the time required for differentiation. However, when explants were taken from
donors at the end of the prepupal stage they developed at a rate comparable to
that found by Gottschewski, the time-lag rarely exceeding 24 h.
Reducing the size of the hanging drop from 0-008 to 0-005 ml resulted in a
considerable reduction in differentiation time: from 8-12 days to 5-6 days.
However, the accumulation of detrimental metabolic wastes apparently increased at an even faster rate since an explant kept for four days in a smaller
drop usually showed more signs of degeneration than an 8 or 9-day explant in
a larger drop. The above also held true for explants cultured in Gottschewski's
medium. (It should be noted here that partial renewal of the culture medium
every third or fourth day increases the survival time of an explant by as much
as a week or more; however, differentiation is greatly retarded or ceases altogether if such a renewal is made.) Since there is no evidence whatsoever for
evagination of eye disks explanted in vitro at the end of the third larval instar,
l8
JEEM 15
278
I. SCHNEIDER
nor any indications from the extensive work on transplantation that transplanted disks can evert in vivo, reports of evagination of larval disks explanted
in vitro must be regarded with skepticism. On the other hand it is possible that
disks explanted at the close of the prepupal period, near the time of normal
eversion, might be found in an evaginated condition when observed in vitro.
SUMMARY
1. A description is given of the histological development of eye-antennal
disks and cephalic ganglia of Drosophila melanogaster cultured in vitro for
varying lengths of time.
2. The eye-antennal disks do not evaginate but move only to a position
normally attained in the late prepupal stage.
3. The relative competence of the various cephalic organs to undergo
differentiation is as follows (descending order): antennal disks, eye disks, brain
lobes, ventral ganglion. Possible explanations for this difference in differentiation capacity are given.
4. In general, if a developmental sequence takes place in vivo within 24 h
after puparium formation, the same sequence occurs in vitro without a serious
time-lag. After this period, however, an interval of from 3 to 9 days is necessary
for comparable development.
RESUME
Histologie des disques oculo-antennaires et des ganglions cephaliques
de la larve de Drosophile, en culture in vitro
1. Le developpement histologique des disques oculo-antennaires et des
ganglions cephaliques, cultives in vitro pendant des temps varies, est decrit chez
Drosophila melanogaster.
2. Les disques oculo-antennaires n'accomplissent pas l'evagination mais
n'atteignent que la position normalement acquise au stade prepupe avancee.
3. La capacite relative de differenciation des differents organes cephaliques
decroit dans l'ordre suivant: disques antennaires, disques oculaires, lobes
cerebraux, ganglion ventral. Des explications possibles de cette difference dans
la capacite de differenciation sont fournies.
4. En general, si une sequence du developpement se produit in vivo dans les
24 heures suivant la formation de la pupe, cette meme sequence apparait in
vitro sans retard notable. Cependant, apres ce stade du developpement, un
retard de 3 a 9 jours est observe pour obtenir un developpement comparable.
I wish to thank Prof. D. F. Poulson and Drs S. J. Counce and W. W. Doane for their
comments and criticisms of the manuscript. The technical assistance of Mrs Betsy Jeavons is
also acknowledged. The major part of this investigation was supported by N.S.F. grant
GB 1718.
Histology of cultured explants
279
REFERENCES
J. (1956). Culture in vitro d'ebauches imaginales de Dipteres. Ann. des Sc. Nat. Zool.
lleserie, 18, 155-61.
FERRIS, G. F. (1950). External morphology of the adult. In The Biology of Drosophila, ed.
M. Demerec. New York: Wiley.
FUGIO, Y. (1962). Studies on the development of eye-antennal discs of Drosophila melanogaster in tissue culture. II. Effects of substances secreted from cephalic complexes upon
eye-antennal discs of eye mutant strains. Jap. J. Genet. 37, 110-17.
GOTTSCHEWSKI, G. H. M. (1958). Ober das Wachstum von Drosophila-Augen-ImsLginalscheiben in vitro. Naturwissenschaften, 45, 400.
GOTTSCHEWSKI, G. H. M. (1960). Morphogenetische Untersuchungen an in vitro wachsenden
Augenanlagen von Drosophila melanogaster. Wilhelm Roux Arch. EntwMech. Org. 152,
204-29.
GOTTSCHEWSKI, G. H. M. (1962). Die Entwicklung des Chiasma externum bei Drosophila
melanogaster. 1. Europdischen Anatomen-Kongresses, Strassburg 1960. Jena: Gustav
Fischer.
GOTTSCHEWSKI, G. H. M., & QUERNER, W. (1961). Beobachtungen an explantierten friihen
Entwicklungsstaden der Augenanlage von Drosophila melanogaster. Wilhelm Roux Arch.
EntwMech. Org. 153, 168-75.
KURODA, Y. & YAMAGUCHI, K. (1956). The effects of the cephalic complex upon the eye discs
of Drosophila melanogaster. Jap. J. Genet. 31, 98-103.
MILLER, A. (1950). The internal anatomy and histology of the imago of Drosophila melanogaster. In The Biology of Drosophila, ed. M. Demerec. New York: Wiley.
ROBERTSON, C. W. (1936). The metamorphosis of Drosophila melanogaster, including an
accurately timed account of the principal morphological changes. /. Morph. 59, 351-99.
SCHNEIDER, I. (1964). Differentiation of larval Drosophila eye-antennal discs in vitro. J. exp.
Zool. 156, 91-104.
SHATOURY, H. H. El (1956). Differentiation and metamorphosis of the imaginal optic
glomeruli in Drosophila. J. Embryol. exp. Morph. 4, 240-7.
STErNBERG, A. G. (1943). The development of the wild type and Bar eyes of Drosophila
melanogaster. Canad. J. Res., Sec. D., 21, 277-83.
DEMAL,
{Manuscript received 10 November 1965)
18-2