Department of Biology, Indiana University, 1001 East Third Street, Bloomington, IN 47405, USA
* Author for correspondence (e-mail: kaufman{at}bio.indiana.edu)
Accepted 1 March 2005
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SUMMARY |
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Key words: even-skipped, Oncopeltus, Milkweed bug, RNAi, Segmentation, Growth zone, Convergent extension, hunchback, Krüppel, Pair-rule, Evolution, Short germband
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Introduction |
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The Drosophila mode of segmentation is not representative of all
insects and is actually evolutionarily derived. Evolutionarily basal insects
undergo what is termed `short' or `intermediate' germband development where
only the anterior segments are initially specified with the posterior body
regions arising later in a sequential anterior to posterior progression
(Davis and Patel, 2002;
Krause, 1939
). The sequential
nature of posterior segmentation in short and intermediate germband insects
implies that the underlying mechanisms that govern the production of posterior
segments potentially differ from Drosophila. Given the importance of
pair-rule genes in producing the first periodic gene expression patterns in
Drosophila, they make a logical choice for understanding posterior
segmentation in short and intermediate germ insects. Here, we focus our
attention on the pair-rule gene even-skipped.
even-skipped (eve) was originally identified in
Drosophila as a member of the pair-rule class of segmentation genes
because hypomorphic alleles produced embryos that lacked the denticle band and
adjacent cuticle from even-numbered segments (odd-numbered parasegments)
a canonical `pair-rule' phenotype
(Nusslein-Volhard et al.,
1984). The gene encodes a homeodomain-containing transcription
factor and acts as a transcriptional repressor
(Biggin and Tjian, 1989
;
Macdonald et al., 1986
).
even-skipped is initially expressed in a broad blastoderm domain that
first resolves into a striped primary pair-rule pattern with a two segment
periodicity (a pattern that correlates well with the hypomorphic eve
phenotype). This primary pair-rule pattern then matures into a secondary
segmental one (Frasch et al.,
1987
; Macdonald et al.,
1986
).
Previous studies of eve in other insects have found its expression
to be variable, with patterns similar to Drosophila, or having only
the pair-rule or segmental phases, implying that the role of
even-skipped may be highly plastic during insect evolution
(Binner and Sander, 1997;
Grbic et al., 1996
;
Grbic and Strand, 1998
;
Kraft and Jackle, 1994
;
Miyawaki et al., 2004
; Patel
et al., 1992
;
1994
;
Rohr et al., 1999
;
Xu et al., 1994
). However, the
vast majority of these studies have examined only the expression but not
function of the gene. Outside of Drosophila, eve function has only
been examined in the beetle Tribolium castaneum where its pair-rule
function is consistent with its pair-rule expression pattern
(Schroder et al., 1999
). Thus,
the evolution of even-skipped function within the insects, especially
among more basal groups, is not at all clear.
In order to gain insight into the evolution of even-skipped function in the insects, we have examined the expression and function of even-skipped in the milkweed bug, Oncopeltus fasciatus (Hemiptera:Lygaeidae) an intermediate germband insect. We find that in this insect, even-skipped does not act as a pair-rule gene, but rather is expressed in a segmental pattern and is required for proper growth and patterning of nearly all segments.
Moreover, all previous studies have only examined the role of striped eve expression, ignoring the earlier broad domain seen in almost all insects. The Drosophila eve phenotype does not suggest any function for this early domain, and has therefore been largely ignored. Here, we present the first report that this earlier domain actually has an important function during segmentation. Our results indicate that in Oncopeltus, this early domain is required for proper expression of the gap genes hunchback and Krüppel.
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Materials and methods |
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Embryo fixation, in situ hybridization and antibody staining
Embryo fixation, probe synthesis and in situ hybridization were carried out
as previously reported (Liu and Kaufman,
2004a). The final color development step was carried out
essentially as described by Liu and Kaufman
(Liu and Kaufman, 2004a
),
except for two-color in situs using BCIP/NBT and BCIP/INT where the first AP
antibody was inactivated by heating to 70°C for 30 minutes in Tris-EDTA
followed by additional fixation for 2 hours before continuing with the second
AP antibody.
RNAi
Double-stranded RNA used in parental and embryonic RNAi was in vitro
transcribed from template prepared one of two ways. Plasmid containing the
insert of interest was linearized by restriction digest, or template was
prepared from a PCR where T3 and T7 phage promoter sequences were added to the
primers. Sense and antisense RNA was synthesized in two separate reactions
using the MEGAscript kit (Ambion). Following in vitro transcription and DNase
treatment, the transcription reactions were immediately mixed in a single tube
and annealed. The RNA was annealed in a PCR machine set to incubate at
94°C for 3 minutes, then set to quickly reach 85°C followed by a slow
cooling to 25°C over the course of 1 hour. We removed unannealed
single-stranded RNA by digestion with RNase A (Ambion) for 15 minutes. A small
amount of annealed RNA was analyzed on an agarose gel to confirm successful
annealing and digestion and we found that the RNase A treatment resulted in
much less smearing of the dsRNA on the gel when compared with previous
methods. Injections for embryonic and parental RNAi was performed as
previously reported (Liu and Kaufman,
2004a).
Image capture and processing
Images of blastoderms and RNAi embryos were captured using a Nikon SMZ1500
stereomicroscope with attached Nikon DXM1200 digital camera. As these samples
were relatively large, a single focal plane was not sufficient to capture all
the detail of the entire embryo. Therefore several focal planes were taken for
each sample and were combined into a single composite image in Photoshop
(Adobe). Images of germband-stage embryos were captured on a Zeiss Axiophot
microscope with attached Nikon DXM1200 digital camera.
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Results |
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Using this strategy, we were able to isolate a 1038 bp fragment of the Of'eve transcript encoding a polypeptide of at least 236 amino acids. As there is no in frame stop codon in the 5' sequence prior to the first methionine codon, it is possible that this fragment does not include the entire open reading frame. However, alignments of the predicted Oncopeltus polypeptide with other insect eve sequences show very strong conservation at the N terminus, suggesting that our clones represent most of, if not the entire, open reading frame. Alignments with other insect eve sequences show sequences similar to the homeodomain, the Groucho co-repressor interaction domain, and an additional region of similarity at the N terminus (Fig. 1).
|
Oncopeltus even-skipped transcript first appears during the
blastoderm stage 20-24 hours after egg lay (AEL). At this stage,
Of'eve transcript accumulates as a broad band covering the posterior
two thirds of the blastoderm (Fig.
2A) and is reminiscent of early eve expression in
Drosophila, where it also first accumulates in all nuclei before the
appearance of its later striped expression
(Frasch et al., 1987
;
Macdonald et al., 1986
).
Shortly thereafter, at 24-28 hours AEL, the Oncopeltus pattern
becomes weaker on the ventral surface (not shown). This is not likely to
reflect a role in determining the dorsal/ventral axis, but probably reflects
the distribution of embryonic and extra-embryonic cells. Indeed expression of
hunchback, Krüppel, and Deformed are also weaker on the
ventral blastoderm surface (Liu and
Kaufman, 2004a
; Liu and
Kaufman, 2004b
) (P.Z.L., unpublished). Given the extreme embryonic
movements during development, the embryo actually rotates twice during
embryogenesis relative to the eggshell. In the interest of consistency, we
orient all blastoderm images as if the egg is held constant and the embryo
moves within it. A consequence of this is that blastoderm cells that are near
the dorsal surface of the egg are actually fated to become ventral in the
embryo.
|
In Drosophila, gap genes such as hunchback and
Krüppel regulate the position and spacing of the primary
even-skipped stripes (Clyde et
al., 2003; Frasch and Levine,
1987
; Small et al.,
1992
). We wished to know if expression of these same genes
correlated with the even-skipped stripes on the Oncopeltus
blastoderm. To this end, we performed double in situ hybridizations for
Oncopeltus even-skipped with hunchback (Of'hb) and
Krüppel (Of'Kr). Of'hb is expressed in two
broad domains in the blastoderm, a weaker anterior band which is anterior to
the mandibular segment and corresponds to the anterior head, and a stronger
central one corresponding to the posterior of the mandibular through labial
segments (Liu and Kaufman,
2004a
). Of'Kr is expressed in a broad posterior domain in
the blastoderm, corresponding to the thoracic segments
(Liu and Kaufman, 2004b
).
Of'eve stripes 1-3 (mandibular through labial) coincide with the
strong central domain of hb, while stripes 4-6 (thoracic) underlie
the Kr domain (Fig.
2H,I). This is in contrast with Drosophila, where
hb and Kr only span two stripes each.
Oncopeltus even-skipped expression in the germband
Oncopeltus embryos undergo `germband invagination' during which
cells of the late blastoderm migrate to the posterior pole of the egg and dive
into the interior of the yolk mass to contribute to the formation of the
germband (Butt, 1947;
Liu and Kaufman, 2004a
). This
process results in cells that originally occupied the posterior tip of the
blastoderm ending up as part of the posterior growth zone of the early
germband.
During germband invagination and throughout the remainder of germband
growth and segmentation, Oncopeltus even-skipped is continuously
expressed in both the mesoderm and ectoderm of the posterior growth zone
(Fig. 2J-K;
Fig. 3), reminiscent of
even-skipped expression in the grasshopper Schistocerca
(Patel et al., 1992).
Additionally, there are a few stripes of expression directly anterior to this
growth zone domain. As Oncopeltus is an intermediate-germ insect and
posterior segments are specified sequentially in an anterior-to-posterior
progression, these even-skipped stripes do not correspond to any
particular segments but rather are always expressed in the chronologically
youngest (most posterior) ones.
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Of'eve stripes also seem to be generated from the growth zone in a
segmental fashion. In Drosophila the secondary segmental stripes
arise de novo after the primary stripes are refined, while in other insects
with both primary pair-rule and secondary segmental patterns, the secondary
stripes are generated from `splitting' of the broader primary stripes
(Binner and Sander, 1997;
Macdonald et al., 1986
;
Patel et al., 1994
). Thus, the
pair-rule nature of expression is revealed by the dynamics of stripe formation
a broad primary stripe of expression followed by narrower segmental
secondary ones. In Oncopeltus, early stripes close to the growth zone
often have a characteristic `V' shape at the midline where they remain
contiguous with the growth zone (Fig.
3B,E). These stripes seem to `peel' off of the growth zone in a
segmental register as they maintain their width as they mature (compare
chronologically younger and older stripes in
Fig. 3B,C2) and do not `split'
to form secondary stripes.
Moreover, early growth zone expression often shows three or four stripes of Of'eve within the unelongated growth zone (Fig. 3A2). These stripes may correspond to anterior abdominal stripes that migrate into the rest of the germband as the germband elongates. If this is the case, then the growth zone may become patterned before actual elongation. At any rate, these stripes also do not appear to be any broader than the abdominal stripes to which they then give rise. Thus, the dynamics of Of'eve stripe formation reveal no obvious pair-rule phase of expression.
Oncopeltus even-skipped RNAi
With Of'eve expression suggesting roles in segmentation and growth
zone function, we wished to functionally test its developmental role.
We therefore used RNAi to specifically knockdown even-skipped
function in Oncopeltus in order to gain insight into its role in
milkweed bug embryogenesis. We directly injected double-stranded RNA into
early Oncopeltus embryos (termed embryonic RNAi, eRNAi)
(Hughes and Kaufman, 2000),
and also injected double-stranded RNA into the abdomens of adult females
(termed parental RNAi, pRNAi) (Liu and
Kaufman, 2004a
), and both yielded equivalent knockdown phenotypes.
We found that occasionally, the first clutch from a given injected female
would contain wild-type embryos, while later clutches would then show the
even-skipped phenotype. This is probably because in the developing
oocytes that give rise to these early broods, the egg chorions were likely
already deposited, preventing the entry of the dsRNA. We therefore excluded
these clutches from our analysis.
We also injected two different dsRNAs corresponding to two non-overlapping
regions of the Of'eve transcript and both regions produced identical
knockdown phenotypes (Table 1).
RNAi of other genes in Oncopeltus results in phenotypes that range in
severity and the resulting hypomorphic series often aids in the interpretation
of the phenotype (Angelini and Kaufman,
2004; Liu and Kaufman,
2004a
; Liu and Kaufman,
2004b
). We took advantage of this and injected dsRNA to
Of'eve in a range of concentrations
(Table 1). Based on their
phenotypic severity, the RNAi embryos were categorized into three classes,
ranging from the strongest (class I) to the mildest (class III).
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|
The mild class III embryos constituted only 1.7% of the total affected pRNAi embryos and as noted were produced only from injection of low concentrations of Of'eve dsRNA (Table 1). Several abdominal segments were formed, but appeared much smaller than normal (Fig. 4B1-B3). The mandibular and maxillary stylets were present, although not extended (not unexpected because the uncoiling of the internal stylets usually occurs at hatching). Segmental grooves of the thorax were occasionally less prominent giving the thorax a smoother appearance. The thoracic legs appeared slightly deformed, although this may be due to steric deformation within the confines of the eggshell rather than reflecting defects in patterning. Importantly, any weak disruptions in the thorax affected all segments. Any bias in sensitivity seemed graded, increasing towards the posterior, with no evidence of any skipping of segments. In order to more clearly examine the phenotype, we used Oncopeltus engrailed expression as a convenient segmental marker in germband stage RNAi embryos. Fig. 4D1,D2 shows late-stage class III germband stained for engrailed and shows that the abdomen is reduced with highly disorganized segmentation (compare Fig. 4D1 and D2 with Fig. 3D1) while the head and thoracic segments appear normal. Thus, class III embryos have defective abdomens but relatively normal heads and thoracic segments.
|
The moderate class II embryos were also rare, making up only 2.1% of the RNAi embryos and were also only produced at lower dsRNA concentrations (Table 1). When compared with the milder class III embryos, these embryos show stronger abdominal defects as well as defects in the thorax the abdomen is severely reduced and thoracic segmentation is defective (Fig. 4E1,E2). In these embryos, posterior thoracic legs are either reduced or missing but anterior structures are left relatively unaffected. Germband stage embryos corresponding to this phenotypic class stained for engrailed reveal strong disruption of posterior growth and patterning but relatively normal anterior segmentation (Fig. 4F). As with class III, these embryos also seem to show a gradient of defect, stronger in the posterior and weaker in the anterior, without any pair-rule like defects.
Class I constituted 81.8% of the RNAi embryos (Table 1). These embryos are characterized by a very large deletion of almost the entire body (Fig. 4G-H2). Given this severity, it is difficult to capture all aspects of the phenotype photographically, so we will describe their morphology based on observations of several class I embryos. These embryos lack most of the body, with no apparent gnathal, thoracic or abdominal segments (as the intercalary segment is so small, we cannot determine its presence or absence). Antenna, eyes and a labrum can still be found and seem morphologically normal, albeit smaller in size (Fig. 4G). Mandibular and maxillary stylets are missing, suggesting that the deleted region spans these segments as well. Late-stage RNAi germbands stained with engrailed probe show that although anterior head elements do form, the mandibular through abdominal regions are entirely missing (Fig. 4H1,H2), consistent with what is seen in hatching-staged embryos. Thus, strong suppression of Of'eve results in loss of almost the entire body, from the mandibular segment to the posterior of the animal.
even-skipped RNAi results in gap gene misexpression
The severe Of'eve phenotype, complete loss of the mandibular
through abdominal segments, was much stronger than we had expected. Although
loss of abdominal segments may be explained by disruption of patterning in the
growth zone, we noticed that loss of the gnathal and thoracic segments
correlated well with the early broad blastoderm domain of expression
(mandibular to the posterior of the blastoderm). We reasoned that as the head
and thorax are normally specified during the blastoderm stage, blastoderms
that are depleted for Of'eve function might become repatterned to
reflect loss of the deleted regions.
We first noticed that RNAi embryos showed defects in germband invagination. In normal 48-52 hour embryos, germband invagination is nearly complete with the site of invagination at the posterior pole of the blastoderm (arrow in Fig. 5A). Of'eve RNAi embryos at a similar stage show a failure of germband invagination with a mislocalization of the invagination site to a more ventral position on the blastoderm (arrow in Fig. 5B). When dissected, these embryos do not have an elongating germband in the yolk (not shown), which is consistent with loss of the entire body in the class I animals. These defects suggest that depletion of Of'eve function may repattern the blastoderm.
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In uninjected animals, Of'Kr is expressed in the posterior third
of the blastoderm, corresponding to the thoracic segments
(Fig. 5E) (Liu and Kaufman, 2004b).
Of'Kr expression in Of'eve RNAi blastoderms is highly
reduced, so that only a tiny patch of expression remains at the posterior tip
of the blastoderm (Fig.
5F1,F2). This suggests that Of'eve is also required for
proper expression of Of'Kr in the blastoderm.
Of'eve is expressed in a dynamic pattern during the blastoderm stage, with an early broad domain covering the posterior two thirds of the blastoderm and a later striped phase of expression. Temporally and spatially, it seems most likely that it is this early broad blastoderm domain (rather than the later striped expression) that is responsible for these gap gene-regulating functions. Thus, in the absence of Of'eve function, the early blastoderm becomes re-allocated to represent only the anterior head.
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Discussion |
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even-skipped does not act as a pair-rule gene in Oncopeltus
One of the characteristics of Drosophila even-skipped and most of
the other pair-rule genes is that they are expressed in the blastoderm in a
series of seven transverse stripes with a two-segment periodicity
(Carroll et al., 1988;
Carroll and Scott, 1986
;
Gergen and Butler, 1988
;
Grossniklaus et al., 1992
;
Hafen et al., 1984
;
Harding et al., 1986
;
Macdonald et al., 1986
).
even-skipped expression has been examined in a number of insects, and
in many species, a primary pair-rule pattern is followed by a later segmental
one. For example, in Drosophila, eve primary stripe expression is in
odd numbered parasegments, and then later minor stripes arise de novo in the
even numbered parasegments (Frasch et al.,
1987
; Macdonald et al.,
1986
). In both the long germ honeybee Apis mellifera and
short germ beetle Tribolium, secondary stripes appear through
`splitting' of the primary pair-rule stripes
(Binner and Sander, 1997
;
Brown et al., 1997
;
Patel et al., 1994
). The
Oncopeltus eve expression dynamic shows none of these pair-rule
patterns, instead initiating in a segmental manner.
Function of even-skipped had previously only been examined in
Drosophila and Tribolium, and was found to have a pair-rule
requirement, reflecting the pair-rule expression pattern in both of these
insects (Nusslein-Volhard et al.,
1984; Schroder et al.,
1999
). In Oncopeltus, there is no apparent pair-rule
phenotype. Instead, there seems to a gradient of sensitivity, with posterior
segments being more sensitive to RNAi depletion. We should note that as
Of'eve is expressed in a broad blastoderm domain, in the growth zone
and in segmental stripes, teasing apart the functions of each of these
individual domains is difficult without more sophisticated genetic techniques.
Because we can assay for a pair-rule phenotype only in the thorax, we cannot
rule out a hidden pair-rule function in the abdominal segments. Nevertheless,
as neither the expression nor functional analyses reveal an apparent pair-rule
role, even-skipped is probably not acting as a pair-rule gene in this
insect.
Divergent regulation of the segmentation genes in Oncopeltus
The non-pair-rule role of Oncopeltus even-skipped suggests that in
this insect, the genetic paradigm regulating the segmentation gene cascade
must differ from Drosophila in several respects.
First, regulation of striped expression of Of'eve by the upstream
gap genes must be divergent. In Drosophila, even-skipped is directly
regulated by gradients of gap gene proteins that bind to stripe-specific
enhancers in the eve promoter
(Clyde et al., 2003;
Frasch and Levine, 1987
;
Small et al., 1992
). The
anterior Drosophila hunchback domain covers eve stripes 1
and 2, while the Krüppel domain covers stripes 3 and 4. This is
in contrast to Oncopeltus, where during the blastoderm stage,
Of'hb spans the first three Of'eve stripes and
Of'Kr spans stripes 4, 5 and 6. Moreover, the stripes in
Drosophila are pair-rule but in Oncopeltus, are segmental in
register. Although Of'eve stripes are expressed in a manner
consistent with their potential regulation by the gap genes, the precise
mechanism governing this regulation is likely to be fundamentally different
from that in Drosophila. Moreover, during germband elongation,
Of'eve stripes are generated sequentially out of the growth zone, a
dynamic very different from Drosophila. Given these differences, the
cis-regulatory elements that govern Of'eve regulation should prove to
be very interesting.
Second, the overall pair-rule mechanism is likely to show fundamental
differences in Oncopeltus. In Drosophila, the primary
eve stripes act within the context of a pair-rule network
(Carroll and Vavra, 1989). But
given the segmental register of Of'eve, this network of
cross-regulation is likely to be significantly different. Moreover in
Drosophila, striped eve expression initiates in the
pre-cellular blastoderm, where these early primary stripes each act as
morphogenetic gradients to regulate the other pair-rule genes
(Fujioka et al., 1995
). In
Oncopeltus, cellularization of the blastoderm nuclei occurs at around
17 hours AEL (Butt, 1947
), well
before the initiation of Of'eve striped expression at around 32
hours. The lack of a prolonged syncytial blastoderm stage in
Oncopeltus suggest that morphogenetic gradients are not involved in
the same fashion in the regulation of Of'eve or in its regulation of
other pair-rule genes.
The third aspect which may differ between Drosophila and
Oncopeltus is the regulation of engrailed by
even-skipped. The expression dynamics of these two genes strongly
suggest that Of'eve regulates Of'en. Of'eve expression
slightly precedes and becomes coincident with expression of Of'en
during both the blastoderm and germband stages. Thus, Oncopeltus
even-skipped is temporally and spatially poised to regulate
engrailed. However, the details are again likely to differ between
Oncopeltus and Drosophila. In Drosophila, both the
odd- and even-numbered engrailed stripes are initiated solely through
action of the primary eve stripes, while the role of the minor
stripes is unclear (Fujioka et al.,
1995; Jaynes and Fujioka,
2004
). It may be that engrailed activation in
Oncopeltus is more similar to the activation of either the odd- or
even-numbered engrailed stripes in Drosophila and that all
engrailed stripes are generated using the same mechanism. It is
interesting that the Of'eve pattern has more affinity to the late
Drosophila (14 stripe) pattern, and may indicate that the minor
stripes in Drosophila are an evolutionary vestige of a previous
function.
eve expression has been found to be surprisingly variable in
several insects, with some insects showing pair-rule only, segmental only,
both, or neither patterns (Fig.
6A) (Binner and Sander,
1997; Grbic et al.,
1996
; Grbic and Strand,
1998
; Kraft and Jackle,
1994
; Miyawaki et al.,
2004
; Patel et al.,
1992
; Patel et al.,
1994
; Rohr et al.,
1999
; Xu et al.,
1994
) (S. Noji, personal communication). Additionally,
eve expression has been examined in a crustacean and found to have no
obvious pair-rule pattern (Davis and
Patel, 2003
). Thus, it is not clear what the ancestral state was
in insects. Perhaps what this variability in striped expression is telling us
is that we should be focusing on what inherent architectural features in the
pair-rule network are allowing such easy change. This will require in-depth
functional analysis of multiple pair-rule genes in an insect such as
Oncopeltus, as well as other arthropods.
|
That a supposedly downstream pair-rule gene regulates supposedly upstream
gap genes is not entirely without precedent. The Drosophila pair-rule
gene runt is also required for proper expression of some of the gap
genes (Tsai and Gergen, 1994).
Drosophila runt is initially expressed in an early broad blastoderm
domain, before the appearance of the characteristic pair-rule stripes and it
is this broad initial domain that is responsible for proper gap gene
expression. In Oncopeltus, the initial broad blastoderm expression of
Of'eve may serve a similar function. It may therefore be the case
that the early broad blastoderm domain regulates the gap genes, while the
later striped expression is in turn regulated by them. This would mean that
Of'eve occupies both upstream and downstream positions in the
segmentation gene hierarchy (Fig.
6B).
Speculations on non-striped even-skipped function
Given that the gap-like function of Of'eve is novel and has not
been reported for other insects, we have much less context in which to discuss
its implications. We therefore offer some speculation that we feel is
important to discuss explicitly.
First, eve in several other insects is also expressed in a similar
initial broad domain, but as this domain has no apparent function in
Drosophila, its potential role in segmentation has been previously
largely ignored (Fig. 6A)
(Binner and Sander, 1997;
Grbic et al., 1996
;
Grbic and Strand, 1998
;
Patel et al., 1994
). In light
of our results, this assumption may need to be re-examined. It may be that
ancestrally, eve had an important gap-like function that was
subsequently lost in the lineage leading to Drosophila.
Second, function of the early broad domain may provide clues to function of the later growth zone domain. Both can be viewed as different manifestations of a similar underlying pattern. Recall that Of'eve expression does not fade from the blastoderm completely, but is maintained as a posterior patch in the blastoderm at the outset of germband invagination and as the germband invaginates, eventually contributes to the posterior growth zone. Therefore, the growth zone domain is a direct continuation of the early broad blastoderm expression. Moreover, the early broad domain fades from the blastoderm surface in an anterior to posterior direction, leaving behind segmental stripes of expression. The growth zone expression can also be thought of as following the same dynamic: the posterior growth zone maintains expression of Of'eve but as it extends in an anterior to posterior direction during germband growth, expression of segmental stripes seem to be left behind. This potentially equates the function of the early broad domain with function in the growth zone. As the segmentation hierarchy proceeds through gap, pair-rule and segment polarity levels, it is possible that this expression in the growth zone indicates that it is being held at a `higher' or `earlier' state, much as the early gap-like domain precedes the later striped expression.
Third, in addition to the role of eve in patterning the growth
zone as discussed above, it is also possible that Of'eve is required
for its growth. The abdomen of short and intermediate germ insects
dramatically elongate during embryogenesis. As the Oncopeltus growth
zone narrows as the germband elongates, it may be that cell rearrangements
contribute to germband growth (Fig.
3A2,C2,D2). In Drosophila, germband extension is chiefly
due to cell rearrangements that are termed `convergent extension'
(Edgar et al., 1989;
Hartenstein and Campos-Ortega,
1985
). It turns out that several segmentation genes, including
even-skipped and hunchback, play important roles in
convergent extension (Irvine and
Wieschaus, 1994
). Thus, it is intriguing that both
even-skipped and hunchback are so strongly expressed in the
Oncopeltus growth zone and that RNAi of these genes results in a
failure of posterior growth (Liu and
Kaufman, 2004a
). This raises the possibility that in addition to
posterior patterning, Of'eve and Of'hb may also be required
for a process similar to convergent extension in Oncopeltus. However,
it is also possible that elongation occurs through increased cell
proliferation. Although no convincing increase of mitotic activity has been in
found in the growth zones of several insects, including Oncopeltus,
we cannot rule out cell proliferation as a source of germband elongation
(Brown et al., 1994
) (P.Z.L.,
unpublished; N. Patel, unpublished). But the growth zone shape changes during
abdominal growth suggest that cellular rearrangments may at least be one
component to posterior elongation
Interestingly, it has been recently shown that RNAi of the posterior gene
caudal in the short germband beetle Tribolium castaneum, the
intermediate germband cricket Gryllus bimaculatus, as well as in the
crustacean Artemia franciscana results in a loss of posterior growth
and segmentation (Copf et al.,
2004; Shinmyo et al.,
2005
). In strongly affected Tribolium embryos, only the
pregnathal head was formed, a phenotype very similar to the Oncopeltus
even-skipped phenotype. In all three organisms, caudal RNAi
leads to weakened, abnormal expression of even-skipped. Additionally,
caudal RNAi in the cricket also leads to loss of hunchback
and Krüppel expression, again similar to the situation to
Oncopeltus even-skipped. Taken together, this suggests that both
caudal and even-skipped are involved in similar functions in
posterior growth and patterning, and that possibly, some functions of
caudal may be mediated via even-skipped.
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ACKNOWLEDGMENTS |
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REFERENCES |
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