Department of Biology, Indiana University, 1001 East Third Street, Bloomington IN, 47405, USA
* Author for correspondence (e-mail: kaufman{at}bio.indiana.edu)
Accepted 16 June 2004
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SUMMARY |
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Key words: Krüppel (Kr), Gap gene, Short germband, Segmentation, Oncopeltus, Milkweed bug, RNAi
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Introduction |
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Although the developmental genetics regulating Drosophila
segmentation is well understood, what we have learned from Drosophila
cannot be universally applied to the other insects. In many ways, embryonic
development in Drosophila is not representative of most insects. For
example, insects can be categorized as being `short', `intermediate', or `long
germband' with the classifications based largely on the number of segments
specified before gastrulation (Davis and
Patel, 2002; Krause,
1939
; Sander et al.,
1985
). Since Drosophila specifies its entire body plan
essentially simultaneously, Drosophila is classified as a long
germband insect. In short and intermediate germband segmentation, only the
anterior segments are specified at the blastoderm stage. The remainder of the
segments arises later during embryogenesis from disproportionate growth of the
posterior, from a region described as the `posterior growth zone'. This region
occupies the posterior-most portion of the elongating germband and growth of
this region gives rise to the posterior segments which are specified
sequentially in an anterior to posterior progression as the germband
elongates. Thus, while long germband insects pattern their entire bodies via
successive subdivision of the blastoderm, short and intermediate germ insects
allocate their blastoderms into only their anterior-most segments and then
produce the remaining segments during a later phase of posterior growth.
(Since the short and intermediate forms of segmentation are conceptually so
similar, for convenience sake we will henceforth refer to both the short and
intermediate forms as `short'.)
In Drosophila, the gap genes are expressed in broad domains in the blastoderm, each of which encompasses several contiguous body segments. Reflecting this expression pattern, Drosophila embryos mutant for gap genes show segmental deletions spanning several contiguous segments. Thus the gap genes are early patterning genes involved in the initial subdivision of the blastoderm. Since one of the essential differences between short and long germ segmentation lies in how the early blastoderm is allocated into broad body regions, comparing the action of the gap genes between long and short germ insects should serve as a good starting point for better understanding the differences between these two modes of insect segmentation.
The Drosophila gap gene Krüppel (Kr) is
required for proper formation of the central portion of the fly embryo.
Krüppel encodes a transcription factor that contains four
zinc-finger motifs that are important for its DNA-binding function
(Gaul et al., 1989;
Rosenberg et al., 1986
). Null
alleles of Krüppel result in embryos that have a canonical `gap
phenotype' and lack the first thoracic through fourth abdominal segments with
the fifth abdominal segment partially deleted. Additionally, the posterior
boundary of this deleted region is frequently marked by the presence of a
mirror image duplication of the sixth abdominal segment
(Gloor, 1950
;
Wieschaus et al., 1984
).
The region deleted in Drosophila Krüppel mutants spans
segments that in short germband insects are specified both during the
blastoderm stage and also later during germband growth. For example, in the
intermediate germband insect Oncopeltus fasciatus (Hemiptera,
Lygaeidae), only the mandibular through the third thoracic segments are
specified during the blastoderm stage. It is later, during germband
elongation, that the abdominal segments become specified
(Butt, 1947;
Liu and Kaufman, 2004
). Thus,
using Drosophila as an analogy, the putative region of Kr
function in Oncopeltus would span segments specified during the
blastoderm stage (thoracic) as well as segments specified during the germband
stage (abdominal). Since nothing is known about Krüppel activity
at the functional level in the context of short germ segmentation and given
the striking differences in segment formation between the blastoderm-derived
and germband-derived body regions, it is difficult to imagine how
Krüppel would act in both of these regions of the
Oncopeltus embryo.
Would Kr function as a canonical gap gene in only the anterior segments, leaving the posterior segments untouched or would Krüppel action span both body regions? In order to shed light on this question and also to better understand segmentation in short germband insects in general, we investigated the developmental role of Krüppel in the milkweed bug, Oncopeltus fasciatus. We isolated the milkweed bug homolog of the Drosophila gap gene Krüppel and report its expression pattern during milkweed bug embryogenesis. Then using RNA-mediated interference (RNAi), we depleted Krüppel activity, which allowed us to examine its function in Oncopeltus segmentation.
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Materials and methods |
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GenBank accession numbers for submitted sequences are: Of'Kr: AY627357, Of'pb: AY627358, Of'Ubx: AY627359, AY627360, Of'abd-A: AY627361.
Embryo fixation, in situ hybridization, and RNAi
Embryo fixation and in situ hybridizations were performed as previously
reported (Liu and Kaufman,
2004). We found that it was much easier to dissect the germband
stage embryos out of the yolk before carrying out the in situ hybridization
procedure. In order to do this, embryos removed from the eggshell but
undissected from the yolk balls were first rocked in a SYTOX solution (a
fluorescent DNA dye; Molecular Probes) for 2 hours. These embryos were then
dissected in PBT under a fluorescence stereomicroscope. Both embryonic (eRNAi)
and parental RNAi (pRNAi) RNA-mediated interference were carried out as
previously reported (Hughes and Kaufman,
2000
; Liu and Kaufman,
2004
).
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Results |
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During the germband stage, the remaining posterior body segments that were not specified during blastoderm stage are now produced through elongation of the posterior portion of the germband, the `growth zone'. Progressive abdominal segment specification can be tracked by observing the appearance of abdominal en stripes developing in an anterior to posterior direction (Fig. 1E-H). This biphasic mode of segmentation, with anterior segment specification during the blastoderm stage and subsequent posterior patterning during germband growth, marks Oncopeltus as an intermediate germband insect.
Isolation of Oncopeltus Krüppel
We took a RT-PCR-based approach to isolate the Oncopeltus homolog
of Krüppel. First, degenerate primers were designed to conserved
regions of previously isolated Krüppel homologs. PCR on milkweed
bug embryonic cDNA allowed us to recover a short fragment corresponding to the
zinc-finger region of Oncopeltus Krüppel (Of'Kr). This
short initial fragment allowed us to design exact primers for 5' and
3' RACE and subsequently isolate both 5' and 3' fragments of
the gene. Together, our 5' and 3' clones include the entire
Of'Kr open reading frame. Of'Kr is predicted to encode a
33.5 kDa protein with a total of four zinc-binding fingers. Additionally, the
Of'Kr protein contains sequences similar to the A- and B-boxes found in
Drosophila Krüppel (Fig.
2).
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|
Of'Kr expression in the germband
The Oncopeltus Kr germband expression pattern is partially a
continuation of expression initiated during the blastoderm stage but also
includes novel domains in the mesoderm and developing nervous system. During
the initiation of germband invagination, Kr expression reflects the
movement of blastoderm cells across the outer surface of the yolk mass. Recall
that during germband invagination, blastoderm cells migrate towards the
posterior pole of the egg and upon reaching it, dive into the interior of the
yolk mass to form the growing germband. These cellular movements can be seen
by tracking the movement of Kr-expressing cells across the
blastoderm. For example, a patch of Kr arises in the anterior of the
blastoderm at the initiation of germband invagination. Shortly thereafter,
this patch has migrated to the middle portion of the blastoderm, coincident
with the germband increasing in length (see
Fig. 3D1-E2). The movement of
this patch of Kr expression highlights the movement of the blastoderm
cells across the yolk mass.
As described above, in late blastoderms Krüppel expression retreats from the posterior pole just before germband invagination begins. This clearing is evident in very early germbands still undergoing invagination (Fig. 3E3). Just after the completion of germband invagination, Kr is expressed in the central region of the germband and is excluded from the posterior growth zone. The exclusion is maintained throughout germband growth (Fig. 4A-G). Thus this lack of Kr expression in the germband growth zone is an apparent continuation of the posterior clearing seen in late blastoderms.
|
Of'Kr expression in mesodermal and neural domains
Shortly after the germband finishes invagination and during elongation,
segmentally reiterated mesodermal expression of Of'Kr begins
(Fig. 4). This mesodermal
expression begins in the thoracic segments, underlying the ectodermal
expression and during germband extension, expands both anteriorly and
posteriorly to encompass more of the germband than the previous ectodermal
gap-like domain. This anterior expansion can be seen as expression of
Kr first in the labial then maxillary and finally in the mandibular
segment (arrowheads in Fig.
4H-K). Although the en stripes of this region are already
present, this anterior expansion of the Kr domain may reflect the
anterior and posterior morphological differentiation of segments that starts
from a region called the differentiation center, which in many insects is
located in the presumptive thorax (Krause,
1939). In some insects, thoracic en stripes do indeed
appear before the gnathal expression of this gene
(Patel et al., 1989
). As
noted, this anterior expansion of mesodermal Of'Kr expression is the
reason that the early germband expression extends further than the earlier
blastoderm expression. In sum, continuation of the blastoderm pattern results
in ectodermal expression corresponding to the first through third thoracic
segments and additional Kr transcript accumulates in the underlying
mesoderm of the thorax with this mesodermal expression expanding anteriorly as
development proceeds.
During the formation of the abdominal segments, mesodermal Kr
expression also expands posteriorly (arrows in
Fig. 4H-K), but is absent from
the overlying ectoderm (Fig.
4D1-E2). However, the posterior boundary of ectodermal Kr
expression remains in the third thoracic segment, consistent with the pattern
established during the blastoderm stage. This abdominal mesodermal expression
proceeds in an anterior to posterior progression and probably reflects the
anterior to posterior maturation of the abdominal segments
chronologically older (more anterior) segments express Kr
mesodermally, while younger segments (closer to the growth zone) do not.
Inspection of this mesodermal expression reveals no apparent regional
differences in the abdomen, and it merely appears in a segmentally reiterated
pattern (compare Fig. 4D2 and
E2). Thus developing germbands express Kr in an
ectodermal gap-like pattern in the central portion of the germband reflecting
continued expression of the blastoderm domain. Kr is also expressed
mesodermally beginning with the central portion of the germband and expanding
to eventually encompass the mesoderm throughout the entire germband. This
mesodermal expression of Kr seems to be shared with other insect
species (Gaul et al., 1987;
Sommer and Tautz, 1991
).
In addition to the ectodermal and mesodermal expression domains described
above, Oncopeltus Kr transcript also appears to accumulate in a
neural-like pattern. In mid-germband stage embryos, an orderly grid-like
pattern of dots appears in the middle region of each segment
(Fig. 4F1,F2) and this pattern
is consistent with known Kr expression and function in the
Drosophila nervous system (Gaul
et al., 1987; Isshiki et al.,
2001
). Like the mesodermal expression described above, this
neural-like expression also progresses in an anterior to posterior direction,
most likely reflecting progressive maturation of each body segment
(Fig. 4E-G).
Morphological analysis of Kr RNAi
In order to determine the functional role of Kr in
Oncopeltus segmentation, we used RNA-mediated gene interference
(RNAi) to deplete Kr transcript and assayed the resulting embryos for
the knockdown phenotype. RNAi is a technique that has been used in many
organisms, which allows specific suppression of gene function via the
introduction of double-stranded RNA (dsRNA) corresponding to the gene of
interest into the developing embryos (Brown
et al., 1999; Fire et al.,
1998
; Hughes and Kaufman,
2000
; Miyawaki et al.,
2004
; Schoppmeier and Damen,
2001
). It has been reported previously that direct injection of
dsRNA into early embryos, termed embryonic RNAi (eRNAi) and also injection of
dsRNA into the abdomens of mothers, termed parental RNAi (pRNAi) yield
knockdown phenotypes in Oncopeltus
(Bucher et al., 2002
;
Liu and Kaufman, 2004
). Since
pRNAi does not produce any injection artifacts, we largely used pRNAi in our
analysis but also included eRNAi as a confirmation of the phenotype. We
injected three different dsRNA fragments corresponding to different regions of
the Of'Kr transcript: a 460 bp 5' fragment, a small 150 bp
3' fragment, and a larger 1.7 kb 3' fragment, which completely
spans the smaller 3' piece and also includes an additional 250 bp of
3' untranslated sequence (Fig.
2). All of these dsRNA fragments yielded the same qualitative
phenotype. Injection of dsRNA at different concentrations resulted in embryos
that ranged in phenotypic severity from strongly affected to completely wild
type (Table 2). This
hypomorphic series of Kr depletion allowed us to categorize the
embryos into three phenotypic classes based on final embryo morphology. All
affected phenotypic classes showed a gap phenotype that included the thorax
and anterior abdomen, with milder classes showing more limited regions of
deletion than the more severe phenotypic classes. Oncopeltus embryos
seem to be very sensitive to Kr depletion, as mothers injected with
the high concentration of dsRNA solution (2 µg/µl) did not give class I
or wild-type embryos. It was only upon injection with very low dsRNA
concentrations (0.004 µg/µl) that we were able to obtain the full range
of severity (Table 2).
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|
Putative class I germband stage embryos stained for en show a small deletion of the anterior abdomen along with defects in meso- and metathoracic segmentation (Fig. 6B), consistent with the late-stage morphological phenotype. In stronger RNAi embryos, this region of defect expands to include more thoracic and abdominal segments. In putative class II germband stage embryos, en expression shows that thoracic segments are more defective and fewer abdominal segments are present than in the class I embryos (Fig. 6D). The weaker phenotypic classes show that the anterior abdominal segments are most sensitive to Kr depletion and as Kr function is further suppressed, the gap expands in both anterior and posterior directions. By counting the number of en stripes on class III germband stage embryos, we determined that severely affected RNAi embryos lack a total of six segments (compare Fig. 6A and D) and that the deleted region seems spans the mesothoracic through fourth abdominal segments.
|
In some RNAi depleted embryos, defects in segmentation were seen to be
discontinuous abnormal en expression seemed to `skip'
segments. This `skipping' is reminiscent of discontinuous defects produced by
weak alleles of Kr in Drosophila
(Wieschaus et al., 1984). This
discontinuity in Oncopeltus Kr action was seen in all phenotypic
classes and was not associated with any particular segment.
Figure 6E shows an example of a
putative class III embryo where the usual central gap of the mesothoracic
through fourth abdominal segment is associated with an additional partial loss
of the sixth abdominal en stripe.
Figure 6B shows an example of a
putative class I embryo with a defective third abdominal en stripe
that is bounded by apparently normal en stripes.
Hox gene expression in Kr RNAi embryos
We wished to extend our analysis of the Kr RNAi embryos by
confirming the identity of the remaining segments using molecular markers. The
homeotic (Hox) genes are a group of genes that are expressed in, and are
thought to be required for, segmental identity in all arthropods including
insects (for a review, see Hughes and
Kaufman, 2002). Thus they make convenient molecular markers for
segmental identity in Oncopeltus. In wild-type embryos, the
Oncopeltus Hox gene Deformed (Of'Dfd) is expressed
in the mandibular and maxillary segments and associated limb buds
(Fig. 7A). In class III RNAi
embryos, the mandibular and maxillary expression is normal, confirming the
identity of these segments. However, Of'Dfd is ectopically expressed
in the first thoracic legs (Fig.
7B). This suggests that in Oncopeltus, Kr represses
Of'Dfd expression posterior to its normal domain. Oncopeltus
proboscipedia is expressed in the labium [but not in the maxillae as in
most insects (Rogers et al.,
2002
)] in wild-type embryos. This expression appears intact in
RNAi embryos, confirming the identity of this segment
(Fig. 7C,D). In the case of the
Tribolium jaws mutation, which is most likely a lesion in the
Tribolium homolog of Kr, proboscipedia
(maxillopedia in Tribolium) is ectopically expressed in the
thorax (Bucher, 2002; Sulston and
Anderson, 1998
). In contrast, we do not detect any ectopic
expression of proboscipedia in Oncopeltus Kr RNAi
embryos.
|
We also examined the expression of the posterior Hox genes, Ultrabithorax (Of'Ubx) and abdominal-A (Of'abd-A) in Kr-depleted animals. In wild-type animals, Of'Ubx is strongly expressed throughout the first abdominal segment and weakly in a neural-like pattern in the entire abdomen (Fig. 7G). This neural-like expression remains intact in RNAi depleted animals, but the segmental expression is not detectable (Fig. 7H). Since the neuronal expression is weak relative to the segmental expression, detection of the neuronal expression suggests that the lack of segmental expression is not merely the result of lack of sensitivity of the in situ hybridization technique. Rather, the lack of segmental expression along with the en expression described above indicates that at least the first abdominal segment is deleted. Of'abd-A is normally expressed in the abdomen, from the posterior of the first abdominal segment and extending posteriorly through the remainder of the abdomen (Fig. 7I). Krüppel RNAi depleted animals show fewer abd-A-expressing abdominal segments, consistent with a large deletion of the anterior abdomen (Fig. 7J,K). Additionally, weak ectopic patches of Oncopeltus abd-A can be seen in the labial and first thoracic segments (Fig. 7K). This ectopic expression of abd-A suggests that in Oncopeltus, Krüppel normally represses abd-A in anterior segments. Thus in Oncopeltus, Krüppel acts to repress expression of both anterior and posterior Hox genes in the central portion of the animal.
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Discussion |
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Kr expression shows both conserved and divergent aspects
During the blastoderm stage, Oncopeltus Kr is expressed in a broad
domain in the posterior one third of the blastoderm which corresponds roughly
to the posterior of the first thoracic through third thoracic segments. This
segmental register is maintained during germband invagination and results in
the continuation of this thoracic pattern in the ectoderm of early germbands.
During germband elongation, Kr is also detected in the mesoderm
underlying the thoracic ectoderm. As germband elongation proceeds, this
mesodermal expression expands both anteriorly and posteriorly to eventually
encompass the mesoderm of the entire body.
The segmental register of Krüppel's central gap-like pattern
differs from its expression in Drosophila melanogaster. In fruit
flies, Kr is expressed in the blastoderm from the mesothoracic
segment to approximately the third abdominal segment
(Gaul and Jäckle, 1989;
Knipple et al., 1985
). In
Oncopeltus, the gap-like domain of Kr covers the posterior
of the first through third thoracic segments but does not extend into any
abdominal ectoderm. Thus relative to the fruit fly, the posterior boundary of
Oncopeltus Kr is shifted anteriorly by about three segments. This
expression domain is more similar to that of the red flour beetle
Tribolium castaneum, where Kr is expressed in only the
thoracic segments and not in the anterior abdomen
(Bucher and Klingler, 2004
;
Sommer and Tautz, 1993
). Given
the similarities between the Oncopeltus and Tribolium
expression patterns, this pattern would appear to represent the ancestral
state for Kr expression at least within the paraneopteran
insects.
Krüppel expression clears in the very posterior of the late
blastoderm just before germband invagination. This may represent either loss
of activation or the initiation of suppression of Kr in these cells.
This posterior clearing of Krüppel expression is maintained
through germband invagination and is manifested in the early germband as a
growth zone devoid of Kr expression. This exclusion from the growth
zone is preserved throughout germband elongation. Thus with regard to
Kr expression, the posterior growth zone appears special in some way.
Indeed, other segmentation genes are specifically expressed in the
growth zone in both Oncopeltus and other short and intermediate germ
insects (Dearden and Akam,
2001; Liu and Kaufman,
2004
; Patel et al.,
1992
; Wolff et al.,
1995
). While the characteristics of the insect growth zone are not
well understood, the fact that several segmentation genes are expressed in or
excluded from this region suggests that there is something special about this
portion of the germband. Krüppel's exclusion from the growth
zone can be directly traced to the posterior clearing first seen in the
blastoderm and suggests that this region may be specified before the
actual formation of the germband and may actually begin to acquire its unique
identity during late blastoderm and seems consistent with observations in
other short/intermediate germ insects
(Schroder et al., 2000
).
Spatial discrepancy between the Krüppel expression pattern and its phenotype
Since the Kr gap expression pattern covers only the thoracic
segments and not the anterior abdomen, deletion of part of the abdomen raises
the important issue of discrepancy between the ectodermal gap expression
pattern and phenotype. A possible explanation for this discrepancy may lie in
technical limitations of determining the precise extent of gap gene products.
For example, Drosophila Krüppel protein expression was initially
reported to span 54-39% of egg-length, but using more sensitive techniques,
was later found to be larger and span 60-33% of egg-length
(Gaul and Jäckle, 1987;
Gaul and Jäckle, 1989
).
In fact, based on genetic evidence, the Drosophila Kr protein
gradient may extend even further still
(Pankratz et al., 1989
). Thus
our failure to detect transcript in the anterior abdominal ectoderm may be
because of experimental limitations rather than be a reflection of biological
significance. However, Krüppel expression in another short germ
insect, Tribolium castaneum, has been examined and has also been
found to accumulate in the thorax but not in the abdomen
(Bucher and Klingler, 2004
;
Sommer and Tautz, 1993
). If
Oncopeltus Kr was indeed expressed in the ectoderm of the milkweed
bug abdomen, this would further imply that Kr expression differs
significantly even between two short/intermediate germ insects and changes in
its expression would have to be highly evolutionarily labile. Since the
Oncopeltus blastoderm becomes cellularized very early in development
(around 17 hours after egg lay), it seems unlikely that a
Krüppel protein gradient can be utilized to specify the anterior
abdominal segments. Rather it may be that the gap genes pattern the
germband-derived segments via cell-cell signaling or via some other long-range
effect (Bucher and Klingler,
2004
; Davis and Patel,
1999
; Eckert et al.,
2004
).
It is a formal possibility that the mesodermal expression, which extends into the abdomen, has a direct function in segmentation. However, Krüppel RNAi resulted in deletion of only the thoracic and anterior abdomen. If the mesodermal expression is important for formation of the anterior abdominal segments, it would require segmentation function to be limited to only these segments and not the rest of the body. This region-specific function seems unlikely and since we detect no qualitative differences between the mesodermal expression of the anterior abdomen as compared with the rest of the body, it seems probable that the ubiquitous mesodermal expression is not involved in segmentation. For these reasons, we attribute the segmentation role to the ectodermal domain in the thorax with the implication that the anterior abdominal segments are deleted as a result of a long-range requirement for Kr in these segments.
Oncopeltus Kr is a bona fide gap gene
We have shown that strong Kr RNAi depletion results in deletion of
the mesothoracic through fourth abdominal segment. This gap phenotype is in
contrast to the RNAi phenotype of the Oncopeltus homolog of another
gap gene hunchback (Liu and
Kaufman, 2004). hb RNAi results in a terminal phenotype
in which the segments of the head are followed by several segments with
abdominal identity. By analysis of the RNAi hypomorphic series, it was
apparent that instead of a true gap phenotype, the hb phenotype is
really due to a combination of anterior homeosis towards abdominal identity
coupled with defective segmentation of the posterior germband resulting in
posterior compaction. Our analysis of a Kr RNAi hypomorphic series
shows that in mildly affected animals, the anterior abdominal segments are
deleted, and the remaining segments are intact with no overt evidence of
homeosis or posterior compaction. As the RNAi depletion becomes more severe,
the deleted region expands both anteriorly into the thorax and posteriorly to
cover more of the abdomen until the terminal phenotype is reached. In strongly
affected animals this results in a large gap spanning the second thoracic
through fourth abdominal segments, but leaving behind normal anterior and
posterior segments. This hypomorphic series in Oncopeltus is similar
to the phenotypes obtained in a Krüppel allelic series in
Drosophila where weak and moderate alleles delete a smaller region of
the body than amorphic alleles (Wieschaus
et al., 1984
). The lack of homeosis or posterior compaction, along
with the gap-like ectodermal expression pattern suggests that in
Oncopeltus, Kr is a bona fide gap gene. Interestingly, a probable
mutation in the Tribolium Kr homolog, jaws, has already been
isolated and mutant embryos show a homeotic transformation of the thorax and
first abdominal segment towards gnathal identity as well as a large deletion
of almost the entire remaining abdomen (Bucher, 2002;
Sulston and Anderson, 1996
).
Therefore, although the expression pattern of Oncopeltus Kr is more
similar to the Kr expression in Tribolium than in
Drosophila, the Oncopeltus Kr phenotype is more similar to
the Kr phenotype in Drosophila than in Tribolium.
Figure 8 shows a comparison of
the expression domains and loss-of-function phenotypes of the Kr
homologs in Oncopeltus, Tribolium and Drosophila.
|
The Krüppel phenotype and the evolution of insect segmentation
We would like to make explicit three observations that may have
implications for the evolution of insect segmentation. First, as we discussed
above, some of the molecular underpinnings may be shared between
blastoderm-derived and germband-derived segments. However, in
Tribolium, another short germ insect, the abdominal gap gene
giant does not act as a canonical gap gene. Instead, Tribolium
giant may have a more general role in segmentation, suggesting that some
aspects of abdominal segmentation have diverged
(Bucher and Klingler, 2004).
Nevertheless, Oncopeltus Kr does act as a true gap gene, suggesting
that at least some of the mechanisms underlying segment formation may be
shared between the milkweed bug blastoderm and germband as well as with
Drosophila.
Secondly, Kr RNAi embryos ectopically express Dfd in a
posterior domain and abd-A in an anterior domain. Oncopeltus
Kr is not only required for the formation of segments in the middle
portion of the embryo, but also regulates anterior and posterior genes.
Kr may directly regulate these Hox genes or instead, Kr may
regulate other gap genes as is the case in Drosophila and these may
in turn regulate the downstream Hox genes
(Jäckle et al., 1986;
Kraut and Levine, 1991
;
Mohler et al., 1989
). Thus in
Oncopeltus, Kr seems to act as a sort of `spacer' both to specify
central segments and to prevent central expression of anterior and posterior
genes.
Lastly, although loss of Krüppel function results in a
deletion of anterior abdominal segments, posterior abdominal segments appear
normal. Since all abdominal segments are normally produced through elongation
of the posterior germband, presence of normal posterior abdominal segments in
Kr RNAi embryos means that for these segments, the segment
formation function of the growth zone was not disrupted. Although
called a `growth zone', this region has not yet been well studied in insects,
and it has yet to be shown to share characteristics with the growth zones of
other arthropods such as spiders
(Stollewerk et al., 2003).
Nevertheless, our results imply that the segment formation function
by the growth zone can, to some degree, be decoupled from the actual
number of segments that it produces.
The above observations suggest a possible (and admittedly speculative) mechanism for transition between short, intermediate and long germ forms of segmentation. For instance, our results show that alterations in activity of a gap gene can change the number of segments that are normally specified at the blastoderm stage. Evolutionarily, this can perhaps be accomplished by decreasing the width of the Krüppel domain on the blastoderm while maintaining the number of segments that it specifies. This would allow more posterior genes to be expressed on the blastoderm and would serve to pack more gap domains (and therefore body regions) on the blastoderm fate map. We have shown that although the number of segments the growth zone produces can increase or decrease, the segment formation ability of the growth zone seems to be largely independent, and the remainder of the posterior segments would be generated as usual, via germband growth. This would result in shifting the relative number of segments generated at the blastoderm stage versus the germband stage in effect converting a shorter germ insect into a longer germ insect. The above scenario is highly speculative and no doubt overly simplistic but is attractive because it offers a mechanism for evolving the mode of segmentation. At this point, it is clear that further work needs to be done. Functional analysis of the segmentation genes in short germ insects has only begun but should provide a greater understanding of these questions and conundrums in insect segmentation.
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ACKNOWLEDGMENTS |
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