Compartments and Polyclones
   in Insect Development

Clones made in early development keep within certain
      fixed boundaries in the insect epithelium.

F. H. C. Crick and P. A. Lawrence

In this article our aim is to describe re-
:ent work on the development of intact
spithelia and in particular the important
results and ideas of Professor Antonio
Garcia-Bellido (1) and his group in Madrid
which are not yet widely known. We try
to explain as clearly as possible what these
ideas are and what sort of experiments
have been done to support them. Some of
the more obvious questions arising from
tbe results and how the new concepts may
relate to other ideas such as "gradients"
are listed.

Development of DrosopLiln

The development of an adult Drosophila
is a complex process. The nucleus of the
fertilized egg divides a number of times to
form a compact mass of about 250 nuclei,
near the center of the egg, without cell
walls. These nuclei then migrate outward
to the inner surface of the egg where for
the first time cell membranes are formed.
The cells divide several more times to form
a single layer of cells, about 4000 in all, lin-

Drs. Crick and Lawrence work in the Cell Biology
Division of the Medical Research Council Laboratory
of Molecular Biology, Cambridge, CB2 2QH. En-
gland.

340

ing the inside of the egg. This is called the
blastoderm. Behaving as a sheet of cells,
the blastoderm undergoes complex folding
movements generating a multilayered
germ band, which soon becomes visibly
segmented. The egg hatches after 24 hours
and the animal then goes through three lar-
val stages each separated by a molt. After
these larval stages, lasting in all about 96
hours, the animal then pupates and meta-
morphoses into the adult fly.

This adult is formed mainly from special
groups of cells in the larva which them-
selves take little or no part in larval devel-
opment or function. These are the histo-
blasts and the imaginal discs. There are 19
of the latter (nine pairs of discs plus the
single genital disc). We shall concentrate
mainly on one pair of these, the so-called
wing disc. The left wing disc, within the left
side of the larva, produces the left wing of
the insect and that part of the dorsal left
side of the thorax next to the wing.

The wing disc is seen in the first larval
stage as a small patch of embryonic epider-
ma1 cells (2). These cells remain diploid,
while the surrounding larval epidermal
cells become polyploid (3). There are prob-
ably only about 15 to 30 cells forming the
wing disc at this early stage (46). During
the course of larval growth these disc cells

divide in all about 10 or I I times (on aver-
age) to give a total of some 50,000 cells (5).
Shortly after puparium formation cell divi-
sion of the disc stops. The disc has now a
characteristic size and shape, being some-
what like a flattened and heavily folded
balloon (7).

At metamorphosis a complicated set of
cell movements occurs, and these result in
the disc being turned inside out so that it
can form the adult structure. The wing it-
self, for example, is first formed as a bag.
The bag is then collapsed to form the adult
wing, which thus becomes a single sheet of
epithelial cells folded and collapsed to
form a double layer of epithelial cells.

Basic Ideas of Clonal Analysis

For the purposes of exposition we now
temporarily leave the wing and describe a
hypothetical sheet of "white" epithelial
cells on the adult fly. We imagine that we
have at our disposal a special technique
that enables us to mark (say black), at ran-
dom, a single cell in a developing disc. The
mark is such that it does not interfere in
any way with the normal development of
the animal. Moreover, all the descendants
of this marked cell retain the mark and can
be recognized in the adult. The method of
marking has the advantage that we can
choose fairly precisely when, in devel-
opment. we mark the cell; but it has the
disadvantage that we cannot mark a par-
ticular cell at that time, but only one cho-
sen at random, and in early stages we usu-
ally mark only one cell in any one individ-
ual. If we assume that the significant fea-
tures of the process are effectively the same
in all individuals, we can piece together
what is happening in development by com-
bining experiments on many different indi-
viduals.

What do we find? Naturally, we see a set
of black cells in the adult, but how many of
them are there, and how are they ar-
ranged?

The first observation is what might be

             SCIENCE. VOL. 189


Fig. 1. A clone descending from a cell marked prior to the formation of the compartment border (XY). The clone is smooth at the edge of the structure
(a) but rough elsewhere (6).  Fig. 2. A clone descending from a cell marked after the formation of the compartment border.   Fig. 3. The clones
made by the two daughter cells of that cell generating the clone shown in Fig. 1.  Fig. 4. The clone made by a cell marked prior to the formation of
the compartment horder, both of whose daughter cells give rise to clones within one subcompartment.

expected. In general, the earlier a cell is
marked in development, the more black
cells we find in the adult. A cell marked
early leaves more descendants than a cell
marked late.

The next obvious question is: What frac-
tion of the total cells are marked? By the
total cells we mean the number of cells in
that portion of the adult epithelium which
has come from the set of cells under con-
sideration in the larva (for example, the
50,000 epithelial cells that come from a
single wing disc).

The number of black cells produced by
marking at a fixed time is not exactly con-
stant, but the variation is such that we can
usefully calculate its average value. If the
average number of black cells in the adult
is, say, a tenth of the total then making cer-
tain reasonable assumptions there were, on
average, about ten cells in the larval set at
the time they were marked (8). As the time
of marking gets later and later in devel-
opment this fraction gets smaller and
smaller, and the frequency of marked
clones produced increases.

From the arrangement of the black cells
we can learn something about their move-
ment during the interval between irradia-
tion and observation. For instance, if there
is a pepper and salt mixture, the cells must
have been intermingling; while a coherent
patch suggests that all the daughter cells
have remained in contact during growth.
The shape of the patch is also informative.
For example, if it is long and thin this may
result from the cell divisions being pre-
dominantly oriented in one direction. In
the case of the wing disc, it is found that
the patches are usually both coherent and
elongated so that the long axes of the
patches are parallel to the long axis of the
wing (4,5. 9).

We must next ask: Even though a patch
is irregularly shaped, is the shape the same
in different individuals? The experimental
results show that it is not so. Consider a set
of experiments in which the mark was
made at more or less the same time in the
development of a number of different indi-

I AUGUST 1975

viduals. Then it is found that the patches
produced, when all drawn on the surface of
a single idealized adult, do not neatly cover
the entire epithelial surface, without either
overlapping or leaving spaces (like a jigsaw
puzzle). On the contrary, if two patches
from separate individuals have ended up in
roughly the same place, then it is always
found that each partly overlaps the other
and are usually of different size. This result
shows that the cell lineage in Drosophila
epithelium is not strictly determined in the
same way in all individuals.

After all these preliminaries we can now
approach the important result. Let us as-
sume that our hypothetical piece of epithe-
lium is smooth in outline, as shown in Fig.
I. Then perhaps it is not too surprising to
learn that a black patch near the borders of
this piece of epithelium has itself a rather
smooth outline where it follows the bound-
ary of the area but has a rough outline,else-
where. We assume that at the earliest
stage of marking (that is, when the disc
is first formed) a black patch can be pro-
duced anywhere within our area. In partic-
ular it may have the size and shape shown
in Fig. I. We now ask: Suppose the mark
is made a little later, say, one cell genera-
tion later, what will the patch be like?
Naturally, it will, on an average, be half
the size, and we expect it to have an ir-
regular outline except where it touches the
area border. But now in some cases a new
and totally surprising restriction appears.
When all the results from many different
patches are combined, it is found that a
rather smooth line (marked Xv) can be
drawn, dividing our hypothetical area into
two distinct parts, such that no black
patch, made at this later time, will ever
cross this line. Moreover, the outline of a
patch touching this line is smooth where it
runs along the line but rough elsewhere
(Fig. 2). And this in spite of the fact that a
patch marked one cell generation earlier
can cross this special line.

The surprising nature of this result can
be seen by going back and considering the
entirely irregular patch illustrated in Fig.

I. We drew this particular patch (marked
at the earlier stage) across the special
boundary XY. We now ask: What would
Fig. 1 look like if instead of just marking
that particular cell we had been able to put
a different mark on each of its two daugh-
ter cells, produced one generation later?
We should now find two adjacent patches,
each with an irregular outline except where
the patches touched. Along the line of con-
tact their outlines would be smooth and
fairly straight (Fig. 3). This result is true
only if the double patch crosses the special
line XY. Otherwise the contact outline of
the two daughter patches would be irregu-
lar (Fig. 4).

Garcia-Bellido, Ripoll, and Morata (10)
have called an area bounded by these
special demarcation lines a compartmenf.
The progeny of a cell marked at about the
time of the drawing of boundary lines
never fills a compartment completely, but
often occupies an appreciable proportion
of it. A compartment is thus made by the
descendants of a small group of cells. We
propose to call the cells in the compart-
ment apolyclone. Just as a clone is a group
of cells which are all, without exception,
the descendants of a single cell, so a poly-
clone is a group of cells that are descended
from a certain (small) group of cells-the
founder cells-which were present in the
embryo at an earlier time. Moreover, in
our terminology they are all the (surviving)
descendants of that small group. This last
point is vital since necessarily all the cells
in a compartment are, for example, de-
scendants of the fertilized egg. The dis-
tinction is that some of the descendants
of the egg make up other parts of the
body; that is, they end up in other com-
partments. The members of a polyclone,
however, all fall within one compartment
and account for all the cells in that
compartment.

This point can be made more sharply.
Consider the small group of cells, the
founder members of the polyclone, and
then consider their immediate ancestors.
Then (except in rare cases) this earlier

341


group will not form a polyclone for the
compartment under consideration. That is,
we will usually find that some of the de-
scendants of these cells end up outside the
compartment we are considering. The cells
in the compartment are necessarily all de-
scended from this smaller group, but they
are not all the surviving descendants.
Therefore, this earlier group are not the
founder members of the polyclone for that
compartment.

The other side of the idea must also be
mentioned: a compartment is never a
clone, except perhaps accidentally in rare
cases. That is, for most cases, the cells in a
compartment cannot be traced back to any
single cell, all of whose descendants fall
within the compartment. This idea, which
implies that for these properties cells are
switched not singly but in groups, is impor-
tant (I [).

We thus see that the idea of a com-
partment and the idea of a polyclone are,
at the moment, intimately connected. As
things stand at present we have no other
reliable criterion for the sharply defined
region we call a compartment except that
a marked clone produced after a cer-
tain time in development will never cross
over the compartmental boundary and in-
clude any part of any other compartment;
whereas clones formed earlier may well do
so. Reciprocally, we cannot say that a
group of cells form a polyclone unless we
first define the compartment to which the
polyclone refers.

We must now consider the second major
fact about certain compartments, namely
that as time goes on they become subdi-
vided. Let us call a certain compartment
camp I; at a later time it will be subdivided
into two compartments which we may call
camp IA and camp 1B. These two sub-
compartments are not necessarily equal ei-
ther in area or in number of cells but to-
gether they add up exactly to camp 1.

By definition all these compartments are
polyclones. That is, the ancestors of all the
cells contained in each compartment can
be traced back to a founder group, early in
the embryo, all of whose descendants end
up in a compartment being considered. It
is an experimental fact that one marked
clone of cells, started from a single cell at
a certain early stage, may stay entirely
within camp I and yet go across the border
between camp 1A and camp IB. A marked
clone made at a slightly later stage, how-
ever, will never cross this boundary. This
implies that in any particular case the cells
that are the founder members of the poly-
clone for camp I form three classes: those
whose descendants will fall (i) wholly with-
in camp IA, (ii) wholly within camp lB,
and (iii) partly into camp 1A and partly
into camp IB.

342

It is this third class which explains why
early clones can cross a subcompartment
boundary whereas later ones cannot. How-
ever, at a slightly later stage in embryogen-
esis some further developmental step must
take place, since at that time the descend-
ants of the founder members of camp I
will fall strictly into the first two classes
listed above. No cell will then be found
with the properties of class iii. Every cell
in this enlarged group will be either a
founder member for camp IA or a founder
member for camp IB. In short, whereas
before only one polyclone existed, that
polyclone can now be considered as the
sum of two distinct polyclones.

The work of Garcia-Bellido and his col-
leagues shows that this process of forming
subcompartments  within larger com-
partments can happen several times in suc-
cession. The data suggest, but do not
prove, that the division takes place each
time into just two parts.

The Methods

We shall now illustrate the methods
used in clonal analysis by describing in
outline the techniques employed by Gar-
cia-Bellido er 01. (IO) in their detailed stud-
ies of the wing disc of Drosophila melano-
gosfer. The wing disc is strictly called the
dorsal mesothoracic disc. There are two of
them in each larva, one for each side of the
adult animal. Each disc produces the entire
epithelium for a wing and that part of one
side of the thorax near the wing. The dor-
sal part of the thorax is called the notum
and the lateral part the pleura.

The method used to mark a clone is
mitotic recombination produced by x-rays
delivered at a chosen time in development,
usually during the larval stages. The ge-
netic makeup of the animal is designed so
that certain mitotic recombinants will be
phenotypically different. For example, if
the animal is heterozygous for the reces-
sive gene yellow (y/ +) then mitotic recom-
bination may produce two daughter cells.
One of these (+/+), will be phenotypically
wild-type and therefore indistinguishable
from unaltered cells, but the other will be
homozygous for yellow b/y). All the de-
scendants of this cell will also be (y/y). If
such a descendant in the adult is colored at
all then it will be yellow rather than the
normal darker color.

An ideal genetic marker would be easily
scored in all types of cell, have complete
expression, and be cell-autonomous. That
is. the phenotype would depend only on the
genetic makeup of the cell in question and
not at all on that of neighboring cells. Un-
fortunately few such markers are known.
Markers often used are: multiple wing

hairs fmwh) which produces groups of two
to five hairs (trichomes) on the wing in-
stead of one per cell as in the wild type; and
forked v) and singed (sn), which produce
deformed bristles and hairs. To assist rec-
ognition, the mutant allele with the most
extreme phenotype among those available
is usually used; and to minimize mistakes
more than one marker is often employed.
Double marking also allows the degree of
expression  and cell autonomy to be
checked.

The markers used so far in this work do
not allow a marked cell to be recognized
when it is first produced in the imaginal
disc, or even after a few divisions. The cell
phenotypes employed can only be scored
by the observer at the adult stage when the
cells have digerentiated. Moreover, only
cells that form (or can be induced to form)
hairs or bristles can be scored at all easily,
so that if these are lacking or sparse in
some particular area it is often difficult to
find the exact edges of a marked clone in
such regions. Fortunately most of the wing
disc derivatives, being covered with hairs,
are relatively easy to score.

If the growing disc in the larva is irra-
diated at the early stages of development,
there will be few target cells and most indi-
viduals examined will not show any mutant
patches. This cannot be overcome by in-
creasing the x-ray dose (which is usually
loo0 roentgens) as too big a dose will inter-
fere with development. One simply has to
examine a fairly large number of flies. If
the x-rays are given later in development,
more mutant clones are produced (since
there are more target cells); but the aver-
age size of each clone will be smaller since
a cell altered at a later stage produces few
descendants. This small clone size means
that it is more difficult to recognize com-
partment boundaries since most of the
clones will be in the middle of a com-
partment rather than near its edge and
even those at the boundary, being small,
will not display the boundary so graph-
ically. This is somewhat offset by the
subdivisions making the compartments
smaller as time goes on but in spite of this
it becomes progressively more difficult to
recognize compartment boundaries. It
would in any case involve much more work
if enough patches are to be scored to make
an apparent boundary statistically signifi-
cant.

However, Garcia-Bellido er al. devised a
method of overcoming this difficulty.
There exist a series of dominant Minute
loci (12) which are lethal when homo-
zygous. When heterozygous, the insects
grow slowly and the bristles are small.
They needed a mutant which (after mitotic
recombination) would make the marked
clone grow faster than the unaltered cells

SCIENCE, VOL. 189


          , 05mm ,                      , 05mm ,
Fig. 5 (left). Drawings of Drosophila wing to show the position of the aotero-posterior compartment border.   Fig. 6 (right). Outline drawing of Dm-
sophila wing to show the area covered by a typical M+/M+ clone in a M/M+ background.

and thus produce a much bigger patch.
Morata and Ripoll (13) showed that
homozygous wild-type cells (M+ /M +)
produced by mitotic recombination di-
vided more rapidly than the slow-growing
heterozygous Minure (M/&f+) back-
ground, which was the effect they needed.
in addition, for reasons which are obscure,
the frequency of mitotic recombination for
(M/M+) larvae after irradiation is appar-
ently increased (14). This is especially use-
ful in the early stages of development when
the normal rate is inconveniently low. A
second somewhat unexpected result was
that in spite of the (M+/M f) clones being
much larger than normal, the overall size
and shape of the wing was not altered (13).
This implies that there are special mecha-
nisms to regulate size and shape which can
cope with differential cell division rates-
an important result in its own right. These
mechanisms can also regulate for the loss
of cells both due to x-rays and the forma-
tion of M/M cells.

The Results

Having given an indication of the meth-
ods used in this type of clonal analysis we
must now mention some of the earlier re-
sults. Becker (15) was the first to use x-rays
to produce clones at particular stages of
development, in his study of the Drosoph-
ila eye. Later Garcia-Bellido (9) noted
that clones produced after the 1st instar
larva never crossed from dorsal to ventral
on the wing; and Bryant and Schneider-
man (8) that they were confined to single
leg segments when larvae older than early
third-stage larva were irradiated. Bryant
(4) made the important observation that
clones in the wing disc could cross from
dorsal to ventral if produced early enough,
but not when produced late. Similarly, in
OncopelU.s (16-18) up until the late
blastoderm stage, clones may extend to
two or more abdominal segments, but after
that stage clones are strictly confined to a

I AUGUST 1975

single segment. These observations all
show that within three different discs of
Drosophila and in the Oncopeltus abdo-
men the "anlagen are represented by sepa-
rate populations of proliferating cells" (4).

The most detailed results so far have
been obtained by Garcia-Bellido and his
colleagues studying the development of the
wing disc. As might have been expected the
earliest clones (irradiation of first-stage
larvae) are contained exclusively within the
fairly large area of the adult cuticle pro-
duced by the entire disc. This shows that
effective separation of the wing disc from
the other discs producing the adult epithe-
lium must have occurred before the first
larval stage. However, even at these early
times a compartment boundary is appar-
ent within the disc. This was first clearly
demonstrated by the Madrid school using
the Minute technique. The boundary,
which separates anterior regions from
posterior regions, runs along the middle of
the wing between the third and fourth vein.
The actual demarcation line is near the
fourth vein but is distinct from it (Fig. 5).
The line runs along both surfaces of the
wing and continues on the body where it
divides the notum into two distinct areas.
Even a very large clone (Fig. 6) will ob-
serve this demarcation line although at
this stage it may well cross the wing mar-
gins, thus appearing on both dorsal and
ventral surfaces and extending onto the
notum.

The edges of the clone are somewhat ir-
regular except where they run along the de-
marcation line. It is not very likely that
this line marks the frontier where two ini-
tially remote and separate groups of cells
have moved together, since both anterior
and posterior regions are within the same
nascent imaginal disc and thus proba-
bly fairly close together (6). Since about
twice as many clones appear in the an-
terior compartment as in the posterior
one, it is surmised that at this early time
there are about twice as many anterior
as posterior cells. That is, the antero-

posterior division is not exactly into two
equal parts but more like a 2 : 1 ratio (IO).

Some time later, during larval devel-
opment (the exact time is not quite clear),
each of these two compartments is found
to be divided into four parts, giving eight
compartments in all. The demarcation
lines divide dorsal from ventral areas and
wing from thorax. The final size of these
compartment areas varies somewhat (from
IO' cells to 10' cells or less). The evidence
that late clones really observe these demar-
cation lines is very strong. They are ob-
served by very large clones, which in some
cases make up as much as 90 percent of a
compartment. Such clones may border a
demarcation line for as many as a thou-
sand cells. Nor is the effect solely due to
the fact that clones are often elongated in a
direction roughly parallel to a demarcation
line. The main axis of these clones meets
the demarcation line at various angles,
sometimes even perpendicularly. Nor on
any simple model can the demarcation
lines be lines of fusion of quite separate
groups of cells if only for the fact that
marked clones made at a slightly earlier
stage will go straight across these lines.

As development proceeds the recogni-
tion of new subcompartments again be-
comes somewhat more difficult because the
effects of differential growth (due to
M+/M+ cells in a M/M+ background)
have less time to produce larger clones.
Garcia-Bellido, Ripoll, and Morata sug-
gest that there may be two further demar-
cation lines formed about the same time.
On the adult fly these separate two areas
on the body, one dividing the notum into
two parts and the other the pleura. These
compartments were all discovered by the
use of MJM+ flies, but similar experi-
ments on non-Minute flies (which have, of
course, smaller clones), show that the de-
marcation lines are also observed in this
more normal situation. The Minute flies
thus serve to make the subcompartments
more easy to observe: the phenomenon it-
self is not peculiar to them alone.

343


Further Problems

Having now described the results on
compartments in outline we must ask how
widely the idea is applicable and what are
its limitations. One limitation is that the
evidence obtained so far relates only to epi-
dermal structures. This is mainly because
in insects they are so easy to observe and
so rich in detail. Internal structures, for ex-
ample, the exact arrangement of the mus-
cles, cannot be studied satisfactorily with-
out the use of more difficult experimental
methods.

However, the properties of internal tis-
sues may be partly imposed by the pattern
of the enclosing epithelium (19) and they
may well also be compartmented.

With regard to compartments in imagi-
nal discs, there are a series of outstanding
questions that need answering. Are all sub-
divisions binary? We have seen how the
first division of the wing disc, after the very
early antero-posterior divisions, appears to
yield four parts rather than two. It is natu-
ral to ask if this is really two separate bi-
nary steps in quick succession, and this
question focuses attention on the exact
timing of the subdivisions. Even for an ob-
viously binary step one can ask whether
the decision is an abrupt one or is spread
over a period. Does it necessarily require
cell division? Are compartment boundaries
always smooth? The edge between the dor-
sal and ventral surface of the wing is very
well defined, and clones that border it are
smooth to the nearest cell (4, 5). but is this
true for all boundaries?

The problem of how a compartment
boundary is formed and how it gets so
straight appears to be a dithcult one. Fac-
tors that may have to be considered are
strictly oriented mitoses near the boundary
(17), straightening effects due to differ-
ential cell affinities, and possibly cell death
for cells which get themselves into the
wrong places, so that the compartment
edges are trimmed. It is claimed (20) that
extensive cell death is unlikely because oth-
erwise clone size near the boundary would
be smaller, which is apparently not the
case.

Nor is it completely clear where the
process of the subdivision of compart-
ments stops. Even the technique for spot-
ting compartment boundaries, using rel-
atively fast-growing marked clones, has
its limitations as, at later times, even these
clonal patches are rather small. How can
we be sure that these are not further sub-
compartments? Even the definition of a
compartment becomes difficult at this
point. Although formally, for example,
the descendants of a single bristle mother
cell [for example, the trichogen, the tor-
mogen, the sense cell, and the neu-
rilemma cell making up a bristle in On-
cope/rus (21)] which are most certainly
a clone and which stay together, could
perhaps be regarded as a compartment,
we feel that this is stretching the term
too far. It would seem sensible to restrict
the term "compartment" for the moment
to fairly large groups of cells and to those
groups which form a polyclone rather
than a clone.

Fig. 7. A metathoracic appendage from a Drosophila carrying an extreme allele for bifhorox. The
posterior haltere develops normally @) while the anterior haltere is transformed into an apparently
normal and complete anterior wing compartment.

344

Other Possible Characteristics of

Compartments

We have seen that, at the moment, a
compartment is defined by its boundaries
and these alone, since clones, made after a
certain time in development, never cross
them. Are there other properties that allow
us to identify a compartment?

One such property may be the area af-
fected by a homeotic mutant. There are
mutants that shift an imaginal disc, or part
of an imaginal disc, into another devel-
opmental pathway. For example, oris-
tapedia (ssa) transforms part of the an-
tenna into leg segments (22).

It is rather rare for a mutant to turn one
whole disc into another whole disc. Possi-
bly such a drastic change would be lethal
and thus escape observation. It is more
common for a part of one disc to be turned
into part of another one. Even in these
cases the transformation is not always
complete, because of partial and variable
expressivity. We can, however, ask the gen-
eral question: In such cases do the (maxi-
mum) boundaries of the transformation
coincide with a compartment boundary
found by the clonal method?

Morata and Garcia-Bellido (23) have
shown by clonal analysis that the haltere
disc (the metathoracic disc) has within it
an antero-posterior boundary; but locating
it precisely is difficult because of the ab-
sence of suitable landmarks on the haltere.
It has been known for many years (24) that
various mutants in the bithorax system
turn various parts of haltere into wing (or
vice versa) with different degrees of ex-
pressivity. A number of mutants appear to
respect the antero-posterior boundary of
wing with some precision and probably
also of the haltere although here the pre-
cision is more difficult to judge. For ex-
ample, an extreme allele of bithorux (bx')
turns the anterior part of the haltere into
anterior wing while leaving the posterior
part of the haltere (which is much smaller)
unaltered. The boundary of this trans-
formed half-wing is very close or identical
to the antero-posterior boundary found by
clonal methods in the wild-type wing (Fig.
7). Another mutant in this complex locus
(postbifhorax) also delineates this bound-
ary because its effect is restricted to the
posterior part of the haltere.

The gene engruiled also delineates the
boundary, and in an especially interesting
way. In flies mutant for engruiled the pos-
terior part of the wing is transformed and
resembles a mirror image of the anterior
part (25). The Minute technique has re-
cently been used to show that the realm of
action of the engrailed gene precisely coin-
cides with the posterior compartment,

SCIENCE. VOL. Is9


there being no effect on the anterior: If
large engmifed (en/en) clones are made in
a wide-type wing (en/+) they may fill the
anterior compartment right up to the an-
tero-posterior boundary but never cross it.
They are completely without effect on the
pattern. However, all engrailed clones in
the posterior part express the phenotype
(26) and, as discussed later, may cross the
antero-posterior boundary.

Another possible correlation is between
gradient discontinuities and compartment
boundaries, These discontinuities can be of
at least two kinds. The first has a discon-
tinuity in the value of the gradient but not
its slope, as shown in Fig. 8. The other has
no discontinuity in the value but a change
of slope, in particular a change of sign of
the slope to give the mirror-image situ-
ation shown in Fig. 9.

The first of these is found between the
segments of the insect cuticle in Rhodnius
and Oncopeltus. Lawrence (16, 18) has
shown in Oncopeltus that marked clones
do not cross the intersegmental boundary,
so here at least we have one clear case
where a clonal boundary coincides at least
approximately with a gradient disconti-
nuity (27). Another possible case is sug-
gested by the mutant engruiled mentioned
above. Since this produces a rough mirror
image across the antero-posterior com-
partment boundary of the wing, one might
be tempted to think that the underlying
gradient (or "prepattern") might have the
mirror image form shown in Fig. 8 both in
the mutant and the wild-type. Otherwise
the experimental evidence for this possible
correlation is either scanty or absent.

There are several other properties which
we can speculate about. Experiments de-
signed to show how mixtures of cells from
imaginal discs appear to sort out show
clearly that cells from different discs will
segregate, suggesting rather strongly that
they have different surface properties (28).
Moreover, such segregation also occurs be-
tween marked cells from different parts of
the same disc. For example, cells from the
anterior part of the wing disc will segregate
from those of the posterior part (29). This
obviously suggests the generalization that
each compartment has characteristic cell
surface properties, different from every
other compartment, which allow cells from
any two compartments to segregate. Thus
the normal development and maintenance
of the antero-posterior boundary in the
wing might depend on the confrontation of
cells of a different type, that is "anterior"
with "posterior" cells. If so, one might ex-
pect that boundary to be malformed or
nonexistent in engrailed flies where the
posterior cells are partially transformed
into those of the anterior type. Clonal
I AUGUST 1975

A /v

    (8)            (9)
Fig. 8. The probable gradient situation in
Drosophila wing; the slope, but not the altitude.
changes near the antero-posterior compartment
border.   Fig. 9. The probable gradient situ-
ation in two adjacent abdominal segments of
Hemiplera. The step probably coincides with
the intersegmental compartment boundary.

analysis of engrailed flies has recently
shown that clones do frequently cross the
line where the border normally is (26).
This never happens in flies wild-type for
the engmiled locus, a result that strongly
supports the idea that the role of the en+
allele is both to control the development
of the posterior pattern and to instruct
the cells so that they do not intermingle
with cells of the neighboring anterior
we.

An additional possibility is that there is
a gradient of cell surface properties within
each compartment. This is certainly sug-
gested by the observation (30) that in the
epidermis of Oncopelrus a graft takes bet-
ter if it is from the same level in the seg-
mental gradient, even if from a different
segment, than if moved to a different posi-
tion in the gradient in the same segment.
These speculations go far beyond the ex-
perimental data now available, but they do
suggest that direct methods of character-
izing cell surface properties, preferably in
situ, would be very valuable. If such a
method could be developed it would have
the enormous advantage that it might
work for the cells of the developing imagi-
nal disc so that one could spot com-
partments and their boundaries at the mo-
ment, or soon after, they are formed.

It is also possible that. even though all
the epithelial cells of a disc appear very
similar, the compartments within them
could differ by a particular enzyme or set
of enzymes. For this reason there is a case
for testing all the imaginal discs, both in
their mature and their developing states,
by as many histochemical tests as are
available. A beginning has already been
made in this approach by Janning (32) us-
ing a test for aldehyde oxidase.

Another histological feature that may
correlate to some extent with com-
partments is the distribution of nerve
axons. Hasenfuss (32). studying the epi-
dermis of Galleria and Rhyacophila. no-
ticed that the nerve axons of the sensillae
in the abdominal epidermis were collected
into groups each of which went to one seg-
mental ganglion only. He suggested that
this was because each group came only

from a single epidermal segment, This,
however, was true only of the axons since
the dendrites were observed to extend over
considerable distances and thus could not
be confined within one segment. A similar
phenomenon has also been observed by
Lawrence (33) in the abdomen of Onco-
peltus. In this case. the intersegmental
boundaries are clearly delineated by color
and cell shape. No axons have been ob-
served to cross these boundaries, although
they do cross the midline. (It is known that
the midline is formed in the embryo by the
fusion of two separate groups of cells.)

One is thus led to the speculation that
the fields outlined by well-defined groups
of nerve axons may perhaps coincide in
certain cases with compartments or sub-
compartments. This might be because
compartmentalization may often occur be-
fore the separation of the neuroblasts from
the presumptive epidermis, so that any cell
surface differences or other labels asso-
ciated with a compartment may be shared
by both the epidermal cells and the neu-
rons.

The hypothetical properties so far dis-
cussed would be possessed by all or most of
the cells within a given compartment or
subcompartment. They could be described
as area properties. Another rather differ-
ent property would be one which charac-
terized boundaries between compartments,
that is. an edge property. For example, the
cells on one side of the intersegmental
boundaries in Oncopelrus are markedly
elongated in the direction of the boundary
(17). Do all compartment boundaries have
this property? For the antero-posterior
wing boundary it seems that the adult cells
have no unusual appearance; but never-
theless a detailed scrutiny of several such
boundaries might be worthwhile. Another
obvious hypothesis is that whereas there
may be free diffusion of certain chemicals
within compartments it may be greatly re-
stricted across compartment boundaries.
This suggests that compartments might
not be electrically coupled to each other,
but a direct test across the intersegmental
boundary in Rhodniw (34) showed cou-
pling to be normal. Moreover, a careful cy-
tological study by electron microscopy has
shown no observable difference in the vari-
ous types of cell junctions (gap junctions,
septate desmosomes, attachment desmo-
somes) for the corresponding intersegmen-
tal boundary in Oncope/fus (35). One is
thus not exactly encouraged to look for
these same differences at compartment
boundaries in structures from imaginal
discs. Nevertheless, it would be surprising
if there were not some important cytolog-
ical difference at compartment bounda-
ries.

345


Possible Mechanisms for Compartment

Formation

We must now consider the nature of the
step which partitions the cells that are the
ancestors of one compartment in such a
way that some of them become the founder
cells of one subcompartment while the oth-
ers become the founders of the other sub-
compartment. As we have seen, this step is
often a partition into two parts (rather
than three, four, or more), and it is possible
that this is always the case. For the mo-
ment we will only consider the case of bi-
nary partition.

At present, little can be said about any
underlying biochemical mechanism, but
we can usefully discuss the problem at the
cellular level. Unfortunately, we have
rather few facts to go on. In view of the
existence of size and shape regulation (as
shown by the experiments in which a rela-
tively fast-growing clone within a com-
partment does not alter its dimensions), it
is not obviously a requirement that the
partition need be always exactly the same,
since any variation, if it is not too big, can
probably be corrected by subsequent
growth. We consider three possible types
of mechanism.

1) The partition of daughrers. All the
cells divide once, one daughter of each di-
vision being  allocated to one sub-

compartment and one to the other.
2) Random allocation. The cells are al-

located at random, with a fixed probability
which we shall assume to be about one-
half. Because of the number of cells in-
volved, the chance of all the cells being ac-
cidentally allocated to one subcompart-
ment is so small that it can be ignored (for
example, for 20 cells this chance is 1 in 2rp
or about 2 in 106). Even if all cells but one
are allocated to one subcompartment, the
single cell allocated to the other could,
conceivably, compensate for this numeri-
cal handicap by an increased rate of multi-
plication.

3) Geographical partition. The patch of
epithelial cells is divided. the dividing line
separating the founder cells of one sub-
compartment from those of the other.

The difficulty with the first two mecha-
nisms is that, in order to get the cells of
each subcompartment together in one
patch, a certain amount of relative cell
movement would have to take place. Since
the partitioning into subcompartments
takes place several times in succession, one
would not expect marked clones to stay in
one piece, as they usually do. Thus these
two mechanisms seem unlikely, except per-
haps for the first of the several partitioning
steps.

The mechanisms can be saved to some
extent by an additional hypothesis; that

  346

any cell which is surrounded by cells of the
other type commits suicide. It is difficult to
make this model precise, but it would ap-
pear to lead to a fair amount of cell death.
Moreover, the cells which migrated would
still have to move to the correct place in
the epithelium relative to other surround-
ing tissues.

The third proposed mechanism-geo-
graphical partition-seems to us to be by
far the most likely one, especially as it does
not need to be extremely precise. Consider,
say. a patch of 20 cells. Let each cell divide
once to give 40 cells. Each of these cells
will be surrounded in the epithelium by
several other cells (the average number is
usually a little above five), one of which
will be its sister cell. Now draw an arbi-
trary (but moderately straight) line par-
titioning the patch into two parts. This line
will separate some cells which are sisters.
The problem is to estimate the fraction
(averaged over many cases) of the original
20 cells which will have daughters sepa-
rated by the line. It is only these particular
cells that can produce a clone of descend-
ants which will go across the boundary
between the subcompartments.

Several approximate estimates have
been made by Ripley (36) using various
simplifications. The fraction defined above
can be written as equal to C/N"2 when N
is thenumber of cells at the time the line is
drawn (40 in the example above) and C is a
parameter which is approximately con-
stant. The values of C found were not far
from 0.55. Thus for N = 25 the fraction is
about 11 percent. This calculation shows
rather clearly that on this simple mecha-
nism the existence of clones which cross
the subcompartment boundary will not
be a rare event if they are marked one
generation before the compartment is
divided.

A more detailed mathematical study of
this problem would be worthwhile since it
is important to compare the detailed ex-
perimental data (what fraction of clones
crosses a border, what fraction runs along-
side one, and the like, as a function of ex-
act time of irradiation) with what would be
expected on the various theoretical models.

Conclusion

We have seen that the work of Garcia-
Bellido and his colleagues has clearly
brought out the formation of compart-
ments and successive subcompartments in
the epithelium produced by the wing
disc of Drosophila and that there is evi-
dence that a similar process occurs in the
production of other regions of the insect
epithelium. We have also seen that the
phenomena, although clearly demonstrat-

ed in outline. need further detailed study,
especially quantitative study. The mecha-
nism that produces these subcompart-
ments is obscure although a plausible
model can be suggested for the general
nature of the process,

It is therefore pertinent to ask what is
the novelty of these ideas, viewed from the
general perspective of development stud-
ies. To do this we must ask what the ex-
periments show does not happen.

We are not talking about the determina-
tion of cell type in the usual sense-for ex-
ample, a muscle cell as opposed to a fibro-
blast-but about cell position. In this sys-
tem the determination and differentiation
of cell types-for example, bristles as op-
posed to epithelial cells-probably comes
later and may well also be dependent on
the compartment to which the cells belong.
What we are concerned with is geographi-
cal position in the organism and, more-
over, not about exact geographical posi-
tion but whether a cell is somewhere within
one well-defined region or another one.

What has been demonstrated in this sys-
tem is that once a major developmental
step of this type has been taken by a cell it
is not reversed in the progeny of that cell,
at least in normal development. If reversal
was possible, a cell which had been deter-
mined for the dorsal side of the wing and
which found itself on the ventral side could
be reprogrammed to be a ventral cell.
What clonal analysis has shown is that this
never happens. Either such a cell cannot
get to the wrong side of the wing, or, if it
does so, it must either move back to the
right side or be killed. The exact mecha-
nism is obscure. Whatever it is, it is clearly
of interest, even though the basic concept,
the irreversibility of major developmental
steps, is not in itself especially novel.

But it would be both novel and exciting
if it turns out that the compartments and
subcompartments are used by the orga-
nism as units for the control of shape and
size; if gradient systems meet at com-
partment boundaries, if cell surface prop-
erties changed abruptly there, if size regu-
lation  occurred partly independently
within each of these domains, and so forth.
It may be that the normal development of
each imaginal disc can usefully be divided
into a precise succession of major steps
each of which produces a set of new com-
partments. If so, by studying compartment
formation, one could both enumerate these
steps and determine their times of action.
On this picture each compartment would
be specified by a unique combination of a
small number of controlling genes [selec-
tor genes (I)] that are active in it. (The
steps that follow-for example, the deter-
mination of a bristle in a particular posi-
tion within a compartment-may be of a

SCIENCE, VOL. 189


somewhat different and more complex
character.) For the first time there is the
real prospect of understanding the logic
behind gene deployment in pattern forma-
tion. As we have seen, the speculative ideas
about compartments in this section are not
supported by hard evidence. The best we
have so far is a series of hints. But it is ex-
actly this possibility, that compartments
may have a wider significance, which
makes the study of them at the present
time so important and so interesting.

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373 (190,.

9. Ripley, personal communication.
We thank Professor A. Garcia-Bellido and Dr. G.
Morata and our collcagues in the MRC Labora-
tory for many helpful cornmenu and criticisms.