Experimenid

THE

EFFECT
EYE

Crll

OF 5-FLUOROURACIL
PIGMENTARY

EPHESTIA

Restwch

37, S&6 I (1965)

ON THE

SYSTEM

IN

K UHNIELLAI
W. MUTH2

Department

of Biology,

University
Received

qf Rochester,
February

Rochester,

N.Y.,

U.S.A.

24, 1964

THE development

of the eye pigments in insects has proven to be of advantage in studying

the relation
between
gene-controlled
processes
and the
appearance
of morphological
phenes. However,
\vhile workers
in this field
have succeeded
in the elucidation
of metabolic
pathways
leading to the
formation
of the pigments and their dependence on the genetic constitution,
our knowledge
of the mechanisms
controlling
the timing of gene-controlled
processes and of turning on and off the machinery
provided
by the suitable
genetic constitution
is far from being understood.
In order to attack this
problem,
some experiments
were undertaken
\vith the hope to detect and
time some of the steps in the ontogeny of the final phene.
The use of antimetabolites,
in particular
of halogenated
pgrimidines,
which during the past years have proved to he useful tools in the study of
nucleic acid and protein biosynthesis,
appeared to be a promising
method
for an attack on the problem of timing in development
since their activity
in metabolism
is relatively well understood.
Since it may be presumed
that
the initiation
of a developmental
process proceeds under rapid synthesis
of
enzymes and specific structures,
antimetabolites
might be expected to interfere with these processes in specific ways. In the formation
of eye pigments
in insects it is knoxvn that the pigments themselves
are tryptophane-derived
ommochrome
pigments
and pteridines,
and that their deposition
involves
the formation
of RNA containing
precursor
granules which show enzymatic
activity [5, 11, 181. It was therefore decided to study the influence of 5-fluorsince 5-fluorouracil
may be expected
ouracil on developmental
processes,
to interfere with the synthesis of normal RNA.
1 This work has been supported
by contract
AT
mission.
z Present address: Institut
ftir Entwicklungsphysiologie,
Experimental

Cell Research

37

(30-l)-2902

of the U.S.
Universitgit

Atomic

zu Kiiln.

Energy

Com-

Effect

of 5-fluorouracil

MATERIALS

on eye pigmen tnry system

AND

METHODS

A wild-type
Ephestia
strain, originally
obtained from North Carolina (NCR) and
inbred for 47 and 48 generations
in this laboratory,
was used. The larvae were fed
on yellow cornmeal and raised in rectangular
plastic jars in a constant temperature
room at 2%24C.
In order to obtain pupae of exactly known age, fully grown larvae at the beginning of the prepupal
stage were collected
and transferred
to Petri dishes with a
small amount of cornmeal.
At intervals
of 6 hr these dishes were checked and the
freshly molted pupae removed. They were treated at the following
times:
%nd day: 30-36 and 42-48 hr after
1st duy: 6-12 and 1X-24 hr after pupation;
pupation;
3rd day: 54-60 and 66-72 hr after pupation;
4th day: 78--84 hr after pupation; Sth day: 102-108 hr after pupation;
6th day: 126-132 hr after pupation;
7th day:
150-156 hr after pupation.
5-Fluorouracil
(5-FU) was a gift from Hoffmann-La
Roche, Inc., nutley,
N.J.
Its application
was simple: At the desired age the etherized
pupa was punctured
with sharp watchmaker
forceps between the 5th and 6th abdominal
segment just
above the heart tube, and a small crystal (about $ mm in diameter)
was pushed into
the abdomen. The wound was sealed with hot paraffin. All operations
were done at
10 times magnification
under a stereomicroscope.
Although
the dose of 5-FU administered
in this way was inaccurate,
the experiments yielded precise and completely
reproducible
results without
any variation.
Apparently
the amount
applied constituted
the saturation
dosis with respect to
the phenomena
studied.
In order to estimate the interval from the time of implantation
to the time when
5-FU reaches the sites of action, the following
tests were carried out: crystals were
implanted
into 24-hr-old
pupae and every 15 min thereafter
three animals were
examined.
After 45 min about + to 2 of the crystal was dissolved and after 16 hr
no crystal could be recovered. The distribution
of a dye was used to obtain a rough
estimate of the rate of transport.
Small crystals of fast green, implanted
into 6-12hr-old pupae, led to coloration
of the eyes after 30 min and after 2 hr to coloration
of the wing-lacunae.
In S-day-old pupae, I hr passed until the dye could be demonstrated in the basal parts of the legs (it was not visible in the eyes because of their
dark pigmentation)
and after 4 hr it reached the tips of the wings.
In spite of its obvious disadvantages
this method of application
of the drug was
chosen, because it made possible the application
of 5-FU without
additional
complications
such as osmotic shock, liquid pressure and salt effects, so that the death
rate could be kept down close to zero. After treatment
the pupae remained
in Petri
dishes on filter paper at the same temperature
as before. At the end of the experiment the eyes were drawn, the heads cut off and prepared for paper chromatography.
Five, 10 or 20 heads per group were ground up in 0.2-0.4 ml methanol/HCl
(2 per
cent HCl) and after centrifugation
the extracts were transferred
t.o Whatman
No. 1
filter paper strips and chromatographed
in ascending
direction
with formic acid
(85 per cent)/methanol/HCl
(10 per cent) in ratio 80:15:0.5
[14].

Experimental

Cell Research

37

RESULTS

General effects of treatment.--Under

the conditions
employed,
the pupal
stage in Ephestia lasts 13 days. Only a few late-comers
hatch on the 14th day.
Owing to this insignificant
variability
it was sufficient
to use only 5 pupae
per group in the first control experiments.
These animals were xvounded in
the same region at difrerent times and sealed in the same way as the experimental pupae, without receiving a crystal of &FU. In contrast to the ohservations made on growing
larvae [lS, 161 the adults emerging from these
wounded
pupae hatched at the same time as uninjured
controls, regardless of
the time at which the wound was made. All developmental
changes which
were examined occurred
at the same time in both groups.
Implantation
of .5-FLJ affects these processes
drastically.
Implantation
during the lst-3rd
day causes almost immediate
cessation of most visible
developmental
changes. Dissection
on the 12th day reveals that the body
c,avity is still filled with liquefied fatbody and tissue debris. No outgrowth
of scales has taken place and the hypodermis
has remained firmly attached
to the overlying
chitin. On the 6th and 7th day small brown spots appear
within the still transparent
wings and legs, the first indication
of beginning
degeneration.
Nevertheless,
the animals survive until at least the 13th day
after pupation
as indicated by their continued
ability to move and by their
respiratory
activity.
A sudden change in response to 5-W treatment
occurs between the 3rd
and 4th day after pupation. Upon implantation
on the 4th pupal day development continues normally
up to the 11th pupal day, when the pigmentation
of scales sets in. The pupa is able to develop into a complete,
normally
shaped moth with fully outgrown
scales which can easily be pulled out of
the pupal case. Its scales, however,
are unpigmented.
These animals are
not able to hatch by themselves
and implantation
up to the 9th day still
prevents hatching.
The distribution
and spreading of the eye pigment.-The
eye is the only
organ that shows
some continuing
development
even after implantation
into 6-12-hr-old
pupae. At this time the eye is colorless and transparent,
so
that the tracheae running through the head are clearly visible. Only in the
dorsocaudal
corner appears a faint orange tinge due to a few pigment spots
(Fig. 1). During normal pupal development
the pigmentation
and differentiation process of the disc proceed from this region in concentric
circles spreading over the eye 113, 171. The progress of pigmentation
with increasing
age
of the pupa is shown in Fig. 1, upper row and in Fig. 2. The drawings
in
Experimental

Cell Research

37

Effect of A-fluorouracil

66-72

78-M

on eye pigmentary

102-

106

system

126 - 132

57

f50-

156

Fig. l.-The
progressive
pigmentation
of the eye. The numbers
on top indicate
the age of the
pupa when 5-FU was implanted.
The upper row of drawings
shows the state of pigmentation
at
the time of treatment;
the lower row shows the state finally
reached.

Fig. 1 are based on the examination

of 5 animals per class in the control
group (upper row) and of 10 animals per class in the experimental
group
(lower row). In normal development,
the pigment remains restricted
during
the first 3 days to a halfmoon-shaped
region in the dorso-caudal
area of
the eye and consists of small red spots easily visible under the binocular.
Its outer boundary
is presented by a sharp bend in the surface of the eye.
Experimental

Cell Research

37

58

11. illufh

Fig. 2.-Development
of normal
(c) 126-132
hr; (d) 174-180
hr.

eye pigment

in the

Ephestia

pupa.

((I) 42-48

hr; (h) 90-96

hr;

Between
60 and 66 hr the pigmented
area begins to expand beyond this
border and tiny red orange dots appear in the more distal parts. While
these cover more and more of the surface of the eye, the dorso-caudal
part
gradually
turns into a darker red to red-brown
color, a process that subsequently
extends also to the distal area. Between
108 and 126 hr the pigment reaches the anterior edge of the eye. While the most distal part still
darkens,
the eye at 150-156 hr becomes filled all over simultaneously
with
a dark pigment, thus leading to the final homogeneously
black coloration.
On the 8th day these processes which can be observed by surface inspection
Experimental

Cell Research

37

Erect

of 5-fluorourucil

Fig. S.-Result
of implantation
of 10 days. (a) Implantation
hr; (d) implantation
at 54-60

on eye pigmentury

59

system

of 5-F1J crystals
into pupae of known
ages. All observed
at age
at 6-12 hr; (b) implantation
at 18-24 hr; (c) implantation
at 30-36
hr.

are completed.
(For the histological
changes correlated
with the pigmentation, [see 13, 171.)
The effect of 5-FU on the pigmentation
process.-If
applied at early stages,
5-FU blocks the expansion
of the pigmented zone, the level reached depending on the time of implantation
(Fig. 1, lower row and Fig. 3). In general,
the older the animal is at time of implantation,
the farther the pigmentation
does proceed. After implantation
into 6-12-hr-old
pupae the pigment remains
restricted
to the halfmoon-shaped
region in the dorso-caudal
area, but during
Experimental

Cell Research

37

60

W. Muth

the first day following

treatment the pigment belt reaches the caudal edge of
the eye and from the second day on a darkening
of the originally
orange
pigment sets in, leading through bright and dark red to a brown color which
remains unchanged
after the 4th day. Implantation
12 hr later allows the
pigment to trespass the sharp anterior boundary
and in animals treated at
42-48 hr more than half of the eye becomes finally pigmented.
Treatment
with 5-FU at 66-72 hr does not stop the spreading
process;
the pigment
reaches the anterior edge on the 5th day.
In all animals treated between 6 and 72 hr, the pigment finally present
is of a brown color instead of black as in the controls and is concentrated
in
small spots distributed
evenly over the pigmented
zone. After the 8th day
neighboring
spots fuse, forming
smaller or bigger masses of pigment, thus
disturbing
the original uniform
pattern. Probably
this is due to a lack in
the normal histological
differentiation.
Though implantation
into pupae 72
hr old does not stop the spreading
of pigment before it reaches the edge of
the eye, it is still capable of inhibiting
the development
of a homogeneous
black coloration.
However,
eight out of ten animals treated 12 hr later
showed the normal evenly black coloration
on the 8th pupal day, while the
other two had the brown
spotted pigmentation
characteristic
for those
implanted
at 66-72 hr. From this time on 5-FU no longer interferes
with the
pigmentation
potencies of the eye, although these are realized 3 to 4 days
after the treatment.
Effects of 5-FU on the composition
of the pigment.-The
pigment of the
adult Ephestia eye can be separated by paper chromatography
into at least
three components
[13, 141: III, a yellow pigment with the highest R,-value;
I, a carmine red pigment; and 0, a violet pigment closest to the starting line.
A fourth component
(II) has been described as present in very low amounts.
All four components
could be demonstrated
in the material
employed
in
these investigations,
II as a very weak band. 0, I and II shared the characteristic reaction of ommochromes
on being sprayed with diluted acetic acid
solutions
of KaSO, and NaNO, (red and yellow or brown coloration,
respectively).
The most rapidly
migrating
substance,
III, however,
which
has
been shown by Kuhn and Egelhaaf [14] to be xanthommatin
gave no reaction with acidic NaSO, solution, and until this time attempts to extract it
from our strain according
to the methods described
by Butenandt
et al.
[2, 31 were unsuccessful.
Thus the question as to the nature of the compound in strain IVCR occupying the position of xanthommatin
remains open.
Spot 0 is constituted
by ommin,
and spot I by an ommatin of unknown
constitution
[ 131.
Experimental

Cell Research

37

Effect of 5fluorouracil

on eye pigmentnry

61

system

On chromatograms
of extracts from twenty normal heads component
III
could be observed on the 2nd pupal day, though in very low amounts,
but
during the following days it increased gradually. Component
I was first visible
as a faint red streak on the 4th day, also with subsequent increase in quantity.
The violet pigment 0 did not appear before the 7th day and was present in

Component

TABLE

I.

Normal
day
appearance

of

Formation
by 5-FU

inhibited
before day

2
4

I
0

III

large amounts one day later. Component

II was omitted in this study, since
its concentration
is very low.
Eyes of animals treated with 5-FU during the lst-3rd
pupal day and killed
on the 10th day contained both components
I and III but showed no trace
of component
0. The amounts formed appeared to be reduced as compared
to untreated eyes of the same age, probably
in part due to the restriction
of
pigment carrying cells to limited regions of the eye (see Figs. 1 and 3). Even
in animals implanted
at 6-12 hr after pupation
and killed on subsequent
days, the occurrence
of both pigments could be demonstrated
at exactly the
same age at which they appeared in the controls. The formation
of component
0, on the other hand, was completely inhibited by implantation
of S-FU up
to 72 hr, but not by implantation
into older pupae. Upon treatment at 78-84
hr the animals were capable of forming so much pigment, that it was possible to recognize
all components
by chromatography
of the contents
of
single heads squashed on the starting line. Ten animals examined in this way
on the 10th day contained a high amount of component
0, comparable
to
that found in the controls.
Implantation
into later stages also had no effect
on the development
of eye pigmentation,
as far as could be judged by the
appearance
of the eye and by chromatography
of the pigments.
Table I
summarizes
the results of the chromatographic
studies.

DISCUSSION

From the results presented it appears that 5-FU separates the development
of the eye pigmentary
system into two separate phases. The first phase is
marked
by a spreading process, which enables the cells taken over by it to
Experimental

Cell Research

37

form two pigment components,

spots I and III. As can be seen by comparing
the upper and lower row in Fig. 1, the final pigmentation
covers a larger
area of the eye than it had reached at the time of implantation.
Obviously
the process blocked is not the pigmentation
process itself but the spreading
of a latent state prec.eding the deposition
of pigment, which provides
necessary conditions
for the final synthesis
of the pigment. This latent spreading
process starts immediately
after pupation
and reaches the anterior edge at
72 hr. The pigmentation
process itself, however,
does not cxpantl
from
the dorso-caudal
corner until 60 hr after pupation.
Then it proceeds synchronously
in both control and <S-FL treated animals, but it stops in espcrimental animals treated before 66 hr before reaching the anterior border of
the eye, indicating
the level that has been reached by the S-FL: sensitive
process.
The finding, that both the expansion
of the pigmented
area and the appearance of the individual
pigment components
take place at the proper
time, indicates that WC are dealing here with the realization
of normally
acquired
potcncics
rather than with phenomena
like those described
by
Anders
and I:rsprung
[l!, where clumps
of ommoc.hrome
pigment were
produced
as a result of tissue damage following
mechanical
injury or L\irradiation.
Since the pigments in Ephestia as \vell as in other arthropods
arc
deposited
on small cytoplasmic
granules which contain ribonucleoprotein
[A, 3, 11, 181, it appears tempting to suppose that the first process, the spwading over the eye of a state preparatory
to the formation
of pigment, may
consist in the progressive
establishment
of new precursor
granules.
The second phase is concerned
with creating
conditions
which
make
possible the synthesis of component
0. It ends between 7X and 84 hr, 3 days
before the actual synthesis
of the pigment takes place. It is noteworthy
that
this time coincides with that at which the spreading process has just covered
the whole eye disc, while the deposition
of the pigment components
I and
III is still under way. Thus it seems that the 5-W blocked properties,
necessary for the production
of component
0 are gained suddenly
throughout
the whole eye after the end of the first phase, upon completion of the spreading
process.
Treatment
with 5-FU is, then, able to divide the development
of eye
synthesis
of the two ommatins
pigmentation
into two separate processes:
I and III, and synthesis of ommin. That these two processes are independent
of each other is indicated also by the fact that they occur in normal development at different times, and that the formation
of the ommin (component
0)
is selectively inhibited by the gene mutant br described
by Kiihn and EgelExperimental

Cell Research

37

Effect of 5fluorourncil

on eye pigmentnry

system

63

haaf [IA]. It can furthermore

be concluded
that 3-K
does not affect the
differentiation
of the pigment itself, but a preparatory
process which may
This process, in the case of the ommatins,
be compared
to determination.
spreads
gradually
over the eye in the course of 60 hr, preceding
the dcposition
of pigment.
In the case of ommin,
it seems to take place in all
cells of the eye at the same time, about 3 days before the actual appearance
of the pigment.
The \vay in \vhich 3-K
affects the preparatory
process for pigment formation is of interest, because it would permit some insight into the nature of the
several different possibilities
which cannot
process. There remain, however,
be distinguished
on the basis of the present experiment.
Since neither treatment with 5-bromouracil
nor with uracil or fluorophenylalanine
gave results
comparable
to the effects of 3-FU, it may he concluded
that the effects are
characteristic
for this substance. As an analogue of uracil it may he expected
to be incorporated
preferentially
into RXA, as has been she\\-n to he the
case in bacteria [12], viruses [9] and mammalian
cells [A]. But since insects
show some peculiarities
with respect to metabolic
paih\vays
(for example,
review by Gilbert and Schneidermann,
[S] it cannot be taken for granted
that findings obtained with other groups of organisms
can be applied to
insects. If, however,
this is assumed, two possibilities
deserve consideration:
S-176 might he incorporated
into newly formed
messenger
KNA and iw
activate it; or it might be incorporated
into the HNA of the precursor
granules,
modifying
their structure
in such a way that the normal attachment
of the
enzymes active in pigment synthesis is disturbed,
or that the enzymes themselves become abnormal
[lo].
h second possible effect of 5-K
to be kept in mind is its inhibitory
action
on DNA-synthesis
upon its conversion
into 5-fluorodeoxyuridine
by the
inhibition
of thymidylate
synthetase
[7]. It may be supposed that this activity
\vould play a prominent
role in processes \vhich are accompanied
by cell
divisions.
Since, according
to Umbach
[17] little cell division
takes place
in the eye after pupation,
and the dividing cells are restricted
to the distal
layers of the eye disc which are poor in pigment-forming
cells, this mechanism
of 5-FU action on eye pigmentation
appears more remote.

SUMMARY

The action of 5-fluorouracil on the development of the eye pigmentation

in the pupa of Ephestia has been studied descriptively and by means of
paper chromatography.
It was observed that treatment with ;I-FU up to 60
Experimental

Cell Research

37

hr after pupation
blocks
a spreading
process
which
is necessary
for the
synthesis
and deposition
of the two pigment
components
which
appear
first
in development.
Treatment
with 5-W up to the fourth
tlay after pupation
suppresses
completely
the formation
of a third
component
of the pigment,
while
after this time S-FU has no effect on the formation
of the eye pigments,
even though
the third pigment
component
appears
only at the 7th clay after
pupation.
The results
are discussed
with respect
to possible
mechanisms
untlerlping
the effects of 5-FU on the processes involved
in pigment
formation.
I wish to express my gratitude to Dr Ernst W. Caspari for the hospitality
I received
in his laboratory
and for his continued
encouragement
during the course of these
investigations,
and to Mr Nicholas
Cohen for his help in the preparation
of the
photographs.
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G. and URSPRU~-G,
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A. and NEUBERT,
G., Z. Physiol.
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A., SCHIEDT,
U., BIEKERT,
E. and KORNMAX,
P., I.iebigs
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(1954).
CASPARI,
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Experimental

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37