Download The Segmentation and Homeotic Gene Network in Early Drosophila

Cell, Vol. 51,
689698,
December
4, 1987,
Copyright 0
1987 by Cell Press
The Segmentation and Homeotic Gene
Network in Early Drosophila Development
Matthew P. Scott and Sean B. Carroll*
Department of Molecular, Cellular
and Developmental Biology
University of Colorado
Boulder, Colorado 80309
The Temporal
In 1980, Nijsslein-Volhard
and Wieschaus reported the
first results of a systematic screen of an entire metazoan
genome for genes that control a particular set of patternforming events, the segmentation of the fruit fly embryo
(Nusslein-Volhard
and Wieschaus, 1980). In total, they
described about twenty zygotically active genes (including some that had previously been found in other ways),
which they assigned to three different classes: those in
which mutations cause multiple adjacent segments to be
missing from the embryo (“gap” genes); those in which
mutations cause alternate segment-size units to be missing (“pair-rule” genes); and those in which mutations delete part of every segment and replace the deleted part
with a mirror image of some of the remaining structure
(“segment
polarity”
genes).
Niisslein-Volhard
and
Wieschaus proposed that the three classes of genes act
to subdivide the embryo into progressively smaller developmental units. A fourth class of genes, the homeotic
genes, controls segment identity; mutations in these
genes cause parts of the fly to follow an incorrect developmental pathway and form structures normally found in another segment.
Many of the segmentation loci have now been cloned
and analyzed at the molecular level. These studies, and
accompanying genetic studies, have revealed several important features about the segmentation and homeotic
gene network. First, there is a temporal order to the expression and function of the various classes of genes.
Second, the genes are expressed in precise spatial patterns that are achieved through the regulatory influence
of previously acting genes and interactions with other
genes of the same class. Third, these precise spatial patterns provide sufficient complexity to assign unique identities to cells at each position along the anterior-posterior
axis. Fourth, many genes act at multiple times and/or in
multiple germ layers to control pattern formation. Finally,
many of the gene products share protein motifs that indicate functional similarities and perhaps evolutionary relationships among the loci.
Our purpose here is to discuss some of the central issues and ideas in this field, not to provide a comprehensive review of all that is known. (For further information,
readers should consult Scott and O’Farrell, 1988; Gehring
and Hiromi, 1986; Duncan, 1987; Akam, 1987; Peifer et al.,
1987; Niisslein-Volhard
et al., 1987.)
* Present address: Laboratory
consin, Madison, Wisconsin
of Molecular
53706.
Biology,
UniVerSity
Of WiS-
Order of Regulatory
Review
Gene Expression
Embryonic pattern formation begins during oogenesis,
when the oocyte is packed with maternally encoded
mRNAs and proteins. Some of the maternally active
genes are segmentation genes, as revealed by maternaleffect mutations that alter the segmentation pattern in the
progeny of homozygous mutant female flies (e.g., Niisslein-Volhard, 1979; Boswell and Mahowald, 1985; Schiipbath and Wieschaus, 1986; Frohnhofer and NiissleinVolhard, 1986; Degelmann et al., 1986; Lehmann and
Niisslein-Volhard,
1986; MacDonald and Struhl, 1986;
reviewed in Niisslein-Volhard
et al., 1987). Some of these
genes encode products that are asymmetrically distributed in the oocyte, in two cases in graded amounts (Mlodzik et al., 1985; MacDonald and Struhl, 1986, Figure la;
Frigerio et al., 1986; Mlodzik and Gehring, 1987). Presumably these genes participate in the initial events that
trigger position-specific activation of zygotic gene expression. Experiments with ligated embryos suggest that position-specific activation of zygotically active segmentation
genes does not depend upon precisely localized determinants that are placed in position during oogenesis
(Schubiger and Newman, 1982). Rather, a dynamic process involving communication between cells (or perhaps
nuclei) in different parts of the developing embryo is
viewed as the more likely mechanism for establishing
positional information.
The first zygotically active genes to be transcribed include the segmentation
genes. Transcripts of the gap
genes are among the earliest detected; KfiipPe/ (Kr)
mRNA, for example, can be observed shortly after the
eleventh nuclear division, when the embryo is still a syncytium (Knipple et al., 1985). Transcripts of the pair-rule
genes that have been examined, including fushi t8rezu
(ftz, Hafen et al., 1984a), hairy (II, lngham et al., 1985)
paired (prd, Kilchherr et al., 1986) and emen-skipped (eve,
Harding et al., 1986; MacDonald et al., 1986), are all detected slightly later than the Kr transcripts. The mRNAs of
the segment polarity genes, such as engrailed (en, Kornberg et al., 1985; Fjose et al., 1985), gooseberry (gsb,
Bopp et al., 1986) and wingless (wg, Baker, 1987) appear
later, during the thirteenth
nuclear division. Finally,
homeotic gene transcripts are initially detected at the cellular blastoderm stage of embryogenesis
(e.g., Levine et
al., 1983; Akam and Martinez-Arias, 1985). In general, the
site of the highest level of RNA accumulation
for each
gene corresponds to the inferred site of gene function as
determined from mutant analyses.
The developmental profiles of the protein products of Kr
(Gaul et al., 1987, Figure lb), ffz (Carroll and Scott, 1985,
Figure lc), eve (Frasch et al., 1987) en (DiNardo et al.,
1985, Figure le), and the three homeotic genes Ultrabithorax(Ubx, White and Wilcox, 1984; Beachyet al., 1985, Figure If), Antennapedia
(An@, Carroll et al., 1986a; Wirz et
al., 1986) and Sex combs reduced (Scr, Mahaffey and
Cdl
690
Kaufman, 1967; Riley et al., 1987; Figure lg) have been examined using antibodies. The protein patterns generally
correspond to the RNA patterns. The most notable exception is Kr (Gaul et al., 1987); Kr transcripts in certain parts
of the embryo are not translated due to an unknown control mechanism that may involve alternative forms Of the
Kr RNA. The timing of homeotic protein synthesis also appears to be controlled; the proteins are first detected hours
after the transcripts are completed. This lag may be due
to inefficient translation or to posttranscriptional
regulation of homeotic RNAs.
Drosophilaembryogenesis
is complete in less than one
day, and the blastoderm stage, when the segmentation
and homeotic genes become active, is reached in just the
first few hours (see Campos-Ortega
and Hartenstein,
1985). During this early period, the patterns of expression
of the segmentation genes change rapidly. The ftz and h
transcripts, for example, are initially found in most or all
cells spanning the length of the embryo, but these “solid”
patterns evolve, in about 30 min, into patterns of seven
transverse stripes. The change occurs before cell membranes are completely formed (Hafen et al., 1984a; Weir
and Kornberg, 1985). It has been suggested that the pattern refinement is due to rapid turnover of the RNA in all
cells, combined with a shut-off of transcription
in the
nuclei that will come to be outside the stripes (Edgar et al.,
1988). A different type of pattern change is observed for
eve RNA (MacDonald et al., 1988) and protein (Frasch et
al., 1987), and for prd RNA (Kilchherr et al., 1988). Both
of these pair-rule genes are expressed at the blastoderm
stage in the transverse seven-stripe pattern, but shortly after gastrulation acquire new fourteen-stripe (one per segment) patterns. In the case of eve, seven new stripes are
initiated; in the case of prd, the seven initially broad
stripes are split by the disappearance
of transcripts from
cells central to each early stripe. The observed changes
in the pattern of expression of segmentation genes presumably arise in response to input from other genes in the
network.
Figure 1 shows the patternsof expression of representative examples from the different classes of segmentation
and homeotic genes. The simpler patterns (Figure la-c)
seen at the blastoderm stage, when the embryo is made
up of a monolayer of about 8000 cells (Figure Id), are
characteristic of the segmentation
genes. At the later
germ band elongation stage (Figure lh), as the metameric
divisions of the embryo appear, the later-acting segmentation genes (Figure le) and the homeotic genes (Figures
lf,g) are expressed in more refined patterns.
The Emergence of Precise
through Gene Interactions
Gene Expression
Patterns
In order to study the interactions between genes involved
in segmentation, molecular probes have been used to detect the product(s) of one gene in embryos mutant for
other segmentation or homeotic gene functions. Altered
spatial patterns of gene expression indicate that the missing gene function controls the product being assayed. In
this way, it has been shown that gap, pair-rule, and seg-
ment polarity genes interact. Generally, each gene class
is influenced by the action of earlier-acting genes that control larger units of pattern and by some members of the
same class. Thus, gap genes influence the pattern of pairrule (Carroll and Scott, 1986; lngham et al., 1988), segment polarity (Ingham et al., 1986) and homeotic genes
(White and Lehmann, 1986; Riley et al., 1987), while
segment-polarity
genes do not (so far as has been tested)
affect gap or pair-rule genes. Some gap genes are mutually negative regulators of each other (J&ckle et al., 1988).
Maternally active genes have also been shown to regulate
the ftz pair-rule gene (Carroll et al., 1988b; Mlodzik et al.,
1987). It is also useful to note the lack of any change in
the product, which indicates, within the limits of detection,
that the mutation tested does not interfere with the expression of the gene whose product is being assayed.
All four zygotically active gap genes and some of the
pair-rule genes are required for the ftz pair-rule gene to
be activated in its normal striped pattern (Carroll and
Scott, 1986). Mutations in each of the gap genes alter
some of the ffz stripes while leaving the other stripes apparently unaffected. In contrast, the pair-rule genes that
regulate ftz affect all the stripes. Of the seven pair-rule
genes other than ftz, only the eve, h, and runt genes influence the ftz striped pattern. The pair-rule gene interactions are hierarchical in at least some cases. For example,
h regulates ftz, but ftz does not regulate h (Howard and
Ingham, 1986). Furthermore, the three genes that regulate ftz do not simply shut off or turn on ftz: the response
of the ftz gene in individual cells to abnormal eve, h, or runt
function depends upon the position of the cell in the embryo, and therefore upon the activities of other genes that
may simultaneously
regulate ftz. This sort of multipleinput scenario is likely to be a general phenomenon.
Although the formal regulatory relationships among the
genes are becoming clear, the mechanisms involved are
still mysterious. It is not known how the gene interactions
result in patterns such as stripes and, in particular,
whether the interactions are direct or mediated through
other genes. The mechanisms must be both precise, in
that adjacent cells must in some cases behave very differently, and flexible, in that the number and exact arrangement of cells is different in different embryos. The mechanisms cannot work through counting cells, since embryos
with drastically different numbers of nuclei can still form
stripes of the normal size (Sullivan, 1987). Furthermore,
the simplicity of the striped patterns conceals regulatory
subtlety. Different stripes depend on different sets of upstream functions; not all ftz stripes, for example, require
the same gap gene function.
The en segment polarity gene is expressed in the
posterior part of each segment (Kornberg, 1981; Kornberg
et al., 1985; DiNardo et al., 1985). Because the en expression pattern of fourteen stripes (Figure le) emerges after
the pair-rule patterns, it seems likely that the en stripes are
derived from the preexisting wider stripes; it is not necessary to invoke a system for forming stripes de novo as it
is for the pair-rule genes. (The broad gap gene stripes, as
in Figure lb, could provide only some of the pair-rule
stripe boundaries.) Indeed, the normal striped en pattern
Review:
691
Segmentation
and Homeotic
Gene
Network
Figure 1. Expression
Patterns of Segmentation
and Homeotic Gene Products
a-c, e-g. Whole mount embryos were permeabilized
and stained with antibodies against segmentation
and homeotic gene products. Anterior is
to the left and ventral is down in each panel. The embryo is about 0.6mm long. a-d show embryos at the blastoderm stage (25-35 hr of development)
when the approximately
6C06 cells form a monolayer covertng the yolk. e-h show embryos at the elongated germ band stage (5-6 hr of development),
at the time when the homeotic protein products become detectable. The wrapping of the segments around the posterior brings the most posterior
segments to their location behind the head. The plane of focus in each case shows only the epidermal expression of each gene; the internal tissues
have related but different patterns. PS: parasegment
(see Figure 2).
(a) Expression of the caudel gene product (MacDonald
and Struhl, 1966). a nuclear protein that is distributed in a gradient in the early blastoderm
embryo. Staining is with peroxidase,
so the protein signal appears black. Photograph courtesy of P MacDonald and G. Struhl. (b) Expression
of
the Kriippel gap gene nuclear protein (Gaul et al., 1967) detected with peroxidase stain. The broad band corresponds
approximately
to PS4-6. Photograph courtesy of U. Gaul and H. Jackie. (c) Expression of the lushi farazu pair-rule gene product in the blastoderm stage embryo, detected by immunofluorescence
so the nuclear protein signal appears white. The anterior edges of the stripes mark the anterior edges of the parasegments
(Lawrence
et al., 1967); the extent of PS2 is indicated. (d) Diagram of an early blastoderm stage embryo. bc, blastoderm cells; y, yolk; pc, pole cells (the primordial
germ cells). (e) Expression of the nuclear protein encoded by the engreiled segment polarity gene (DiNardo et al., 1966) in an embryo at the elongated
germ band stage. The stripes mark the posterior part of each segment primordium, and therefore the anterior part of each pamsegme nt. The tracheal
pits (tp) are visibte. PS2 includes the posterior maxillary and anterior labial compartments.
(f) Expression
of the U/tmbIthorax
homeotic gene as
a nuclear protein (White and Wilcox, 1964; Beachy et al., 1965) at the extended germ band stage. PS6 and PSI3 are indicated. (g) Expression
of
the Sex oontba reduced homeotic gene product, also a nuclear protein (Riley et al., 1967; Mahaffey and Kaufman, 1967) at the extended germ band
stage. This embryo is turned relative to the embryos in (e) and (f); the arrowhead marks the ventral midline. The major site Of Sex combs reduced
expression
at this stage is in PS2. Later, as the germ band retracts, Sex combs reduced becomes active in PS3 as well. (h) Diagram of an extended
germ band stage embryo. The parasegments
are numbered. tp, tracheal pits.
Cell
692
forms only if the gap and pair-rule genes function properly. Different en stripes require input from different upstream genes, just as different ftz stripes require different gap genes for their formation. Alternate en stripes do
not appear in the absence of ftz function (DiNardo and
O’Farrell, 1987); the other set of alternate en stripes require
the pair-rule gene hairy (Howard and Ingham, 1988); and all
of the en stripes require ewe function (Harding et al., 1988;
MacDonald et al., 1986). The products of all three of the
segment polarity genes that have been studied, in contrast to pair-rule gene products, persist throughout embryogenesis, therefore overlapping with the next class of regulatory gene products, encoded by the homeotic genes.
Homeotic gene transcripts are first detectable at the
blastoderm stage as the cell membranes form (e.g., Akam
and Martinez-Arias, 1985; Levine et al., 1983); this is when
the segmentation gene products are at their peak abundance. In three cases, it has been demonstrated that
homeotic gene expression is initially controlled by some
of the boundaries set up by the segmentation genes. The
hunchback (hb) and Kr gap genes (White and Lehman,
1986; lngham et al., 1986), and the ffz pair-rule gene (Duncan, 1986; lngham and Martinez-Arias, 1988) all regulate
the spatial patterns of homeotic gene expression in embryos. Wild-type hb function prevents Ubx expression in
the anterior of the embryo; Kr mutations appear to alter
Ubx expression by altering pair-rule gene expression; and
ffz acts as a positive regulator of certain aspects of
homeotic gene transcription. Whether all segmentation
genes regulate homeotic genes is an open question.
Although the initial patterns of expression of the homeotic genes appear to be set up by the segmentation
genes, the maintenance of those patterns requires the actions of at least two other classes of genes: the homeotic
genes themselves, and the Polycomb-like class of genes
(Hafen et al., 1984b; Struhl and White, 1985; Wedeen et
al., 1988). For example, extra sex combs (esc) gene function is required to keep homeotic genes off where they
should be off after the initial pattern is set (Struhl and
Akam, 1985). Homeotic genes are expressed at high levels in the parts of the embryo where their functions are
most easily detected genetically and, in at least some
cases, at lower levels in more posterior regions of the embryo. For example, An@ RNA (and protein) is concentrated
in the posterior Tl and anterior T2 compartments (also
known as parasegment 4; parasegments are described in
Figure lh and Figure 2) and is present at lower levels in
more posterior regions, while Ubx products are expressed
at their highest levels in posterior T3 and anterior Al, (i.e.,
parasegment 6; Figure If) and in lower amounts in the rest
of the abdomen. The regulated levels of expression in the
posterior regions appear to be maintained by the negative
influences of other homeotic genes that are expressed
more posteriorly. For example, if the Ubx gene is inactivated, the high level of An@ expression extends back
through the region where Ubx is normally at a high level
(Hafen et al., 1984b; Harding et al., 1985; Carroll et al.,
1986a). The homeotic genes that act even further posteriorly, abdominal A and Abdominal 6, are negative regulators of Ubx (and abdominel A also negatively regulates
An@) in the posterior regions (Struhl and White, 1985;
Hafen et al., 1984b). All of these interactions may serve to
maintain and elaborate patterns that are initially set up by
the actions of segmentation genes upon homeotic genes.
In addition to regulatory interactions among the segmentation and homeotic genes, some of these genes presumably control other types of genes that carry out differentiation. Potential downstream target genes include
growth-controlling
genes, genes encoding cell surface
components, and genes encoding specialized products
such as cuticle components or neurotransmitters.
Other
apparently independent regulatory networks, such as the
sex determination gene network (Maine et al., 1985; Belote et al., 1985) presumably also converge on some of the
same target genes, since segmental differentiation in the
fly is also sexually dimorphic.
Control
of Tleeueepeciflc
Gene Expression
The actions of segmentation and homeotic genes are not
limited to the control of epidermal precursor cell fates.
High levels of segmentation and homeotic gene products
have been observed in the central nervous system and,
for some genes, in the mesoderm and peripheral nervous
system (reviewed in Doe and Scott, 1988). The expression
of homeotic genes is differentially controlled in neural ectoderm, epidermal ectoderm, and mesoderm (Akam and
Martinez-Arias,
1985; Martinez-Arias,
1986; MartinezArias et al., 1987). Similarly, segmentation genes can be
differentially activated in different tissues. The proteins
encoded by ftz (Carroll and Scott, 1985) and eve (Frasch
et al., 1987) are found in subsets of the cells in the central
nervous system after their blastoderm patterns of stripes
have disappeared.
Furthermore,
these two pair-rule
genes are expressed in each segment of the developing
nervous system, as opposed to the alternate parasegmental pattern in the blastoderm stage embryo. These observations suggest that the control of ftz and eve may be
affected by multiple tissue-specific cis-acting control elements and corresponding
Pans-acting factors.
The cis-acting control elements involved in ftz expression have been dissected using what have become standard Drosophila molecular genetic techniques:
transposon-mediated
transformation
and promoter-reporter
gene fusions (Hiromi et al., 1985; Hiromi and Gehring,
1987). By joining different parts of the ftz gene to the E.
coli 8galactosidase
(lacz) gene, at least four regions have
been identified that control different facets of ftz gene
expression. The “zebra” element is responsible for the
striped distribution of halactosidase
and is within 0.62 kb
of the translation initiation site. Most or all of the segmentation genes that affect ftz act upon the zebra element. A
second region further upstream of the “zebra” element is
responsible for activation of ftz in the ventral nervous system. A third region 3’ of the gene has a positive influence
upon the quantitative expression of the gene. An enhancer
found at -6.1 to -3.4 kb upstream of the ATG is necessary
to elevate ftz expression to the normal level in the ectoderm, and this sequence appears to respond to ftz protein
itself; the enhancer has no effect in an embryo that lacks
Review: Segmentation and Homeotic Gene Network
693
Figure 2. The Relationships
between
Segments,
Parasegments,
and Compartments
A segment is composed of an anterior and a posterior compartment.
Parasegments
are also composed of compartments,
but are offset from seg
merits by one compartment
(Martinez-Arias
and Lawrence, 1985). Compartments
are defined by boundaries
respected by cell lineages (GarciaBellido ei al., 1973; Crick and Lawrence,
1975). At the blastoderm
stage. posterior compartments
are approximately
one cell wide in the anterior-posterior
axis, while anterior cumpartments
are about three cells wide. The first visible metameric divisions to appear in the embryo, while
the germ band is elongated, are the paraeegments.
The divisions of the body seen later in development
are segmental. Md, mandibular segment;
Mx, maxillary segment; Lb, labial segment; Ti-T3, thoracic segments; Al-A9, abdominal segments.
ftz function. Thus, ftz expression in the ectoderm involves
a positive autoregulatory
loop that is required for maintenance, not initiation, of the striped pattern. In contrast,
neural expression of ftz does not appear to depend on ftz
function. A fourth region has been identified using certain
constructs lacking upstream sequences; these transformants express figaiactosidase
stripes in the head, where
ftz is not normally expressed.
Structural Themes among the Homeotic and
Segmentation Gene Products
Clues about the molecular mechanisms used by the segmentation and homeotic gene products have come from
sequence analysis. Certain amino acid sequences appear as recurrent motifs in many of the proteins. The first
such theme to be recognized was the homeodomain
(McGinnis et al., 1984b; Scott and Weiner, 1984), a 61amino-acid sequence common to the protein products of
at least six homeotic genes (Harding et al., 1985), at least
five segmentation genes (ffz, eve, prd, en, and gsb [Sopp
et al., 1986; C&e et al., 19871), and at least two maternally
active genes (bicoid, Frigerio et al., 1986; and caodal,
Mtodzik et al., 1985; MacDonald and Struhl, 1986). The
similarity between part of the homeodomain and sequences
in bacterial DNA-binding
proteins (Laughon and Scott,
1984) and in transcriptional
regulators encoded by the
MAT locus in yeast (Shephard et al., 1984; Laughon and
Scott, 1984) suggests that homeodomain-containing
proteins may be transcriptional
regulators. Sequence-specific DNA binding by a homeodomain
has been observed
in vitro (Desplan et al., 1985), but the importance of DNA
binding to the functions of homeodomains
in vivo has yet
to be proven.
A second structural motif is the “zinc finger” structure
that was first proposed for the Xenopus transcription factor IIIA (Miller et al., 1985) and has now been found as an
apparently related sequence in the Drosophila gap genes
Kr (Rosenberg et al., 1986) and hb (Tautz et al., 1987), and
agroup of genesof unknown function (Schuh et al., 1986).
Again, a transcriptional
regulatory function is suggested,
and the possibility of a role in posttranscriptional
ContrOl
is also raised. Translational control in Drosophila embryos
has been observed for the RNA products of at least two
genes, the segmentation gene caudel (MacDonald and
Struhl, 1986) and the gap gene Kr (Gaul et al., 1987).
A third type of repeat is the paired box, a region of homology of 128-135 amino acids found in paited and at
least two other loci (Bopp et al., 1986). The paired box appears to be unrelated to any previously determined protein
structure.
A fourth recurrent theme is the abundance of poiyamino
sequences in many of the regulatory gene products. The
most striking are the “opa” or “M” or CAG repeat (McGinnis
et al., 1984a; Wharton et al., 1985; Laughon et al., 1985)
which encodes poiyglutamine in several homeotic genes;
the paired (PRD) repeat, which encodes a sequence of alternating histidines and prolines and is found in paired
and other genes (including bicoid, an important regulator
of embryonic polarity; Frigerio et al., 1986; Frohnhilfer
and Nilsslein-Volhard,
1986); and the pen repeat (GGX
triplets), which has been found in Ubx (Beachy et al.,
1985), in a maternal-effect homeotic gene (fsfl)h), and in
many genes of unknown function (Haynes et al., 1987).
The discovery of these protein motifs has prompted
speculation on their origins and functions. Several possibilities seem reasonable. The genes containing similar sequences could ail be derived from a common ancestral
gene. This would imply that genes with apparently very
different roles in the control of development-for
example,
the maternal-effect polarity control genes, segmentation
genes, and the homeotic genes-ail
evolved from the
same ancestral gene, since some genes in each of these
classes contain homeoboxes.
Another possibility is that different types of genes were
assembled from repetitive (and nonrepetitive)
components, and then evolved to perform different functions depending in part on how the components were combined.
This type of evolutionary pathway could have involved
transposition of repetitive components (e.g., homeoboxes)
and their subsequent
assembly into functional genes
through deletions or other chromosomai rearrangements.
Homeoboxes are often found as exon units (although additional 3’ coding sequence is usually also present in the
same exon), which is consistent with a piecemeal gene assembly model. This sort of model is also supported by the
finding that different combinations of protein themes occur in different proteins. For example, the f&red segmen-
Cell
694
tation gene contains a homeobox and a PRD repeat
(Frigerio et al., 1988), the bicoid polarity control gene contains a homeobox and a PRD repeat (Bopp et al., 1988)
the Antp gene contains a homeobox and a CAG repeat
(but no PRD repeat) (Schneuwly et al., 1988; Stroeher et
al., 1988; Laughon et al., 1986) and some of the genes
of unknown function that contain a PRD repeat also contain CAG repeats (but not known homeoboxes).
Whatever the evolutionary history of these elements, it
is clear that while most segmentation genes are dispersed
throughout
the genome, the clustered
homeoboxcontaining loci (Regulski et al., 1985) of the Antennapedia
complex (ANT-C, Kaufman et al., 1980) and bithorax complex (6X-C Lewis, 1978) probably are a classic example of
gene duplication and divergence. Not only the homeodomain sequences have been conserved during evolution,
however. The protein encoded by the Deformed (Dfd)
homeotic gene, a gene in the ANT-C, has been found to
be strikingly related in sequence to proteins encoded by
a frog and a human homeobox gene (Regulski et al.,
1987). Therefore certain aspects of homeotic protein
structures other than the homeodomain
appear to have
predated the divergence of vertebrates and invertebrates,
and to have been conserved since then,
The cross-hybridization
of the DNA sequences encoding the related protein domains has proven to be extremely useful in allowing the isolation of genes that control early development or that are good candidates for
doing so (e.g., McGinnis et al.,1984a; Fjose et al., 1985;
Harding et al., 1986; Schuh et al., 1986; Bopp et al., 1986;
Frigerio et al., 1986; MacDonald et al., 1986; MacDonald
and Struhl, 1986). This success implies that the protein
themes, and presumably the biochemical functions they
encode, are specific to genes that serve as central regulators during development. The data so far tend to support
this idea, although there is still no direct proof.
The Determination
of Cell Identities
Segmentation
Genes
by Pair-Rule
Three of the pair-rule segmentation genes, ffz (Scott and
Weiner, 1984; McGinnis et al., 1984a), eve (Harding et al.,
1986; MacDonald et al., 1986) and prd (Frigerio et al.,
1986) contain homeoboxes. Sequences for the five other
genes in this class have not yet been reported. The ffz,
eve, and prd genes are each expressed in stripes and no
two of them appear to have stripes in phase. If homeodomain-containing
proteins are transcriptional
regulators,
then different cells along the anterior-posterior
axis of the
blastoderm would contain different arrays of them.
The striped patterns of the pair-rule gene products
therefore lead to a simple model for how each blastoderm
cell is instructed to be different from its immediate neighbors just anterior or posterior (Gergen et al., 1986). At the
blastoderm stage (Figure Id), a segment primordium is
about four cells wide in the anterior-posterior
axis, so
stripes in alternate segment-size (four cell) units define a
repeat unit of eight cells (four “on,” four “off”) (in some
cases, the stripes become narrower after they first appear,
Figure lc). Because the stripes of one gene product are
offset from stripes of others, each cell in the repeat unit
will express a particular combination of gene products. In
theory, only four different gene products expressed in outof-frame stripes (with three to four cells per stripe) would
suffice to distinguish each cell in the repeat unit from each
of the other cells. There are at least eight pair-rule genes,
so ample genetic complexity for such a system exists.
Some of the pair-rule genes control the expression of
others, so a simple combinatorial
model cannot be the
whole story. The pair-rule genes are not all equivalent.
Some may serve only to control other segmentation
genes, helping to direct the formation of their expression
patterns, and may play no role in directly controlling the
downstream target genes (whatever they may be) that actually carry out differentiation processes. It is also possible that some of the pair-rule genes control other segmentation genes and control downstream
target genes.
Among the target genes are the segment polarity genes,
at least three of which, en, gsb, and wg, are expressed in
about fourteen stripes, one per segment. If most of the
other segment polarity genes are expressed in offset
stripes, then the eight cells in the repeat unit could be distinguished both by the transient action of the pair-rule
gene products and by the longer-lived segment polarity
gene products. The determination of individual embryonic
cells may thus be viewed as the activation (or repression)
of certain sets of regulatory genes in precise spatial patterns that together direct the cell into a particular developmental pathway. The embryonic fate map may now be
drawn based upon knowledge of the spatial domains of
regulatory genes.
After the initial expression of genes in stripes, cell interactions are likely to occur that result in further pattern
refinements (discussed in Scott and C’Farrell, 1988). The
nonautonomous
action in the embryo of the segment
polarity gene wingless (Baker, 1987) suggests that some
of the segment polarity genes may be involved in communication between cells. Cell-cell communication may lead
to the formation of smoothly graded and complete cuticular patterns. Some of the segment polarity genes may
therefore be involved in the sensing by cells of the character of neighboring cells.
All of the genes discussed here are regulators of anterior-posterior
patterning. If a set of genes with similar
behaviors exists for the dorsal-ventral axis, then each cell
or small group of cells could be determined by position
along the two axes, that is by the array of anteriorposterior and dorsal-ventral
regulators activated within it.
A substantial number of genes controlling dorsal-ventral
differentiation have been found (e.g., Simpson, 1983; Anderson and Nijsslein-Volhard,
1984) but whether they act
by the same set of molecular mechanisms as the anterior-posterior
genes is not yet known.
How Do Homeotic
Genes
Make Cells Different?
Homeotic genes are cell-autonomous
in their actions
(e.g., Garcia-Bellido
and Lewis, 1978); a cell will behave
according to its homeotic genotype, regardless of the
genotype of the surrounding cells. In the extreme case, a
Review: Segmentation and Homeotic Gene Network
695
single cell that has an active Ubxgene would develop into,
for example, a T3 bristle, even if the surrounding
cells
lacked Ubx function and made a l2 pattern of bristles.
However, a cell does need positional information to respond appropriately to the homeotic signal. A cell in one
part of a segment might respond to the presence of, for
example, Ubx protein by making a bristle, while a cell that
also contains Ubx protein but is in a different position
would respond to the Ubx signal by elongating in a T3-like
way. A cell must integrate information about its position,
presumably information derived from segmentation gene
functions, with the information about its segmental identity that comes from homeotic genes.
Cells expressing the same homeotic gene(s) in different
parts of the same segment primordium could behave
differently if: 1) the homeotic proteins are made at different
levels in different cells; 2) different combinations
of
homeotic proteins are made in different cells within a segment (or parasegment);
3) there are variant forms of
homeotic proteins made in cells in different positions (if
these variants are transcription factors, they could control
different target genes or differentially affect the same target gene); and 4) the segmentation gene products that
acted earlier, or that continue to be active, modify the effects of homeotic gene products.
It seems likely that all four mechanisms are used. Substantial cell-to-cell variations in the levels of homeotic proteins have been observed. Notably, minor differences in
Ubx (Lewis, 1978) or Scr (Kaufman et al., 1980) gene dosage are sufficient to change the cell phenotype. It is also
clear that many cells contain more than one homeotic protein (e.g., Carroll et al., 1988a) and, at least in the case of
Ubx, that variant forms of the protein exist (Beachy, 1988).
The persistence of some segmentation gene products
through the time when homeotic gene products are made
is consistent with the idea that some type of interaction between them is important for determining cell fate. In some
compartments
there appears to be a relatively uniform
amount of, for example, Ubx protein in all of the epidermal
primordial cells (White and Lehmann, 1988; Figure If). If
the cells do, in fact, all contain the same amount of the
same Ubx protein, or the same mix of Ubx isoforms, cells
in the primordium must behave differently according to
their position, and they “know” their positions as a result
of the earlier actions of segmentation genes. Because the
pair-rule segmentation genes are only transiently active,
one possibility is that the early action of pair-rule (or other)
genes could leave a lasting imprint on the cell, possibly
through a transcription priming mechanism that renders
certain target genes responsive to the homeotic proteins
that appear later. Alternatively, the cell “memories” could
be due to the continued activities of segmentation genes.
eng&ed(Kornberg
et al., 1986; DiNardo et al., 1985) and
wingless (Baker, 1987) segment polarity gene products,
for example, persist throughout embryogenesis.
These
and other proteins could modify the effects of the
homeotic proteins. Thus in deciding cell fates within a
segment-in
deciding which cells should make bristles
and which should not in forming the stereotypical segmental pattern-cells
appear to respond both to the
homeotic genes active within them and to the positional
information that has been provided by the segmentation
genes.
Speculations
of Homeotic
on the Evolution
Genes
and Function
A possible series of evolutionary events that may have led
to the formation of homeotic genes is suggested by the location of the ftz gene in the ANT-C (Wakimoto and Kaufman, 1981), the strong similarity between the homeodomains in the ffz protein and the homeotic proteins (Scott
and Weiner, 1984; McGinnis et al., 1984b), the autoregulation of ffz (Hiromi and Gehring, 1987), and the activation
of homeotic genes by ftz (Ingham and Martinez-Arias,
1988). The development of the largely identical segments
of an annelid-like insect ancestor may have depended on
the function of a gene similar to fiz (or eve) that was expressed in stripes and directed the formation of repeating
pattern elements. The homeotic genes could have arisen
by duplication and divergence of the ftz-like gene-an
extension of the proposal by E. Lewis (1951) that homeotic
genes arose by duplication and divergence. This would be
consistent with the clustering of the Drosophila homeotic
genes in two groups, the ANT-C and BX-C, with one group
including ftz. The existence of the two groups may have
resulted from a splitting apart of one ancestral cluster. Indeed, in a beetle species, the ANT-C- and BX-C-like
homeotic genes appear to be clustered together (Beeman, 1987).
If the ancestral ftz-like protein had positively regulated
its own gene, like the present day ftz (Hiromi and Gehring,
1987), it would also have positively regulated the duplicated gene(s). The ffz gene has been shown to activate
three homeotic genes, Scr Antp and Ubx, in their positions of highest expression and major function, i.e.,
parasegments
2, 4, and 8, respectively (Ingham and
Martinez-Arias,
1988). These parasegments are regions
where ftz is normally expressed; thus the homeotic genes
are active at their highest levels in places that correspond
to part of the ftz pattern. But what prevents the homeotic
genes from being expressed in all of the places where ftz
is active? The newly reduplicated genes could come to be
expressed differently in different segments (thereby taking a step toward becoming homeotic genes) by acquiring
c&-acting elements that turn off some of the stripes.
These elements could receive signals from regionally expressed earlier-acting genes such as the gap genes. A
current example might be the repression of the Ubx
homeotic gene in the anterior of the embryo by the hunchback gap gene (White and Lehmann, 1986).
While frz is also regulated by gap genes (Carroll and
Scott, 1986), the result is a reiterated striped pattern, not
expression in only certain segments. How difficult would
it be for the ftz striped expression pattern to change into
a more homeotic sort of pattern? A clue is provided by recent experiments showing that the ftz upstream sequences, specifically the enhancer element mentioned
earlier, can direct a homeotic pattern rather than an evenly
striped pattern if the enhancer is merely inverted (Hiromi
Cell
696
and Gehring, 1987). The new pattern seen is similar to that
of transcripts from one of the Antp promoters (Ingham and
Martinez-Arias, 1988)-that
is, most of the expression is
detected in a single stripe in parasegment 4. These experiments with ftz demonstrate that it does not necessarily require elaborate changes in c&acting elements to go
from a striped pattern to a modified striped pattern in
which one of the stripes is much stronger than the others.
The change observed, moreover, was shown to depend
upon the function of the gap gene Kr (Hiromi and Gehring,
1987), again implicating the gap genes in region-specific
expression. Thus in the evolution of homeotic genes, the
responses to the gap genes would merely have to be
modified from the existing responses of a ftz-like gene; it
would not be necessary for an entirely new kind of gene
interaction to evolve.
Segmental organization is a relatively simple repeating
pattern, but homeotic genes need to direct the formation
of quite complex patterns. The DNA sequences that regulate homeotic genes in cis appear to be very complex (see
Peifer et al., 1987), which perhaps is another reason for
the large size of these genes. One homeotic gene, Anfp,
appears to have evolved by acquiring a second promoter
that controls the same protein-coding sequence (Schneuwly
et al., 1988; Stroeher et al., 1988; Laughon et al., 1988).
In this way the same protein can be regulated by two sets
of cis-acting sequences, allowing divergence of functions
similar to that generated by gene duplication. The Antp
promoters are differentially controlled by tmns-acting regulators (Ingham and Martinez-Arias, 1988), and are differentially expressed in imaginal discs (Jorgensen et al.,
1987); thus, in this case, two different sets of cis-acting sequences are in fact used. Gene duplication,
multiple
promoters, the acquisition of large arrays of cis-acting elements, and changes in protein sequences all appear to
have played a role in the evolution of the homeotic genes.
Conclusions
The advanced understanding
of systems as diverse as
phage assembly and yeast mating type has relied upon
the systematic isolation of mutations that affect most or all
components of the system. The same approach to Drosophila development, as first applied by a few pioneers
such as E. Lewis, C. Niisslein-Volhard,
and E. Wieschaus,
has also been successful. We anticipate that the continued detailed study of genes that control embryonic pattern formation will yield many additional ideas about the
biochemical nature of positional information and the molecular basis of cell determination. In particular, we expect
that the most provocative information will come from the
analysis of the expression and function of maternal gene
products, the identification of the molecular mechanisms
controlling interactions between the different tiers of the
zygotic gene hierarchy, and the characterization
of cisacting elements of pattern-regulating
genes that respond
to specific spatial, temporal, and tissue-specific regulatory proteins.
Acknowledgments
We thank the other members of the laboratory for stimulating discussions. Thanks to Drs. Robert Boswell, Margaret Fuller, David Gubb,
and William Wood for comments
on the manuscript.
We are grateful
to Drs. Paul MacDonald, Gary Struhl, Ulrike Gaul, and Herbert Jbkle
for donating photographs,
to Dn. Steve DiNardo, Pat O’Farrell, and
Rob White for gifts of antibodies, and to Alice Bernat for artwork. Our
research is supported
by NIH grant HD-18183 to M. P S., a Searle
Scholar Award to M. F! S., and an NIH postdoctoral
fellowship to
S. B. C. Thanks also to Cathy lnouye for preparing the manuscript,
again and again.
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