Department for Biology II, Ludwig-Maximilian-University Munich, Luisenstraße 14, 80333 Munich, Germany
Author for correspondence (e-mail:
klingler{at}biologie.uni-erlangen.de)
Accepted 6 January 2004
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SUMMARY |
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Key words: giant, Short germ, Long germ, Segmentation, Tribolium, Gap gene, Abdomen, Krüppel, jaws, Drosophila, Evolution, Morpholino, Parental RNAi
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Introduction |
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The red flour beetle, Tribolium castaneum, is a short germ insect
amenable to functional analysis via genetic, transgenic and RNAi approaches
(Beeman et al., 1989;
Berghammer et al., 1999
;
Brown et al., 1999
;
Bucher et al., 2002
;
Maderspacher et al., 1998
;
Sulston and Anderson, 1998
). A
number of segmentation genes have been isolated from this species. The segment
polarity genes engrailed and wingless
(Brown et al., 1994b
;
Nagy and Carroll, 1994
), as
well as several pair-rule genes (Brown et
al., 1994a
; Patel et al.,
1994
; Sommer and Tautz,
1993
) were shown to be expressed in corresponding patterns in
Tribolium and Drosophila, suggesting that their functions
are largely conserved. Although conservation of pair rule activity within
arthropods is a matter of ongoing debate
(Davis and Patel, 2003
),
mutant phenotypes indicate pair rule action in Tribolium
(Maderspacher et al., 1998
;
Sulston and Anderson, 1996
).
In addition, several homologs of gap genes have been cloned from
Tribolium. Orthodenticle (Tc'otd-1), hunchback,
Krüppel and tailless are expressed in the blastoderm in a
similar anterior to posterior order (Li et
al., 1996
; Schroder et al.,
2000
; Sommer and Tautz,
1993
; Wolff et al.,
1995
). While the gap genes Tc'otd-1 and
Tc'hunchback are active in similar segment primordia as in
Drosophila, they appear to play a more prominent role in anterior
specification than in Drosophila
(Schroder, 2003
). The function
of the posterior Tc'tailless domain, however, is not conserved, as
abdominal segments arise at a time when Tc'Tailless protein has long
disappeared (Schroder et al.,
2000
). The apparent conservation of pair rule functions and the
non-conservation of at least some gap genes prompted us to investigate the
role of the abdominal gap gene giant.
giant is a transcription factor of the basic leucine zipper family
which so far has been investigated only in Drosophila
(Capovilla et al., 1992;
Hewitt et al., 1999
;
Strunk et al., 2001
).
Dm'giant expression appears during the early blastoderm in two broad
domains. The anterior domain subsequently resolves into several stripes, the
most posterior of which is located in the maxillary segment
(Eldon and Pirrotta, 1991
;
Kraut and Levine, 1991b
;
Mohler et al., 1989
). Also at
later stages of development, Dm'giant remains expressed in a complex
pattern in the embryonic brain. The posterior domain initially covers the
posterior pole of the blastoderm embryo, but later retracts and covers the
primordia of abdominal segments 5-7. Shortly after cellularization, this
domain disappears. In mutant embryos, the labial engrailed stripe is
deleted leading to a fusion of the labial with the first thoracic segment in
cuticles (Petschek and Mahowald,
1990
; Petschek et al.,
1987
). In addition, the engrailed domains of abdominal
segments 5-7 fuse. In cuticles, the anterior compartments of these segments
are deleted while the remnants fuse
(Petschek and Mahowald, 1990
).
Dm'giant exerts repressive functions on gap, pair rule and Hox genes.
Mutual repression of giant and Krüppel has been shown
to be crucial for the refinement of their expression domains
(Capovilla et al., 1992
;
Kraut and Levine, 1991b
). The
patterns of pair rule genes are disturbed in head as well as in abdominal
regions in Dm'giant mutant embryos
(Langeland et al., 1994
;
Petschek and Mahowald, 1990
;
Small et al., 1991
). Direct
interaction of Dm'giant with one of its pair-rule target genes,
even-skipped (eve), has been studied in great detail
(Small et al., 1992
;
Wu et al., 1998
). Bound to
regulatory DNA, Giant functions as a short range repressor that acts over
distances of 100-150 bp (Gray et al.,
1995
). The protein contains an interaction domain for the
co-repressor CtBP but exerts its repressive function in part through
CtBP independent mechanisms
(Strunk et al., 2001
).
In this work, we describe the isolation and analysis of Tribolium giant, the first ortholog of giant in a species outside of Diptera. Similar to Drosophila gap genes, Tc'giant functions in both segmentation and Hox gene regulation. However, expression and functional analysis of Tc'giant clearly show that it plays a role that fundamentally differs from the well understood function of its Drosophila ortholog.
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Materials and methods |
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Sequence analysis
Alignment of Dm'Giant and Tc'Giant was done using Clustal W 1.5
(Higgins et al., 1992) using
default settings except for a gap open penalty of 30 and a gap extension
penalty of 0.1. For the phylogenetic analysis we conducted a BLAST
(Altschul et al., 1997
) search
with the leucine zipper domain of Tc'Giant to identify all closely related
sequences in the database. Of these, a representative range of species was
selected and these sequences were aligned by the Clustal W program (BLOSUM
matrix, default values). The PUZZLE algorithm
(Strimmer and von Haeseler,
1996
) as implemented in PAUP 4.0
(Swofford, 1998
) was then used
for a phylogenetic analysis using default settings. Bootstrap analysis was
performed with PAUP 4.0, using standard settings and 500 replicates. A search
in the Conserved Domain Database (CDD) at NCBI did not identify any conserved
protein motives apart from the leucine zipper.
Histology
Whole-mount in situ hybridization was performed according to established
protocols (Tautz and Pfeifle, 1989). For double staining, fluorescein- and
digoxigenin-labeled probes were detected using alkaline phosphatase and
ß-galactosidase, the latter after signal enhancement via biotin
deposition (Prpic et al.,
2001). A detailed protocol is available from the authors. For in
situ hybridization of injected RNAi embryos, embryos were removed from the
microscope slide using a fine brush soaked with PEMS buffer. Fixation was done
as usual, but embryos were devitellinized manually. As extended exposure of
embryos to room temperature resulted in mRNA degradation, the bulk of the
embryos were refrigerated, while small batches were devitellinized using fine
insect needles (Original EmilCarlt Insect Pins 0.1mm).
RNAi
For embryo injections, sense and antisense RNAs were synthesized from a
full-length Tc'giant cDNA plasmid using the T7 Megascript Kit
(Ambion), using T7 RNA polymerase (Ambion) and T3 RNA polymerase (LaRoche).
Annealing was performed in injection buffer (potassium phosphate 20 mM, sodium
citrate 3 mM pH 7.5) (Fire et al.,
1998). Different concentrations of resulting dsRNA
(Tc'giant: 2000 ng/µl, 750 ng/µl, 75 ng/µl and 7.5 ng/µl,
Tc'dll: 2 µg/µl) were supplemented with Phenol Red to 0.05%
(Sigma) and filtered (Ultrafree 0.45 um, Millipore) prior to injection.
Tribolium eggs were collected for 1 hour at 25°C and kept for
another hour at 33°C to improve injection survival. The embryos were then
dechorionated using `Klorix' bleach, washed in water and mounted on microscope
slides without applying glue. They were injected in air at an intermediate
anteroposterior position to minimize damage to egg poles where maternal
morphogens may be localized and where the growth zone will develop. After
injection, embryos were allowed to develop for four days at 33°C in a
humid chamber. Fully differentiated embryos/larvae were embedded in Hoyer's
medium and cleared at 65°C. Thirty-two percent of the injected eggs
differentiated cuticles, and of these, 56% displayed Tc'giant
phenotypes. Of the three concentrations tested, the two higher ones resulted
in similar frequencies of RNAi phenotypes, while the lowest concentration
produced mostly wild-type cuticles. As a control, we injected dsRNA from a
gene of known function, Tc'distalless
(Beermann et al., 2001
). The
resulting embryos displayed distalless-specific leg defects with high
frequency, while segmentation defects were not observed.
For parental RNAi, mature female pupae were fixed to a microscope slide, ventral side up using rubber cement (`Fixogum', Marabu). To avoid interference with eclosion, only the posteriormost portion of the abdomen was allowed to contact the rubber cement. We generated dsRNA from a PCR template whose primers had T7 promoter sequences at both ends. After precipitation with NaAc/ethanol the dsRNA was dissolved in injection buffer. Approximately 0.15 µl of dsRNA (2000 ng/µl and 750 ng/µl) was injected between abdominal segments three and four, at a ventrolaterally position (in order not to damage the CNS). About 30 eclosed females were mated to untreated males, and eggs were collected beginning 1 week after injection. All embryos in the first egg-lay displayed Tc'giant phenotypes in cuticle preparations. During the following 2 weeks, eggs were fixed for histochemistry using standard procedures. Three weeks after injection, the portion of embryos displaying Tc'giant phenotypes dropped to 40% and egg collection was discontinued. Therefore, at least 40% of embryos used for histochemistry were expected to display Tc'giant phenotypes.
Morpholino oligonucleotides
A morpholino oligo (Gene-Tools) was designed to cover both possible
starting ATGs (5'CCATCGCAAATTCTGCTTTTTCCAT-3').
Injection of 1 mM and 0.66 mM concentrations (in injection buffer) resulted in
premature termination of development of all embryos. With lower concentrations
(0.4 and 0.2 mM) the proportion of fully differentiated embryos (32%) and
cuticles displaying phenotypes (42% of differentiated embryos) was similar to
our embryonic RNAi experiments. Morpholino injections essentially gave the
same results as with RNAi experiments. However, in only 9% (n=32) was
the transformation of maxilla to T1 complete. Morpholinos stoechiometrically
compete with translation, whereas dsRNA is thought to involve an enzymatic
reaction. Probably, the enzymatic RNAi mechanism knocks down gene function
more effectively, resulting in residual giant function in
morpholino-injected animals.
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Results |
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Tc'giant expression during embryogenesis
We used in situ hybridization to see if the expression of giant is
conserved in Tribolium (Fig.
2). In freshly laid eggs, putative maternal transcripts are
distributed homogeneously throughout the syncytial blastoderm. Later,
expression retracts from both poles and intensifies along the posterior edge
of this domain (Fig. 2A-C).
Eventually a circumferencial stripe is formed, which persists into the germ
rudiment stage (Fig. 2D,F),
while the intensity of the remaining domain decreases. A second
Tc'giant domain arises de novo at the posterior pole of the embryo at
the posterior pit stage (Fig.
2D). Cells lining the posterior pit express Tc'giant,
while cells in the center of the invaginating pit remain unstained
(Fig. 2E). In the germ
rudiment, Tc'giant staining becomes more intense at the anterior
boundary of this posterior domain (Fig.
2F). During early germ band elongation, this domain splits into
two stripes (Fig. 2G) while
expression in the head stripe ceases. As the germ band continues to grow, the
first of the posterior stripes also fades, followed somewhat later by the
second. Meanwhile, the low-level expression in the head condenses into a
complex and dynamic pattern of brain cell clusters
(Fig. 2I-K). Tc'giant
expression ceases altogether before the germ band has fully elongated, and no
staining was detected in subsequent embryonic stages.
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In Drosophila, the gap gene Krüppel (Dm'Kr) is
positioned exactly between the two giant domains and negatively
interacts with both of them (Capovilla et
al., 1992; Kraut and Levine,
1991a
). Tribolium Krüppel appears at the posterior
pole at a time when the anterior Tc'giant domain has just retracted
from this region and well before the posterior Tc'giant domain
emerges. Therefore, Tc'giant and Tc'Krüppel are
expressed mutually exclusively prior to the posterior pit stage
(Fig. 3F) as in
Drosophila. In the germ rudiment, Tc'Krüppel becomes
restricted to a sharply demarcated band initially covering segment primordia
T2 and T3 and eventually extending into T1 (data not shown). The posterior
Tc'giant domain arises within this Krüppel domain,
overlapping in the T3 segment during the whole course of expression
(Fig. 3G,H). This data
indicates that the posterior Tc'giant domain is not negatively
regulated by Krüppel as in Drosophila.
Tc'giant determines the identity of gnathal segments
To investigate the function of Tc'giant during segmentation, we
applied both embryonic and parental RNAi and morpholino oligos to reduce
Tc'giant activity (Bucher et al.,
2002; Brown et al.,
1999
; Fire et al.,
1998
). Although the strength of the phenocopies depends on the
amount of injected dsRNA and morpholino oligos (Figs
4,
6), the majority of embryos
share three characteristics: (1) the total number of body segments is reduced;
(2) the number of segments with thoracic morphology (i.e. leg-bearing
segments) is increased to four or five; and (3) the gnathal appendages maxilla
and labium are missing. Other head structures, i.e. antenna, labrum and
mandible, are not affected.
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The role of Tc'giant in homeotic segment specification could be
either direct, similar to the function of the non-Hox homeotic gene
spalt or it could indicate a role in regulating homeotic genes. One
potential target Hox gene is maxillopedia (mxp), the
Tribolium ortholog of Dm'proboscipedia
(Shippy et al., 2000). Loss of
this gene leads to the transformation of maxillary and labial palps into legs.
To see if mxp expression is indeed regulated by Tc'giant, we
used parental RNAi (Bucher et al.,
2002
) to generate embryos with reduced Tc'giant activity
and stained them for mxp by in situ hybridization. Indeed we find
that expression of mxp in the appendages of the maxillary and labial
segments is reduced or absent (see Fig.
5I,J). This confirms that giant is involved in Hox gene
regulation in the gnathocephalon. However, the homeotic phenotype of
Tc'giant probably involves mis-regulation of additional homeotic
genes, because only the palps are transformed in mxp-null mutants,
not the complete maxillary and labial segments as in Tc'giant RNAi
embryos. A detailed analysis of the Hox genes involved in the transformation
will be published elsewhere.
Tc'giant is required for segmentation of thorax and abdomen
Depending on dsRNA concentration, up to nine body segments are deleted in
Tc'giant RNAi embryos (Fig.
6A-D). Using morpholino oligonucleotides to knock down
Tc'giant gene activity, we achieved similar phenotypes with deletions
of up to seven segments (Fig.
6E). As morpholinos are structurally different from dsRNA, and are
thought to knock down gene function by a different molecular mechanism, the
RNAi results outlined below are indeed specific for reduced Tc'giant
activity. Although the abdomen is affected in most RNAi larvae injected with
high concentrations of dsRNA, leg bearing segments are deleted only in 24% of
these larvae: 17% and 7% have four and three leg bearing segments,
respectively, instead of five. In the following, we will refer to particular
segments according to their wild-type identity and not the identity resulting
from homeotic transformation.
Owing to the uniform morphology of abdominal segments, it is difficult to ascertain which segments exactly are deleted in a given larva. Only in larvae displaying very weak phenotypes, where remnants of all segments are still present, is it possible to unambiguously identify the affected segments. In four such embryos, segments T2, A2, A6 or A7 were found to be partially deleted, respectively. This suggests that sensitivity to Tc'giant depletion is distributed rather evenly throughout the thorax and abdomen. The last two abdominal segments bear pairs of specialized appendages (which later during development fuse with the telson): the dorsal urogomphi (segment A9) and the ventral pygopods (A10, see Fig. 4A). Of these, the urogomphi are missing in 70% of all RNAi embryos. This identifies A9 as the posteriormost affected segment that additionally appears to be very sensitive to lack of Tc'giant activity. The pygopods, however, are usually not affected.
The anterior limit of Tc'giant requirement for segmentation is T1. This is not obvious from the inspection of larval cuticles, but can be clearly seen in the pattern of the segment-polarity gene engrailed in RNAi embryos (Fig. 5). As mentioned earlier, none of 52 such embryos showed any patterning disturbance in gnathal segments. However, partial or complete deletions of engrailed stripes were frequently observed throughout thorax and abdomen. Notably, the T1 segment is reduced or deleted in 39% of these embryos. In many engrailed-stained embryos, the T1 stripe is more severely affected than the T2 stripe, and quite frequently the T1 stripe is deleted completely, leaving an increased distance between the last gnathal and the first thoracic engrailed stripe. Later, however, cells are rearranged within the fused segment such that the labial and T2 engrailed stripes reach wild-type distance while the segment broadens laterally. Some of the excess cells contribute also to enlarged appendage primordia (Fig. 5E, white arrowhead; compare with more anterior appendages that are at the same stage of development or slightly older, black arrowhead). During subsequent development, this defect is further corrected for such that no disturbance in the first thoracic segment is apparent on the cuticular level. However, this segment actually represents a fusion of T1 and T2.
This regulative propensity of the embryo indicated that analysis based on cuticles might underestimate the defects elicited by RNAi. To determine the number of affected engrailed stripes prior to repair or elimination of partially deleted segments, we analyzed 28 germ bands just after they had completed segmentation, as indicated by the presence of an invaginated proctodeum. Most of these (61%) lacked four to six segments, and in a sizable fraction (18%) seven to eight segments were missing. This share of severe defects is indeed somewhat higher than in cuticle preparations. Taken together, the analysis of germband and cuticle phenotypes indicates that Tc'giant is pertinent for formation of 12 body segments, i.e. T1 through A9. Although we never obtained a larva lacking all these segments, these 12 segments all have a certain probability to be missing in Tc'giant RNAi larvae. In Drosophila, no more than four contiguous segments are affected in giant mutations. Thus, Tc'giant appears to have a different, and potentially much more central role in segment patterning than Dm'giant.
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Discussion |
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giant expression is conserved in the head but not in the abdomen
Expression of Tc'giant reveals both conserved and diverged
aspects. In Tribolium and in Drosophila, giant is active in
the maxillary segment, and later in a highly dynamic pattern in the brain.
Therefore, this expression was probably present in the last common ancestor of
all holometabolous insects. Another similarity is the appearance of a second
expression domain in the posterior blastoderm. However, although in
Drosophila both domains appear simultaneously, the posterior domain
appears later than the anterior one in Tribolium. This could simply
reflect the anterior to posterior sequence of segment formation in the beetle.
However, relative to emerging segment primordia, this domain is located five
segments more anterior. In Drosophila, the abdominal segments A5 to
A7 arise right under the posterior giant domain
(Kraut and Levine, 1991b;
Petschek and Mahowald, 1990
)
while in Tribolium the anlagen of segments T3 through A2 are covered
by the posterior Tc'giant domain. This shift in expression must
reflect a fundamental change in gene function: either the Tribolium
and Drosophila giant orthologs function by different mechanisms to
pattern the same segments, or alternatively, if they act through a similar
short range gradient mechanism, they must specify different segments.
The Tc'giant expression pattern also indicates divergent
interactions with other segmentation genes. As the posterior domain arises in
the late blastoderm, it is probably under zygotic control. This is in contrast
to the situation in Drosophila, where the maternal genes
caudal and bicoid cooperate to activate posterior
giant expression (Rivera-Pomar et
al., 1995). Second, the posterior Tc'giant domain appears
right within the Krüppel domain, and co-expression of both genes
is observed in segment T3 for an extended time period. This again is in
contrast to Drosophila, where strong mutual repression with
Krüppel is crucial for regulation and proper function of
giant (Kraut and Levine,
1991a
). However, inhibitory interactions between
Tc'Krüppel and the anterior giant domain could still be
conserved, since these domains are mutually exclusive also in
Tribolium. Finally, we note another intriguing feature: maturation of
all three Tc'giant stripes (maxialla, T3 and A2) occurs in identical
relation to the pair-rule gene register. In fact, the split of the posterior
domain into distinct stripes concurs with pair rule patterning rather than
preceding it (Fig. 3B,C). This
raises the possibility that in later stages Tc'giant may be regulated
by pair rule genes. In contrast to this, Dm'giant expression precedes
pair rule activation and the gene unambiguously acts on a higher hierarchical
level. Evidently, it is not only the position, but also many aspects of the
regulatory network involving giant that differ between
Tribolium and Drosophila. Our functional data confirm this,
and in addition reveal that the function of the anterior domain has diverged
in both insects.
Required for identity but not formation of head segments?
Like other Drosophila gap genes, Dm'giant functions in
positioning pair-rule stripes, and its role in defining the anterior border of
eve stripe 2 has been studied in much detail. Lack of giant
function leads to expansion of this stripe
(Arnosti et al., 1996;
Small et al., 1992
;
Small et al., 1991
) and to
concomitant loss of the labial engrailed stripe
(Eldon and Pirrotta, 1991
;
Petschek and Mahowald, 1990
).
By contrast, we did not detect any defects in head segmentation, and the
labial engrailed stripe was unaffected in RNAi embryos. The most
anterior segmentation defect that we observed was the deletion of the T1
engrailed stripe. The primordium of this stripe arises at a distance
of one segment width to the posterior but two segment widths to the anterior
Tc'giant domain. It seems more likely, therefore, that formation of
this segment depends on the posterior rather than the anterior domain, which
may not be involved in segmentation at all.
However, a crucial role of the anterior Tc'giant domain in
homeotic specification is established by our experiments. Tc'giant
RNAi larvae display a coordinated two-segment shift of all thoracic identities
towards anterior. Generally, maxillary and labial segments are fully
transformed towards T1 and T2 respectively, while the T1 segment adopts T3
identity, followed by two segments with identities ranging between T3 and
abdomen. The shift of several segment identities implies that
Tc'giant directly or indirectly regulates several Hox genes (i.e.
those required for the identities of at least maxilla, labium, T1 and T2).
Homeotic function of Tc'giant is not surprising, because
Drosophila gap genes are known to regulate homeotic genes, and
Dm'giant, specifically, defines the anterior border of
Antennapedia (Reinitz and Levine,
1990). However, these functions in the regulation of homeotic
genes are usually not evident from Drosophila gap gene phenotypes, as
the homeotically affected regions are missing in the developed embryo because
of the segmentation defects.
The homeotic two-segment shift in Tc'giant RNAi embryos follows
`all or nothing' kinetics: We never observed a homeotic shift across one
segment width. This argues against a simple mechanism where a gradient of
Tc'Giant protein emanating from the anterior domain would directly position
gnathal and thoracic Hox genes. In addition, partial transformation of maxilla
or labium is extremely rare even though RNAi or morpholino knockdown
experiments should produce many intermediate levels of residual gene function.
Therefore, the coordinated regulation of several Hox genes by
Tc'giant appears to rely on a mechanism involving tight thresholds.
Interestingly, the phenotype of jaws
(Sulston and Anderson, 1996),
a mutant in the Tc'Krüppel gene (A. Cerny, G.B. and M.K.,
unpublished) displays a homeotic transformation that is opposite to
Tc'giant phenocopies. In jaws larvae, thoracic and anterior
abdominal segments are transformed to alternating pairs of maxillary and
labial segments, while in Tc'giant RNAi embryos, maxilla and labium
are transformed to T1 and T2, respectively. This suggests that
Tc'giant and Tc'Krüppel have opposing functions in
regulating the same set of thoracic and gnathal Hox genes. This may indicate
mutual inhibition of Krüppel and the anterior giant
domain as in Drosophila (but in contrast to the posterior
Tc'giant domain). In addition, the homeotic phenotypes of both
Tc'Krüppel (jaws) and Tc'giant display double
segmental effects, suggesting the involvement of pair-rule genes in homeotic
segment specification.
In Tribolium, giant has a long-range effect on abdominal patterning
Even though our RNAi and morpholino knock down experiments may not have
achieved complete inactivation of the Tc'giant gene product, we
frequently obtained segmentation phenotypes much more severe than those of
Dm'giant null-mutations. In Dm'giant mutant embryos, the
loss of the posterior domain results in a fusion of the engrailed
stripes corresponding to segments A5 to A7, which are the segment primordia
covered by this domain (Petschek and
Mahowald, 1990; Langeland et
al., 1994
). Drosophila gap genes are expressed in domains
whose diffuse boundaries function as short-range morphogenetic gradients that
position pair-rule stripes (Hulskamp and
Tautz, 1991
; Rivera-Pomar and
Jackle, 1996
). Accordingly, both Dm'giant domains
regulate pair-rule stripes in this manner
(Langeland et al., 1994
;
Reinitz and Sharp, 1995
;
Small et al., 1991
;
Wu et al., 1998
). However, the
rather severe patterning defects observed at the pair-rule level are to some
extent repaired during later stages of development
(Klingler and Gergen, 1993
),
resulting in a less serious larval phenotype. By contrast, in
Tribolium embryos displaying strong Tc'giant phenocopies,
segmentation is disturbed in a region comprising twelve segments, ranging from
T1-A9.
Intriguingly, the phenotype of Tc'giant knock-down larvae is not
only stronger than that of Dm'giant mutants, but it also differs in
the spatial and temporal relationships between expression domain and affected
segments. The posterior domain of Tc'giant appears at the posterior
pole of the blastoderm embryo at a time when the primordia of the first
thoracic segments are patterned in this region. By this time, cellularization
has most likely occurred (Handel et al.,
2000). Thus, if thoracic and anterior abdominal defects of
Tc'giant RNAi larvae reflect a short-range regulation comparable to
that of Dm'giant, diffusion of the Tc'Giant protein across
cell membranes would be required. However, the secondary Tc'giant
stripes actually resemble pair-rule stripes in width and spacing, in addition
to the way they arise near the growth zone (see.
Fig. 2F-H,
Fig. 3B-D). It is therefore
possible that these two stripes regulate pair-rule stripes in a manner more
typical of pair-rule interactions in Drosophila, i.e. by direct
activation and repression within precise boundaries.
In any case, the Drosophila paradigm cannot explain why very
posterior abdominal segments require giant function in
Tribolium, as these segments are formed at a large distance
(spatially and temporally) from the posterior Tc'giant domain(s). The
segment A9, for example, is frequently deleted in giant RNAi larvae,
but arises six segments posterior to Tc'giant expression and long
after expression has ceased (Fig.
2K). At this point, we can only speculate how Tc'giant
exerts this long-range effect. For example, Tc'giant could be
involved in setting up and/or starting a segmentation process in which a
`chain of induction' mechanism (involving gap or pair-rule genes) would
pattern the growing abdomen (Meinhardt,
1982). Alternatively, Tc'giant may jump-start an
oscillator machinery analogous to that underlying somitogenesis in vertebrates
(Pourquie, 2001
). In both
cases, loss of Tc'giant would lead to improper setup and subsequent
breakdown of the machinery, which could then result in defects in distant
segments. However, one could also envisage the role of Tc'giant to be
a rather general one. Tc'giant expression in the early growth zone
may simply be required for making a proper growth zone, and reduction of
Tc'giant activity may result in aberrant behavior of the affected
cells during later growth, leading to segmentation defects in an indirect way.
Evidently, more data are needed to distinguish between these disparate
possibilities, including data about other posterior gap genes.
Gap genes in long and short germ embryos
The Drosophila blastoderm is evenly covered by seven overlapping
gap gene domains, which provide ample positional information for the
regulation of pair-rule stripes (see Fig.
7A). Our findings on giant, together with data for
several other Tribolium genes, suggest that the positions of gap gene
domains are conserved anteriorly, but have changed fundamentally in posterior
body regions (compare Fig. 7A with
7B,C). For example, Tc'otd-1 and Tc'hunchback
are expressed in and are required for the formation of head and thoracic
segments in both Drosophila and Tribolium
(Finkelstein et al., 1990;
Hulskamp and Tautz, 1991
;
Li et al., 1996
;
Royet and Finkelstein, 1995
;
Schroder, 2003
;
Wolff et al., 1995
). In
addition, Tc'tailless is expressed in similar head regions as in
Drosophila (Mahoney and Lengyel,
1987
; Schroder et al.,
2000
; Weigel et al.,
1990
), and we have shown in this paper that the anterior stripe of
Tc'giant covers the maxillary segment in both beetle and fly.
Anterior conservation is also observed in more basal insect taxa: In the
grasshopper Schistocerca, hunchback is also expressed in gnathal and
thoracic segments (Patel et al.,
2001
). At the level of the head gap genes, otx/otd
similarities are observed even between Drosophila and vertebrates,
suggesting conserved principles of head patterning among distantly related
bilaterian animals (Reichert and Simeone,
1999
).
|
Although the expression and function of pair-rule genes in the
Tribolium abdomen appears to be largely conserved
(Brown and Denell, 1996;
Maderspacher et al., 1998
;
Sommer and Tautz, 1993
), we
show that for gap gene orthologs this is not the case. If the beetle mode of
short germ embryogenesis indeed represents the ancestral mode
(Tautz et al., 1994
), our data
suggest that it is changes in the abdominal gap gene system that lie at the
heart of the evolution from short to long germ embryogenesis. Although the
exact role of the Tribolium gap gene orthologs remains to be
elucidated, they have clearly experienced more evolutionary change than the
pair-rule and segment polarity networks.
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ACKNOWLEDGMENTS |
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Footnotes |
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