AMER. ZOOL., 29:617-652 (1989)
Mitotic Domains Partition Fly Embryos,
Reflecting Early Cell Biological Consequences of
Determination in Progress1
VICTORIA E. FOE
Friday Harbor Laboratories, University of Washington,
Friday Harbor, Washington 98250
AND
GARRETT M. ODELL
Department of Zoology, University of Washington,
Seattle, Washington 98195
SYNOPSIS. We link classical observational developmental biology with modern techniques
and concepts of cell biology, focusing on early development in the fruitfly,Drosophila
melanogaster. In contrast to the first thirteen synchronous nuclear division cycles following
fertilization of the egg, cell division in the fourteenth cycle partitions the embryo into
mitotic domains. These are bilaterally symmetric clusters of cells, spatially and temporally
patterned, invariantly, embryo-to-embryo. Cells in a given mitotic domain share the same
mitotic schedule, different from that in neighboring domains. In addition, cells in at least
some mitotic domains share distinct attributes such as special cell morphologies, spindle
orientations, morphogenetic movement behaviors, and eventual differentiated tissue fates.
We argue that a mitotic domain differentiates its control of the fundamental cell cycle
process as a consequence of its having embarked on a developmental pathway different
from that of neighboring domains. In embryos of flies with certain mutations at earlyacting genetic loci, perturbations of the normal pattern of mitotic domains forecast the
final mutant phenotype. Mitotic domains are visible by non-invasive light microscopic
observation of live developing embryos, or by staining fixed embryos with fluorescently
labeled antibodies to microtubules—methods that will likely work well on embryos of
phylogenetically diverse type. We document the methodological and phylogenetic generality of mitotic domains with micrographs of Calliphora (blow-fly) embryos; the homology
to Drosophila is obvious. We present a theoretical framework for thinking about the process
of embryonic cell determination as a gradual dynamical process and argue that, to learn
most about determination, we should correlate the earliest perturbations we can inflict
with the earliest phenotypic consequences we can assess: perturbations of the mitotic
domain pattern serve this purpose perfectly.
these groups of cells "mitotic domains"
In this paper we try to link classical because cells in a given group share the
observational developmental biology with s a m e mitotic schedule, different from that
modern techniques and concepts of cell i n neighboring groups. The cells in at least
biology. After a general introduction, we s o m e mitotic domains share more than a
will focus almost entirely on the cell biol- common cell cycle length. They share
ogy of early development, including gas- attributes such as special cell morpholotrulation, in Drosophila. We will concen- gies> spindle orientations, morphogenetic
trate on a recently discovered phenomenon movement behaviors, and eventual tissue
that begins just after the mid-blastula tran- fates.
sition, in which some mechanism finely Control of the cell cycle, and of mitosis,
partitions the blastula/gastrula into sepa- a r e among the most fundamental of cell
rate groups of embryonic cells. We call biological processes in all cells (see A. Murray's [1989] essay in this volume). We argue
that a mitotic domain differentiates its con.
trol of this fundamental process as a con-'
INTRODUCTION
1
From the Symposium on Science as a Way of Knowi_ i J
J
i
c • i
ing-Cell and Molecular Biology presented at the Annual sequence of its having embarked on a develMeeting of the American Society of Zoologists, 27- opmental pathway different from that of
30 December 1988, at San Francisco, California.
neighboring domains. That is, we believe,
617
618
V. E. FOE AND G. M. ODELL
different mitotic domains comprise cell
groups on their way toward determination
as the primordia of different larval structures, and the distinct characteristics shared
by cells in a mitotic domain reflect this.
The fruits of genetic mutant screens,
carried out by Niisslein-Volhard, Wieschaus, Jiirgens, and their co-workers,
seeking early-acting genes in Drosophila, are
now available as an extensive menagerie of
mutant flies whose embryos exhibit developmental defects, as assessed by examining
the cuticle2 secreted by larvae (Tearle and
Niisslein-Volhard, 1987). We include in this
paper preliminary evidence that, in at least
some of these mutants, very early perturbations of the normal pattern of mitotic
domains forecast the perturbations of cuticle pattern that the larva produces much
later.
Mitotic domains are visible by non-invasive light microscopic observation of live
developing embryos or, more vividly, by
fluorescently labeled antibodies to microtubules that dramatically illuminate, in
fixed specimens, mitotic spindles of dividing cells. These methods for visualizing
mitotic domains will likely work well in
examining early pattern formation in
embryos of phylogenetically diverse type;
they rely neither upon the formidable array
of molecular probes that bind to specific
gene products, such as have been developed recently for Drosophila, nor upon
stocks of genetic mutants. Thus, with minimal preliminaries, one may be able to
examine quickly, and compare, the earliest
development of diverse organisms by visualizing mitotic domains.
We have no idea how general the partitioning of early embryos into mitotic
domains may be phylogenetically, but
mitotic domains are certainly not unique
to Drosophila embryos. In this paper we
publish preliminary evidence that, as
gauged by cell biological criteria in general, and the sequence and pattern of
mitotic domain partitioning in particular,
the early development of Calliphora vomitoria (blow-fly) embryos is nearly identical
to that of Drosophila melanogaster (fruit fly)
embryos. Each is an advanced dipteran fly;
Drosophila maggots eat decaying fruit while
Calliphora maggots eat decaying flesh.
The two genera diverged some 60 million
years ago (Shaw and Meinertzhagen, 1986).
To facilitate observational comparison
between Drosophila and Calliphora embryos,
some figures below show Drosophila and
Calliphora micrographs side by side. We
suggest that readers ignore the Calliphora
micrograph panels until they reach our discussion of Calliphora toward the end of the
text.
Since the Science as a Way ofKnowing proj-
ect aims to catalyze the active participation
of students in biological science—students
who may not enjoy access to the expensive
equipment essential for many aspects of
cell biological research—we emphasize that
the phenomena we discuss below are within
the experimental grasp of anyone equipped
with a decent compound microscope, a fiber
optics lamp (or a flashlight), some mineral
oil, Scotch brand double-stick tape, and a
bit of patience. For this reason, we take the
unusual step of including a brief appendix
on "materials and methods" in this review
paper; we advocate the use of Calliphora
embryos because they are so large3 that
even mediocre optics yield compelling
views. Because the technology required is
minimal, students equipped additionally
with merely the courage to try experiments
that might fail—seeking mitotic domains
in the early development of previously
unstudied organisms—may well make truly
important research discoveries at the junction, described below, between cell and
developmental biology.
How Do INITIALLY IDENTICAL
TOTIPOTENT CELLS BECOME
DIFFERENTLY SPECIALIZED AS
AN EGG BECOMES AN ORGANISM?
Macroscopically, the classical issue of
developmental biology is to understand
how an egg becomes an organism: what is
8
The larval cuticle is the exoskeleton of the larva,
which is densely covered by denticles, hairs, and other
visible morphological features by which one can distinguish some segments from others.
3
1.5 mm long, which is thrice the length of Droiophxln embryos, containing blastoderm cells 27 times
the volume of Drosophila cells.
MITOTIC DOMAINS PARTITION FLY EMBRYOS
the sequence and what are the mechanisms
by which its various tissues and organs arise
and cohere. Examples of how to address
this question experimentally are: (i) perturb the process early and assess the consequential alteration of the morphology of
the resulting phenotype; (ii) produce fate
maps: ablate a part of the blastula or gastrula and discover what the ablated part
would have become by finding out what
the resulting organism lacks; (iii) cause
genetic mutations and screen organisms for
phenotypic changes of behavior or morphology. In all of these examples, we wait
until the organism's constituent cells
undergo "terminal differentiation" before
we assess the changes that experimental
manipulations caused.
From a microscopic vantage, the puzzle
of development is how the myriad cells that
spring from a single ancestral cell become
so rapidly and wildly different—as assessed
by cell biological criteria—in beautifully
choreographed temporal and spatial patterns that build organs and tissues that cohere. What mechanism directs a cell, that
potentially could have become any cell in
the finished organism, to become precisely
one specialized (differentiated) cell with
such narrowly restricted properties that a
histologist can tell at a glance that, for
example, "this is an endodermal epithelial
cell"? If propagated in tissue culture, it and
its progeny remain endodermal epithelial
cells. How is this long-term cellular memory, which characterizes a determined cell,
maintained? We emphasize here that the
developmental process of cell determination is a continuous process, not just an end
result. It is a process that unfolds separately
and gradually in each cell, involving ever
greater restriction of the cell biological
properties that cell can express. (See J. A.
Moore's [1989] section 71 in this volume.)
Moreover, each cell and its progeny will
exhibit numerous specialized behaviors
during embryogenesis, not just one final
differentiated cellular phenotype.
The concepts and techniques of modern
cell biology make it possible to re-phrase
classical macroscopic questions of developmental biology on a microscopic singlecell scale, and make it possible to ask those
questions much earlier during develop-
619
ment. We inflict the same kinds of perturbations mentioned above, but instead of
waiting until differentiated cells exhibit
their final tissue type, we can assess transient changes in the cell biological behavior
of cells in developing embryos, while the
mechanism of determination is acting. The earlier we assess the consequences of the perturbations we inflict, the more likely we
are to understand the process of determination. In this paper we concentrate upon
studies of the cell division cycle during early
development as a way to reveal early commitment of cells just after the mid-blastula
transition.
Virtually all organisms that develop regulatively do so in two qualitatively different
stages. In the first stage, the single oocyte
nucleus commences a sequence of relatively rapid division cycles (typically 10 to
13, which are often synchronous) fueled
by energy stores, and controlled by proteins and mRNAs that the mother installed
in the egg. As the single original nucleus
becomes many, some mechanism partitions the egg's cytoplasm so that every
nucleus has a cytoplasmic surround to
interact with. In some embryos (e.g., frogs)
cytokinesis accompanies each nuclear division to subdivide the oocyte into many separate cells with one nucleus each. In others,
(e.g., birds and arthropods) the oocyte
remains a syncytium4 through the earliest
nuclear division cycles, and only later partitions each nucleus and its cytoplasmic surround into a separate cell. The second stage
begins at the mid-blastula transition with a
dramatic slowing of the division cycles and
the initiation of significant zygotic transcription, that is, the production of proteins coded by the embryo's own genes. It
is in this second stage that the embryo's
genes begin to direct its own development.
That is, starting in this stage, some interaction between each nucleus and its surrounding cytoplasm determines which of
its genes to transcribe and which to repress.
This mid-blastula transition occurs after 12
division cycles in the frog Xenopus, and after
13 in the dipteran flies Drosophila and Cal-
4
A single cell with many nuclei.
620
V. E. FOE AND G. M. ODELL
liphora. Starting with this stage, the future
fates of many cells are determined.
A CELL BIOLOGICAL PORTRAIT OF
THE NORMAL DROSOPHILA Embryo
Prior to, and Just After,
the Mid-blastula Transition
Virtually all investigations of development involve assessing changes in the normal course of events caused by some experimentally inflicted perturbation. To assess
changes, we need an accurate description of
the normal situation. This section gives a
brief sketch of the normal course of events
in t h e fruit fly Drosophila melanogaster, con-
centrating mostly on mitotic cycle issues,
Full details may be found in Foe and Alberts
(1983) for events up to the mid-blastula
transition, and in Foe (1989) for events following
Prior to the mid-blastula transition, mitoses occur
Slobal synchrony
During the first 13 nuclear division
cycles, most of the Drosophila melanogaster
egg is a single syncytial cell with many nuclei
that divide nearly synchronously. Figure 1
and its caption summarize the complex cell
biology of the Drosophila embryo prior to
the mid-blastula transition.
with
FIG. 1. Schematic drawing of the embryonic stages leading up to gastrulation in D. melanogaster, reproduced
from Foe and Alberts (1983). Each embryo's anterior end is up. Solid black circles represent nuclei, stippled
regions represent yolk, and the white space denotes yolk-free regions of cytoplasm. From stage 2 until cycle
10, the egg is a single syncytial cell with many nuclei. In cycle 10 the germ cells form, while the rest of the
egg remains syncytial until stage 14B. At all times, each nucleus sits in the center of a yolk-free island of
cytoplasm. The numbers beside the embryos denote their developmental stages, and count their division
cycles. A stage begins with the start of interphase and ends with the conclusion of mitosis. Stage 1 is the
fertilized zygote during its first interphase and mitosis. For example, the upper right embryo is in the 4th
cycle which terminates with the 4th nuclear division. The 8 nuclei in a cycle 4 embryo result from the 3 prior
division cycles.
In stages 1-6, nuclei multiply exponentially in successive synchronous mitoses in the central region of the
eggIn late stage 7, about 85% of the nuclei in their cytoplasmic islands begin migrating to the egg's cortex,
leaving behind the future yolk nuclei. These yolk nuclei divide in approximate synchrony with the others
during cycles 7-10.
In cycle 9, the first of the migrating nuclei reach the cortex at the posterior pole of the egg. These nuclei
organize the posterior cytoplasm and plasma membrane, forming cytoplasmic protuberances, called pole buds.
Early in cycle 10, the remainder of the migrating nuclei reach the cortex, and there distribute themselves
in a monolayer, organizing somatic cytoplasmic buds, one per nucleus, over the entire egg cortex. The pole
bud nuclei at the embryo's posterior divide again in synchrony with the nuclei in the somatic buds, and then
quickly cellularize. The resulting cells, progenitors of germ cells, are called pole cells. They will continue to
divide, but after this stage lose mitotic synchrony with the cortical and yolk nuclei.
In cycle 11, the yolk nuclei cease dividing, but, with continuing DNA replication cycles, become polyploid.
During cycles 10-13, the syncytial nuclei in their somatic buds at the cortex divide with near synchrony.
During these four cycles their mitosis is metachronous, usually starting first in nuclei near both the anterior
and posterior poles, then spreading wavelike towards the equator. Thus, the blastoderm nuclei near the
anterior and posterior poles are the first to begin and the first to conclude division; earlier cycles (1-9) are
more synchronous.
During cycle 13, the yolk particles and cytoplasm segregate at the cortex, creating a dramatic increase in
the depth of the yolk-free cytoplasm layer there. The mitoses that conclude cycle 13 are the last globally
synchronized mitoses in the embryo and they are the final syncytial mitoses; every \4th division involves
cytokinesis as well as nuclear division.
During stage 14A, cellularization occurs: plasma membranes invaginate between the cortical nuclei, to
generate separate cells (Fullilove and Jacobson, 1971; Turner and Mahowald, 1976). The nuclei elongate in
concert with the increasing depth of invaginating membranes, the result of which is creation of about 6,500
elongated blastoderm cells that occupy the entire depth of yolk-free cortical cytoplasm. The figure depicts
stage 14A at both early (no cell membranes evident) and late (cellularization just completed) times. As
cellularization proceeds, one sees in live embryos a dark line, parallel to the egg's cortex, move inward. This
is called the "yolk furrow," and comprises the lined up grooves of invaginating membranes. Cellularization
is complete when this yolk furrow stops moving inward and darkens as the membranes pinch off individual
cells from the yolk, which remains a single interior cell with multiple polyploid yolk nuclei—the "yolk cell."
In stage 14B, immediately following cellularization, gastrulation movements begin. The infolding of cells,
about one-third of the distance down from the anterior pole, depicts a section of the cephalic fold.
MITOTIC DOMAINS PARTITION FLY EMBRYOS
14A
I
/
/.„
,II,,I i:
\l
> cellular
blastoderm
621
14B
622
V. E. FOE AND G. M. ODELL
MITOTIC DOMAINS PARTITION FLY EMBRYOS
623
Panels A and B of Figure 2 document
the near synchrony of the early mitoses,
showing an embryo all of whose nuclei are
nearly simultaneously in metaphase of the
cycle 13), these genes turn on uniformly
everywhere between 15% and 70% egg
length. In cycle 14, expression of each pairrule gene resolves into seven stripes, each
ISth division cycle. For any given cycle, all spanning one pair of segments. The evennuclei have about the same cycle length even skipped (eve) gene is an example. Third, and
though cycles 10 through 13 show a pro- well into cycle 14, after sharp pair-rule gene
gressive increase in length—the durations patterns have resolved and cellularization
of cycles 10, 11, 12, and 13 are about 8, is nearly complete, very narrow stripes of
10, 13, and 16 min, respectively, at 25°C. "segment-polarity gene" expression, just
Earlier cycles are shorter—probably too one cell wide, turn on. The engrailed gene
short for any significant zygotic transcrip- discussed below is an example of a segment
tion to occur. Cycle 14 is not only much polarity gene.
longer than previous cycles, but also of
The development of spatial patterns of
markedly different lengths in different segmentation gene expression occurs in
regions of the embryo, as we discuss in rings perpendicular to the anterior-postedetail below.
rior axis. Simultaneously, the products of
During cycles 12, 13, and 14, zygotically another group of genes—the dorsal-ventranscribed gene products accumulate in tral genes—turn on sequentially, eventuprecise spatial patterns. Products of mater- ally generating bands of expression parnal-effect5 genes installed in the egg by the allel to the anterior-posterior axis. Dorsalmother modulate expression of early-act- ventral genes of both maternal-effect and
ing zygotic genes. For example, the mother zygotic types are involved. Dorsal is an
installs mRNA of the bicoid gene, localized example, discussed below, of a maternalat the anterior tip of the egg. Protein trans- effect gene, whose products the mother
lated from this mRNA accumulates in a gra- installs in the egg. Examples of zygotically
dient, highest at the anterior end, lowest transcribed dorsal-ventral genes are snail
at the posterior. See Frohnhofer and Niis- and twisted gastrulation. We discuss these
slein-Volhard (1986), Driever and Niis- below.
slein-Volhard (1988), Berleth et al. (1988).
There is good evidence that spatial patThis bicoid protein gradient orchestrates terning of these segmentation and dorsalpatterning along the anterior-posterior axis ventral genes directs the larva's morphoof three tiers of zygotic segmentation genes logical subdivision since mutants lacking
that "turn on" in sequence (Driever and one or several of these genes exhibit speNusslein-Volhard, 1989). So-called "gap cific morphological defects in segmentagenes" constitute the first tier, and they tion or in the dorsal-ventral arrangement
turn on in broad bands, each of which spans of specific tissue types, respectively.
many segments in the hatched larva. The
During cycle 14, when segmentation
Kriippel gene is an example. Next, the genes turn on sequentially in ever sharp"pair-rule" gene pattern arises. Initially (in ening bands, cells form, creating an epithelial sheet surrounding the large elliptical yolk cell. As soon as cellularization is
5
A maternal-effect gene is one whose presence or complete, about 55 min after cycle 14
absence in the embryo is immaterial, but whose presence or absence in the mother affects development begins (at 25°C), the embryo commences
gastrulation, which internalizes the mesoin all of the mother's eggs.
Fic. 2. Micrographs of Drosophila (panels A and B) and Calliphora (panel C) eggs, stained with fluorescently
labeled antibody to /3-tubulin, show all nuclei nearly synchronously in metaphase of the \2>th division cycle.
Panel A of an entire Drosophila egg and panel C of an entire Calliphora egg are at the same absolute magnifications to dramatize the enormous size difFerence between the eggs: the Calliphora egg is 2.9 times longer
than the Drosophila egg, and nearly 27 times the volume. Panel B shows a higher magnification view of the
same Drosophila egg to document the near synchrony of its nuclei in metaphase.
V. E. FOE AND G. M. ODELL
624
23
u
14
MITOTIC DOMAINS PARTITION FLY EMBRYOS
dermal and endodermal germ layers to
establish the basic larval body plan. Gastrulation begins about 85 min after zygotic
transcription starts. In this short interval,
patterned gene expression organizes the
regional specializations of cellular morphology and function that are responsible
for the complex events of gastrulation.
Against the background of earlier synchronous
divisions, mitotic domains dramatically
partition the gastrulating embryo
Early in gastrulation, a complex mitotic
pattern occurs which is quite unlike the
metachronous mitotic pattern observed for
cycles 10-13 (Poulson, 1950; Sonnenblick,
1950; Madhavan and Schneiderman, 1977;
Turner and Mahowald, 1977; Foe and
Alberts, 1983; Hartenstein and CamposOrtega, 1985). This subsection and the next
four summarize briefly studies reported in
detail in Foe (1989) on this complex mitotic
pattern of the cycle-14 cells. Embryos
stained with fluorescently labeled antibodies against tubulin reveal the configuration
of the microtubules and therefore the phase
of the nuclear division cycle in each cell.
The detailed atlas of mitotic pattern shown
in Figure 3 summarizes a study of many
micrographs, taken at different stages of
gastrulation, arranged in temporal
sequence. Figures 5A and B show sample
micrographs. Time-lapse movies of live
gastrulating embryos confirm the temporal sequence deduced from these studies of
fixed antitubulin-stained embryos.
625
What are mitotic domains and what are
their characteristics?
Discrete groups of cells enter mitosis
nearly synchronously, but out of synchrony
with the surrounding cells. We call each
such group of nearly synchronously dividing cells a "mitotic domain." Except for
those mitotic domains that symmetrically
straddle the dorsal or ventral midlines, each
domain on one side of the embryo has a
mirror image partner on the other side,
separate from it, but mitotically synchronized. On each side of the bilaterally symmetric embryo, most mitotic domains comprise only adjacent cells; however, a few of
these domains consist of disjoint cell clusters that are metamerically repeated. The
general shape of individual domains is constant from embryo to embryo, even though
many domains have a complex and irregular shape.
In each mitotic domain of contiguous
cells, mitosis appears to start in one or a
small number of central cells and to spread,
wavelike, in all directions until it stops at
the domain's boundary. Thus, except for
the slight metachrony associated with these
waves, all cells within a mitotic domain terminate their cycle 14 at the same time.
Except for the slight metachrony associated with the thirteenth mitotic wave
described in Figure 1 that sweeps from the
poles to the equator, all cells within a mitotic
domain begin their cycle 14 at the same
time. Thus, all cells in a given mitotic
domain share approximately the same
FIG. 3. Atlas of the mitotic domains projected onto a 75 min embryo—at the start of germ band elongation,
adapted from the color-coded atlas in Foe (1989). This figure summarizes the positions of the various domains
ascertained by studying many hundreds of micrographs such as Figure 5, each of which revealed only a few
domains by virtue of the mitotic synchrony of their constituent cells. The upper, middle, and lower panels
show dorsal, ventral, and mid-sagittal section views, respectively. Numbers designate the domains of synchronously dividing cells according to the sequence in which they enter mitosis. Note that at 75 min, mitosis
has begun only in the first seven mitotic domains; we project the later-dividing domains back to the positions
they had at 75 min. N and M denote the two domains of asynchronously dividing cells. A and B denote the
domains of non-dividing cells. In the dorsal and ventral views, stippling and shading emphasize the unity of
disjoint, but metamerically reiterated, domains: (16, 17, 21 and 25).
The embryo heads face left, and in the cross sectional view, the dorsal surface faces the top of the page.
The cephalic fold is an early, transient, morphological feature of Drosophila (and Calliphora) embryos easily
visible through a dissecting microscope. In the atlas, it appears as a darker line running between domains 5
and 6, and between domains 5 and A in the dorsal view. In the ventral view, the cephalic fold (cf) appears
to separate domain 2 from M and N. In the bottom panel's sectional view, the cephalic fold (cf) is the crease
formed anteriorly in domain 14. Domain 10, shaded in the bottom panel, is the mesoderm, internalized by
invagination of the ventral furrow.
626
V. E. FOE AND G. M. ODELL
duration of the 14th interphase and this
duration is different for cells in adjacent
domains. In the figures and text, we time
all events from the start of interphase 14
at 25°C. Mitotic domain 1, the first to complete interphase 14 and to enter mitosis,
does so at about 70 min on this scale, while
the cells of mitotic domain 25 do not enter
their \4th mitosis until about 115 min. In
the left-hand panel of Figure 4 we display
a time line showing when the various
domains begin their 14th mitosis.
The mitotic domains exhibit considerable variation in size. For example, prior
to its fourteenth division, mitotic domain
10 consists of over 600 contiguous cells,
while mitotic domain 25 consists of 14 cell
pairs—28 single cells—each occupying
disjoint but precisely specified positions on
the embryo surface.
Each mitotic domain gets a number
according to its time of entry into mitosis.
These are the numbers that label domains
in Figure 3. These numbers also order
domains according to increasing durations
of cycle 14. We combine these sequential
domain numbers with a subscript number
that identifies the nuclear cycle in question.
For example, d,46 names the sixth cluster
of cells to begin their 14th mitoses and is
an abbreviation6 for "mitotic domain 6."
Asynchrony during mitosis 15 partitions
<9I46 into two sub-domains, designated by
d,46 -> 3,51 and d,46 -• di52, respectively.
This nomenclature extends to other subdomains and later cycles in the obvious way.
The embryo also contains regions where
cells never divide again, and others in which
the cells divide asynchronously during long
intervals spanning 40 min or more. These
regions are not, by definition, "domains of
synchronous mitosis," but are, on the basis
of their morphological properties, unified
groups of cells; we designate them with letters. Cells in domains A and B do not divide
during the period studied. In the cells of
ventral domains M and N (abbreviated dl4M
and <9i4N), the length of cycle 14 varies
6
We use the symbol "d" to abbreviate "domain"
because, as students of calculus will remember, d is
the symbol that denotes "partial differentiation."
from 95 to 135 min, and 140 to 190 min,
respectively, and during this interval (95190 min), cells in many other domains complete their Ibth cycle. The first domain to
complete its 15th cell cycle is d,410, which
enters mitosis 15 at about the same time
that d,425 enters mitosis 14 (see Fig. 4).
Domain mitoses occur on a rapidly
deforming cell sheet
Morphogenetic movements progressively transform embryonic organization
during the hour immediately following cellularization (Turner and Mahowald, 1977;
Fullilove and Jacobson, 1978). It is clear
that the specialized behaviors of certain of
the mitotic domains, some of which we
describe below, organize this transformation. Figure 3 depicts diagrammatically the
positions that the cells in each mitotic
domain occupy at about the time when the
first embryonic cells enter mitosis 14, even
though, at that time, only seven domains,
3141—3,47, actually exhibit the mitoses that
define them. A single embryo, stained to
reveal mitosis, would never resemble Figure 3; there is, obviously, no single time
when the cells of all the mitotic domains
are dividing simultaneously. Thus Figure
3 is an abstraction—a forecast. Moreover,
the flow of gastrulation movements continuously alters the positions of the domain
boundaries. In Figure 4, we present a time
line that summarizes the schedule of mitosis and the major morphogenetic movements that occur during gastrulation. In
order to emphasize the dynamic nature of
the embryo during gastrulation and germ
band extension, we schematize in the right
panel of Figure 4 the way four mitotic
domains (di44,3147, d1410, and <3H14) change
their shapes and positions.
The schedules of mitotic domain timing and of
gastrulation movements are tightly coupled
Gastrulation and germ band extension
involve rapid and extensive mechanical
deformations of the blastoderm cell sheet.
Thus the shape of each domain changes
significantly with time, both before and
after its cells divide, and the relative positions of the domains must change, too. For
MITOTIC DOMAINS PARTITION FLY EMBRYOS
a given domain, any embryo-to-embryo
variation of its time of mitosis relative to
the timing of gastrulation movements
would cause significant variation in the
shape of its dividing patch of cells as
revealed by anti-tubulin staining. There is
no such variation. There is, however, significant variation in developmental timing.
We have measured the time schedules of
early development for dozens of individual
embryos at 25°C using time-lapse movies.
Compared to the "average embryo," a particular embryo can accelerate and decelerate its developmental schedule by about
±7 min. That is, it may produce a cephalic
fold 5 min earlier than average, and then
that same embryo may open its cephalic
fold 6 minutes later than average. The
times of developmental events in the left
panel of Figure 4 are averages, each with
standard deviations of about 2-3 min.
There is the same kind of variation in the
timing of mitotic domain sequence. It is as
if development is a movie in which the pictures show the domain geometry while the
sound track tells the schedule of gastrulation deformations. Each embryo projects
the movie at a rate that wavers, but the
627
sound track and the pictures remain always
synchronized. What mechanism in the
embryo enforces this synchrony? We do
not know, but the tight coupling between
the mitotic, and blastoderm-deformation,
schedules suggests that interaction between
various domains may play some causative
role in driving gastrulation movements.
Alternatively, some "master clock" mechanism may drive both.
Mitotic domains are in precise register with the
striped expression pattern of the
segmentation gene engrailed
We stained embryos with antibody
against engrailed protein simultaneously
with anti-tubulin and saw that, along the
anterior-posterior axis, certain domains are
in perfect register with stripes of engrailed
expression that mark the posterior part of
each future segment (data shown in Foe,
1989). This is important because location
of certain domains in register with particular engrailed stripes identifies those
domains as primordia of specific segments
(see Kornberg et al. [1985] and DiNardo et
al. [1985]). A few domains comprise serially repeated cell clusters: d1416, d H 17,
Fie. 4. Time line diagram, adapted from Foe (1989). Spread out along the vertical developmental time axis,
the drawings in the left panel schematize the pattern and sequence of mitotic-domain division times. Spread
along the same time axis in the right panel, cartoons illustrate the sequence of deformations and motions
imposed by the morphogenetic movements of early embryogenesis upon four selected mitotic domains (4, 7,
10, and 14). The time axes, identical in both panels, measure minutes from the start of the 14//J interphase
at 25°C. The time axes also indicate the developmental "stages," numbered 5 through 10, of Bownes (1975
and 1982) as modified by Campos-Ortega and Hartenstein (1985); horizontal dotted lines mark these stage
boundaries.
• Domain 4, stippled in the right panel, is the telson which forms the posterior tip of the germ band. During
stages 9 and 10, the posterior two-thirds of the forming embryo, called the germ band, extends more than
twofold, carrying the telson around the posterior pole (the left end) of the egg. Domain 4 divides during
early stage 8. Thereafter, continuing extension of the germ band pushes domain 4 under the amnioserosa,
out of sight by about 100 min (at 25°C).
• Domain 7, shown hatched with horizontal lines, forms parts of the anterior and posterior walls of the
cephalic fold, so it disappears from view when that fold forms, as shown in the stage 6—7 embryo. It divides
while hidden during stage 8. When, at the end of the rapid germ band elongation stage, the cephalic fold
partially opens, domain 7 emerges posterior to the fold and later, at stage 10, the cells it comprises evaginate
to form the maxillary lobe, the primordium of a mouthpart.
• Domain 10, the presumptive mesoderm, invaginates permanently during stage 6, then divides during stage 8.
• Invagination of domain 10 draws the rest of the blastoderm cell sheet toward the ventral midline, as indicated
by the flow arrows in the stage-5 embryo. This brings the paired strips that constitute domain 14, shown
hatched with diagonal lines, into contact at the ventral midline. Domain 14 divides during stage 8, while
germ band extension is doubling its length. The arrows in the stage-10 embfyo show this domain ingressing
to form part of the ventral nerve cord (Thomas et al., 1988). This occurs between 275 and 330
628
V. E. FOE AND G. M. ODELL
Stages
^Domain mitosis times (min from start
of cycle 14
at 25' C).
Cellularization
of the
5
blastoderm
Gastrulation-
Rapid
germ band
elongation
Slow
germ band
elongation
Gnathal and
clypeolabral
lobe formation
Key to the left panel: T h e numbers, 1,2
25 and N and M, fanned out in the left panel, reference the
mitotic domains shown in Figure 3. Lines run from the time on the developmental time axis when division
begins in a particular domain, through the number identifying that mitotic domain, to end at a dot showing
the location of that domain in a cartoon version of the atlas in Figure 3.
Mitoses occur for extended periods in domains N and M, and the skewed bell-shaped curves at the bottom
of the left panel are histograms that graph mitoses per unit time in domains N and M. The mitotic rate is at
its peak value in domain X, for example, at 105 min.
MITOTIC DOMAINS PARTITION FLY EMBRYOS
629
•Stages
min from start of cycle 14
at 25' C.
(Inward growing membranes reach
{base of nuclei.
Cell membranes reach full depth.
(Transplantation experiments imply cell
I fates are determined
(The cephalic fold and ventral furrow
I start to form.
(The ventral furrow closes tc the depression
<carrying pole cells shifts dorsally. Buckling
' o f amnioserosa creates 2 dorsal folds.
(Antibodies reveal the first 14 engrailed
i stripes.
(The posterior midgut invaginates
I internalizing pole cells: 65—73 min.
First cells enter mitosis 14
Germ band elongation begins
(Polar mitosis of domain 9 internalizes
(brain neuroblasts: 80 - 90 min.
(The amnioserosa becomes squamous and
{the dorsal folds disappear: 95-100 min.
•Germ band elongation carries the
<telson out of sight under the
^amnioserosa: 95—105 min.
(Domain M cells change shape:
M00-105 min.
Domain 8 invaginates: 100—110 min.
(The ventral portion of the cephalic
(fold opens.
First wave of ventral neuroblast
(ingression complete
Antibodies reveal a 15—th engroiled stripe
across the telson (105 min) and four head
potches appear sequentially: 105-140 min.
" F i r s t cells enter mitosis 15.
The clypeolabral lobe begins to form
(The clypeolabral lobe is well formed;
/ ( t h e gnathal lobes begin to form.
—'
rThe mesectodermal cells
/*
(ingress after 170 min.
Key to the right panel: The column at the far right lists selected developmental events, and the times when
they occur, during the three hours following the start of cycle 14.
In the five cartooned embryos in the right panel, arrows indicate the deformational flow of the blastoderm
cell sheet during gastrulation and germ band extension. These deformations carry mitotic domains along, as
indicated by the trajectories of four domains (4, 7,10, and 14). The parts of the caption highlighted by bullets
on the previous page describe the deformational flow these domains exhibit.
630
V. E. FOE AND G. M. ODELL
d, 4 21, and 31425 are examples. We identify
these repeated cell clusters as members of
a common domain because they divide at
the same time and because they occupy
homologous positions in the presumptive
segments of the embryo as seen by their
alignment with respect to the engrailed
stripes.
The mitotic domains form a much more
complex pattern than does the engrailed
protein. In the posterior two-thirds of the
embryo, where fourteen engrailed stripes
appear during cycle 14 and encircle the
embryo, the domains reveal a dorsoventral
partitioning of the embryo not reflected in
the engrailed stripes; the dorsal and ventral
boundaries of the mitotic domains mark
out a pattern of longitudinal strips on the
surface of the germ band (shown diagrammatically in Fig. 3). Thus, it appears that
genes of the dorsal-ventral and the segmentation type act together to orchestrate
the mitotic patterns of cycle 14.
domains, the cells all exhibit unique morphogenetic and cell biological features that
are distinct from those of cells in adjacent
domains; from a cell biological vantage, different domains appear to function differently as units in early embryogenesis. We
emphasize that many domains that exhibit
some unique morphogenetic performance
do so before their cells divide synchronously.
Almost certainly, mitotic synchrony is
merely a superficial, but visible, symptom
that cells in a domain share deeper determining traits—a symptom that appears significantly later than the determination
mechanism previously acted to partition the
domains. The next four paragraphs give
examples of observable traits that distinguish domains.
The cells of some mitotic domains adopt
unusual shapes. For example, just as gastrulation movements begin (at about 55
min) and all cells are still in interphase of
cycle 14, every cell in d,48 becomes cuboidal while other blastoderm cells retain a
columnar shape. At about 95 min, the cells
Cells of specific mitotic domains exhibit
of domain A become squamous, while,
characteristic morphogenetic behaviors and
about 10 min later, those of domain M
give rise to specific structures
elongate in a direction perpendicular to
A "fate map" catalogs what each region the long axis of the embryo. The cuboidal
of the early embryo will become after dif- d,48 cells are the precursors of the anterior
ferentiation in the normal course of events.7 part of the gut. The squamous cells of
We have inferred the fates of the domains domain A become the extra-embryonic
mentioned below from earlier investiga- amnioserosa. The elongated cells of domain
tions of normal development, and of devel- M will produce both cuticle-secreting cells
opment following tissue ablation and trans- of the ventral hypoderm and neurons of
plantation (Lohs-Schardin et al., 1979; the ventral nerve cord.
Poulson, 1950; Technau and CamposAll cells in d,210 and dI422 invaginate
Ortega, 1986a, b; Underwoods al., 1980). abruptly by infolding as coherent sheets of
Since the domains partition the embryo cells. These two domains invaginate at difmore finely than the fate maps deduced in ferent times, but both invaginate while their
these earlier studies, the exact destiny of constituent cells are in interphase 14, that
many domains is at present uncertain. is, before the division that identifies each
Studies by one of us (VEF), now underway, group as a specific mitotic domain. Immein which cells in specific domains are diately prior to their invagination, the apimarked and followed, will assess more pre- cal tips of the cells of <91410 decrease their
cisely the larval fates of the various diameter, changing the shape of these cells
domains. See Foe (1989) for current best from tall columnar to wedge-shaped, with
guesses.
the apices facing the embryo exterior
It is striking that in some of the mitotic (Poulson, 1950; Turner and Mahowald,
1977). The cells of d1422 behave similarly.
Just prior to their division, the cells of
7
Note that the existence of a fate map does not dI414 lie in two continuous rows, each primarily one cell wide, on either side of the
imply when cells become determined.
MITOTIC DOMAINS PARTITION FLY EMBRYOS
ventral midline (Fig. 5B). They retain this
organization during mitosis of cycle 14 and
for about 240 min afterwards. These cells
are precisely those that express the products of the gene called single minded (Crews
et ai, 1988). Later, all the daughter cells
of d1414, but not those of neighboring
domains, will enter the embryo interior by
individual ingression, giving rise to glial
cells and to the midline neurons of the ventral nerve cord (Thomas et ai, 1988).
The cells of d148 and d,49 also generate
daughters destined for the embryo's interior, but these cells arrive in the interior
by yet another mechanism—an oriented
division. During mitosis of cycle 14, the
cells of these domains divide along axes
normal to the surface of the embryo, unlike
the cells of all other domains which divide
along axes parallel to that surface. Figure
7 documents the oriented divisions of d,49.
As a consequence of this oriented division,
d149 produces one set of daughter cells on
the surface and the other inside what will
be the embryonic head. The internalized
cells provide a major contribution to the
developing brain while the surface cells
likely generate external cuticle-secreting
631
cells and sensory structures. The anticipated fates of 88 cells are discussed below.
Are the classical ectodermal, endodermal, and
mesodermal germ layers separated before
mitotic domains partition the embryo more finely?
J. A. Moore (1987) discusses how, classically, the first and primary partitioning
of the blastula's cells is supposed to delineate three primary germ layers: ectoderm,
endoderm, and mesoderm. Subsequent
subdivision of each germ layer is presumed
to determine the primordia for the tissues
and organs that that germ layer comprises.
The concept of germ layers arose first in
the study of vertebrate embryos, and was
then generalized to apply to invertebrates
(see Moore, 1987). In Drosophila, for example, d1410 cells would constitute the classical mesodermal germ layer whose cells form
the connective tissues, muscles, fat bodies,
circulatory system including the blood, and
all parts of the gonads except the germ
cells. d1422 cells will give rise to the epithelial lining of the posterior part of the
gut, so 31422 is a part of the endodermal
germ layer. An example of an ectodermal
mitotic domain is d,4l 1. It will give rise to
FIG. 5. Mitotic domains in wild-type Drosophila embryos. In these micrographs, and in all micrographs below,
the anterior ends of embryos face left. In all micrographs, fluorescently labeled antibodies to ^-tubulin stain
the bipolar mitotic spindles in dividing cells that otherwise appear clear black; non-dividing cells appear gray
since, during interphase, the stained tubulin lies in the cytoplasm around, above and below the nucleus. The
nucleus occupies the tubulin-free area in the center of each interphase cell.
Panel A shows a tilted dorsal view (as in the top panel of Fig. 3) of an 80 min embryo. Panel B (adapted
from Foe, 1989) shows a ventral-lateral view (as in the middle panel of Fig. 3) of a 90 min embryo, cf marks
the cephalic fold. Here, and in the micrographs that follow, numbers indicating the order in which the
domains enter mitosis mark each mitotic domain. In panel A, cells in domains 1, 3, 4, 5, and 6 are in some
phase of mitosis. Except for domain 3, which occupies the dorsal midline at the anterior tip, each domain
comprises two cell clusters, one on the right and one on the left side of the embryo. In panel B, cells in
domains 9, 11, 14, and 16 are in some phase of mitosis. Domain 2 cells, having completed mitosis earlier, are
now in interphase of cycle 15. For help in locating the various domains compare the micrographs with the
atlas in Figure 3.
FIG. 6. Mitotic domains in wild-type Calliphora vomitoria embryos. This figure faces Figure 5 to facilitate
comparison of Calliphora embryos with Drosophila embryos. The upper panel is a dorsal view and the bottom
panel is a ventral view. To further facilitate comparison, the Drosophila embryos in Figure 5 appear at about
three times greater magnification than the Calliphora embryos in this figure so that their apparent sizes match;
note the scale bars in each figure. Domain homologies between Calliphora and Drosophila embryos are evident
from comparing this figure to Figure 5. For example, the telson, labeled as domain 4 in Drosophila (Fig. 5A),
has a homolog in Calliphora. Anterior to the cephalic fold (cf), dorsal domains are evident in Calliphora that
are homologous to domains 1, 3, and 5 in Drosophila. In Figure 6B ventral domains in Calliphora equivalent
to Drosophila domains number 11 and 14 are evident. Differences are apparent as well; see text for further
explanation.
632
V. E. FOE AND G. M. ODELL
MITOTIC DOMAINS PARTITION FLY EMBRYOS
SSS
634
V. E. FOE AND G. M. ODELL
the cuticle-secreting cells that form the
dorsal surface of the larva.
The domain atlas in Figure 3 shows many
fine scale subsets of cells on the blastoderm
surface; these subsets are fantastically more
complex than expected for three germlayer zones. It is quite possible that the
progeny of cells in a given domain will end
up building (or contributing to) a structure
that is entirely ectodermal, or entirely
endodermal, or entirely mesodermal. If so,
it would be possible to construct a simplified fate map of the Drosophila blastoderm
that distinguishes the three classical germ
layers, in which each mitotic domain lies
entirely within one of the three germ-layer
zones. If so, one might try to argue that
the three germ layers are first determined
and subsequently partitioned into the many
mitotic domains.
Our guess, however, is that spatial patterning in Drosophila does not start with an
initial segregation into three germ layers.
We believe, instead, that the domains of Figure 3 partition the blastula into many more
subsets than three prior to segregation of the.
three classical germ layers. From the start of
ers. This is a prediction. The next paragraph outlines a proof that pivots on the
outcome of experiments in progress.
As a consequence of their oriented division, the daughters of dl48 form two parallel sheets. The superficial layer of daughter cells of d,48 later invaginates almost
certainly forming part of the cuticularized
foregut. In arthropods only ectoderm
secretes cuticle, delineating endodermal
and ectodermal contributions to the gut.
Thus, according to the terminology of classical embryology, the superficial layer of
di48 daughter cells belongs to the "ectodermal germ layer." We believe, tentatively, that the internalized layer of daughter cells of d,48 contributes to the epithelial
lining of the anterior noncuticularized gut.
Confirmation of this hypothesis awaits
analysis of experiments, now in progress
by one of us (VEF), in which we track the
trajectories and fates of individually marked
d,48 cells to see if any ends up in the anterior midgut. If they do, the interior layer
of <9148 daughter cells would be part of the
"endodermal germ layer" according to the
terminology of classical embryology. This
would mean that the 14th division of cells
in dI48 produced daughter cells destined
for two different germ layers! It would mean
that the ectodermal and endodermal germ
layers of classical embryology cannot be
separate in Drosophila until d,48 divides.
This, then, would be a clear case in which
at least seven mitotic domains (d,4l to d,47)
segregate prior to delineation of the classical endodermal and ectodermal germ layers.
zygotic transcription, before any morphogenetic movements start and even prior to
cellularization, the stripes of segmentation
gene expression along the anteroposterior
axis, and the bands of dorsal/ventral genes
along the orthogonal axis, together define
a very complex checkerboard pattern of
gene expression over the blastoderm surface. We believe that this checkerboard
pattern of overlapping gene expression
subsequently determines the many mitotic
domains. And we believe that all this occurs
Although it may be appealing from an
prior to segregation of the three germ lay- abstract conceptual vantage, grouping the
FIG. 7. Enlarged ventral-lateral views of the region in front of the cephalic fold in Drosophila and Calliphora
embryos. The cephalic fold (cf) is the crease running from the lower left to the upper right of each embryo.
The ventral surface of the embryos is visible at the lower left of each panel. In the Drosophila embryo in panel
A (adapted from Foe, 1989), the cells of mitotic domain dlt9, indicated by the arrow, divide with their spindle
axes perpendicular to the embryo's surface (note the star-like appearance of spindles seen end on). This
feature, in addition to their mitotic synchrony, distinguishes cells in 3,49 from cells in neighboring domains.
At this stage, the adjacent domains have mostly completed their 14M mitoses.
The lower panel, B, shows a similar view of a Calliphora embryo, at about three times lower magnification
(the scale bars in each panel measure 50 iim). Calliphora exhibits a mitotic domain with polar divisions (endon spindles) which, while we do not yet know whether, in Calliphora, it is the ninth domain to initiate mitosis,
is clearly homologous to Drosophila's d,,9. The arrow points to this domain. Note the spindles oriented parallel
to the embryo's surface in the surrounding domains.
The " B " in each panel indicates a non-dividing domain, homologous in the two embryos.
MITOTIC DOMAINS PARTITION FLY EMBRYOS
@S8
636
V. E. FOE AND G. M. ODELL
organ primordia into the three classical
germ layers is, we believe, mere semantic
convenience and oversimplification, not a
mechanistic description of how cell determination proceeds in embryos.
Mutants that exhibit cuticle-pattern or
morphogenetic movement defects show corresponding mitotic domain defects
Large scale genetic screens have revealed
many of the genes that function either in
the mother or in the new zygote to establish pattern in the early embryo (for reviews
see Mahowald and Hardy, 1985; Akam,
1987). Many of these genes act sequentially
in tiers (see for example, DiNardo and
O'Farrell, 1987; Akam, 1987; Edgar et al,
1989). That is, products of maternal-effect
genes (e.g., bicoid), expressed early in spatial
patterns, orchestrate subsequent patterning of gap genes (e.g., Kruppel), which
orchestrate, in turn, subsequent patterning of pair rule genes (e.g., fushi tarazu)
that, in turn, organize segment-polarity
gene patterning (e.g., engrailed). To illustrate how mitotic domain perturbations
correlate with other known defects in the
development of embryos mutant at earlyacting genetic loci, we discuss in this section the Drosophila maternal-effect mutants
bicoid and dorsal, and the zygotic mutants
Kruppel, snail, and twisted gastrulation).
Embryos from mothers carrying more
(4), or fewer (1), than the normal 2 copies
of the maternal-effect gene called bicoid,
exhibit dramatic global shifts along the
anterior-posterior axis of virtually all early
spatial pattern (see Frohnhofer and Niisslein-Volhard, 1986; Driever and NiissleinVolhard, 1988; Berleth et al., 1988). Extra
copies of bicoid shift the cephalic fold toward
the posterior and cause enlargement of all
head parts at the expense of abdominal
parts that contract (see Fig. 8A). In contrast, a deficit of bicoid gene copies shifts
the cephalic fold towards the anterior,
increasing the size of the presumptive thorax and abdomen at the expense of the
head (see Fig. 8B). All the mitotic domains
are present in these two mutants, but the
numbers of cells per domain differ compared to wild type (compare Fig. 8A and
B with Fig. 5B).
These extreme perturbations are apparently transient; that is, the embryos become
apparently normal adults (Berleth et al.,
1988). We have examined the larvae that
hatch from 1-copy and 4-copy embryos, and
these too appear superficially normal.
Therefore, to correlate the genetic perturbation with the resultant phenotypic
variation, one must look at early stages of
development.
Figure 9A shows the extraordinary
transformation caused in embryos from
mothers carrying zero copies of the bicoid
gene. The anterior cells that would normally form head and thorax structures form
instead an extending germ band, with a
second telson and pole cell cup (devoid of
pole cells) at the front. Only mitotic
domains that constitute the telson and
abdomen form in these mutants. Domains
that divide after 3144 are not shown, but
appear in the normal sequence. Domains
characteristic of the head and thorax
apparently cannot form in the absence of
bicoid expression. Figure 9 shows a d,44 in
mitosis in both the anterior and posterior.
Embryos from mothers in which the dorsal gene is absent exhibit no ventral structures (Anderson and Niisslein-Volhard,
1984); for example du 10 is altogether missing, no ventral furrow forms, and no cells
FIG. 8. Drosophila embryos whose mothers installed too much and too little bicoid product. In both panels,
numbers indicate some of the mitotic domains shown in the atlas in Figure 3. The top panel shows a ventral
view of an embryo with 4 copies of the maternal-effect gene bicoid, which is twice the normal number of
copies. The lower panel shows a ventral view of a mutant with one-half the normal number of bicoid copies—
1 copy. Compare these ventral views to the ventral view of the wild-type embryo in Figure 5B to see how
four copies shift the cephalic fold (cf) farther posterior and one copy shifts it farther anterior. All domains
are present in each embryo, but head domains in the 4-biroirl-capy embryo (top panel) are relatively larger
than, and have more cells than, their counterparts in the 1 -bicoid-copy embryo (bottom panel). Surprisingly
these perturbations are transient; adults with normal proportions result (Berleth et al., 1988).
MITOTIC DOMAINS PARTITION FLY EMBRYOS
§37
638
V. E. FOE AND G. M. ODELL
of mesodermal type develop. Mitotic
domains that, in wild-type embryos, span
only a limited dorsal-ventral extent, expand
to form ring-shaped domains that circumnavigate the entire embryo. Figure 9B
shows two such mutant domains, both in
the head region.
Many of the genes transcribed by the
zygotic nuclei during, and prior to, gastrulation can affect individual mitotic domains.
For example, in the normal embryo, the
dorsal-most cells of the blastoderm (domain
A in Fig. 3) do not undergo further cell
division, but become the large, squamous,
polyploid cells of the amnioserosa. During
morphogenesis, this sheet of amnioserosa
cells folds up accordion-like in front of the
elongating germ band (see cartoons of
stages 8 and 9 in right panel of Fig. 4). In
embryos homozygous mutant 8 for the gene
called twisted gastrulation, the cells of the
amnioserosa remain columnar, the
amnioserosa never exhibits the usual
accordion folds, and the tip of the elongating germ band deflects into the interior
of the embryo (Zusman and Wieschaus,
1985). The consequences are lethal.
A second example concerns the ventralmost cells of the blastoderm, d1410, which
develop apical constrictions and participate together in the formation of an invaginating cell sheet in the normal embryo.
Once internalized, these cells constitute the
primordium of all larval cells of mesoder-
8
Embryos from parents, each of whom have one
good copy of a gene and one mutant copy, can inherit
0, 1, or 2 good copies. The 259£ of the embryos who
inherit 0 good copies are called "homozygous mutant."
mal type. In embryos homozygous mutant
for the zygotically transcribed gene snail,
d1410 is aberrant: its cells do not divide on
schedule, do not invaginate, and do not
produce muscles or the other mesodermal
tissues (Kavita Arora, personal communication).
Embryos in which the zygotically transcribed gap gene Kriippel is absent exhibit
a larval cuticle phenotype lacking the second and third thoracic segments and the
first 5 abdominal segments (Wieschaus et
ai, 1984). These embryos show perturbations of the mitotic domains in the region
this gap spans (data of VEF not shown).
For example, in wild-type embryos, d 14 ll
comprises 5 repeats (see Fig. 3) and gives
rise to dorsal ectoderm spanning the second thoracic to the eighth abdominal segments. 5146 occurs anterior to 5,411 and
includes the primordia of the first thoracic
segment. In Kriippel mutants, d 14 ll has
only two repeats (of about normal size). <9146
enlarges posteriorly, encroaching into the
territory occupied by the anterior of
domain d 14 ll in wild-type embryos.
EMBRYOS OF CALLIPHORA VOMITORIA
BLOW-FLIES EXHIBIT THE SAME
KIND OF MITOTIC DOMAINS
SEEN IN DROSOPHILA
Drosophila and Calliphora embryos appear
to exhibit very similar early development
even though they diverged some 60 million
years ago (Shaw and Meinertzhagen, 1986).
Calliphora eggs are about three times longer
than Drosophila eggs. With a length-towidth ratio of 2.9, Calliphora eggs are proportionally longer and thinner than Drosoph-
Fic. 9. Drosophila embryos from mothers lacking the bicoid gene, or the dorsal gene, lack certain mitotic
domains. Here we see micrographs of two embryos with dim futures. The top panel shows how, in an embryo
without any of the bicoid gene's product installed by the mother, no head parts or cephalic fold forms. Instead
the embryo makes two posterior tips, with telsons (domain d u 4) at both ends. Pole cells (pc) develop only at
the natural posterior tip.
The lower panel shows how, in an embryo without any of the dorsal gene's product installed by the mother,
no ventral structures develop. Some mitotic domains that used to occupy a limited dorso-ventral extent in
wild-type embryos become ring-shaped domains that circumnavigate the rforsa/-deficient embryo. The two
circumferential domains shown here dividing likely result from a dorsal-to-ventral expension of d,,l and d M 3.
A ventral view would show that no ventral furrow forms.
The embryos in both panels havejust reached the stage to begin germ-band-elongation (see Fig. 4). Obviously
this process proceeds with striking abnormality in both of these lethal mutants.
MITOTIC DOMAINS PARTITION FLY EMBRYOS
639
640
V. E. FOE AND G. M. ODELL
ila eggs, which have a 2.4 length-to-width
ratio. Figure 2 dramatizes the differences
in size and shape. Drosophila and Calliphora
eggs have about the same number of cortical nuclei (about 6,500) when they cellularize in the 14th interphase. Each mitotic
spindle organizes about the same fraction
of the eggs' cortical cytoplasmic volume,
and this means each Calliphora spindle and
nucleus occupies about 27 times the volume of their counterparts in Drosophila
eggs. Roughly speaking, Calliphora embryos
appear to be photographic enlargements
of Drosophila embryos, developing on the
same temporal schedule.
The first life history stages of the two
species are similar in that both exploit an
ephemeral larval resource (rapidly decaying fruit or carrion). In this case one expects
evolution to select for the fastest possible
egg-to-crawling-larva development time,
because competition between larvae means
that slow developers risk starvation (Ives,
1988). It is likely that this is why, in both
species, the first 13 division cycles preceding the mid-blastula transition race at the
fastest rate known in the animal kingdom.
The procedures used on Drosophila for
removing the chorions and vitelline membranes en masse, for fixation, and for staining with anti-tubulin (see Foe, 1989), also
work on Calliphora. With the protocol for
revealing mitotic domains in Drosophila in
hand, and with an understanding of them,
a comparative analysis of mitotic domains
in Calliphora vs. Drosophila proceeds very
quickly. Refer to Foe (1989) and to the
Appendix for experimental techniques
simplified for classroom use.
The first 13 division cycles are similarly
synchronous in both Calliphora and Drosophila embryos (see Fig. 2), and against
this prior uniformity embryos of both
species exhibit the same kind of mitotic
domain partitioning in the \4th cycle just
following the mid-blastula transition. Figures 5 and 6 illustrate the surprising
homology, as assessed by comparing mitotic
domains, between Calliphora and Drosophila embryos. Figure 7 documents, with
higher magnification micrographs of the
developing head, the existence of domains
in Calliphora homologous to polar-dividing
d149, and to non-dividing domain B, in Drosophila.
There are a few differences in mitotic
domains between the two species, too. For
example, domains d1416 and d 14 l7, which
consist of separate metamerically reiterated clusters, occur in Drosophila only in
the abdomen, but their apparent homologs
in Calliphora extend throughout the abdomen and thorax. As another example, the
homolog of Drosophila's d,43 occupies a
relatively larger area of the blastoderm in
Calliphora than in Drosophila. Minor differences in the timing of division in homologous domains exist as well. At 25°C the
\4th mitoses appear to be compressed into
a shorter time interval in Calliphora than
in Drosophila.
Many authors have attempted to classify
morphological structures in terms of evolutionary derivation. Matsuda (1965) and
Rempel (1975) among others argue that
evidence for such evolutionary derivation
should be based primarily on embryological data. The mitotic domains constitute
the earliest embryonic primordia so far
recognized. We predict that it will prove
much easier and less ambiguous to study
homology between structures of different
insect species by comparing their embryonic mitotic domains than by comparing
hatched larvae, especially as regards head
structures, which often differentiate into
fantastically baroque larval parts that defy
simple comparison.
THE DETERMINATION PROCESS
VIEWED AS A DYNAMICAL SYSTEM
Elucidation of the complex stereotyped
pattern of nuclear division in the early Drosophila embryo raises a number of questions central to developmental biology.
Mitotic synchrony in each domain seems
to be indicative of some deeper underlying
property, yet to be determined, that the
cells of any one mitotic domain share and
that distinguishes them from the cells of
adjacent domains. This may be the same
property that results in the commitment of
cells to distinct developmental pathways. It
is, after all, during cycle 14, when this complex mitotic pattern first appears, that 111—
mensee (1978) and Simcox and Sang (1983)
MITOTIC DOMAINS PARTITION FLY EMBRYOS
demonstrated the existence of cells whose
larval fates were determined.
Classically, to discover experimentally
whether a cell is determined one would
transplant a suitably marked cell to a new
location and, after significant development
and differentiation, assess whether the
transplanted cell differentiates as it would
have in its original location. If it does, it is
called determined at the time it was transplanted. If it does not—that is, if it differentiates into tissue appropriate to its new
location, forgetting its original fate—then
the cell was not determined when it was
transplanted. (See Simcox and Sang [1983]
and J. A. Moore's [1989] statement 71 in
this volume.) We do not yet know whether
the mitotic domains delineate cell groups
with distinct cell determination identities
in this classical sense. We do not even know
in detail how the mitotic domain atlas in
Figure 3 correlates with Drosophila fate
maps.
We agree that this classical definition of
determination is the proper definition of
the outcome of the process of determination, but we believe that the process itself
is more interesting than the outcome. We
need to understand the mechanism that
orchestrates this process.
Dynamical systems theory is a conceptual
framework for thinking biochemically
about the process of determination
In this section we discuss a way of thinking about cell determination as a continuously graded dynamical process. We discussed above the observation that zygotic
transcription during cycles 13 and 14,
guided by maternally installed information, leads to accumulations of the products of many segmentation and dorsal-ventral genes which, taken together, mark out
a checkerboard pattern on the blastoderm.
The many genes involved interact in a
tightly coupled network in which various
genes influence the transcription rates of
others that, in turn, regulate them. This
patterning starts with a very coarse scale,
then gradually generates ever finer scale
and sharper resolution. If one or another
gene in the network is mutant or absent,
then it can profoundly alter the spatial pat-
641
tern that the network generates. This
resultant perturbation of the spatial accumulation pattern of gene products causes
perturbations in the visible pattern of
mitotic domains during cycle 14 (as several
examples above illustrated) and, later on,
causes perturbations of the larva's morphology. We want now to introduce a way
of thinking about the mechanism of embryonic cell determination as governed by
networks of interacting genes that act as a
"decision-making" mechanism. We want
to think of these networks as constituting
"switches" that are capable of flipping to
any of several settings (determination), and
are, once set, capable of remaining stably
set (cellular "memory").
Waddington (1966) introduced an "epigenetic landscape" metaphor for differentiation. He likened a totipotent embryonic cell to a ball at the top of a hillside.
Many parallel gullies run down the hillside,
bifurcating. When the ball starts rolling
down the hill, it can follow any of many
possible gullies. Infinitesimal forces can
deflect it, sideways, from one gully to
another. Rolling into a particular gully
restricts its future choices to the gully it is
in, or to ones that bifurcate from it farther
down the hill. There is a sequence of leftor-right choices for the ball to make as it
rolls down, and each choice it makes
becomes rapidly irreversible because, having taken the right fork, there is no (natural) way to back up and go left. In the
epigenetic landscape metaphor, the gullies
are shallow at the top where the ball starts,
but become ever deeper farther down the
hill. While the ball is traversing a gully with
low banks, a minor perturbation could
throw it out of one gully and into another.
Farther down the same gully, when it is
deep, the same magnitude perturbation
would have no effect. The gullies lead to
stable termini at the bottom of the hill at
any of which the ball could come to rest;
these represent determined or differentiated cell states.
Waddington's landscape provides a nice
way to think about determination. It evokes
the dynamical nature of the process, the
early plasticity, the sequential choices, and
the eventual stability to perturbations. But
642
V. E. FOE AND G. M. ODELL
what real biochemical mechanism could
underlie it? To a surprising degree, the
dynamical behavior of very simple networks of interacting genes, in which the
products of each gene modulate transcription of the others, matches Waddington's
metaphor. To see the match, it is necessary
to make a mathematical model of how several genes interact, then, with a computer,
calculate the dynamical behavior of the
model and graph it in a special way. We
will do this very briefly here, and refer the
reader interested in the details to the excellent general discussion by Slack (1983) of
"dynamical systems theory" as applied to
the puzzle of developmental determination.
We will consider here a generalization
of a detailed model by Edgar et al. (1989)
of three Drosophila segmentation genes—
the pair rule genes9 hairy, eve, and runt—
interacting as a network during cycles 13
and 14. That model aimed to explain how
the interaction of the three genes leads to
"bi-stable switch" behavior of the network
acting in the determination process of each
blastoderm nucleus, and to explain how
the myriad nuclei in the blastoderm cell
sheet could, as a consequence of this switch,
express sharply resolved stripes of runt and
fushi tarazu alternating with stripes of hairy
and eve. We sketch that model here, generalize it, then show how its behavior is an
instance of Waddington's metaphor, hairy,
eve, runt, and fushi tarazu are examples of
the early-acting Drosophila genes that Nusslein-Volhard, Wieschaus, and Jiirgens
identified in their mutant screen (see references in Tearle and Niisslein-Volhard,
1987); we expect mutations in these genes
to alter the mitotic domain pattern,
although we do not yet know what the
altered domain atlas may look like in
embryos mutant at these loci.
Edgar et al. (1989) constrain their network model to negative interactions between
hairy, eve, and runt. That is, they assume
that the product of the hairy gene represses
transcription of runt, and that, in turn, runt
protein represses transcription of hairy.
This is an important difference between
the model discussed below and those discussed by Slack (1983) in which the crucial
mechanism in each model is positive feedback autocatalysis. It is a sequence of drug
injection experiments (Edgar et al., 1989)
that forces the constraint of allowing only
negative interaction between the pair-rule
genes. Very roughly described, these
experiments showed that:
• The mRNAs transcribed from hairy, eve,
runt, and fushi tarazu are all very unstable
with half lives of about 6 min.
• The fushi tarazu protein is unstable with
a half life of about 5 min.
• Following injection of protein synthesis
inhibitors into Drosophila eggs in early
cycle 14, pair-rule gene stripes disappear
because every nucleus in the germ band
transcribes hairy, eve, runt, and fushi tarazu at higher than normal rates.
These experiments imply that, unless
actively repressed by specific proteins
(which are unstable and disappear shortly
after a cycloheximide injection halts protein translation), each nucleus transcribes
each pair-rule gene at the maximum rate.
Each gene's products appear in bands only
because some protein factor represses its
transcription in a spatially patterned fashion—in the interbands. Figure 10 depicts
9
They are called segmentation genes because they
the network model that Edgar et al. (1989)
play a causative role in determining the segmented
morphology of the larva. They are called pair-rule proposed to account for known interacgenes because, as judged by cuticle defects in larvae
tions between hairy, eve, and runt. In this
lacking one of the genes, they act on segment pairs;
essay, we generalize this network to include
mutants show cuticle defects in every other segment.
the possibility of repressive interactions
The peculiar names that biologists have given to four
(of the eight) pair-rule genes discovered so far are: between eve and hairy. The resulting nethairy, runt, eve (which is short for even skipped), and//z work is very simple, but can exhibit very
(which is short for fushi tarazu). Because these genes
complicated dynamical behavior.
have been cloned, molecular probes exist for each of
To understand the behavior of the gene
them: radioactively labeled anti-sense RNA strands
that hybridize with their mRSA in \itu, as well as antinetwork in Figure 10, we have stated its
bodies to their proteins.
assumptions in the mathematical language
MITOTIC DOMAINS PARTITION FLY EMBRYOS
of differential equations. The equations
constitute a dynamical system of the general
sort that Slack (1983) discusses, and they
allow us to compute how the concentration
of each gene's product evolves with time
(starting with a very low initial concentration) as a consequence of the gene's expression rate being a function of the concentrations of the other two gene products.
See Edgar et al. (1989) for some of the
mathematical details. By adjusting parameters that characterize the strength by
which each gene represses the other two,
we can explore the qualitatively different
kinds of behavior the network can
exhibit—kinds of behavior one could not
guess intuitively, but which emerge from
the mathematical analysis. It turns out that
not just any kind of behavior is possible,
but only a few kinds, of which we consider
just one here: tri-stable switch behavior. The
network has built into it, as determined by
the repressive strengths each gene exerts
on its competitors, three dynamically stable equilibrium points. These are three different sets of concentrations:
• runt high and both hairy and eve low;
• hairy high and both runt and eve low;
• eve high and both hairy and runt low.
Under each of these conditions, the decay
and synthesis rates of each gene's product
exactly balance so that the concentrations
of the three proteins do not change. There
are other equilibrium states, but only three
are stable to perturbations. That is, if something (an experiment, say) perturbs the
network somewhat after it has come to rest
at one of these equilibrium points, it returns
to the same equilibrium point. These three
points represent stable states of determination (as regards those characteristics of
the cell controlled by the expression levels
of the three genes); once the cell
approaches any one of the three stable
equilibria, it will stay there, unless very
greatly disturbed.
The existence of these three equilibrium
points depends on parameters in the system. Thus, changes in the rate constants
by which the gene products bind to DNA
can cause changes, even elimination, of
each of the three equilibria. Changes in the
643
FIG. 10. Schematic diagram of the mutual-repression pair-rule gene network. The products of each
gene in the network represses expression of the other
two, as indicated by arrows marked with minus signs.
Unless repressed, each gene is expressed constitutively. Each gene's products decay and have the short
6 min half lives Edgar measured. The network behaves
dynamically as a switch only if each gene's protein
acts cooperatively to repress transcription of the other
genes. Mathematically, the requirement is that, at low
concentrations, doubling the concentration of hairy
product, for example, quadruples the strength of its
repression of runt transcription. This will be the case
if hairy product acts by making dimers that then bind
to DNA sites to repress transcription. The network
does nothing interesting unless repression is cooperative. See Edgar et al. for a discussion of the necessity of cooperativity. Edgar et al. (1989) analyzed a
slightly simpler network that did not include the hairy
— eve and hairy — eve interactions shown by dashed
arrows in the figure.
constitutive rate of transcription of any of
the three genes can have similar effects.
The interesting feature of the network's
behavior is not that it has these three stable
states. The interesting feature is that,
regardless of its initial concentrations, a
cell must go eventually to, and remain at,
one of those states. This means that the
process of determination, as modeled in
this dynamical systems framework, is "canalized." Cells cannot be determined in just
any state, but only in one of several quantized, distinct, stable equilibria. Regardless
of what an experimenter inflicts, each cell
will end at a state that is meaningful in an
autonomous cell-biological sense, although
it may be a state that does not mesh with
the states of its neighbors. Figure 11 por-
644
V. E. FOE AND G. M. ODELL
MITOTIC DOMAINS PARTITION FLY EMBRYOS
trays in phase portraits the trajectories a
cell can follow.
The words above describing the "gullies" of Waddington's epigenetic landscape
could equally well describe the bifurcating
rivers of trajectories seen in Figure 11. Note
especially what happens to the group of
trajectories that start near the phase portrait's coordinate origin (corresponding to
low initial concentrations): they rise
together as the concentrations of all three
genes' products increase at similar rates,
In the center of the diagram, this group of
645
trajectories splits, or bifurcates, into two
rivers. One runs up to the equilibrium at
which runt is at high final concentration
while hairy and eve are low. The other river
falls (that is, the concentration of runt
decreases), and then it, too, bifurcates into
one river running to the {hairy high, eve
and runt low) equilibrium, and another
river running to the {eve high, hairy and
runt low) equilibrium. That is, the phenomenon of making sequential binary
decisions arises naturally from very simple
repressive interactions between three
FIG. 11. Phase portraits of the dynamical behavior of the three-gene switch network. Every panel shows
how the concentration of three gene's products (of hairy, eve, and runt) might change with time as the product
of each gene represses the expression of the other two. At any given time, t, each gene's product will have
certain concentrations, (H(t), E(t), R(t)), and these are used as cartesian coordinates to locate a point in the
3-dimensional phase space whose coordinate axes are labeled (HAIRY, EVE, RU) in the figure. As time advances,
the (H(t), E(t), R(l)) concentrations change and thus the point in the phase space will move. A computer
program plots the trajectory of the moving phase point as a continuous line that starts at coordinates corresponding to the initial concentrations and ends at a final stable equilibrium point (state). Trajectories such
as these could be drawn to fill up the entire phase portrait densely; one goes through every point. But in the
top and middle panels we have drawn only a few, whose initial concentrations begin near the coordinate
origin. Arrowheads show the direction of movement with increasing time. The phase portraits in the center
and lower panels show, in 3-D stereo-pair images, several such trajectories from which the routes of the others
can be inferred. Instructions for viewing stereo pairs appear below.
Top panel: The drawings show mono images of three groups of trajectories that come to rest at three
different equilibria. All trajectories start near the coordinate origin, which corresponds to zero initial concentrations. The left drawing shows a group of trajectories that start with slightly elevated concentrations of
R(0) and all progress to a final stable equilibrium point with a high value of R, and low values of both H and
E. The middle drawing shows a group of trajectories that start with a slightly elevated concentration of H
and lower concentrations of E and R. These all run to a final stable equilibrium point with a high concentration
of H and low concentrations of R and E. The right drawing shows a group of trajectories that start with
slightly elevated concentrations of E and lower concentrations of H and R. These all run to a final stable
equilibrium point with a high concentration of E and low concentrations of R and H.
Center panel: The stereo pairs show the three groups of trajectories in the top panel merged. There are
three possible equilibria at which trajectories can come to rest. Regardless of what the initial concentrations
are, the system will eventually attain one of these equilibria. The interesting thing about this phase portrait
is that trajectories that start near the coordinate origin (H = 0, E = 0, R = 0) first diverge, then converge to
one of these three possible destinations. The destinations represent three determined states; the initial conditions near the origin represent cells with a slight initial bias, but cells that, if very slightly perturbed near
the origin, could end up in any of the possible destinations. See text for further explanation.
Bottom panel: This stereo pair shows the same groups of trajectories in the center panel, but from a different
vantage, and with several other trajectories added. These other trajectories document the great stability of
the three final states. The set of all points in the phase space from which trajectories would run to a given
equilibrium point is called the "attraction basin" of that equilibrium point. From the several trajectories, one
can judge the size and shape of the attraction basins of each equilibrium. After a perturbation from one of
the equilibrium points to any point in the corresponding attraction basin, the trajectory will return to the
same equilibrium point.
Instructions for viewing the stereo pairs in the center and bottom panels. Only 7% of human beings cannot
fuse stereo views. Without using any mechanical device, but by slightly crossing your eyes, you should be able
to fuse the left and right images together into a central three-dimensional picture that gives a convincing
illusion of depth. If you have trouble fusing the stereo pairs, try focusing both eyes on the tip of your index
finger held about halfway between your eyes and the page. This will force fusion of the images behind your
finger, although they will be out of focus. The left and right images on the page will remain in your visual
field, flanking a central fused image; ignore the side images. Then pull your finger out of the way while
keeping your eyes aimed through the point where your finger was. Concentrate steadily on the central image
until you achieve focus and fusion. Persistent concentration is the key. It is really worth trying to master this
technique because, when you succeed, the illusion of three-dimensional reality is incredible. You cannot dodge
responsibility by claiming to be among the unlucky 7% until you try for at least 5 min with no progress.
646
V. E. FOE AND G. M. ODELL
genes. Think of each trajectory in the mid- considered is certainly wrong in detail, but
dle panel of Figure 11 as representing a that does not matter. The mathematical
different cell that feels slightly different model of "the real gene network" will have
influences from earlier acting genes and/ the same general properties as the one we
or maternally installed factors that mod- just discussed.
ulate transcription. As a consequence of
If this framework for understanding the
the network interactions, these cells, that process of commitment is correct then the
start at almost the same place, rapidly process of differentiation is most interesting
diverge, forming three different "rivers" precisely where it is most labile—early on.
of trajectories. The trajectories that form As soon as you try to understand the proeach river converge inexorably as each river cess of determination in this framework,
runs to one of the three possible deter- the classical distinctions (see Slack, 1983)
mined states of stable equilibrium.
between "specialized," "determined," and
For our present purposes, the most "differentiated" start to melt. What
interesting property of the dynamical remains is a process in which cells become
behavior of the model is this: cells on tra- ever less plastically determined, in which a
jectories near their starting points near the late-acting perturbation must have great
coordinate origin are already "deter- force to cause a shift of fate that a slight
mined" to run to one particular terminus perturbation could have caused by acting
even though they have very similar states sufficiently early. This means that it does
initially. They are "determined," but any not matter much whether the boundaries
slight perturbation, in the form of influ- of mitotic domains coincide with boundences from neighboring cells, experimen- aries between cells that are differently
tal manipulations, etc., can shift any cell irreversibly determined in the classical sense.
from one trajectory to any other. That is What matters is that they are almost cerbecause the trajectories are initially very tainly a very early cell biological reflection
close together. As time goes on, however, of some degree of divergence along traand cells move out along their diverging jectories headed for different final states.
trajectories, it takes ever greater perturbations to shift a cell onto another trajecCONCLUSION
tory that goes to a different equilibrium
If the above theoretical framework
than it is already destined to reach. That describing cell determination is accurate,
is, the attraction basins of the three equi- then to probe the mechanism responsible
libria intersect near the coordinate origin for orchestrating cell commitment, it is
(see Fig. 11). It is easy to jump from one essential to look as early in the process as
attraction basin to another when the con- possible. To "look early" means to inflict
centrations of all three gene products are early experimental perturbations, or mutalow, and all cells must start with low con- tions of early-acting genes, then to assess
centrations of zygotically transcribed genes. the earliest resultant alterations one can
Three is a small number of genes. We detect. The nature of the process is that,
consider only three in order to be able to with time, it becomes ever more resistant
make a comprehensible diagram. To imag- to perturbation. After trajectories diverge,
ine what really happens during embryonic this plasticity fades; a perturbation that
determination, imagine a phase portrait moves a cell from one trajectory to a neighsuch as the one in Figure 11 but with boring one makes no change in final deshundreds of coordinate axes, one for each tination. It changes only the route the cell
gene involved. Imagine this high-dimen- takes to that destination.
sional phase space invested with myriad
Molecular probes are now available that
distinct stable equilibria—the destinations make it possible to localize in situ the
in Waddington's landscape—and imagine expression of both the mRNA and protein
rivers of trajectories running out toward for many Drosophila segmentation and dorall of them, bifurcating and bifurcating and sal-ventral genes. One can do this at any
again bifurcating. The particular model we stage of development, and it has been done
MITOTIC DOMAINS PARTITION FLY EMBRYOS
impressively; the volume of such information recently discovered is overwhelming (see, for example, Akam, 1987). Surely,
alterations in the spatial and temporal patterning of genes, which the gene probes
will reveal, must occur earlier than changes
in the pattern of mitotic domains. Having
just emphasized that, to understand the
embryonic determination mechanism, we
should look as early as possible, why should
we bother with assessing perturbations of
mitotic domains? The reason is that to
understand embryogenesis we must comprehend (at least) two puzzles:
1. what mechanisms control the developmental sequence and spatial pattern of
differential gene expression?
2. how does differential gene expression
control the physical actions of cells to
direct them to reshape and build the
embryo?
These are questions of mechanism—
mechanisms that are intimately coupled.
To begin to answer them, we must correlate accurate descriptions of both the gene
expression patterns and the cellular
responses to them. Using mutants, we must
correlate changes in one with changes in
the other.
The last section of this paper on the
application of dynamic systems theory to
gene networks relates to item 1. A mitotic
domain comprises cells exhibiting synchronized actions directed by the gene expression patterns that gene networks produce.
That is, to lay a foundation of data relevant
for approaching item 2, we advocate mitotic
domains as easily observable, phylogenetically general,10 early delineations of cell
groups newly launched along diverging
determination pathways, which, along the
way, express different, developmentally
meaningful, cell actions. We do not yet
know to what extent membership in a particular mitotic domain correlates with the
647
number of determination "decisions"
already made by a cell. However this turns
out, it is certain that, in order to probe the
mechanism responsible for determination,
one must use cell biological characteristics
of cellular "phenotype" to assess how far
cells have proceeded along the diverging
trajectories that lead eventually to classically defined determination end states. It
is, further, essential to conceive of determination as a gradual process. It starts with
initial totipotent identity of many cells. It
advances through fragile stages of slight
differences between cells, during which
dynamic gene switching, and possibly cellcell communication, gathers adjacent cells
into cohorts of common fate or splays them
apart. Eventually, the process terminates
at diverse, imperturbably stable, final states
of determination. We know already, experimentally, what the previous sentence
means. We want to know what its predecessor means mechanistically and experimentally. Much of the scientific language
of classical developmental biology seems
biased to conceive of development as a discontinuous process. It seems to say that
each cell makes the trip from initial totipotency to terminal differentiation in a few
discontinuousjumps, stopping only at a few
named stations. To understand developmental biology, we need to understand the
navigation scheme underlying the continuous trip, not the names of the stops.
Toward this aim, we need to devise a new
language of process, in which there are
infinitely divisible degrees of "determination," not just undetermined cells and
determined cells.
We need, moreover, a more plastic
notion of differentiation. It is obviously
wrong to think of embryonic cells as amorphous, without differentiated character,
right up until the moment they suddenly
differentiate and "do something specific."
It is certain that most somatic cells end up
terminally and irreversibly differentiated,
for example, as a striated muscle cell doing
nothing other than retracting the larva's
10
To comprise two dipteran fly species is not won- mouth hook, with no capability whatever
derful phylogenetic generality. Note, however, that to suddenly participate in an epithelium.
Calliphora is the first and only species we have scanned That mesodermal cell's lineage started,
for mitotic domains other than Drosophila; this is a
however, with some cell in mitotic domain
tantalizing hint of generality.
648
V. E. FOE AND G. M. ODELL
d1410. That precursor and its progeny had
to adopt several very precisely tuned, albeit
transient, cell biological morphologies and
functions en route to becoming a striated
muscle cell. It had to specialize very precisely to function morphogenetically as an
epithelial-like cell in the mesodermal cell
sheet that invaginated to form the ventral
furrow, internalizing the mesodermal germ
layer. That ventral furrow formed because
of carefully coordinated apical constrictions in all the cells of d1410 during a particular nine-minute time window just after
cellularization (see Odell etal, 1981). The
cytoskeletal architecture required for the
morphogenetic performance of those cells
(then apparently epithelial) required no
small degree of differentiation to assemble
and control. At least temporarily, those cells
exhibited sufficient specialization, and specific cell-cell interaction behavior, to qualify them as "differentiated," had they been
part of a fully developed organism. We
know that zygotically transcribed genes
orchestrated that specialization because
embryos lacking the gene snail, for example, have no group of cells that behave like
d,410 cells do in wild-type embryos. Meanwhile, the cells in d,49 behave utterly differently than the cells in d,410 even though
they divide only a bit earlier and lie nearby.
Regardless of the degree to which these
cells are determined in the classical sense,
they are, by cell biological criteria, differentially specialized, and this differential
specialization is sufficiently significant that
its failure is lethal.
sive genes yields tri-stable switch behavior in which (totipotent) nuclei, with
nearly identical initial conditions, run
along diverging trajectories toward
eventually stable equilibria at one of
three determined states. In this model,
early perturbations of small magnitude
can shift a cell between trajectories
leading to opposite determination states;
late perturbations of equal magnitude
have no long-term consequence. If this
is correct, we will learn most about the
mechanism of determination by correlating the earliest perturbations we can
inflict with the earliest phenotypic consequences we can assess.
2. We summarize a recently completed
work by Foe (1989) of the cycle 14
mitotic domain atlas in Drosophila as a
case study of how to use subtle differences in cell biological behavior during
early embryogenesis to reveal the first
morphological consequences of determination very shortly after it occurs.
3. We sketch preliminary results of ongoing studies of how mutations at earlyacting genetic loci in Drosophila affect
mitotic domains. This opens an exciting
research direction: if we can assess in a
cell biological way the changes in the
mitotic domain atlas, and changes in
action and interaction of domains, in
the many mutant Drosophila embryos
now available in which known early-acting genes are missing or aberrant, then
we will attach specific genetic handles
to specific developmental cellular functions. We emphasize that mitotic
Our point is that the techniques of moddomains
provide a spatial "map" of the
ern cell biology permit us to assess trangastrula that is more finely divided than
siently differentiated characteristics of
any fate map yet made for Drosophila
developing cells, and we should make full
embryos,
and which is as sharply
use of this to study the process of cell deterresolved and as constant, embryo to
mination as it unfolds, because it is likely
embryo, as existing gene expression
that these transiently expressed charactermaps revealed by antibody or in situ
istics require no less precise regulation of
hybridization probes.
gene expression than does terminal differentiation. Our main foci in this essay are 4. To the extent to which mitotic domains
four:
turn out to be phylogenetically general—and the existence of mitotic
domains in blow-flies hints at general1. We advocate the concept that embryity—they provide a short cut method
onic cell determination is a continuous
for investigating and comparing the
process. We describe how a simple model
early development of diverse organnetwork of just three mutually repres-
MITOTIC DOMAINS PARTITION FLY EMBRYOS
isms, for assessing homology, without
the investment of the many thousands
of person-years it has taken to produce
the truly astonishing collection of
mutant stocks, cloned genes, in situ
hybridization probes, and antibodies
now available for Drosophila.
We close with a personal note, concerning
determination, to any totipotent reader
who chances to scan these pages. We hope
you can see the wonder through the verbiage. To make a career of biological science, you must present your work in a serious style; long strange specialists' words
convey meaning efficiently, so we use them.
The essay we write, however, bears the
same resemblance to a developing embryo
as a stuffed falcon does to a diving peregrine: it is merely accurate—as far as it
goes. That is, translating our observations
of the living cells of a developing embryo
into written form is, for us, like taxidermy;
there is a sense of loss and museum mustiness to it because we cannot convey in a
paper the sense of wonder and astonishment that floods up through a microscope
aimed at a living embryo. We can perhaps
convey our testimonial that there is at this
time no career on planet earth more emotionally thrilling, day after day, than basic
research on living cells. Calliphora blowflies have the same kind of mitotic domains
as Drosophilal We discovered this six weeks
prior to this symposium. We call this work,
but nothing we ever did as play—in high
school or college—was as astonishing or as
much fun.
ACKNOWLEDGMENTS
We thank Christianne Niisslein-Volhard
for the generous gift of mutant stocks. We
are grateful to Kavita Arora for sharing
pre-publication results of her study of the
snail mutant. We thank Deanna Lickey for
traveling hundreds of miles on Christmas
eve to print the photographic plates in this
article. We thank Ingrith Deyrup-Olsen,
Ulrike Gaul, and Bruce Alberts for critical
readings of the manuscript and valuable
suggestions that improved it. The Natural
Products Branch, Division of Cancer
Treatment, NCI, gave us taxol. Michael
649
Dickinson gave us in November (a difficult
season to find flies) five female, and two
male, blow-flies and told us how to nurture
them as founders of our Calliphora colony.
V.E.F. gratefully acknowledges funding by
NIH grant GM36263 and by ACS equipment grant NP-478.
REFERENCES
Akam, M. 1987. The molecular basis for metameric
pattern in the Drosophila embryo. Development
101:1-22.
Anderson, K. V. and C. Nusslein-Volhard. 1984.
Genetic analysis of dorsal-ventral embryonic pattern in Drosophila. In G. M. Malacinski and S. V.
Bryant (eds.), Pattern formation: A primer in developmental biology. Macmillan Publishing Co., New
York.
Berleth, T., M. Burri, T. Gudrun, D. Bopp, S. Richstein, G. Frigerio, M. Noll, and C. Niisslein-Volhard. 1988. The role of localization of bicoid
mRNA in organizing the anterior pattern of the
Drosophila embryo. The EMBO Journal 7:17491756.
Bownes, M. 1975. A photographic study of development in the living embryo of Drosophila melanogaster.]. Embryol. Exp. Morph. 33:789-801.
Bownes, M. 1982. Embryogenesis. In R. Ransom (ed.),
Handbook of Drosophila development, p p . 66—94.
Elsevier, Amsterdam.
Campos-Ortega, J. A. and V. Hartenstein. 1985. The
embryonic development q/TJrosophila melanogaster.
Springer-Verlag, Berlin, Heidelberg, New York,
and Tokyo.
Crews, S. T., J. T. Thomas, and C. S. Goodman.
1988. The Drosophila single-minded gene encodes
a nuclear protein with sequence similarity to the
per gene product. Cell 52:143-151.
DiNardo, S. and P. H. O'Farrell. 1987. Establishment and refinement of segmental pattern in the
Drosophila embryo: Spatial control of engrailed
expression by pair rule genes. Genes and Development 1:1212-1225.
DiNardo, S., J. M. Kuner, J. Theis, and P. O'Farrell.
1985. Development of embryonic pattern in D.
melanogaster as revealed by accumulation of the
nuclear engrailed protein. Cell 43:59—69.
Driever, W. and C. Nusslein-Volhard. 1988. A gradient of bicoid protein in Drosophila embryos. Cell
54:83-93.
Driever, W. and C. Nusslein-Volhard. 1989. The
bicoid protein is a positive regulator of hunchback
transcription in the early Drosophila embryo.
Nature 337:138-143.
Edgar, B. A., G. M. Odell, and G. Schubiger. 1989.
A genetic switch, based on negative regulation,
sharpens stripes in Drosophila embryos. Dev.
Genet. 10. (In press)
Foe, V. E. 1989. Mitotic domains reveal early commitment of cells in Drosophila embryos. Development. (In press)
650
V. E. FOE AND G. M. ODELL
Foe, V. E. and B. M. Alberts. 1983. Studies of nuclear
melanogaster (Meigen) In M. Demerec (ed.), Bioland cytoplasmic behavior in the five mitotic cycles
ogy of Drosophila, pp. 168-274. John Wiley and
that precede gastrulation in Drosophila embryoSons, New York. Reprinted in 1965, Hafner, New
genesis. J. CellSci. 61:31-70.
York and London.
Frohnhofer, H. G. and C. Nusslein-Volhard. 1986. Rempel.J.G. 1975. The evolution of the insect head:
Organization of anterior pattern in the DrosophThe endless dispute. Quaest. Entomol. 11:7-25.
ila embryo by the maternal gene bicoid. Genes & Shaw, S. R. and I. A. Meinertzhagen. 1986. EvoluDevelopment 1:880-890.
tionary progression at synaptic connections made
Fullilove, S. L. and A. G. Jacobson. 1971. Nuclear
by identified homologous neurones. Proc. Natl.
elongation and cytokinesis in Drosophila montana.
Acad. Sci. U.S.A. 83:7961-7965.
Dev. Biol. 26:560-577.
Simcox, A. A. and J. H. Sang. 1983. When does
Fullilove, S. L. and A. G. Jacobson. 1978. Embryonic
determination occur in Drosophila embryos? Dev.
development: Descriptive. In M. Ashburner and
Biol. 97:212-221.
T . R. F. Wright (eds.), The genetics and biology of Slack, J. M. 1983. From egg to embryo (Chapter 8 espeDrosophila, Vol. 2c, pp. 105-227. Academic
cially). Cambridge University Press, Cambridge,
Press, London, New York, San Francisco.
New York.
Hartenstein, V. andj. A. Campos-Ortega. 1985. Fate Sonnenblick, B. P. 1950. The early embryology of
mapping in wild-type Drosophila melanogaster 1.
The spatio-temporal pattern of embryonic cell
division. Roux's Arch. Dev. Biol. 194:181-195.
Illmensee, K. 1978. Drosophila chimeras and the
problem of determination. In W. J. Gehring(ed.),
Drosophila melanogaster. In M. Demerec (ed.), Biol-
ogy of Drosophila, pp. 62-167. John Wiley and
Sons, New York. Reprinted in 1965, Hafner, New
York and London.
Tearle, R. and C. Nusslein-Volhard. 1987. Tubingen
Genetic mosaics and cell differentiation, Vol. 9, pp.
mutants and stock list. Drosophila Information
51-69. Springer-Verlag, Berlin, Heidelberg, New
Service 66:209-269.
York.
Technau, G. M. and J. A. Campos-Ortega. 1986a.
Ives, A. 1988. Aggregation and the coexistence of
Lineage analysis in embryos of Drosophila melacompeting insects: A theoretical model supnogaster: II. Commitment and proliferative capaported with evidence from the carrion fly combilities of neural and epidermal cell progenitors.
munity. Ph.D. Diss., Princeton University.
Roux's Arch. Dev. Biol. 195:445-454.
Kornberg, T., I. Siden, P. O'Farrell, and M. Simon. Technau, G. M. and J. A. Campos-Ortega. 19866.
1985. The en locus of Drosophila: In situ localizaLineage analysis of transplanted individual cells
tion of transcripts reveals compartment-specific
in embryos of Drosophila melanogaster. III. Comexpression. Cell 40:45-53.
mitment and proliferative capabilities of pole cells
Lohs-Schardin, M., C. Cremer, and C. Nusslein-Voland midgut progenitors. Roux's Arch. Dev. Biol.
hard. 1979. A fate map for the larval epidermis
195:489-498.
of Drosophila melanogaster. Localized cuticle defects
Thomas, J. B., S. T. Crews, and C. G. Goodman.
following irradiation of the blastoderm with an
1988. Molecular genetics of the single-minded
ultraviolet laser microbeam. Dev. Biol. 73:239locus: a gene involved in the development of the
255.
Drosophila nervous system. Cell 52:133-141.
Turner, F. R. and A. P. Mahowald. 1976. Scanning
Madhavan, M. M. and H. A. Schneiderman. 1977.
electron microscopy of Drosophila embryogenesis
Histological analysis of the dynamics of growth
I. The structure of the egg envelopes and the
of imaginal discs and histoblast nests during the
formation of the cellular blastoderm. Dev. Biol.
larval development of Drosophila melanogaster.
50:95-108.
Wilhelm Roux's Arch. 183:200-305.
Turner, F. R. and A. P. Mahowald. 1977. Scanning
Mahowald, A. P. and P. A. Hardy. 1985. Genetics
electron microscopy of Drosophila melanogaster
of Drosophila embryogenesis. Ann. Rev. Genet.
embryogenesis: II. Gastrulation and segmenta19:149-177.
tion. Dev. Biol. 57:403-416.
Matsuda, R. 1965. Morphology and evolution of the
Underwood, E. M., F. R. Turner, and A. P. Mahowinsect head. Memoirs of the American Entomoald. 1980. Analysis of cell movements and fate
logical Institute 4, Ann Arbor, Michigan.
mapping during early embryogenesis in DrosophMoore, J. A. 1987. Science as a way of knowing—
ila melanogaster. Dev. Biol. 74:286-301.
developmental biology. Amer. Zool. 27:415-573.
Moore, J. A. 1989. A conceptual framework for biol- Waddington, C. H. 1966. Principles ofdevelopment and
differentiation. Macmillan Publishing Co., New
ogy. Part I. Amer. Zool. 29:671-812.
York.
Murray, A. 1989. The cell cycle. Amer. Zool. 29:
Wieschaus, E., C. Nusslein-Volhard, and H. Kluding.
511-522.
1984. Kriippe\, a gene whose activity is required
Odell, G. M., G. Oster, P. Alberch, and B. Burnside.
early in the zygotic genome for normal embry1981. The mechanical basis of morphogenesis I.
onic segmentation. Dev. Biol. 104:172-186.
Epithelial folding and invagination. Dev. Biol. 85:
Zusman, S. and E. Wieschaus. 1985. Requirements
446-462.
for zygotic gene activity during gastrulation in
Poulson, D. F. 1950. Histogenesis, organogenesis,
Drosophila melanogaster. Dev. Biol. 111:359-371.
and differentiation in the embryo of Drosophila
MITOTIC DOMAINS PARTITION FLY EMBRYOS
APPENDIX
SIMPLIFIED METHODS FOR CULTURING AND
STUDYING BLOW-FLY EMBRYOS
You'll need a population cage. This is any screened
box of approximately 1 cubic foot capacity with a flyproof door. The box should have a hole big enough
to put your hand through while holding a pint jar.
You make the door by attaching to the hole a cloth
sleeve, like a shirt sleeve. You keep the flies caged by
keeping the sleeve tied in a knot except when you put
your arm through it (while making a commotion so
the flies flee the sleeve). The flies lay best when they
are warm, so try to keep them at about 25°C, and
simulate the long day length of summer by keeping
a lamp near the cage (you can leave it on all the time,
or about 16 hr per day, whichever is easiest). Keeping
the lamp on the side of the cage opposite the sleeve
attracts the flies away from the sleeve and reduces
the number of flies that escape when you pull your
arm out.
Buy Calliphora pupae from Biological Supply, Inc.,
Lynnwood, Washington (telephone: (206) 743-4270)
for about $7.00 per hundred. They will not smell and
will hatch into adult blow-flies within about 10 days.
Make sure you don't get Sarcophagaflies.They look
about the same, but the females lay their eggs into
their own abdomens, where the embryos develop and
hatch into maggots that the female then deposits, live
and crawling, into carrion; it's best to leave these
alone. The adult Calliphoraflieswill live in the population cage at room temperature, for about 45 days.
Feed them cane sugar cubes and dry milk powder,
and keep water always available, for they will dehydrate and die within 36 hr without water. A good way
to supply water is to put a piece of cotton cloth on
top of a mason jar filled with water. Then put a petri
dish or saucer on top of the towel and turn this assembly upside down inside the population cage. The cloth
acts as a wick.
If you want eggs to study, you should have started
three days ago. Place a few small pieces of raw vertebrate liver in the cage three days before you want
eggs. The flies will eat some of it, and this "primes"
them to produce eggs. Take out the liver after they
feast for a day. Then, on the day you want eggs (three
or four days later), put small strips of liver into the
cage. We put the liver strips in a large petri dish,
partially covered by a lid because they like to lay eggs
"under things." Watch. Soon, usually within an hour
one fly should start laying, and this act will encourage
many others to start laying, too. What each female
does is deposit about 100 eggs, carefully lined up and
stuck together, in a pile. This takes about 45 min,
and gives you, in a single clutch of eggs, a developmental sequence spanning 45 min. Reach in and discard any clutch of eggs whose deposition time you
don't know. One hundred flies will lay zillions of eggs.
They seem to lay any time of the day when there is
light and it is warm.
If you want to raise an unending supply of blow-
651
flies, it's easy. Get a few hundred eggs (about 3 clutches,
though it's better to take 25 eggs from each of 10
flies to maintain genetic diversity . . .) and put them,
along with about a pound of vertebrate liver (or hamburger, or a dead rat"), into a 1 quart plastic cottage
cheese container (it can be any kind of container that
you won't mind discarding later—and you will want
to discard it, we promise). Cover with a cloth held on
by a rubber band. Let whoever has the strongest stomach inspect the action periodically. The maggots tend
to crawl around in large swarms. They dislike light.
After about three days, when you see vigorously
crawling maggots, pour vermiculite on top of the meat,
filling the container, and now the cloth cover becomes
crucial for proper containment. The container will
begin to smell; keep it in a fume hood if you have
one. Wait about ten days. The maggots will have
crawled up into the vermiculite to pupariate, making
beautiful little brown pupal cases that do not smell.
Sooner than 10 days after pupariation, gather the
pupae with the old vermiculite (or, better, put pupae
in new vermiculite) into a clean container. Put this
into a population cage. Periodically sprinkle water on
the vermiculite and pupae if the humidity is low. The
flies will hatch about 12 days after pupariation. If the
vermiculite is too dry when they hatch, they won't
properly inflate their wings. They will still lay eggs,
but it's nicer for the flies if they have functioning
wings. That's about all there is to raising flies and
getting eggs.
You'll see one of the most amazing sights of your
life if you watch a fly, through a stereo dissecting
microscope, emerge from its pupal case. We won't tell
you in what way it's amazing. It won't escape your
notice, however. No one seems to know, officially,
why blow-flies are called blow-flies; after you watch,
you'll think you know. The first time we saw it, we
stayed up until 4:30 A.M. watching one after another
emerge.
To observe the miracles of early embryogenesis,
put about 20 eggs onto a piece of Scotch brand "double stick" tape on a glass slide. Under a dissecting
scope, arrange them by rolling them with a needle so
that different flies have different orientations—to yield
dorsal, ventral, and lateral views. The respiration tube
on the dorsal surface of the chorion provides a landmark for judging orientation. The ventral surface is
the one with the greatest convex curvature. If, when
rolling the eggs on the tape, the chorion (an opaque
sheath) comes off, so much the better. It obscures the
view a bit and the embryos don't need it, since you
11
Supposedly, it is possible to raise flies on an artificial medium made from brewer's yeast, milk powder,
agar, cholesterol, and other stuff. It is not worth the
trouble because it costs more, smells just as bad, and
produces slow-growing larvae. In our experience, it
is not necessary to coddle these flies. They survive
just fine in dumpsters. We suggest you stick to raw
meat.
652
V. E. FOE AND G. M. ODELL
will cover the eggs immediately with a puddle of mineral oil.12 The mineral oil has the effect of making
the chorion transparent, it prevents dehydration
(replacing the chorion), and it transports plenty of
oxygen. You'll need a good compound microscope
with a 10 x to 20 x non-immersion objective lens.
Using substage transmitted light, you can see the
embryo go through cycles 2 through 13, watch mitotic
waves propagate, and see cellularization during early
cycle 14. At room temperature, this will take 2!/2-3!/2
hr (longer in England). After cellularization, you
should arrange side illumination. The best (but
expensive) way to arrange this is to use a fiber optics
lamp aimed at a low angle (i.e., almost perpendicular
to the optics path). Instead of a $300 fiber optics lamp,
a small-size spot-beam flashlight will suffice (such as
a focusable-beam Techna light). Turn the sub-stage
!
Drugstore variety is fine.
illumination off, of course. The cephalic fold and ventral furrow invaginate dramatically and quickly (about
10 min); one reason you want lots of embryos on the
slide is that things happen fast. It is easy to miss important events. Taking all eggs on a slide from one clutch
insures that the embryos will all be doing the same
thing close to the same time. Gastrulation is a time
lapse movie that plays in real-time. From the time
cellularization is complete, just prior to the start of
the gastrulation movements, you should be able to
detect cells, and you may even be able to see them
divide in domains. The experience will make you
wince, or at least think twice, the next time you swat
a fly.
See the "materials and methods" section in Foe
(1989) for more sophisticated techniques for staining
and observing mitotic domains.
We hope that anyone who tries this on different
species of insects will write to us and tell us what
happens.