J. Embryol. exp. Morph. 82, 1-8 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
Do larval epidermal cells possess the blueprint for
adult pattern in Drosophila?
By MEKKARA MANDARAVALLY MADHAVAN AND
KORNATH MADHAVAN
Department of Biology, College of the Holy Cross, Worcester,
Massachusetts 01610, U.S.A.
SUMMARY
When the diploid histoblasts, the precursors of adult abdominal epidermal cells, of the larva
of Drosophila are deleted by y-radiation, the polytene larval epidermal cells survive metamorphosis and secrete cuticle and cuticular outgrowths. A comparison of the morphology of the
cuticle secreted by the larval epidermal cells in the different regions of the abdominal segments
to that secreted by the histoblasts of the unirradiated animal suggests that the former contain
the blueprint for the pattern of landscape of the adult abdominal cuticle and possibly could
provide this information to the dividing and spreading histoblasts during the normal ontogeny
of the fly.
INTRODUCTION
In hemimetabolous insects, the larval epidermal cells (LEC) which are present
at the time of hatching from the egg and their descendants are responsible for the
different cuticular patterns seen in the nymph and adult. On the other hand, in
the higher holometabolous insects such as Drosophila the distinct and different
cuticular patterns exhibited by the larva and adult have a dual origin: that of the
larva is derived from the LEC, and that of the adult is due to the imaginal cells,
i.e. the progeny of the imaginal discs and histoblasts. Since, in normal development, the LEC die during metamorphosis, we have no information regarding
their potential to form the adult pattern. If the LEC could be rescued from their
programmed cell death, one may be able to witness the cuticular pattern formed
by these cells after metamorphosis. One of the methods to rescue these cells is
by exposing the larva to gamma radiation which selectively kills the histoblasts
(Poodry, 1975). Such irradiated larvae undergo metamorphosis and in the absence of the histoblasts, the LEC take over the function of secreting a cuticle and
cuticular elements. In these animals, it is possible to compare the pattern
established in the adult by the LEC to that formed by the histoblasts of the
unirradiated animal, thus indicating whether or not the adult pattern could be
derived from LEC. In our studies we have used the above procedure to maintain
the LEC and in the following we report the patterns formed by them in different
regions of the abdominal segments.
M. M. MADHAVAN AND K. MADHAVAN
MATERIALS AND METHODS
Adult flies of an Oregon-R strain of Drosophila were raised at 25 °C on a
standard medium, and late third instar larvae were collected from the walls of the
food bottles. These were transferred to smaller food vials and were exposed
to various doses (5, 8, 10 and 12 kR; 320R/min) of gamma radiation from a
cobalt60 source. Subsequently, the animals were allowed to develop at 25 °C.
Stained whole mounts of the epidermis of a few of these animals were made at
22-26 h after pupariation in order to see the condition of the histoblast nests during
this time. At the end of 6-8 days after pupariation all the unemerged animals were
dissected from their puparia. A few of the experimental animals were used to
examine the cuticular morphology of the abdominal segments under the scanning
electron microscope. For this purpose, the animals were dehydrated in an ascending series of acetone, dried in a Samdri-780 critical-point drier using CO2 as the
transitional fluid, carefully mounted on aluminium stubs with double-stick Scotch
tape and sputter coated with gold and palladiumfilmto a thickness of about 20 nm.
Secondary emission scanning electron micrographs of these preparations were
made on an AMR 1000 A SEM using an accelerating voltage of 20 kV. The remaining experimental animals were used to make whole mounts of stained or unstained
preparations of abdominal epidermis and the overlying cuticle according to the
method described earlier (Madhavan & Madhavan, 1980).
RESULTS
Each of the adult abdominal segments of Drosophila is derived mainly from
three pairs of histoblast nests, i.e., the anterior dorsal (ADN), posterior dorsal
Fig. 1. The intersternal region (is) of an adult showing the hairs with bulbous bases.
(X1000).
Fig. 2. Feulgen-stained whole mount of the epidermis of the right side of the fourth,
fifth and sixth hemisegments of a pharate adult (24 h after pupariation) resulting from
a larva which received 8 kR radiation. The number of cells in the anterior (ADN) and
posterior (PDN) dorsal, ventral (VN) and spiracular (SN) nests are considerably less
compared to that of the normal animal at a corresponding stage. (X200).
Fig. 3. Feulgen-stained whole mount of the left hemitergite of an adult resulting
from a larva which received 5 kR radiation. This hemitergite is formed partly by
persisting larval epidermal cells (LEC) (arrowheads) in the lateral region and partly
by adult epidermal cells in the medial region. The nuclei of some of these cells are
not in focus. (X200).
Fig. 4. Feulgen-stained whole mount of the left hemitergite of an adult resulting
from a larva which received 5 kR radiation. Note that the persisting larval epidermal
cells at the level of hairy and bristled regions of the hemitergite secrete cuticle with
hairs and those at the level of intertergal region (it) secrete smooth cuticle. The hairs
secreted by the LEC do not form regular rows and they orient in all directions, while
those by the adult epidermal cells are arranged in regular rows and are posteriorly
oriented. (x250).
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Determination of cuticular pattern in Drosophila
3
(PDN) and ventral (VN), which are present among the LEC (Madhavan &
Schneiderman, 1977; Roseland & Schneiderman, 1979; Madhavan & Madhavan, 1980). During metamorphosis of the insect, the LEC die and are sequentially replaced by the adult epidermal cells derived from these histoblasts. The
details of this orderly cell replacement and the cuticular pattern established by
the progeny of the histoblasts in the adult abdomen have recently been published
(Roseland & Schneiderman, 1979; Madhavan & Madhavan, 1980,1982). When
completed, the adult abdominal tergum appears as a series of sclerotized
cuticular plates (tergites) which are covered with posteriorly oriented cuticular
outgrowths, viz. hairs and bristles. The tergites are separated from each other by
smooth intersegmental (intertergal) regions. Similarly, the adult ventrum
consists of a series of segmental, sclerotized plates (sternites) which are located
in the middle region of the ventrum. The anterior border of the sternites bear a
pair of sensilla trichodea (Wheeler, 1960; Lawrence, Green & Johnston, 1978).
The sternites bear posteriorly oriented bristles and hairs, and the latter as in the
tergite, are arranged in regular rows. Unlike the tergites, the sternites are
separated from each other by intersegmental (intersternal) regions which bear
posteriorly oriented hairs resembling those in the pleura (Fig. 1). Laterally, on
either side of the sternites, the unsclerotized cuticle (pleura) appears as a continuous sheet and bears regularly arranged and posteriorly oriented hairs. The
spiracles are arranged segmentally in the pleura, near the lateral boundaries of
the tergites.
During normal development, by 24 h after pupariation, the ADN and PDN
have fused and the fused dorsal nest and VN of the third, fourth and fifth abdominal segments contain on the average 786 and 479 cells, respectively (Madhavan
& Madhavan, 1980). In those animals which received 5 kR radiation, at 24 h after
pupariation, these nests contained slightly fewer number of cells than in normal
animals. However, when the radiation dosage was increased to 8kR, the ADN
and PDN remained separate (Fig. 2) and these and VN contained on the average
Fig. 5. Feulgen-stained whole-mount preparation of the posterior of the third and
anterior of the fourth hemitergite and their intertergal region of an adult resulting
from a larva which received 5 kR radiation. Note that the LEC which are immediately
posterior to the macrochaetae secrete hairs while those in the intertergal region
secrete smooth cuticle. (x400).
Fig. 6. Feulgen-stained whole mount of the pleura of an adult resulting from a larva
which received 12 kR radiation. Note that the hairs secreted by the persisting
polytene LEC show bulbous bases. (x580).
Fig. 7. The tergopleural border (dashed line) of the left fourth hemisegment of an
adult resulting from a larva which received 10 kR radiation showing two different
kinds of hairs secreted by the tergal (t) and pleural (p) LEC. s, remnant of spiracle.
(x610).
Fig. 8. Third sternite region of an adult resulting from a larva which received 10 kR
radiation, showing LEC bearing hairs. Note that these diversely oriented hairs, like
those on the tergite, are narrow, long and without folds at their bases. (x500).
4
M. M. MADHAVAN AND K. MADHAVAN
only 41,10 and 24 cells respectively (a total of 30 histoblast nests were analysed).
These values decreased further when the radiation dosage was increased to
10 kR, and at 12 kR radiation, the number of cells in the various nests remained
approximately the same as those observed in the late third instar larva (Madhavan & Schneiderman, 1977; Roseland & Schneiderman, 1979).
Animals which received 5 or 8 kR radiation contained more adult epidermal
cells which provided the landmarks of the pattern of the adult tergum such as the
hairs and bristles (micro- and macrochaetae) than those which received still
higher doses of radiation. Hence, we have used whole-mount preparations of
animals resulting from lower doses and to a lesser extent those which received
10 and 12 kR radiation for detailed analysis to compare the cuticular pattern
formed by the larval and adult epidermal cells.
The abdominal segments of animals which received 5 kR radiation, externally,
resembled those of newly emerged normal adults though with reduced number
of bristles, while those that received 8 kR radiation contained segments in which
the bristled area was restricted to a few hemitergites or parts of hemitergites of
segments. In the stained whole mount preparations of animals resulting from
5 kR radiation which failed to produce enough adult epidermal cells to fill the
hemitergum, the LEC remained in those areas not occupied by the adult epidermal cells. When this happened, the LEC situated in the anterior hairy, middle
hairy and bristled (Figs 3 & 4) and posterior hairy (Fig. 5) regions secreted hairs.
The LEC in the intertergal region, which could be identified from the insertion
sites of persisting larval muscles and the newly formed imaginal muscles,
secreted only smooth cuticle (Figs 4 & 5). Thus, the LEC existing at different
Fig. 9. Third sternite region of an adult resulting from a larva which received 5 kR
radiation. Due to radiation, the right ventral histoblast nest was destroyed and as a
result the right half of the sternite is formed by the LEC (enclosed by dashed line),
while the left half is secreted by the adult epidermal cells (enclosed by dotted line).
The latter secrete bristles and hairs which are oriented posteriorly. The LEC bear
hairs which resemble those secreted by the adult epidermal cells, but unlike the
latter, are oriented in different directions and are not arranged in regular rows. Note
the sensillum (arrowhead) among the LEC. (x440).
Fig. 10. Tergopleural region of a normal adult showing the hairs secreted by the
adult epidermal cells of these regions. Note that the hairs in the pleural (p) region
are with broader bases which are thrown into folds similar to those secreted by the
LEC occupying this region in the irradiated animal (see Fig. 7). s, spiracle; t, tergal
region. (xlOOO).
Fig. 11. Sternite region of a normal adult showing the arrangement of hairs and
bristles. The hairs resemble those of the tergite rather than those of the pleura
although both the sternite and pleura are derived from the progeny of the same
histoblast (ventral) nest. (x900).
Fig. 12. Feulgen-stained whole mount of the dorsum of an adult resulting from a
larva which received 8kR radiation. The third and fifth hemitergites contain adult
epidermal cells and bear their cuticular products. The fourth hemitergite is missing
and its intersegmental region (4) is fused with that of the third (J) hemitergite
forming a continuous smooth cuticle in the place of the missing hemitergite. (x 100).
Determination of cuticular pattern in Drosophila
5
levels in the tergal region of a segment showed capacities to secrete cuticle with
hairs or smooth cuticle similar to their adult counterparts characteristic of their
positions in the tergum. The hairs secreted by the LEC, as initially observed by
Poodry (1975), appeared different in their spatial arrangement from those
secreted by the adult epidermal cells.
The hairs of the tergal and sternal regions of the normal adult abdomen resemble each other while those of the pleura display a different morphology
(Madhavan & Madhavan, 1980). Similarly, the hairs secreted by the LEC in
these three regions in the irradiated animal appeared different in that those on
the tergum and sternum were narrow without any folds at their bases, while those
on the pleural region were broad based and the cuticle around their bases were
thrown into folds (Figs 6,7,8). These differences in the morphology of the hairs,
as in a normal adult, help to distinguish the tergopleural and pleurosternal
borders of the segment.
During normal development, the descendants of the ventral histoblast nest
form the sternite and part of the pleura of the adult abdomen (Roseland &
Schneiderman, 1979; unpublished observations). In those instances where the
ventral nest of one side of a segment was destroyed by radiation, the sternite of
the resulting segment was formed partly by the surviving LEC and partly by the
adult epidermal cells. The half containing the LEC secreted hairs and that containing the adult epidermal cells secreted hairs and bristles typical of sternite
region (Fig. 9). The LEC of the pleural and sternite regions secreted hairs which
were distinct from each other (Figs 7, 8) but similar to those secreted by their
adult counterparts (Figs 10, 11).
In those animals which received 8 kR radiation, when compared to those that
received 5 kR, the area covered by smooth cuticle in the dorsum was greater
while that with cuticular outgrowths was less. The larval and adult epidermal
cells in the different regions of the tergum secreted the characteristic cuticle and
cuticular outgrowths as described for animals receiving 5kR radiation.
Fig. 13. Unstained whole mount of the sixth sternite (st) and surrounding pleura (p)
of an adult resulting from a larva which received 8 kR radiation. Note the sensillum
(arrowhead) in the anterior region of the sternite. s, remnant of spiracle. (x200).
Fig. 14. Feulgen-stained whole mount of the left hemisternite of an adult resulting
from a larva which received 8kR radiation. The hairs secreted by the sternal LEC
(s) appear different from those by the pleural LEC (p). (x420).
Fig. 15. Unstained whole mount of the ventral cuticle of an adult resulting from a
larva which received 8 kR radiation showing the posterior of the fourth (st4) and
anterior of the fifth (sts) sternites and the intersternal region (is). Note that the
morphology of the hairs in the sternite appears distinct from that in the intersternal
area, although both are secreted by the persisting LEC. (x400).
Fig. 16. Dorsum of the right fourth andfifthhemisegments of an adult resulting from
a larva which received 10 kR radiation showing the predominant smooth cuticle on
the dorsum with narrow wedge-shaped hairy regions (enclosed by dashed line)
towards the lateral side. The arrow denoted the mid-dorsal line. (xl40).
6
M. M. MADHAVAN AND K. MADHAVAN
Occasionally, we observed that an entire hemitergite was missing except for a
small area with hairy cuticle at its lateral region. Its intersegmental region was
fused with that of the anterior segment resulting in the formation of a continuous
smooth cuticle in the place of the missing hemitergite (Fig. 12).
On the ventral side, the sternites were represented as islands of larval cells that
secrete hairs characteristic of the sternite surrounded by other LEC that secrete
hairs characteristic of the pleural (Figs 13,14) and intersternal regions (Fig. 15).
These sternal islands also contained either paired or single sensilla at their anterior
region (Figs 9,13). A larval epidermal cell was found beneath each sensillum, and
in the vicinity of this larval epidermal cell was a cluster of three small nuclei.
Those animals which received 10 and 12 kR radiation contained mostly or only
LEC in the hemitergum. Since there were no histoblasts to replace the LEC as
in normal development, the decreased number of LEC in the different regions
of the hemitergum of the experimental animals could be due to radiation damage
or metamorphic changes or both. The LEC in the lateral region of the hemitergum secreted hairs. Since the rest of the cuticle in the tergum was smooth, we
conclude that the LEC present underneath the smooth cuticle belong to the
intersegmental region of the hemitergum. This behaviour of the two groups of
LEC resulted in a smooth cuticle with narrow, lateral wedge-shaped hairy
regions in the terga of the abdominal segments (Fig. 16).
DISCUSSION
The ability of the LEC of Drosophila either to secrete or not to secrete
cuticular outgrowths in various positions of the tergum, and their capacity to
produce different kinds of hairs on the tergum versus pleura corresponds very
well to that of the adult epidermal cells occupying similar positions in these
regions. Similarly, the capacity of the LEC occupying the sternal region of the
segment to produce a different kind of hair compared to those occupying the
pleural and intersternal regions also corresponds to the expression of the adult
epidermal cells occupying these different regions. This experimental exposition
of the latent ability of the LEC to express the regional differences seen in the
adult pattern is the first of its kind to be recorded for any higher holometabolous
insects.
Poodry (1975) suggested that the surviving larval epidermal cells during
pupal-adult moult secrete another first instar-like cuticle. During normal
development, the epidermal cells of the dorsum of the abdominal segments of
the first instar larva, except those in the inter-segmental region, secrete fine
unpigmented hairs while those on the ventrum and, dorsum and ventrum of the
second and third instars do not (Lohs-Schardin, Cremer & Niisslein-Volhard,
1979; unpublished observations). As seen in the present study, however, when
the LEC were allowed to survive metamorphosis they secreted hairs not only on
the tergal, but also on the pleural and sternal regions during pharate adult
Determination of cuticular pattern in Drosophila
7
development. This would, therefore, argue against Poodry's suggestion that the
surviving larval epidermal cells are responding to the hormonal milieu by secreting another first instar-like cuticle.
The LEC are different from the adult epidermal cells in that they are not able
to secrete the micro- and macrochaetae. This difference in the activity of the two
kinds of cells may not reflect a true difference in their informational content;
rather, it may reflect their way of life during ontogeny. The precursors of the
chaetae-secreting cells undergo proliferative, and finally, differentiative
divisions during the formation of chaetae as described for both hemi- and
holometabolous insects (Lees & Waddington, 1942; Peters, 1963; Lawrence,
1966), while the LEC remain polytenic and non-dividing throughout their postembryonic life.
The sensilla trichodia found in the anterior region of the sternites appear
similar to those observed in the corresponding region of the normal adults.
Sensilla differentiation has been studied in detail in the hemimetabolous
insect, Oncopeltus fasciatus by Lawrence (1966). His studies showed that
sensilla differentiation involves the same sort of differentiative mitoses observed during bristle formation. In Drosophila although the distribution and
morphology of sensory hairs on the larva and adult have been reported by
Kankel et al. (1980), their development has not been described. In the present
study, we could not determine with certainty whether the larval epidermal cell
underlying the sensillum or the group of three small cells in its vicinity is
responsible for its secretion in the anterior region of the sternites of the
irradiated animals.
Since the LEC which were established in the embryo at the time of blastoderm
formation have a similar ability to produce the adult pattern of hairy and smooth
cuticle as the histoblasts, it is tempting to suggest that during metamorphosis the
LEC could provide their positional information to the dividing and spreading
histoblasts. The histoblasts could interpret this positional information and form
the characteristic pattern of the cuticular landscape of the adult segment during
metamorphosis. If this proves to be true, it remains to be seen how this information transfer occurs from the LEC to histoblasts and their progeny. Also, then
the histoblasts, as in their other characteristics (cell division, growth pattern and
cuticle secretion) may prove to be different with regard to information transfer
for pattern formation, from their counterparts, i.e., the imaginal discs, which
form the adult head and thorax, since several experimental studies (Gehring,
1978) have clearly shown that whole or pieces of imaginal discs are capable of
expressing their pattern autonomous of the LEC. Further, we believe that our
hypothesized information transfer from the LEC to histoblasts as seen in the
renewal and replacement of the epithelium of the adult abdominal segment of
Drosophila could also occur in other instances of cell replacement seen during
the development of many of the internal organs of the adult fly (Bodenstein,
1950; Anderson, 1964).
M. M. MADHAVAN AND K. MADHAVAN
We thank Professor Carroll Williams, Dr Sam Wadsworth and the anonymous reviewers for
carefully reading the manuscript and offering valuable suggestions, Dr William Gelbart for the
use of radiation facilities, Ed Seling for help with scanning electron microscopy and Claire
Erickson for assistance in photography. We also gratefully acknowledge thefinancialsupport
from the White Hall Foundation and Monsanto Company.
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(Accepted 21 March 1984)