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First published online 23 January 2008
doi: 10.1242/dev.018317
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1 Department of Genetics, University of Cologne, Zülpicherstrasse 47, 50674
Köln, Germany.
2 Max-Planck Institute for Evolutionary Biology, August-Thienemannstrasse 2,
24306 Plön, Germany.
Author for correspondence (e-mail:
tautz{at}mpil-ploen.mpg.de)
Accepted 28 December 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Segmentation, Gap genes, Hox genes, Tribolium, Short germ embryogenesis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Maternally expressed Dm'hb polarizes the
Drosophila embryo by forming an anterior to posterior morphogenetic
gradient in the posterior half of the syncytial blastoderm, which is required
for positioning the central and posterior gap gene domains
(Tautz, 1988;
Hülskamp et al., 1990;
Struhl et al., 1992). The
anterior zygotic expression of Dm'hb overlaps spatially and
functionally with the maternal expression and appears to act as a canonical
gap gene being required for the formation of an adjacent set of segments
(Lehmann and Nüsslein-Volhard,
1987).
In addition to the role during segmentation, Dm'hb also controls
the expression of Hox genes. The anterior domain of Dm'hb limits the
anterior borders of the Dm'Ubx and Dm'Antp expression
domains (Irish et al., 1989;
Lehmann and Nüsslein-Volhard,
1987; Qian et al.,
1991; White and Lehmann,
1986; Zhang and Bienz,
1992). However, the effects caused by this ectopic expression of
Hox genes in Dm'hb mutants are often concealed by the segmentation
defects, as the Hox genes are ectopically expressed in the segments that are
deleted in the larvae (Lehmann and
Nüsslein-Volhard, 1987).
The role of hunchback in segmentation has also been functionally
studied in other insects with the aim to elucidate the transition from short
germ to long germ embryogenesis (He et
al., 2006; Liu and Kaufman,
2004; Mito et al.,
2005; Schröder,
2003). Although the expression patterns of hunchback are
well comparable, different functional roles have been ascribed to
hunchback in the different insects. More or less canonical gap
phenotypes were reported for Tribolium
(Schröder, 2003) and
Nasonia (Pultz et al.,
2005). Different phenotypes, including transformations and loss of
trunk segmentation were found in Oncopeltus
(Liu and Kaufman, 2004),
Gryllus (Mito et al.,
2005) and Locusta (He
et al., 2006). In addition, it is clear that the primary regulator
of zygotic hunchback expression in Drosophila, bicoid, is a
late evolutionary acquisition that emerged only in the higher Diptera
(Stauber et al., 2002;
Schröder, 2003). Thus, it
appears that hunchback regulation and function has been subject to
major evolutionary changes even within insects.
Even in Drosophila, the role of hunchback is more complex
than it is often portrayed. Some alleles of Dm'hb produce directly a
combination of homeotic transformations and trunk segmentation defects
(Lehmann and Nüsslein-Volhard,
1987). One allele has originally been identified as Regulator
of postbithorax and hence as a homeotic gene
(Bender et al., 1988). In
addition, given that phenotypic effects are always a combination of loss of
the gene itself and changes in downstream genes, it is often not easy to
recognize possible conserved features.
Here, we re-investigate the hunchback phenotype in
Tribolium and assess its role in regulating the trunk gap genes and
Hox genes. We focus the study on the anterior expression region, as this
reflects the key function for hunchback in organizing the
Drosophila segmentation gene cascade
(Hülskamp and Tautz,
1991). We find that one can identify some core components of
hunchback function that appear to be conserved in all insects. This
includes the role in specifying anterior borders of Hox gene expression and
interactions with other gap genes. However, the major function in segmentation
that leads to the typical gap phenotype in Drosophila appears to be
limited to long germ embryos. Hence, most similarities in hunchback
function appear to exist among insects that represent the ancestral type of
embryogenesis, while more-derived types have developed additional
features.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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RNA interference
Parental RNA interference essays were performed as described by Bucher et
al. (Bucher et al., 2002).
Double-stranded RNA was injected into pupae at a concentration of 2
µg/µl. We found this concentration ideal to obtain maximum penetrance
for most genes. Eclosed females were mated with wild type males and reared
under standard conditions (see above). Knockdown embryos were collected every
second day and one collection per week was kept at 30°C to monitor RNAi
penetrance at the cuticular level. The collections were performed until the
phenotypic effect had decreased significantly. Embryos for in situ
hybridization were taken from females showing the highest penetrance, as
judged by the parallel analysis of cuticle phenotypes.
Embryo fixation
The eggs were washed for 1 minute in 50% bleach solution and for 2 minutes
in running water to remove the chorion. The fixation was performed in a
scintillation vial with 3 ml PBS, 6 ml heptane and 4% formaldehyde for 30
minutes. The eggs were then devitellinized by replacing the PBS with 8 ml of
methanol and by shaking thoroughly for 30 seconds. The eggs that lose the
vitelline membrane become hydrophilic and move from the interphase to the
hydrophilic phase. After several washes with methanol they were transferred to
Eppendorf vials. The remaining eggs were passed through a 0.9 mm needle until
the vitelline membrane was removed.
Expression analysis
The gene expression profile was obtained by whole-mount in situ
hybridization as previously described
(Lehmann and Tautz, 1994;
Tautz and Pfeifle, 1989).
Digoxigenin- or fluorescein-labelled probes were detected using alkaline
phosphatase-coupled antibodies and INT/BCIP (red) or NBT/BCIP (blue)
substrates.
Cuticular preparation
First-instar larvae were digested overnight in a 1:1 lactic acid/Hoyer's
medium solution at 70°C and mounted on microscope slides. The cuticular
autofluorescence in a range of 520-660 nm was detected on a Leica Confocal
microscope, and maximum projection images were created from z stacks
composed of 50 layers scanned four times each.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Schröder (Schröder,
2003) has previously shown that loss of Tc'hb function
(Tc'hbpRNAi) does not affect the pre-gnathal segments
labrum, antenna and mandible. In the strong phenotypes, the remaining segments
bear no appendages and appear to have abdominal identity. As a consequence,
this could be interpreted as a gap phenotype in which the maxillary, labial
and thoracic segments are missing
(Schröder, 2003).
However, a reanalysis of the phenotypic series suggests that the phenotype observed is a combination of transformation and loss of segments. In weak phenotypes, all segments appear to be present, but the maxillary and the labial segments are transformed into abdominal identity (Fig. 1B). In addition, the thoracic segments look also more like abdominal segments, although T2 and T3 retain some appendage stumps, which may even look like developed legs in T3 (arrow in Fig. 1B). In more severe phenotypes, all segments beyond the mandibular one look like abdominal segments (Fig. 1C). Yet there are often more than the normal eight abdominal segments, suggesting that this cannot be interpreted as a simple loss of the remaining gnathal and thoracic segments. Instead, one sees a disruption of more posterior segments (arrowhead in Fig. 1C,D) and an increasing loss of posterior structures (Fig. 1D). Therefore, the Tc'hbpRNAi phenotype can be described as a transformation of gnathal and thoracic segments into abdominal identity combined with a loss of abdominal segments in the more severe phenotypes. In the most extreme phenotypes, only the antennae and mandibles are left, followed by four segmental structures of abdominal identity (Fig. 1D).
Regulation of Hox genes
The transformation of anterior segment identity into posterior segment
identity suggests an ectopic expression of posteriorly acting Hox genes in
Tc'hbpRNAi embryos. In order to test this, we have
compared the wild-type expression of the gnathal Hox genes, Tc'Dfd
and Tc'Scr, as well as the trunk Hox genes Tc'Antp, Tc'Ubx
and Tc'AbdA with their expression in Tc'hbpRNAi
embryos (Fig. 2). To assess the
segmental register in these embryos, we have used the segment polarity gene
gooseberry (Tc'gsb) as segmental marker. Tc'gsb was
chosen instead of engrailed because its expression precedes the
expression of other segment polarity genes
(Savard et al., 2006a).
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). Given that the mandible is not affected in
Tc'hbpRNAi embryos, Tc'Dfd expression was
expected to be retained in this segment. Tc'Scr is required for the
labial segment (DeCamillis et al.,
2001), but given that this is transformed into an abdominal
segment, its absence is in line with the phenotype.
The three trunk Hox genes Tc'Antp, Tc'Ubx and Tc'AbdA are
all expanded towards anterior in Tc'hbpRNAi embryos,
starting to be expressed from the mandibular segment onwards
(Fig. 2E-J). Each of these
genes has specific functions in specifying thoracic and anterior abdominal
segments, in agreement with their specific anterior expression borders
(Lewis et al., 2000). However,
in the abdominal segments, they are all co-expressed in wild-type embryos
(Fig. 2E,G,I) and they are
likely to have a joint function in specifying abdominal segment identity.
Accordingly, the fact that all three are co-expressed from the mandibular
segment onwards in Tc'hbpRNAi embryos is in line with the
observation of the transformations into segments of abdominal identity
(Fig. 1).
We can conclude from these observations that Tc'hb is required for the regulation of at least four Hox genes along the anteroposterior axis, although some of these regulatory effects may be indirect (see Discussion).
Regulation of gap genes
To understand the basis of the segment deletions observed in
Tc'hbpRNAi embryos, we have analysed Tc'Kr and
Tc'gt as possible target genes. In Drosophila, the
hunchback gradient is required to regulate other gap genes, in
particular Dm'Kr, Dm'kni and Dm'gt
(Hülskamp et al., 1990;
Struhl et al., 1992). The
homologues of Krüppel and giant have been functionally
studied in Tribolium (Bucher and
Klingler, 2004; Cerny et al.,
2005) and we have therefore focused on these in the following.
Tc'Kr expression starts already at the blastoderm stage with a
broad domain at the posterior end (Fig.
3A) (Sommer and Tautz,
1993), which covers the three thoracic segment primordia in the
early germband (Fig. 3C)
(Cerny et al., 2005). In
Tc'hbpRNAi embryos, this domain is strongly reduced or
even absent (Fig. 3B,D),
indicating that Tc'hb is required for its activation. There is also a
segmental expression of Tc'Kr, which appears during segment
differentiation (Fig. 3E)
(Cerny et al., 2005). This
expression is not affected in Tc'hbpRNAi embryos, although
fewer segmental stripes are generated (Fig.
3F), in line with the loss of segments in such embryos. Thus,
Tc'hb is required for the activation of the early Tc'Kr
domain. This is also an essential function of hunchback in
Drosophila (Hülskamp et al.,
1990; Struhl et al.,
1992; Schulz and Tautz,
1994), i.e. this regulatory interaction appears to be
conserved.
Tc'gt is initially expressed in a broad domain during blastoderm
stage covering the future head and gnathal segments, but excluding the labium
(Bucher and Klingler, 2004).
The trunk expression appears during germband elongation
(Fig. 4A) and converges to two
stripes over the third thoracic and second abdominal segments
(Fig. 4B)
(Bucher and Klingler, 2004). In
Tc'hbpRNAi embryos, the anterior domain is not visibly
affected (Fig. 4C). The
expression of the trunk stripes, however, is lost (compare
Fig. 4B,D). With further
development, it becomes apparent that the segments that should have expressed
Tc'gt are partially fused, as monitored by the Tc'gsb
expression (Fig. 4D). No
further segments are produced beyond this point, at least in strong
phenotypes. These results suggest that Tc'hb acts formally as an
activator of Tc'gt, which would be different from its role in
Drosophila, where it acts as a repressor
(Struhl et al., 1992).
Regulation by gap genes
As cross-regulatory interactions among gap genes are known in
Drosophila, we have also analyzed the effects of Tc'Kr and
Tc'gt on the expression of Tc'hb.
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). This
domain becomes weaker in the early germband
(Fig. 5A). At later stages,
Tc'hb forms a strong expression domain in the growth zone, remaining
there until the end of segmentation (Fig.
5D). Finally, there is weak expression in segmental stripes
(Fig. 5D). In Tc'KrpRNAi embryos, the blastodermal Tc'hb expression domains appear not to be strongly affected, although it is possible that the head domain is extended towards the posterior pole. Given that this domain develops very dynamically, it is not possible to show this unequivocally. A major effect is seen from early germband stages onwards. The posterior domain develops much earlier and is expressed much more strongly. Its anterior boundary is initially within the maxillary segment (Fig. 5B) and overlapping the normal head domain. This boundary recesses at later stages and the domain is broadly confined to the growth zone (Fig. 5E). Thus, Tc'Kr acts formally as a repressor on the posterior domain, a role that is not known from Drosophila.
In Tc'gtpRNAi embryos, Tc'hb expression is not
significantly changed. There are no visible effects on the anterior domains
(Fig. 5C). The posterior
expression domain in the growth zone is present, but appears to be activated
earlier and in a smaller area (Fig.
5F). This effect could be caused by the segment deletions observed
in these embryos (arrowhead in Fig.
5F). In Drosophila, giant has a role in regulating
secondary anterior Dm'hb expression domains, but has no role for the
posterior Dm'hb domain (Wu et
al., 1998).
Effect on pair-rule genes
Gap genes in Drosophila are directly required for regulating the
primary pair-rule stripes. Accordingly, pair-rule stripe formation is
disrupted in the area of the expression of the respective gap gene. In
Tribolium it seems possible that the pair-rule pattern is set up only
via interactions among the pair-rule genes themselves, whereby runt
and even-skipped have essential functions
(Choe et al., 2006). We have
therefore analyzed the expression of these genes in
Tc'hbpRNAi embryos
(Fig. 6). In wild type, the
first Tc'runt stripe appears in the maxillary segment, the second in
the first thoracic segment. The border of hunchback expression is
within the labial segment, i.e. between the two stripes. Thus, if
hunchback did have a direct effect on Tc'runt expression,
one would expect to see the first two stripes to be affected. This does not
appear to be the case. Stripe 1 and the distance to the second stripe are
practically unchanged in Tc'hbpRNAi embryos
(Fig. 6A,B). Only the formation
of the further stripes is disturbed. They form a large domain rather than
separate stripes (Fig. 6C,D).
At later stages, only a broad domain remains visible in the growth zone
(Fig. 6E,F). The situation is
comparable for Tc'eve, with the complication that the pattern is more
dynamic. The first Tc'eve stripe overlaps the mandibular/maxillary
segments and the second the labial/T1 segments. These then split into
segmental stripes (Fig. 6G). In
Tc'hbpRNAi embryos, these first two stripes are almost
normally formed and only subsequent stripes are less well resolved
(Fig. 6G,H). An additional
difference concerns the stability of the stripes. In
Tc'hbpRNAi embryos they disappear much faster than in wild
type (Fig. 6I-M).
Interestingly, however, separate stripes are still seen in the growth zone
(Fig. 6L,M), suggesting that
stripe patterning is less disrupted for Tc'eve than for
Tc'run.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Cerny et al., 2005;
Savard et al., 2006a). Thus,
regulation of Hox genes and segmentation genes are linked features for the
Tribolium gap genes. In the following, we want to argue that this
core role of Tc'hb can be reasonably well defined and that it is in
fact not so much different between the different insects.
Regulation of Hox genes
The setting of Hox gene expression domains appears to be particularly
sensitive to hunchback function and may be the key feature for
understanding its role. Changes in Hox gene expression are also one of the
hallmarks of the allelic series of hunchback phenotypes in
Drosophila. In hypomorphic class III alleles
(Lehmann and Nusslein-Volhard,
1987), Dm'Ubx is ectopically expressed in the thoracic
segments (White and Lehmann,
1986). In the region where four metameres should have formed
(corresponding to two thoracic and two abdominal segments), only two enlarged
metameres spanning this entire region appear. Owing to a resizing process,
which involves cell death, these two enlarged metameres approach wild type
width later in development (White and
Lehmann, 1986). Because of ectopic Dm'Ubx expression,
they are specified as abdominal segments. Hence, the phenotype is
characterized as a loss of T2 and T3 in the larvae, although the remaining
resized metameres are composed of primordial cells of thoracic and abdominal
segments.
Other hunchback alleles in Drosophila are directly
characterized by homeotic transformations of anterior segments into abdominal
identity (Lehmann and
Nüsslein-Volhard, 1987;
Hülskamp et al., 1994) or
act as dominant regulators of Hox genes
(Bender et al., 1988). Some
Dm'Ubx enhancers have been shown to bind HB protein, i.e. the
regulatory interaction appears to be direct
(Qian et al., 1991). Similar
results were also obtained for Dm'AbdA regulation and enhancers
(Casares and Sánchez-Herrero,
1995; Irish et al.,
1989; Shimell et al.,
2000) and there is evidence that Dm'Scr, Dm'Antp and
Dm'AbdB are also regulated by Dm'hb
(Casares and Sánchez-Herrero,
1995; Riley et al.,
1987; Wu et al.,
2001).
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;
Pelaz et al., 1993).
In Drosophila, hunchback does not act as a repressor on
Dm'Antp. Instead, the secondary blastoderm expression of
Dm'hb, the PS4 stripe expression, acts as an activator of
Dm'Antp in this domain (Wu et
al., 2001). An equivalent of the PS4 stripe expression is missing
in Tribolium (Wolff et al.,
1995) and a conserved regulatory interaction cannot be expected
for this aspect. Thus, the repression effect of Tc'hb on
Tc'Antp is not a conserved feature, at least not in
Drosophila.
A possible direct repressor function of Tc'hb on Tc'Ubx
is not obvious, as Tc'hb expression does not visibly reach to the
anterior border of the Tc'Ubx trunk expression domain. However, the
regulatory effect might be mediated via epigenetic regulation. It was proposed
that Dm'hb initiates the formation of a silencing complex and that
another protein, apparently dMi-2 (Kehle
et al., 1998), takes over the role of Dm'HB protein when Dm'HB
levels start to decline. In Tribolium, this mechanism would imply
that only a few cells in the growth zone, which show no HB protein expression
(Wolff et al., 1995), might
retain the capacity to express Tc'Ubx, even though the actual
transcription may start later.
Regulation between gap genes
The second consistent feature of Tc'hb function is the interaction
with other gap genes, most notably Tc'Kr
(Fig. 7A). In Drosophila,
Krüppel is regulated by many other genes
(Gaul et al., 1987), but the
only activators that were identified are Dm'bcd and Dm'hb
(Hoch et al., 1992;
Hülskamp et al., 1990;
Struhl et al., 1992).
Dm'bcd is a late addition in higher Dipterans
(Stauber et al., 2002), so
only Dm'hb is a candidate for a conserved positive regulator.
Moreover, it has been shown that Dm'hb alone is capable of
establishing a functional Dm'Kr expression domain
(Schulz and Tautz, 1994).
Hence, the finding that the Tc'Kr domain is dependent on
Tc'hb is in line with the activation role of hunchback on
Krüppel observed in Drosophila. Given that
Tc'Kr expression starts already at blastoderm stage at the posterior
pole, in the region where Tc'hb forms a short gradient, it would seem
likely that this effect is direct, i.e. this may be another conserved feature
of hunchback function.
The regulatory interaction with giant, however, is clearly not
conserved. In Drosophila, hunchback is a strong repressor of
giant, i.e. the anterior expression border of the posterior domain is
set by a low concentration of the HB protein gradient at blastoderm stage
(Struhl et al., 1992). By
contrast, in Tribolium, we find formally an activating effect of
hunchback on giant. However, at the time where
Tc'gt becomes expressed in the trunk, there is no contact to the
Tc'hb domain, i.e. this effect is likely to be indirect.
Tc'Kr cannot be the mediator of this effect, as loss of
Tc'Kr alone does not lead to a complete loss of the trunk
Tc'gt stripes (Cerny et al.,
2005). Instead, the effect may be caused by a combination of
Tc'Kr and Tc'mlpt. Tc'mlpt expression in the trunk is
strongly reduced in Tc'hbpRNAi embryos and Tc'gt
expression is lost in Tc'mlptpRNAi embryos
(Savard et al., 2006a). This
combined loss of Tc'Kr and Tc'mlpt in
Tc'hbpRNAi embryos may account for the loss of
Tc'gt expression in the trunk. Thus, we can conclude that the
apparent direct interaction between hunchback and giant in
Drosophila is not a conserved feature of hunchback function,
but has probably been acquired in the lineage towards the higher Diptera.
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) have studied hunchback function in the intermediate
germband insect Oncopeltus (Of'hb) using also a parental
RNAi approach. Their phenotypic series is almost identical to the series we
have found for Tribolium. Only the most extreme phenotype is stronger
in Tribolium. They find also misexpression of Hox genes and conclude
that Of'hb has the same dual role that we find for
Tribolium. Hence, hunchback functions in Oncopeltus
and Tribolium are likely to be very similar, possibly even at the
level of the interactions with the other gap genes. For example, the segmental
defects seen in the abdomen of intermediate strength
Of'hbpRNAi phenotypes appear to correspond to the segments
that are also affected in Tribolium and which may be related to the
loss of the two stripes of trunk giant expression.
Mito et al. (Mito et al.,
2005) have studied hunchback function in Gryllus
(Gb'hb). Although they suggest that there are distinctly different
functions for hunchback in this species, it seems that the core
findings are nonetheless very much in line with our results in
Tribolium and the results in Oncopeltus. Again, ectopic
expression of Hox genes is the first effect seen in weak
Gb'hbpRNAi phenotypes, accompanied with signs of
transformation of thoracic segments. Stronger Gb'hbpRNAi
phenotypes show a progressive loss of abdominal segments. The most severe
phenotypes described by these authors are not as strong as those found for
Tribolium or Oncopeltus, but it is naturally difficult to
ensure that the parental RNAi effect is fully penetrant. These authors have
also studied expression of Gb'Scr and Gb'Kr and find,
similar to our results in Tribolium, that expression is severely
reduced or even absent in Gb'hbpRNAi embryos.
He et al. (He et al., 2006)
have shown parental RNAi phenotypes for hunchback in Locusta
(Lm'hbpRNAi) and conclude that some of these appear to be
different from those found for Tribolium, Oncopeltus and
Gryllus. In the weakest Lm'hbpRNAi phenotype,
they find only abdominal effects, but no anterior transformation effects,
suggesting that the Hox gene misregulation effect is not as sensitive as in
the other species. However, we interpret their most frequent
Lm'hbpRNAi phenotype (class II) as embryos where the head
and thoracic segments are transformed into abdominal segments and where
segmentation stops after this. This would be comparable to the strongest
phenotypes in Tribolium, Oncopeltus and Gryllus, although
even fewer segments appear to be produced in Locusta, possibly
because the germ anlage is so extremely short in this species. The even
stronger Lm'hbpRNAi phenotypes observed by these authors
(class III) appear to be related to a separate function of hunchback
in the extra-embryonic membrane (He et
al., 2006).
Pultz et al. (Pultz et al.,
2005) found that the headless mutant in Nasonia is an
apparent null allele of hunchback (Nv'hb). They find also
misregulation of Hox genes in Nv'hb mutant embryos but the phenotype
is not easily comparable with the ones found for Tribolium, Gryllus
or Oncopeltus. Instead, the Nv'hb mutant phenotype mimics
the Drosophila phenotype in showing a large deletion of anterior
segments, as well as loss of posterior abdominal segments
(Pultz et al., 1999). However,
as hypomorphic Nv'hb alleles are not available, it is difficult to
assess whether these would show a homeotic transformation, as we see it in
Drosophila. The more extensive loss of head segments in
Nv'hb mutant embryos may be explained by the fact that
bicoid is partially redundant with hunchback function in
Drosophila (Hülskamp et al.,
1990), i.e. might rescue some of the phenotypic effects. As there
is no bicoid in Nasonia, this effect would not occur.
The role of hunchback in segmentation
Bucher and Klingler (Bucher and
Klingler, 2004), Cerny et al.
(Cerny et al., 2005) and Choe
et al. (Choe et al., 2006) have
suggested that the action of the pair-rule genes may not be as strongly
coupled to the gap genes in Tribolium as it is known in
Drosophila. Choe et al. (Choe et
al., 2006) have even suggested that the segmentation process may
largely be controlled by interactions among the pair-rule genes themselves.
This inference is also supported by the analysis of Tc'hb function.
If Tc'hb were directly involved in setting segmental boundaries, one
would expect that the major phenotypic effect would occur in or around the
domain where it is expressed. However, the first two pair-rule stripes of
Tc'runt appear to form more or less normally in
Tc'hbpRNAi embryos and disruption of the patterning is
seen only for the subsequent stripes. This is in line with the observation
that four segments are still formed in the most extreme
Tc'hbpRNAi phenotypes
(Fig. 1D), although they are
transformed into abdominal identity. A similar pattern is seen for
Tc'eve, although this is more complex owing to the fast splitting of
the primary stripes and the fast disappearance of the anterior stripes in
Tc'hbpRNAi embryos. Thus, there is no indication that
Tc'hb is directly involved in regulating the anterior pair-rule
stripes. However, it is evident that the regulation of the Hox genes and the
setting of segmental boundaries have to be coupled by some mechanism, but it
is still not known how this is achieved.
The conserved core elements of hunchback function
Short (or intermediate) germ embryogenesis is the ancestral form of
embryogenesis in insects (Tautz et al.,
1994). The hunchback function found in these types of
embryos should be taken as a reference when considering conserved and diverged
features. Interestingly, most details of hunchback function are fully
comparable between Tribolium, Oncopeltus and Gryllus,
although these insects belong to different orders that have a longer
evolutionary separation time than, for example, beetles and flies
(Savard et al., 2006b).
The two key features of hunchback function are clearly the regulation of Ultrabithorax, as well as the activation of Krüppel (Fig. 7A). These features are well documented in Drosophila and it seems now clear that they are ancestral. By contrast, the effect on Antp, Scr and giant appear to be partially indirect and partially not conserved, at least not with respect to the exact type of interaction.
Most intriguingly, the name-giving `gap' function does not belong to the
conserved core elements of hunchback function, but appears to have
evolved independently in Drosophila and Nasonia (e.g. long
germ embryos). The term `gap gene' is therefore not appropriate for the
hunchback function in most insects and appears also not appropriate
for Krüppel and giant in Tribolium
(Bucher and Klingler, 2004;
Cerny et al., 2005). Thus, one
could consider to revive another name that has been used to describe the gap
genes, namely `cardinal genes'. Meinhardt
(Meinhardt, 1986) has proposed
this name in the context of his segmentation model for Drosophila. He
proposed the existence of `cardinal regions' that would be set up by maternal
gradients and the genes expressed in these regions would be required for
regulating pair-rule expression. Interestingly, he concluded that this
mechanism would only be required for long germ embryos, because the sequential
segment formation in short germ embryos could be achieved by pair-rule gene
interactions alone. However, Akam (Akam,
1987) has then pointed out that gap genes regulate both
segmentation genes and Hox genes in broader domains and that the term
`cardinal genes' should reflect both of these aspects. Given that the Hox gene
regulation appears to be the more conserved function of gap genes, it would
indeed seem appropriate to adopt the term `cardinal genes' for this gene
class, at least for other insects.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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