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DEVELOPMENTAL GENETICS 23:1–10 (1998)
REVIEW ARTICLE
Segmentation: Painting Stripes
From Flies to Vertebrates
LESLIE PICK*
Brookdale Center for Developmental and Molecular Biology, Mt. Sinai School of Medicine, New York, New York
INTRODUCTION
This issue of Developmental Genetics focuses on the
topic of segmentation. Included are discussions of this
process in a wide range of organisms, spanning the evolutionary ladder from insects to fish, birds, and mammals. A definition of segmentation is worth considering.
Bateson (1984) defined segmentation as a repetition of
pattern elements along the major body axis. In insects,
this segmental pattern is distinct and clearly visible in
the external structures of both the larva and adult. The
role of segmentation in establishing the vertebrate
body plan, which has been more controversial, is discussed here and in three other articles in this issue.
How are segments established in Drosophila?
The process of segmentation is best understood for
the fruit fly, Drosophila melanogaster, largely as a
result of the work of Christiane Nüsslein-Volhard, Eric
Weishcaus, and co-workers [1980, 1984, 1985, 1994] as
well as Lewis [1978, 1994], who pioneered studies of
Drosophila homeotic genes. The basic segmented body
plan of Drosophila is specified by positional information
laid down in the early embryo by an interacting group
of regulatory genes [for review, Akam, 1987; Gergen et
al., 1986; Ingham and Martinez-Arias, 1992; Lawrence,
1992; Scott and O’Farrell, 1986; St. Johnston and
Nüsslein-Volhard, 1992]. Many of these genes were
identified in massive genetic screens designed to identify lethal mutations that alter the pattern of the larval
cuticle [Nüsslein-Volhard et al., 1985; Nüsslein-Volhard
and Wieschaus, 1980]. The results of these screens
suggested a model whereby the embryo is subdivided
into increasingly specified body regions by the sequential action of regulatory genes. Maternally active coordinate genes specify the polarity of the egg, whereas
zygotically active gap, pair-rule, and segment polarity
genes are required for the development of the segmented body plan of the animal [Nüsslein-Volhard and
Wieschaus, 1980]. The homeotic genes convert equivalent segmental units into ones having unique identities
[Lewis, 1978, 1994]. These regulatory genes chart out
pathways of embryonic development, but are not themselves directly involved in differentiation [GarciaBellido, 1975].
r 1998 WILEY-LISS, INC.
In Drosophila, the division of the anterior-posterior
axis into repeated metameric units is controlled by the
pair-rule genes [Jurgens et al., 1984; Nüsslein-Volhard
et al., 1984, 1985; Nüsslein-Volhard and Wieschaus,
1980; Wieschaus et al., 1984). Mutations in the pairrule genes result in lethality accompanied by deletions
of alternating regions of the body. The periodic deletions caused by the eight different pair-rule genes are
out of frame with each other. Together, these genes act
to direct the development and differentiation of segmental units (parasegments) [Martinez-Arias and Lawrence,
1985] of the larva and adult. How do these genes specify
embryonic pattern? One clue to their function comes
from the fact that these genes are expressed in spatially
and temporally restricted patterns in the embryo. For
the most part, these restricted expression patterns
correlate well with the domains of action of these genes
as reflected by their mutant phenotypes. For example,
the pair-rule gene fushi tarazu (ftz) is expressed in a
striped pattern in the primordia of the even-numbered
parasegments that are missing in ftz mutant embryos
(Fig. 1) [Hafen et al., 1984]. In the absence of ftz
function, these regions of the embryo fail to develop,
whereas the remaining regions develop autonomously,
resulting in approximately half-size embryos that fail
to hatch as larvae (see Fig. 2). Mis-expression studies
have highlighted the importance of the restricted,
striped expression domains of the pair-rule genes.
Expression outside of their striped domains results in
lethality, accompanied by distinct and specific cuticular
defects [Struhl, 1985]. Thus two broad questions must
be addressed to understand the function of the pair-rule
genes: (1) how are their periodic striped patterns established, and (2) once correctly positioned, how do these
gene products provide the information to direct the
growth and differentiation of body segments that form
the basis of the three-dimensional body plan of the fully
developed organism?
*Correspondence to: Leslie Pick, Brookdale Center for Developmental
and Molecular Biology, Mt. Sinai School of Medicine, One Gustave L.
Levy Pl., Bx 1126, New York, NY 10029. E-mail: [email protected]
Received 11 May 1998; Accepted 19 May 1998
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Fig. 1. The Drosophila pair-rule gene ftz is expressed in a striped
pattern. A. In situ hybridization to ftz mRNA shows expression of ftz in
seven stripes in the primordia of the even-numbered parasegments.
The distributions of ftz mRNA and protein are indistinguishable at
this stage. Note that this expression occurs at the blastoderm stage,
well before any morphological signs of segmentation are visible in the
embryo. Transplantation experiments showed that this is the stage
when determination occurs along the anterior-posterior axis. B.
Perdurance of a ftz/lac Z fusion gene [Pick et al., 1990] highlights the
anterior parasegment border. The stripes of endogenous Ftz fade
during germ band extension and are not detectable by the time the
development of segments is apparent. However, b-galactosidase protein is stable in embryos, serving as a fate marker for the cells that
express Ftz early. Obvious here is the off-setting of segment (morphologically visible, long arrow) and parasegment (marked by b-galactosidase, short arrow) borders. Expression of b-galactosidase was with
X-gal [Hiromi et al., 1985]. Embryos are oriented anterior, left; dorsal,
up. A: courtesy of K.Su.
PAIR-RULE STRIPES ARE FIRST INDICATION
OF PERIODICITY IN THE EMBRYO
Genetic studies demonstrated that embryonic regulatory genes act hierarchically to establish the Drosophila body plan. The wild-type function of genes
acting earlier in the hierarchy is necessary for the
correct expression of downstream genes. That is, maternal coordinate gene function is necessary for correct
expression of the gap genes, which are in turn required
for correct pair-rule expression, and so on [see e.g.,
(Carroll and Scott, 1986; Frasch et al., 1988; Gutjahr et
al., 1993; Harding et al., 1989; Howard and Ingham,
1986; Ingham et al., 1988; Ingham and Martinez-Arias,
1986; Martinez-Arias et al., 1987]. Since the pair-rule
genes are the first genes to be expressed in a repeated
pattern, it is thought that they have a mechanism to
‘‘interpret’’ nonperiodic information laid down by maternal and gap genes, which they ‘‘convert’’ into periodic
information provided by their striped patterns. For
some pair-rule genes, this ‘‘interpretation’’ is done by
cis-acting regulatory elements that integrate informa-
Fig. 2. Ftz is required for the development of alternate parasegments. Wild-type larval cuticle preparation is shown on the left.
Characteristic rows of denticles identify each segment, as indicated.
On the right is a ftz mutant embryo, which is missing every other
parasegment. The cells that express Ftz in stripes in the blastoderm
are the primordial cells of those missing here [Hafen et al., 1984;
Nusslein-Volhard and Wieschaus, 1980]. Photograph courtesy of Dr.
J.J. Zhao.
tion provided by graded expression patterns of maternal and/or gap gene products to generate a single stripe
[Hartman et al., 1994; Howard et al., 1988; Howard and
Struhl, 1990; Langeland et al., 1994; LaRosee et al.,
1997; Pankratz and Jackle, 1990; Pankratz et al., 1990;
Riddihough and Ish-Horowicz, 1991; Small et al., 1991,
1992, 1993]. Detailed analysis of one of these regulatory
elements—the eve stripe 2 element—showed that this
stripe is formed by input from maternal and gap genes:
transcription of stripe 2 is activated by bicoid and
hunchback in the anterior region of the embryo and
repressed by the gap genes giant and Kruppel at the
anterior and posterior borders to form a sharp stripe
[Small et al., 1992]. DNA binding, transcription studies,
and mutagenesis experiments in vivo have demonstrated that these effects are direct: the eve stripe 2
element contains binding sites for each of these gene
products that mediate regulation. These studies set a
precedent by demonstrating that aperiodic information
from maternal and gap genes can be interpreted directly by a pair-rule regulatory element to form a stripe
in the embryo. However, even for eve, and also the
pair-rule gene hairy, which has modular, single stripe
regulatory elements, the situation is not so simple.
Although an eve stripe 3 element has been identified
[Small et al., 1993, 1996], single stripe elements have
not been found for all of the eve stripes. Therefore, it is
not clear yet whether the elegant eve stripe 2 story is
the rule and has taught us how generic stripes are
made in the embryo, or whether this story is the
exception among more complex regulatory modes.
The situation for other pair-rule genes such as ftz and
paired (prd) is more complicated [Gutjahr et al., 1993,
SEGMENTATION
Fig. 3. The Drosophila segment polarity gene en is expressed in
stripes. A: En protein is expressed in a characteristic 14 stripe pattern
during early germ band extension [DiNardo et al., 1985], detected here
with a monoclonal anti-En antibody. En defines the posterior compartment of Drosophila segments. B. Expression of alternating En stripes
(brown) overlaps with Ftz stripes (blue). C. Ftz is required for the
expression of alternating En stripes. In ftz mutant embryos, shown
here, the ‘‘Ftz-dependent’’ En stripes are missing. Photographs courtesy of K. Su and Y. Yu.
1994; Hiromi and Gehring, 1987; Hiromi et al., 1985].
The striped patterns of these genes are set up by
modular regulatory elements that direct expression in
seven stripes (‘‘seven stripe elements’’). The most extensive analysis here has been of ftz regulation: Deletions
and mutations in seven stripe elements in the context
of reporter genes in transgenic flies, as well as DNA
binding studies, indicate that multiple activators and
repressors interact to direct expression in stripes. However, these seven stripe elements do not appear to be
composed of a series of single stripe elements analogous
to those found for eve and hairy. The identities of the
trans-acting regulators that establish stripes through
seven stripe elements remain elusive. An early model
proposed that primary pair-rule genes (e.g., hairy, eve),
themselves already expressed in stripes, direct the
establishment of stripes for secondary pair-rule genes
such as ftz or prd, through seven stripe regulatory
elements [Carroll, 1990; Dearolf et al., 1990]. Although
this model has an intrinsic logic that is appealing, it is
clearly an oversimplification that is not supported by
the following experimental observations: (1) ftz and prd
stripes are established in pair-rule mutant embryos.
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Other pair-rule genes affect striped patterns at the
level of refinement and maintenance only [Ingham and
Gergen, 1988; Gutjahr et al., 1994; Yu and Pick, 1995),
and (2) ftz and prd stripes do not arise en masse as
originally speculated, but develop differentially along
the anterior-posterior axis in distinct orders that cannot be explained by a simple positive or negative
pair-rule crossregulation [Gutjahr et al., 1993; Klingler
and Gergen, 1993; Yu and Pick, 1995]. The situation for
the pair-rule gene runt appears to be a hybrid between
the two scenarios described above: both seven stripe
elements and single stripe elements are necessary to
reconstruct wild-type levels of correctly positioned runt
stripes [Butler et al., 1992; Klingler et al., 1996].
Together, studies of these regulatory elements suggest
that the seven stripe elements that establish ftz, prd,
and runt stripes use a novel mechanism for generating
periodicity that remains to be elucidated.
In sum, a general concept has emerged to explain
how periodicity can be established in the embryo
through the refinement of hierarchical information
provided by a network of regulatory genes. However,
despite intensive investigation in a number of laboratories, it is not yet possible to provide a mechanistic
explanation of a complete striped pattern for any single
pair-rule gene. Clearly, much remains to be learned
about how periodicity is generated, even in Drosophila.
Reinitz and Sharp [1995] and Mjolsness et al. [1991]
are taking a novel approach to address the issue of
stripe formation, using a combination of computer
modeling and ‘‘bench’’ experiments. This approach attempts both to quantitate and integrate the huge body
of experimental data obtained from analyzing the evolution of gene expression patterns in wild-type and
mutant embryos. Here, Reinitz et al. [1998] present a
provocative paper which suggests that combinations of
gap gene input can direct the formation of only one set
of stripes in the embryo, and these stripes correspond to
the domains of expression of even-skipped. This conclusion is intriguing in light of the open questions about
various modes of stripe formation for different pair-rule
genes, described above. The future challenge for the
‘‘gene circuit model’’ will be to form testable predictions
about how these other sets of stripes are established in
the embryo.
DOWNSTREAM FUNCTIONS
OF PAIR-RULE GENES
Getting the pair-rule genes expressed in stripes is not
enough: Once expressed in their proper position, the
products of these genes must do something to promote
the development and differentiation of segments. We
know surprisingly little about this aspect of pair-rule
gene function and even less about the cell biology
required for segment differentiation and assembly.
When the pair-rule genes were cloned in the late 1980s,
they were found to encode DNA binding transcription
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factors, as were many of the other genes isolated in the
‘‘Nüsslein-Volhard screen.’’ It seemed straightforward,
therefore, to presume that these genes would directly
regulate the expression of downstream target genes—
the ‘‘realizator genes’’ of Garcia-Bellido [1975]—that do
the job of making segments. However, most of the
target genes identified thus far are other regulatory
genes in the hierarchy and often the clearest targets are
autoregulatory. Clearly, a cascade of transcription factors is not enough to build an embryo.
The identification of target genes for many of these
proteins has been hampered by the fact that they
encode DNA binding proteins that are members of
larger families and that significant DNA binding often
requires unknown protein cofactors. A clear example of
this are homeodomain proteins: DNA binding specificity is relatively low and the binding sites for different
family members are overlapping [reviewed in Mann,
1995]. For proteins such as Runt, bHLH proteins such
as Hairy, and even homeodomain proteins such as Ftz,
physiologically significant DNA binding seems to require partner proteins that are only now being identified [Fisher et al., 1996; Florence et al., 1997; Golling et
al., 1996; Guichet et al., 1997; Ohsako et al., 1994; Yu et
al., 1997]. This has precluded the use of standard in
vitro DNA binding assays for identifying targets, even
for these well-characterized pair-rule gene products.
Thus the identification of direct target genes has required more labor-intensive combinations of molecular,
biochemical, and genetic approaches [see, e.g., Schier
and Gehring, 1992].
What is the general wild-type function of the pairrule genes? Two views have been proposed that are best
summarized by the title of a review by Peter Lawrence
[1987]: ‘‘Pair-rule genes: Do they paint stripes or draw
lines?’’ The ‘‘painting stripes’’ view is a combinatorial
one: the interdigitating/overlapping expression patterns of different pair-rule genes generates a repeated
code along the anterior-posterior axis. Combinations of
different pair-rule products in different cells within this
repeated unit result in assignment of particular cell
fates [Gergen et al., 1986; Scott and O’Farrell, 1986].
The ‘‘painting stripes’’ view proposes that the function
of at least two pair-rule genes (eve and ftz) is to draw a
line that marks the anterior parasegment border. This
border would form a boundary between neighboring
developmental fields, and each parasegment would be
defined from this border, possibly by a gradient of a
diffusable morphogen within each parasegmental zone
[Lawrence, 1987]. Support for the latter view comes
from the finding that Ftz and Eve stripes have sharp
anterior borders that overlap precisely with the anterior borders of engrailed expression [Lawrence et al.,
1987]. Engrailed (en) is a segment polarity gene expressed in 14 stripes in the embryo that define the
posterior compartment of each segment [DiNardo et al.,
1985; Fjose et al., 1985; Kornberg, 1981; Kornberg et al.,
1985; Lawrence and Morata, 1976]. Ftz and Eve are
each responsible for the establishment of alternating en
stripes [see Fig. 3; DiNardo and O’Farrell, 1987; Howard
and Ingham, 1986], and, for Ftz, there is strong evidence that this regulation is direct [Florence et al.,
1997].
A short communication in this issue [Lawrence and
Pick, 1998] tests the hypothesis that graded expression
of Ftz itself within a parasegment defines the parasegment border. According to this model, en is expressed
only in response to high concentrations of Ftz and,
therefore, only at the anterior border of the Ftz stripe.
Two experimental observations presented here clearly
rule out this model. First, the authors show that Ftz
itself is not expressed in a gradient within each stripe.
Earlier presumptions that this is the case were based
upon analysis of b-galactosidase protein expressed
from ftz/lac Z constructs. In this case, it is the perdurance of the protein in late embryos that creates the
appearance of a gradient. Second, varying the dosage of
Ftz in the embryo did not affect en expression, as would
have been expected for the gradient model. This rules
out the possibility that a gradient of Ftz defines the en
border, but leaves open the question of what the
wildtype function of Ftz is.
One extreme view emerging from studies of pair-rule
regulation of en, described above, is that the sole
function of Ftz and Eve in the embryo is to regulate en.
The ‘‘business end’’ of making segments would then be
in the hands of the segment polarity genes. This view
ignores other obvious candidate direct targets of these
genes such as the homeotic genes [Ingham and Martinez-Arias, 1986]. However, the possibility that the
pair-rule genes regulate only other regulatory genes
must still be considered.
At the other end of the spectrum is the more recent
proposal that Ftz and Eve regulate virtually every gene
that is expressed in the blastoderm embryo (Z. Liang
and M.D. Biggin, unpub. data). Support for this view is
based in part upon in vivo UV crosslinking experiments
that suggest that Ftz and Eve bind DNA as promiscuously in embryos as they do in the test tube [Walter et
al., 1994]. Distinguishing between these views, or
finding a compromise between them, will require a
better understanding of how pair-rule proteins regulate
transcription in the embryo.
Two papers in this issue address this question by
examining regulation of target genes by the pair-rule
proteins Runt and Prd. Tsai and Gergen [1994] showed
previously that ectopic expression of Runt protein leads
to repression of the head gap gene orthodenticle (otd).
Here, they show that otd is also a target of Runt in
wild-type embryos since otd expression is ectopically
activated in the posterior of runt mutants [Tsai et al.,
1998]. Whether or not otd is a direct target of Runt
remains to be determined; however, Tsai et al. [1998]
show here that repression of otd by Runt does not
require the conserved VWRPY motif necessary for
interaction with the co-repressor Groucho [Paroush et
al., 1994]. Interaction with Groucho was previously
shown to be necessary for Runt repression of specific eve
SEGMENTATION
and hairy stripes, but not for repression of en stripes
[Aronson et al., 1997]. Thus, Runt appears to have at
least two independent molecular modes of repression in
the embryo by which it regulates genes at three levels of
the hierarchy: gap, pair-rule, and segment polarity
segmentation genes.
Unlike other pair-rule genes, the Prd protein contains two DNA binding domains—the Prd domain and
the homeodomain [Frigerio et al., 1986; Kilcherr et al.,
1986; Treisman et al., 1991; Wilson et al., 1995; Xu et
al., 1995]. Both of these binding domains are essential
for Prd function in vivo and are necessary for activation
of gooseberry (gsb, a segment polarity gene that also
contains a Prd domain) [Baumgartner et al., 1987;
Gutjahr et al., 1993], en and eve, three candidate direct
targets of Prd [Bertuccioli et al., 1996; Li et al., 1993; Li
and Noll, 1996; Miskiewicz et al., 1996]. The fact that
Prd protein itself contains two DNA binding domains
suggested that unlike other homeodomain proteins
discussed above, Prd may have intrinsic DNA binding
specificity and selectivity that override the requirement
for interaction with other cofactors. This idea was
supported by the finding that the two domains bind
cooperatively to an optimized Prd binding site in vitro
[Jun and Desplan, 1996]. These observations raised the
question of whether all Prd targets contain binding
sites for both of these domains and whether cooperative
binding to these sites is what allows for selective
regulation by Prd of downstream genes in the embryo
[Miskiewicz et al., 1996]. Direct activation of eve by Prd
through a target element that contains both Prd domain and homeodomain binding sites [Fujioka et al.,
1996] has provided a model for Lan et al. [1998] to study
these questions in the embryo. Using a combination of
in vivo assays in transgenic embryos and transcription
assays in cell culture, Lan et al. [1998] have designed a
system to study the relationship between the two Prd
DNA binding domains and their relative roles in regulating expression of downstream genes. Alterations in
binding specificity as well as in spacing abrogated eve
activation in the embryo, whereas other targets were
less severely affected. Interestingly, even for eve regulation, in vitro optimized binding sites for a single
binding domain could replace the composite site. These
results suggest that high affinity binding of Prd to DNA
determines target site selection in vivo and that different Prd-responsive regulatory elements may utilize
each DNA binding domain to different extents. Thus,
Prd may indeed require additional cofactors for selection of some downstream targets.
SEGMENTATION IN OTHER INSECTS
Drosophila is a long germ band insect: the entire
germ band and segmental pattern of the body plan is
established simultaneously [Sander, 1976]. Segmentation is established more or less synchronously along the
anterior-posterior body axis by the time cellularization
is completed in the blastoderm embryo [Chan and
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Gehring, 1971]. Thus the pair-rule genes, which establish segments, are expressed in their characteristic
seven stripe patterns at this time and the 14 stripes of
En arise more or less en masse in response to this
uniform expression of pair-rule genes. In contrast,
segments arise sequentially in the short and intermediate germ band insects, as development proceeds. Studies of the molecular basis of segmentation in these
insects has, therefore, been of interest, since morphology would suggest mechanisms that are principally
different from those operating in Drosophila.
Homologues of Drosophila segmentation genes have
been studied in the extreme short germ band grasshopper, Schistocerca americana [Dawes et al., 1994; Patel
et al., 1992] and in short, intermediate, and long germ
band beetles [Patel et al., 1994]. Homologues of the
pair-rule genes ftz and eve have been identified in the
grasshopper, but appear to play roles only in the
nervous system there and not in segmentation. In
contrast, pair-rule genes are expressed in segmentally
repeated patterns in short, intermediate, and long
germ band beetles [Patel et al., 1994]. Gap gene homologues also have been identified [Sommer and Tautz,
1993]. Tribolium homologues of Drosophila hairy [Sommer and Tautz, 1993], ftz [Brown et al., 1994], eve
[Brown et al., 1997; Patel et al., 1994], and runt [cited in
Brown and Denell, 1996] have been identified and all
are expressed in striped patterns in this short germ
band insect.1 In keeping with the sequential addition of
segments to the body plan, stripes for these pair-rule
genes arise one-by-one during the growth phase, suggesting that segments are successively specified as
development proceeds [reviewed in Patel, 1994]. Furthermore, engrailed homologues are expressed in stripes
that mark the posterior segment compartment in a
wide range of insects [Patel et al., 1989], reviewed in
Patel [1994]. These observations suggest that basic
mechanisms directing segment establishment are conserved in different types of insects. However, it should
be noted that the although the Tribolium homeotic
complex (HOM-C) does contain a ftz homologue that is
expressed in stripes, a deletion within the complex does
not generate an obvious pair-rule phenotype [Brown et
al., 1994; Stuart et al., 1991]. This may suggest different functions of the pair-rule genes in different insects,
a higher degree of redundancy in Tribolium than in
Drosophila, or a specific feature of this HOM-C deletion
that obscures identification of pair-rule function.
Sulston and Anderson [1996] carried out a genetic
screen in Tribolium to identify genes responsible for
segmentation in this insect without the bias created
from reliance upon Drosophila segmentation genes for
their identification. From a screen for cuticular defects,
five mutants were identified that caused defects in
1There
has been some discussion of whether Tribolium should be
classified as a short or intermediate germ band insect; see Patel et al.
[1994]; Sommer and Tautz [1993]; Sulston and Anderson [1996] for
various points of view.
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segmentation reminiscent of what is seen in Drosophila. In this issue, these authors have analyzed the
expression patterns of Tribolium segmentation genes
eve and en in some of these mutants [Sulston and
Anderson, 1998]. Both similarities and differences between Drosophila and Tribolium segmentation are
highlighted here. Future analysis of these mutant
phenotypes and molecular cloning of Tribolium segmentation genes will elucidate the molecular basis of segmentation in Tribolium and allow for further comparisons to the Drosophila system.
SEGMENTATION IN VERTEBRATES
The role of segmentation in vertebrate development
has been much more controversial. The most obvious
segmented structures in vertebrates are the somites
[reviewed in Gossler and de Angelis, 1998], as well as
the rhombomeres of the hindbrain. Somites are metameric divisions of the paraxial mesoderm that give rise
to the major segmented structures of the vertebrate
body: the axial skeleton, skeletal musculature, and
dermis. Somites also impose segmentation on the peripheral nervous system, which is derived from neural
crest [Christ and Ordahl, 1995; Keynes and Stern,
1988; Psychoyos and Stern, 1996]. Somites form in
vertebrates from the presomitic mesoderm (PSM) in an
anterior-posterior progression reminiscent of the sequential addition of segments in short germ band
insects. The first somites form in the anterior portion of
the PSM; additional somites form sequentially in an
anterior to posterior direction, while new mesoderm is
added at the posterior end. The exact number of
somites formed and the timing of somitogenesis are
species-specific, e.g., ,30 somites develop in zebrafish
and a new pair is added roughly every 30 minutes,
whereas in the mouse, there are 65 somites (30 in the
tail), which form every 90 minutes. Mature somites
consist of an epithelial layer surrounding undifferentiated mesenchymal cells. Much like Drosophila segments, somites are divided into anterior and posterior
compartments at an early stage. Similarly, the identity
of individual somites is specified according to position
along the anterior-posterior axis. The cells of the somites
differentiate according to their positions relative to the
notochord and surface ectoderm to form the dermomyotome, myotome, and sclerotome, which then give rise to
the skin, muscles, and skeleton of the vertebral column,
respectively [reviewed in Gossler and de Angelis, 1998].
Hox genes have been known to specify positional
identity of Drosophila segments and of somites and
rhombomeres in higher organisms for some time [reviewed in Kenyon, 1994; Krumlauf, 1994]. However,
until quite recently, the establishment of segmentally
repeated units such as somites was thought to be
fundamentally distinct from the segmentation process
in insects [reviewed in Kimmel, 1996]. The recent
characterization of vertebrate homologues of the Dro-
sophila pair-rule gene hairy has raised the possibility
that pair-rule patterning is an evolutionarily conserved
process. A zebrafish hairy homologue, her1, is expressed
in a repeated pattern in the PSM. Stripes of expression
appear before the differentiation of somites and the
expression domains of her1 stripes and alternating
nonexpressing domains, each correspond to the primordia of a single somite [Muller et al., 1996; see also van
Eeden et al., 1998]. Because somitogenesis occurs sequentially, only two to three her1 stripes are observed
at any one point in time: her1 stripes arise in the
emerging posterior PSM as more anterior stripes fade,
before morphological evidence of somite formation is
visible. This mode of expression is very similar to
pair-rule striping in Tribolium.
Equally unexpected was the expression of the chick
hairy homologue, c-hairy1 [Palmerin et al., 1997].
c-hairy1 is expressed in the PSM in cyclic waves along
the posterior-anterior axis. The timing of each wave of
c-hairy1 expression corresponds to the time required
for formation of a single somite (,90 minutes in the
chick). The wave emanates from the posterior tail bud
region and progresses anteriorly to mark each somite
before it has become morphologically distinct. Reiterated waves of expression emerge sequentially suggesting the existence of a ‘‘molecular clock’’ that times the
periodic expression of this pair-rule gene. Thus the
major control of the repeated c-hairy pattern would be
temporal, in contrast to the pair-rule stripes in Drosophila, which are repeated spatially. These results
suggest that pair-rule genes function to specify metameres in vertebrates, as they do in Drosophila. A
further speculation is that pair-rule patterning was
used in a common segmented ancestor [Kimmel, 1996].
Genetic analysis will be required to determine the
function of these vertebrate genes and to identify other
pair-rule-type genes that may not be structurally homologous to the known Drosophila pair-rule genes.
Large-scale genetic screens were carried out by Nusslein-Volhard’s group and others to identify mutations
in the zebrafish that affect embryonic development
[Driever et al., 1996; Haffter et al., 1996; Kimmel et al.,
1989]. Among many mutations identified were two
groups of genes involved in somite formation [van
Eeden et al., 1996]. The first group ( fused somites,
fss-group) fails to establish anterior-posterior somite
boundaries, whereas the second group ( you-group mutants) establishes somites but has defects in somite
patterning [van Eeden et al., 1996]. Interestingly, one of
these, sonic you, encodes a homologue of Drosophila
hedgehog, a segment polarity gene that is widely conserved [Schauerte et al., cited in van Eeden et al., this
issue]. Here, van Eeden et al. [1998] further analyze the
defects seen in these zebrafish segmentation mutants.
They show that irregular somite boundaries that can
form at later stages in fss mutants require the activity
of the you-type genes that have apparent similarity to
Drosophila segment polarity genes. They further use
SEGMENTATION
the her1 gene as a marker to analyze pair-rule patterning in these mutants: all fss-class mutants affect the
expression of her1, although to varying extents. Creating double mutants for two different fss-type genes
effectively abolished her1 expression. Surprisingly, no
pair-rule phenotype (analogous to Drosophila) was
found here. This intriguing result suggests that either
pair-rule stripes play a different role in fish than in
flies, or that redundancy in the vertebrate system
[Nüsslein-Volhard, 1994] compensates for the loss of
one set of pair-rule stripes.
Palmerin et al. [1998] examine, in this issue, the
molecular basis for formation of rostro-caudal compartments in chick somites. Interestingly, genes implicated
in this process in mouse and chick are homologues of
the Drosophila Delta and Notch genes, which play quite
different roles in establishing cell fate in the fly [reviewed in Campos-Ortega, 1995; Muskavitch, 1994]. In
mouse, Delta is expressed in the presomitic mesoderm
and in the posterior compartments of the somites
[Bettenhausen et al., 1995]. In accord with this, loss-offunction Delta mutations cause loss of anterior-posterior polarity in the segments, similar to a Drosophila
segment polarity phenotype [de Angelis et al., 1997].
Here, Palmerin et al. [1998] show that chick Delta and
Notch are expressed in partially overlapping domains
in the presomitic mesoderm and in the posterior compartment of the somites. In contrast to the chick hairy
homologue, discussed above, these genes are not expressed in waves, consistent with different modes of
regulation for genes acting at different levels of the
hierarchy. In vitro culture experiments show that these
expression patterns are maintained in the absence of
overlying ectoderm. However, although repeated patterns of gene expression are still detectable, somite
formation did not occur in the absence of ectoderm. As
discussed above for Drosophila, these results highlight
questions about what is required, once regulatory genes
are correctly positioned in the embryo, in order to direct
the differentiation and growth of three dimensionally
patterned body structures in the animal.
The work by Zachgo et al. [1998] addresses later
aspects of somite patterning in the mouse. The Danforth’s short tail (Sd) mutation was first identified by
Dunn et al. in 1940 and results in abnormalities of the
axial skeleton [see also Gleucksohn-Schoenheimer, 1945;
Gleucksohn-Schoenheimer, 1943]. Recent experiments
have shown that Sd function is required in the notochord for maintenance of notochord integrity: breakdown of the notochord in these mutants appears to be
responsible for the wide range of defects seen in the
somite-derived vertebral column seen in Sd mutants
[Maatman et al., 1997]. Here, Zachgo et al. [1998] show
that an enhancer trap insertion that results in lac Z
expression in the notochord, and a recessive lethal
phenotype, has targeted the Sd locus. Their genetic
analysis suggests that the original Sd mutation is a
gain-of-function mutation. These observations high-
7
light the importance of interactions in patterning the
somites once metamerization is established in vertebrates. With an enhancer trap line in hand, it will be
possible to clone the Sd gene and begin to identify the
molecular basis for patterning of the somites by the
notochord in wild-type embryos.
CONCLUDING REMARKS
Tremendous progress has been made in recent years
in understanding the process of segmentation. Most is
known in Drosophila where saturation screens have
identified most of the genes involved in this process.
However, even in the fly, many basic mechanisms have
still to be elucidated. Although it is clear that the
pair-rule genes are responsible for generating periodicity in the Drosophila embryo, how they do so is not as
clear as it may seem. Much remains to be learned about
how repeated patterns of gene expression are established and about the downstream functions of these
regulatory genes. Recent advances in studies of segmentation in other insects and in vertebrates have revealed
a surprising conservation of the basic mechanisms used
in the fly. Many of the genes involved are the same, and,
most surprisingly, pair-rule stripes are found in higher
vertebrates. Substantially more of the regulatory hierarchy appears to be conserved than was thought just a
few years ago. How far will this functional conservation
go? The stripes of pair-rule homologues suggest functional similarities across phyla, but this has yet to be
tested. Given the conservation of genes at all levels of
the embryonic regulatory hierarchy, a major goal in the
coming years will be to describe the pathways that
define evolutionary divergence.
ACKNOWLEDGMENTS
I thank Tom Lufkin and Ron Kohanski for comments
on the manuscript. This is publication #269 from the
Brookdale Center for Developmental and Molecular
Biology.
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