Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima City 770-8506, Japan
Author for correspondence (e-mail:
noji{at}bio.tokushima-u.ac.jp)
Accepted 14 February 2005
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SUMMARY |
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Key words: Gryllus bimaculatus, Orthoptera, Intermediate germ insect, Gap gene, hunchback, RNAi, Segmentation
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Introduction |
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To understand the similarities and differences in the molecular mechanisms
of short/intermediate and long germ segmentation, we need to compare the roles
of the developmental genes involved in early embryonic patterning. In
Drosophila, gap genes play a central role in the early subdivision of
the blastoderm into broad regions. Maternal gradients lead to the activation
of gap genes a broad domain, and the syncytial environment allows the gap-gene
products to diffuse and produce overlapping short-range gradients
(Hülskamp and Tautz,
1991; Rivera-Pomar and
Jäckle, 1996
). These short-range gradients define the stripe
patterns of the primary pair-rule genes
(Small and Levine, 1991
). Gap
genes are also responsible for providing positional information to regulate
the expression of Hox genes, which assign identities to each segment
(McGinnis and Krumlauf, 1992
).
Although gap genes act in a syncytial environment in Drosophila, the
orthologs of gap genes in short and intermediate germ insects are expressed in
a cellularized environment. The orthologs of the gap genes of these insects
cannot act in the same manner as those in Drosophila. Thus,
elucidating the functions of the gap genes in short and intermediate germ
insects would provide crucial clues to clarify the molecular segmentation
mechanisms.
The gap gene hunchback (hb), which codes for a
zinc-finger type transcription factor, is crucial for anteroposterior
patterning in various insects (Liu and
Kaufman, 2004; Lehmann and
Nüsslein-Volhard, 1987
;
Patel et al., 2001
;
Schröder, 2003
;
Tautz et al., 1987
). For
example, in Drosophila, the loss-of-function alleles for hb
show a canonical gap defect: i.e. deletion of the labial through the
metathoracic segments. The hb RNAi depletion in Tribolium, a
short germ insect, results in deletion of the gnathal and thoracic segments,
suggesting that the canonical gap function of hb is conserved
(Schröder, 2003
). On the
other hand, in the intermediate germ insect Oncopeltus, the
hb (Of'hb) RNAi depletion results in a non-canonical,
gap-like phenotype, i.e. a combination of the transformation of the gnathal
and thoracic regions into an abdominal identity and defects in posterior
elongation and segmentation (Liu and
Kaufman, 2004
). This indicates that Of'hb is required to
suppress the abdominal identity and for proper germband growth and
segmentation (Liu and Kaufman,
2004
). These results suggest that hb function differs
among insects.
To deepen our understanding of the mechanisms of insect segmentation by obtaining data from more phylogenetically basal species, we have focused on the intermediate germ cricket, Gryllus bimaculatus (Gb, Orthoptera), which is more basal than Oncopeltus (Hemiptera). In our study, we isolated Gryllus hb (Gb'hb) and analyzed its functions, using embryonic and parental RNA interference, and found the following: (1) Gb'hb regulates the Hox gene expression to specify a regional identity in the anterior region, as observed in Drosophila and Oncopeltus; (2) Gb'hb controls germband morphogenesis and segmentation of the anterior region probably through the pair-rule gene, even-skipped at least; (3) Gb'hb may act as a gap gene in a limited region between the posterior of the prothoracic segment and the anterior of the mesothoracic segment; and (4) Gb'hb is involved in forming at least seven abdominal segments, probably through its expression in the posterior growth zone, which is not conserved in Drosophila. These findings suggest that Gb'hb functions in a non-canonical manner in segment patterning. We will discuss both conserved and divergent aspects of Gb'hb functions from an evolutionary point of view.
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Materials and methods |
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Whole-mount in situ hybridization
Standard protocols were used for whole-mount in situ hybridization with a
digoxigenin (DIG)-labeled antisense RNA probe, as previously described
(Niwa et al., 2000). In situ
hybridization for double staining was carried out as follows. The antisense
RNA probe was labeled with DIG or fluorescein. Hybridization was carried out
following the standard protocol. After hybridization, anti-fluorescein-AP Fab
fragments (Roche) were added and a color reaction for the fluorescein-labeled
probe was performed using NBT/BCIP as the substrate. The samples were then
washed in a TNT buffer, before re-fixing using 4% paraformaldehyde in PBS at
4°C overnight. After subsequent washing in TNT, anti-digoxigenin-POD Fab
fragments (Roche) were added and then the samples were washed in TNT again.
Treatment for signal enhancement was then carried out using the TSA Biotin
System (PerkinElmer Life Sciences) following the manufacturer's instructions.
The color reaction for the DIG-labeled probe was performed using the Vector
Nova Red substrate kit (Vector).
RNAi
We synthesized double-stranded RNA (dsRNA) using the MEGA-script Kit
(Ambion), and used PCR fragments as the template for in vitro transcription.
The PCR fragments were amplified using upstream and downstream primers that
contained the T7 promoter sequence. The synthesized RNA was extracted using
phenol/chloroform and ethanol precipitated. The RNA was denatured in boiled
water after being suspended in a Tris-EDTA buffer and annealed at room
temperature overnight. The resulting dsRNA was suspended in an appropriate
volume of water after ethanol precipitation. The final concentration of dsRNA
was adjusted to 20 µM for the Gb'hb dsRNA (395 bp, spanning the
four central zinc fingers) and the DsRed2 dsRNA [660 bp, derived from
the pDsRed2-N1 (Clontech)]. The DsRed2 dsRNA was used for negative
control experiments. For embryonic RNAi, the cricket eggs were collected for 2
hours and used within 1 hour of collection. We microinjected the dsRNA in the
posterior end of the egg, as previously described
(Zhang et al., 2002). For
parental RNAi, we injected adult females with a 0.6 µl dsRNA solution in a
ventrolateral position, between segments T3 and A1 (details to be published
elsewhere). Fifteen injected females were mated with untreated males, and the
eggs were collected from 5 to 10 days after injection.
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Results |
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An additional Gb'hb expression domain appeared near the posterior
end of the abdomen (Fig. 2G)
and became more intense as the posterior region elongated
(Fig. 2H). The posterior band
then split into two stripes, and a new band appeared in the posterior region
(Fig. 2J). Double staining for
Gb'hb and Gb'wg indicated that the two intense stripes were
located between the A7- and A9-Gb'wg stripes
(Fig. 2K). In
Drosophila, the posterior hb expression domain is located in
the region of parasegment (PS) 13/14 and is required to form the abdominal
segments A7 and A8 (Tautz et al.,
1987; Lehmann and
Nüsslein-Volhard, 1987
;
Bender et al., 1987
). In the
grasshopper, the hb posterior expression domain spans a region from
the posterior compartment of A7 to the anterior compartment of A9
(Patel et al., 2001
).
We detected Gb'hb transcripts in the ovary and early eggs using RT-PCR (data not shown), which indicated the presence of maternal transcripts. It is unclear, though, when the maternal transcripts are replaced by the zygotic ones.
RNAi analysis of Gb'hb
To investigate the Gb'hb function, we applied both embryonic RNAi
(eRNAi) and parental RNAi (pRNAi) to deplete the transcript
(Bucher et al., 2002;
Miyawaki et al., 2004
). We
confirmed that no qualitative phenotype differences were produced by either
the eRNAi or the pRNAi. We used pRNAi for further analyses using in situ
hybridization, because it does not produce injection artifacts and all of the
developed embryos showed effects of RNAi by our pRNAi for Gb'hb
(Table 1). We were able to
categorize the resulting embryos into three phenotypic classes, based on the
severity, as shown in the embryos just before hatching and in the embryos
stained with a segment marker gene Gb'wg at stage 9
(Fig. 3). The most severe class
I embryos (Fig. 3J-M) showed a
gap-like phenotype, in which the head with the mandible was followed by
several segments without appendages. In the class I embryos, the antenna,
mandible and cercus formed almost normally. In milder phenotypes, the class II
(Fig. 3G-I) and III
(Fig. 3D-F), a part of the
appendages was suppressed. Of the affected appendages, the T3 leg was the most
resistant to Gb'hb depletion. In the class II embryos, all gnathal
and thoracic appendages were suppressed, except for vestigial legs in the T3
segment, and the embryos were shortened as a result of the reduced number of
segments (Fig. 3G).
Segmentation disturbances were frequently observed in all of the phenotypic
classes (Fig. 3F,H,I). Such
phenotypes are reminiscent of those resulting from hb depletion in
another intermediate germ insect, Oncopeltus, in which the gnathal
and thoracic regions are transformed into an abdominal identity, forming a
small abdomen with defective segments (Liu
and Kaufman, 2004
). Even in the most severe class I embryos,
vestiges of T3 legs were observed in 70% of the RNAi embryos (n=28
out of 40, Fig. 3L). Most of
these embryos had only three or four segments posterior to the leg vestiges,
indicating that seven abdominal segments had been deleted in the severe case.
This number may not be maximal because it was often difficult to ascertain the
precise number of deleted segments because of segmentation disturbances. The
class I embryos without leg vestiges showed no crucial differences from
embryos with leg vestiges in their external morphology, indicating that all of
the class I embryos were resulted from suppressing the gnathal and thoracic
identities and growth and segmentation defects in the posterior region.
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Effects of Gb'hb RNAi on regional identities
To further investigate the effect of Gb'hb depletion on segment
identity, we examined the expression patterns of the Hox genes in the
Gb'hb RNAi embryos. In the wild-type embryo at stage 9
(Niwa et al., 1997),
Gryllus abdominal-A (Gb'abdA) is expressed in the posterior
compartment of A1 and the remaining abdominal segments
(Fig. 4A)
(Miyawaki et al., 2004
;
Zhang et al., 2005
), thus
making it an abdominal-marker gene. We observed ectopic expression of
Gb'abdA in the gnathal and thoracic regions in the Gb'hb
RNAi embryos (Fig. 4B,C). This
supports our interpretation based on the morphological observation that
suppression of appendages in the Gb'hb RNAi embryos is a result of
the homeosis of the gnathal and thoracic regions towards an abdominal
identity, even in the most severe phenotype
(Fig. 4D). This is essentially
consistent with the results of an RNAi analysis of Oncopeltus hb
(Liu and Kaufman, 2004
).
|
Effects of Gb'hb RNAi on early embryonic patterning
The Gb'hb RNAi effects extend outside the expression domain of the
gap pattern in the early stages. Effects outside of this expression domain may
be caused through regulation of other segmentation genes and/or Hox genes
during early embryogenesis. To investigate the effects of Gb'hb
depletion on early embryogenesis, we examined the expression patterns of the
Gryllus orthologs of Drosophila segmentation genes
(Krüppel, even-skipped and wingless) and Hox genes
(Antennapedia and abdominal-A).
We isolated a fragment of the gap-gene ortholog Gryllus
Krüppel (Gb'Kr) from the degenerate PCR and 5' RACE,
using cDNA from embryos within 24-72 hAEL. This fragment contained the Type 1
to 3 zinc fingers and part of the Type 4 zinc finger of the five zinc fingers
(Type 1-5) in Drosophila Kr (Fig.
5) (Rosenberg et al.,
1986). In the wild-type embryos at 40-42 hAEL, Gb'Kr is
expressed in a broad domain in the thoracic region and spot-like domains in
the gnathal region (Fig. 6A,
details to be published elsewhere). In Gb'hb RNAi embryos,
Gb'Kr expression was severely affected. The Gb'Kr expression
domain in the thoracic region was reduced in the RNAi embryos
(Fig. 6B,C). In severe cases,
the thoracic broad domain was suppressed, except in the peripheral region
(Fig. 6C). This suggests that
Gb'hb is involved in transcriptional regulation of Gb'Kr in
the thoracic region directly or indirectly. The reduction of the
Gb'Kr expression domain could be partially due to the defect in
embryonic growth, because the Gb'hb RNAi embryos in the early stages
were shorter and wider than wild-type embryos at the same time after
egg-laying. Such a morphological change in the Gb'hb RNAi embryos
suggests that Gb'hb is required for proper development of the
germband.
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To see the effects of Gb'hb depletion in the homeotic segment
specification in early embryos, we examined the expression patterns of the
homeotic genes in the Gb'hb RNAi embryos. Gb'Antp is
strongly expressed in the thoracic region of the wild-type embryos at 38-40
hAEL (Fig. 6J)
(Zhang et al., 2005). The
Gb'Antp domain was reduced in the Gb'hb RNAi embryos
(Fig. 6K). The expression of
Gb'abdA begins at the boundary between the thoracic region and the
abdominal region in the wild-type embryos by 45-47 hAEL
(Fig. 6L)
(Zhang et al., 2005
). The
Gb'hb RNAi resulted in ectopic expression of Gb'abdA in the
gnathal and thoracic regions (Fig.
6M). These results indicate that Gb'hb is involved in
regulating the Hox genes in the gnathal and thoracic regions during early
embryogenesis.
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Discussion |
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Gb'hb functions as a gap gene in the anterior region
In Drosophila hb mutants, the region between the labial and
metathoracic segments is known to be deleted. In Gb'hb RNAi embryos,
we were not able to find such a large deletion in the anterior region.
However, precise analysis of the expression patterns of Gb'wg and
Gb'eve stripes in Gb'hb RNAi embryos revealed that a narrow
region was deleted. In wild-type Gryllus embryos, the Gb'wg
stripes are located at the anteroposterior boundary of each segment
(Miyawaki et al., 2004;
Niwa et al., 2000
), as shown
in Fig. 7B; in the
Gb'hb RNAi embryos the T1 and T2 stripes of Gb'wg were
fused, indicating that the region from the posterior T1 to the anterior T2 was
deleted (indicated by a bracket in Fig.
7B). This is supported by the fact that segmental stripes 4 and 5
of the Gb'eve were not observed in the Gb'hb RNAi embryos.
These results indicated that Gb'hb functions as a gap gene in a
limited region from the posterior T1 to the anterior T2
(Fig. 7D). We can not deny that
more segments would be deleted in more severe Gb'hb phenotype,
because in our RNAi experiments we are unable to confirm a complete knockdown
of Gb'Hb activity. However, the most severe class of the
Gb'hb phenotypes that we observed is likely to result from the
strongest depletion of Gb'hb, because appearance frequency of the
most severe phenotype increased with injected amount of dsRNA for
Gb'hb without change in the phenotype itself (data not shown). This
presumption is supported by the fact that the anterior deleted region was not
extended in Gb'hb RNAi embryos, when more severe effects of RNAi on
abdominal regions appeared.
It is very mysterious that the segmental deletion appears in the thoracic
region where Gb'hb transcripts were not detected. Similar phenomena
were reported for gap gene orthologs of other short/intermediate germ insects
such as Oncopeltus (Liu and
Kaufman, 2004) and Tribolium
(Bucher and Klingler, 2004
).
The following two possibilities might explain the phenomenon: one is that
Gb'hb indirectly regulates the expression of pair-rule genes, such as
Gb'eve in the thoracic region through Gb'Kr and/or other
downstream genes; the other is that very low levels of Gb'hb
expression could directly regulate the expression of pair-rule genes in the
thoracic region. The latter possibility may be supported by the fact that in
grasshopper embryos, transcripts of hb and its proteins exhibit
distinct stepped expression levels in the gnathal/thoracic region, i.e. high
levels in the gnathal region and low levels in the T1 region
(Patel et al., 2001
).
Maternal functions of Gb'hb are unknown
We detected maternal transcripts in Gryllus eggs using RT-PCR.
However, in Schistocerca, which belongs to Orthoptera along with
Gryllus, the Hb protein rather than the hb transcript
appears to be maternally provided (Patel
et al., 2001). It remains unclear whether maternal transcripts or
proteins of Gb'hb functions in the early stages. Because our
embryonic and parental RNAi experiments exhibited no qualitative differences
and the embryonic RNAi yielded the severest class of phenotypes at a higher
rate than the parental RNAi, the Gb'hb RNAi phenotypes we obtained
seem to be a result of the depletion of zygotic transcripts. Thus, we could
not estimate the contribution of the maternal transcripts of Gb'hb or
its proteins to the RNAi phenotypes.
Gb'hb determines anterior identities through regulation of expression of the Hox genes
We found that the maxillary, labial and thoracic segments were homeotically
transformed into abdominal identity in the late stages of Gb'hb RNAi
embryos, while the anterior head and mandibular segment remained intact
(Fig. 7C,D). A similar
transformation has been reported in Of'hb RNAi embryos
(Liu and Kaufman, 2004). As we
observed that the ectopic expression of Gb'abdA in the maxillary,
labial and thoracic segments is induced in early Gb'hb RNAi embryos
not later than the stage when normal expression of Gb'abdA begins in
the abdominal region (Fig. 7B),
Gb'hb probably suppresses the abdominal identity in the maxillary,
labial and thoracic segments by repressing the Gb'abdA expression
during early embryogenesis, which is consistent with a report for
Of'hb.
As the homeotic transformation occurs in a much larger region than the
hb expression domain, it is also mysterious how Gb'hb
regulates the expression of homeotic genes. In Oncopeltus, Of'hb was
speculated to indirectly regulate the expression of Of'abdA, because
ectopic expression of Of'abdA was not detected in the blastoderm
stage (Liu and Kaufman, 2004).
This may be true in the thoracic region of the Gryllus embryos,
because the T3 segment is the most resistant to the homeotic transformation
into abdominal identity caused by Gb'hb RNAi depletion. Because the
expression of Gb'Kr was reduced in Gb'hb RNAi embryos, and
we know that both hb and Kr act as abdA repressors
in Drosophila (Casares and
Sánchez-Herrero, 1995
;
Shimell et al., 2000
),
Gb'Kr appears to be a candidate of Gb'abdA repressor in the
thoracic region, although the precise mechanism for induction of the
Gb'Kr expression by Gb'hb remains to be clarified.
We also observed that Gb'Antp was expressed in the prospective
labial and thoracic segments in the early stages
(Fig. 7A). In the shortened and
widened Gb'hb RNAi embryos, the Gb'Antp expression domain
was severely reduced (Fig. 7A).
Thus, Gb'hb regulates expression of Gb'Antp, directly or
indirectly. The reduced Gb'Antp expression domain may correspond to
the region most resistant to the homeotic transformation into abdominal
identity caused by Gb'hb RNAi depletion, probably overlapping the
Gb'Ubx expression and leading to T3 segment identity
(Zhang et al., 2005). This
should be closely related to the formation of the residual leg-like structures
observed in Gb'hb RNAi embryos
(Fig. 7C).
In the late stages, Gb'hb RNAi depletion also affected the expression patterns of Gb'Scr, Gb'Antp and Gb'Ubx. We found that Gb'hb is required for the expression of Gb'Scr in the labial and T1 segments, for the suppression of Gb'Antp expression in the maxillary segment, and for the suppression of Gb'Ubx expression in the region from the maxillary to T2 segments. Our results suggest that Gb'hb determines the anterior segment identities through regulation of expression of the Hox genes, as found in Drosophila.
Regulatory network among Gryllus segmentation genes
Fig. 7E shows a regulatory
network involving hb in Drosophila and a putative one in
Gryllus. In Drosophila, the prolonged syncytial stage allows
transcription factors to diffuse freely between the adjacent nuclei and exert
their functions by forming diffusion-controlled gradients. Bicoid (Bcd)
activates hb and Kr in the anterior region of the early
Drosophila embryo with its morphogenetic gradient
(Driever and Nüsslein-Volhard,
1989; Hoch et al.,
1991
). In turn, the morphogenetic gradient of the Hb protein
organizes the expression of the other gap genes Kr and
knirps (kni)
(Hülskamp et al., 1990
).
Hb acts as an activator of Kr and a repressor of kni at low
levels, while high levels of Hb repress the Kr expression
(Hülskamp et al., 1990
).
These gap genes define the expression domains of pair-rule genes, such as
eve, to generate their periodic expression patterns. The functions of
hb and Kr as an activator and a repressor, respectively, for
the second pair-rule stripe (stripe 2) formation of eve have been
extensively investigated (Small et al.,
1991
; Stanojevic et al.,
1991
). Additionally, hb regulates other pair-rule stripes
of eve (stripe 3) and other pair-rule genes, e.g. runt and
paired (Gutjahr et al.,
1993
; Klingler et al.,
1996
; Small et al.,
1996
).
In Gryllus (Fig.
7E), it was shown that Gryllus caudal (Gb'cad),
instead of bcd, organizes the gap domains of Gb'hb and
Gb'Kr (Shinmyo et al.,
2005). Our Gb'hb RNAi analysis suggested that
Gb'hb activates Gb'Kr, directly or indirectly, although more
data are needed to establish this regulatory interaction. Furthermore, we
found that Gb'hb directly or indirectly regulates the Gb'eve
expression in the prospective thoracic region of the embryo, suggesting that
the hierarchical relationship between hb and eve is
conserved between Gryllus and Drosophila. Our results also
suggested that the Gb'abdA expression is suppressed by Gb'hb
and Gb'Kr, directly or indirectly, in the anterior region, as in
Drosophila. Thus, although the maternal morphogenetic organizer in
the regulatory network of segmentation may have switched from cad to
bcd during insect evolution, its downstream relationship in the
segmentation and Hox genes appears to have been mostly conserved between
Gryllus and Drosophila
(Fig. 7E).
Evolution of hb functions in insect segmentation
In spite of these conserved aspects of the putative regulatory network, we
found that Gb'hb functions differ considerably from those of
Drosophila hb in segment formation
(Fig. 7D). Although we cannot
deny that more segments might be deleted in Gryllus, as observed in
the Drosophila hb mutant, it is likely that Gb'hb plays the
role of a gap gene that patterns fewer segments than in Drosophila.
Interestingly, in the hb RNAi phenotypes of Oncopeltus, no
segmentation gap was observed in the gnathal and thoracic regions, suggesting
that the requirement of the hb gap function in this species is
minimal, if at all present (Liu and
Kaufman, 2004). On the contrary, Tribolium hb was
reported to have the anterior gap function similar to Drosophila
(Schröder, 2003
). In
Tribolium, (a short germ insect) some of the anterior segments are
patterned under syncytial conditions
(Sommer and Tautz, 1993
).
These lines of evidence suggest that the number of anterior segments regulated
by hb increased during evolution from cellular to syncytial
segmentation, or that the canonical function of hb may have evolved
from the non-canonical functions of an ancestral hb during insect
evolution. Although the anterior hb-expression domain appears to be
fundamentally conserved in insects, the number of pair-rule stripes of the
pair-rule genes regulated by hb in the anterior region may increase
during evolution, probably owing to modification of the
cis-regulatory elements of the pair-rule genes. Comparative analyses
of the cis-regulatory elements of the pair-rule genes would shed some
light on this issue.
In short/intermediate germ insects, the posterior segments form
sequentially from the posterior growth zone during germband elongation. We
found that Gb'hb is required in the formation of at least seven
posterior segments. In addition to our finding, Of'hb was found to be
expressed in the posterior growth zone and involved in growth and segmentation
(Liu and Kaufman, 2004). On
the other hand, in Drosophila, hb is reported to be required only for
the formation of the A7 and A8 segments
(Lehmann and Nüsslein-Volhard,
1987
) (Fig. 7D). These facts suggest that the hb function in the posterior region may
have been reduced during evolution from the short/intermediate to long germ
embryogenesis. As hb is similarly expressed in the prospective A7 to
A9 segments in Gryllus and Drosophila, the hb
functions in these segments may have been conserved in both short/intermediate
and long germ embryogenesis. Recently, Peel and Akam
(Peel and Akam, 2003
) proposed
an assumption that the posterior sequential segmentation in short/intermediate
germ insects is controlled by a Notch signaling-dependent segmentation clock.
If it is the case, Gb'hb might be involved in regulating segmentation
clock. It is also possible that Gb'hb controls posterior growth
through regulation of the morphogenesis of the germband, because the shape of
the elongating posterior region was affected by Gb'hb RNAi depletion
(Fig. 7B). Further precise
analyses of posterior segmentation in short/intermediate germ insects will be
required to elucidate its mechanisms.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Molecular Genetics, Albert Einstein College
of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
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