1 Institute for Biology, Department Developmental Biology,
Friedrich-Alexander-University Erlangen, Staudtstrasse 5, 91058 Erlangen,
Germany
2 Interfakultäres Institut für Zellbiologie, Universität
Tübingen, Abt. Genetik der Tiere, Auf der Morgenstelle 28, 72076
Tübingen, Germany
Author for correspondence (e-mail:
klingler{at}biologie.uni-erlangen.de)
Accepted 7 October 2005
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SUMMARY |
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Key words: Krüppel, Giant, Even-skipped, Dfd, Scr, Antp, Ubx, Short germ, Long germ, segmentation, Gap gene, Abdomen, Jaws, Tribolium castaneum, Drosophila, Evolution, Parental RNAi
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Introduction |
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How is anteroposterior patterning accomplished in fully cellularized
organisms? Somitogenesis in vertebrates has been shown to rely on temporal
regulation for the generation of repeating units along the anteroposterior
axis, based on a segmentation clock involving components of the Notch
signalling pathway (Pourquie,
2001). A segmentation clock involving the Notch system
appears to function in basal arthropods, i.e. spiders
(Schoppmeier and Damen, 2005
;
Stollewerk et al., 2003
) and a
clock mechanism may function in centipedes as well
(Chipman et al., 2004
). Also in
these taxa, as in many insects including Tribolium, the majority of
segments arise by posterior addition of cells to a growing germ band, similar
to vertebrate embryos. In contrast to vertebrates, many orthologs of
Drosophila pair-rule and segment-polarity genes are expressed in
stripes also in these short-germ arthropods
(Chipman et al., 2004
;
Damen et al., 2000
;
Patel et al., 1994
;
Sommer and Tautz, 1993
). It
has been suggested, therefore, that the segmentation clock is an ancient
mechanism to pattern posteriorly growing embryos, and that pair-rule and
segment-polarity genes originally served to transmit the primary clock signal
to the growing and differentiating segments
(Tautz, 2004
). In the
evolutionary line leading to Drosophila, the regulation of stripe
genes then may have come under the control of spatial regulation provided by
those genes that, in Drosophila, represent the upper levels of the
segmentation hierarchy, i.e. gap genes and maternal genes
(Peel and Akam, 2003
).
In the short-germ beetle Tribolium, the embryo elongates by
posterior growth similar to spider and myriapod embryos. However, the
Notch pathway appears not to be involved in anteroposterior
patterning in this insect (Tautz,
2004). Pair-rule genes are expressed and function in
double-segmental units in Tribolium
(Brown and Denell, 1996
;
Maderspacher et al., 1998
),
and an analysis of the Tc'hairy regulatory region provided evidence
for stripe-specific regulation (Eckert et
al., 2004
). In addition to pair-rule genes, homologues of gap
genes are also expressed during germ-band growth in Tribolium, and in
other short-germ insects (Bucher and
Klingler, 2004
; Liu and
Kaufman, 2004a
; Liu and
Kaufman, 2004b
; Mito et al.,
2005
; Patel et al.,
2001
; Schröder et al.,
2000
; Sommer and Tautz,
1993
; Wolff et al.,
1995
). Functional studies using RNAi in these species have led to
diverse interpretations of how similar the role of these short germ gap genes
are compared with Drosophila gap genes.
One problem with RNAi studies is that the true null phenotype of the genes
investigated remains unknown. Unlike many other evo-devo systems, in
Tribolium, developmental genes can be identified and analysed through
the isolation of embryonic lethal mutants. Albeit more laborious, the
mutagenesis approach has the potential of providing more defined, and less
variable, lack of function situations. In addition, this classical genetics
approach allows us to identify short-germ-specific genes that have been lost
in long-germ dipteran species, the sequence of which evolves very fast, or
which in Drosophila are not involved in segmentation. Screens for
embryonic lethal genes identified several putative gap and pair-rule mutations
(Maderspacher et al., 1998;
Sulston and Anderson, 1996
).
Most of these phenotypes differ substantially from those of known
Drosophila mutants. In order to determine if any of the segmentation
genes already molecularly identified in Tribolium is affected in one
of these mutants, we tested putative gap gene mutations for linkage to gap
gene orthologues.
In this paper, we identify the previously identified Tribolium
mutant jaws (Sulston and
Anderson, 1996) as an amorphic Krüppel mutant, and
provide the first detailed analysis of a gap gene null phenotype in a
short-germ embryo. This amorphic Tc'Kr phenotype, as well as weaker
phenotypes generated by RNAi, clearly differ from those of Drosophila
Krüppel (Dm'Kr) mutations
(Wieschaus et al., 1984
),
suggesting a principally different role for this gap gene orthologue in the
short-germ embryo of Tribolium.
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Materials and methods |
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Mapping of jaws relative to Tc'Kr
Sequence polymorphisms in candidate genes were identified by amplifying and
sequencing non-coding fragments (5' UTR, 3' UTR or intronic DNA)
from adult beetles of GA-1, SB and Tiw-1 wild-type strains. Identified
sequence polymorphisms could either be scored directly as PCR fragments on an
agarose gel or were converted into RLFPs. For Tc'Kr, a polymorphism
in the 3'UTR was identified. This polymorphism was amplified as a 205 bp
fragment by primer sequences ACGACTTGGCGGTTAATG and TACGAAAGTAGGCACACAAC. In
Tiw-1, but not in SB, this fragment is cleaved by AseI into
subfragments of 141 and 64 bp (Fig.
2) that were visualized on a 2.5% NuSieve Agarose gel (Cambrex Bio
Science). For mapping, DNA was isolated from single beetles that had been
identified as mutant carriers by scoring the offspring from single matings for
presence of mutant larvae. Detailed protocols concerning our general mapping
strategy, and DNA extraction from beetles and larvae can be provided on
request.
Parental RNAi
Parental RNAi was performed as described
(Bucher et al., 2002). As
template for in vitro transcription, PCR-products with T7 sequences at both
ends were amplified from cDNA plasmids or genomic Tribolium DNA. For
injection, dsRNA was used at a concentration of 1-4 µg/µl.
Harvest of mutant jaws embryos
In order to obtain jaws mutant embryos in large numbers, offspring
from 40 identified jaws/+ parents was sexed as pupae, and virgin
females were crossed to their fathers. One-sixth of the eggs produced by this
father/daughter population will be homozygous for the mutants. Similarly, to
obtain the Tc'gt/jaws `double mutant' phenotype, Tc'gt dsRNA
was injected into the same offspring pupae and eclosed females then were
crossed to identified jaws carrier males.
Confocal images
First instar larvae were cleared in lactic acid/10% ethanol overnight at
60°C. After washing with lactic acid, cuticles were mounted on a slide
under a cover-slip that was supported with rubber gum. This allowed manual
positioning to a ventral-up orientation. Cuticular autofluorescence in the 520
to 660 nm range was detected on a Leica confocal microscope by excitation at
488 nm and maximum projection images were generated from image stacks.
Expression analysis
Single (Tautz and Pfeifle, 1989) and double label
(Prpic et al., 2001)
whole-mount in situ hybridisations were carried out as described.
Tc'Kr-RNAi germband stage embryos are particularly fragile and were
manually devitellinized on double sticky tape: 12- to 18-hour-old embryos were
transferred to ethanol and then gently attached to a double-sided sticky tape.
After replacing ethanol with water, the vitelline membrane tightly adheres to
the tape and embryos can be manually devitellinized using diminutive insect
needles. In order to avoid RNA degradation, devitellinized embryos were
promptly transferred to methanol and stored at -20°C.
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Results |
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While Dm'Kr is expressed in the centre of the blastoderm embryo,
in the Tribolium blastoderm this domain appears at the posterior pole
(Fig. 1A). Relative to the
segment primordia, however, this position is roughly conserved, as
Tribolium is a short-germ embryo
(Sommer and Tautz, 1993). We
used Tc'even-skipped (Tc'eve) as an additional marker to map
the position of the gap domain precisely
(Fig. 1B-E). During germ
rudiment formation, Tc'Kr remains expressed in a broad central
domain. In early germband stages (Fig.
1B,C), the anterior border of Tc'Kr lies within the 2nd
stripe of Tc'eve (`eve2'). When eve2 splits into segmental stripes,
eve2a and eve2b [corresponding to labial and first thoracic segments
(Patel et al., 1994
)], the
Tc'Kr domain abuts the posterior border of eve2a (1D). At this time,
Tc'Kr also fades from the growth zone and a posterior border forms
just anterior to the eve4 stripe as it arises near the growth zone
(Fig. 1C,D). As the segmental
stripes eve3a and eve3b form, the posterior boundary of the Tc'Kr gap
domain coincides with eve3b (Fig.
1E). Accordingly, in germ band embryos the Tc'Kr gap
domain overlaps very precisely the three thoracic segments - which is more
anterior than in Drosophila, where the Tc'Kr domain is
centered over the primordia of segments T2 to A2
(Myasnikova et al., 2001
).
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jaws is closely linked to the Tc'Kr locus
The jaws mutation was originally induced in a GA-1 background
(Sulston and Anderson, 1996).
Preliminary experiments suggested that this mutation had been induced in a
chromosome carrying a RFLP polymorphism in the Tc'Kr gene (`Tiw-1
specific polymorphism') that differs from the corresponding sequence in the SB
wild-type strain (`SB specific polymorphism'). In order to test for close
linkage between jaws and Tc'Kr, we made use of the fact that
a jaws mutant strain had been kept in our laboratory by recurrent
outcrossing to SB females for over six generations [for stock-keeping of
embryonic lethal mutations see Berghammer et al.
(Berghammer et al., 1999
)].
Therefore, in our stock collection, most of the genome in the jaws
strain must have been replaced by SB-specific alleles. Only loci very close to
jaws are likely to still be represented by GA-1-specific alleles,
because presence of the jaws mutant had been selected for in every
generation. When we scored 80 adult beetles from our stock collection that
carried one copy of the jaws mutation, we found that every one of
these animals was heterozygous for both polymorphisms at the Tc'Kr
locus (Fig. 2A). This shows
very close linkage between the jaws mutation and the Tc'Kr
gene and suggested that jaws is a mutation in the Tc'Kr
gene. As a control, we also tested 20 of these animals for polymorphisms in
the Tc'eve gene and found, as expected for a locus not linked to
jaws, that they all were homozygous for a SB-specific Tc'eve
polymorphism.
The first zinc finger of Tc'Kr is altered in jaws
To confirm the identity of the jaws and Tc'Kr loci, we
isolated genomic DNA from homozygous jaws-mutant larvae and
PCR-amplified three fragments from the Tc'Kr locus that cover both
exons. Sequence comparison with control amplificates from the SB and GA-1
strains revealed an amino acid replacement in the Tc'Kr-coding
sequence of mutant animals. This transition changes the second histidine of
the first zinc finger to a tyrosine (Fig.
2B,C). As the Cys-Cys-His-His Zn-finger motive is essential for
the correct structure of the DNA-binding domain, a missense mutation in such a
key amino acid is likely to inactivate the Tc'Kr gene. In this
respect, the jaws mutation, now to be termed
Tc'Krjaws, is similar to an amorphic Krüppel
mutation identified in Drosophila: in the Dm'Kr9
allele, one of the crucial Zn-finger cysteines is converted to serine,
completely abolishing Dm'Kr function
(Redemann et al., 1988).
Below, we provide additional evidence that Tc'Krjaws
indeed does fully inactivate the Tc'Kr locus.
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In Tc'Krjaws embryos
(Fig. 3F), the head is
differentiated as in wild type. The next four segments (thoracic and 1st
abdominal) develop gnathal structures such that the regular maxillary (mx) and
labial (lb) segments are followed by two additional pairs of maxillary and
labial segments. Including the normally developed mandible (md), this results
in a total of seven gnathal segments (md-mx-lb-mx-lb-mx-lb). Posteriorly,
these gnathal segments are followed by one segment of abdominal morphology,
and the posterior end of the embryo is formed by terminal structures similar
to wild type, including urogomphi and pygopodes, the derivates of the 9th and
10th abdominal segments. Hence, the total of gnathal, thoracic and abdominal
segments in Tc'Krjaws embryos is 10 compared with 16 in
wild type, i.e. six segments are deleted, while four segments are homeotically
transformed (Sulston and Anderson,
1996). This phenotype differs from that of strong Dm'Kr
mutants where the thoracic and the first four abdominal segments are deleted
and no homeotic transformations are evident in differentiated mutant larvae.
The ectopic maxillary structures of Tc'Krjaws mutant
embryos deviate somewhat from normal maxillae in that they lack endites (the
mala) and sometimes possess distal claws rather than the sensory structures
characteristic of maxillary palps (this is especially the case for the most
posterior pair of maxillas). In addition, the ectopic labia (as well as the
endogenous labium) are abnormal in that they usually do not fuse ventrally.
Weaker phenotypes obtained by RNAi support the interpretation that these
imperfect gnathal segments in fact are of mixed gnathal and thoracic character
(Fig. 3D,E).
In intermediate strength and weak RNAi phenotypes (Fig. 3C,D), more abdominal segments remain and the transformation of thoracic segments towards gnathal fate is less pronounced. Frequently, the first and third thoracic segments still differentiate legs in embryos whose second thoracic segment already is transformed into labium. This indicates that higher levels of Tc'Kr activity are required for inhibiting labial fate than for repressing maxillary fates. In addition, the additionally present abdominal segments in these embryos usually display homeotic transformations towards a more anterior, i.e. thoracic or gnathal, fate (Fig. 3D). In these abdominal segments, there is also a tendency for alternating maxillary and labial fates, and small irregular appendages can sometimes be observed. In conclusion, the weak Tc'Kr RNAi phenotypes also differ significantly from those of weak Dm'Kr mutants, displaying additional homeotic transformations of abdominal segments towards more anterior fates.
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The homeotic effect of Tc'Krjaws is epistatic over that of Tc'gt RNAi
Interestingly, RNAi knock-down of the Tc'giant gene
(Tc'gt) leads to a homeotic phenotype opposite to that caused by
Tc'Kr inactivation. In Tc'gt RNAi embryos, the maxillary and
labial segments are transformed towards thoracic identity
(Bucher and Klingler, 2004)
(see also Fig. 4C). We wondered
which of these transformations would prevail in a `double-mutant' situation.
To this end, we performed Tc'gt RNAi knock-down in a
Tc'Krjaws mutant background (see Materials and methods).
In this experiment, we obtained Tc'gt knock-down phenotypes in the
majority of embryos while a fraction corresponding to
Tc'Krjaws homozygous animals showed a phenotype very
similar to that of Tc'Krjaws alone
(Fig. 4B). They differed only
from the normal Tc'Krjaws phenotype in that they lacked
one or two additional segments. This is to be expected, because in
Tc'gt RNAi embryos, thoracic and abdominal segments can be deleted
that are not affected in Tc'Krjaws, i.e. the segmentation
phenotypes of these experimental larvae corresponds to a superposition of
Tc'gt RNAi and Tc'Krjaws. However, the homeotic
transformations caused by Tc'Krjaws are clearly epistatic
over those produced by Tc'gt RNAi knock-down. This suggests that the
homeotic transformation of gnathal segments into thorax in Tc'gt RNAi
embryos is an indirect effect (see Discussion).
Expression of homeotic genes in Tc'Krjaws and Tc'gt RNAi embryos
The striking homeotic transformations in Tc'Krjaws
larvae could either be due to misregulation of homeotic genes, or could
indicate a direct role of Tc'Kr in specifying segmental fates.
Previous work already has shown that the Hox gene proboscipedia
(Tc'pb) is ectopically expressed in Tc'Krjaws
mutant embryos (Sulston and Anderson,
1998). However, Tc'pb becomes active relatively late
during development, and only in the maxillary and labial palps, not in
complete segments. Thus, we asked how the expression of Hox genes early active
in the maxillary, labial and thoracic segments would relate to the
Tc'Krjaws phenotype.
The Deformed (Tc'Dfd) gene is expressed in the mandibular
and maxillary segments (Brown et al.,
1999). In Tc'Krjaws embryos, two strong and
one weak additional Tc'Dfd domains are observed that are separated
from each other by gaps approximately one segment wide
(Fig. 5A-D). The two strongly
expressing ectopic domains correspond to the first and third thoracic segments
that, in Tc'Krjaws mutant larvae, develop maxillary
characteristics. The Sex combs reduced (Tc'Scr) gene is
active in the ectoderm of the second parasegment in Tribolium
(Curtis et al., 2001
), which
largely corresponds to the labial segment
(Fig. 5E-G; Tc'Scr
expression is also present in the mesoderm of additional segments). In
Tc'Krjaws embryos (Fig.
5H-J), ectopic activity of Tc'Scr is present in the
primordia that correspond to the second thoracic and first abdominal segments
of wild-type animals, i.e. in those segments that differentiate labial palps
in mutant larvae. Therefore, the gnathal Hox genes Tc'Dfd and
Tc'Scr are active in complementary double-segmental frames in
Tc'Krjaws mutant embryos, which is consistent with the
phenotype of differentiated mutant larvae.
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Together, these data show that the Tc'Krjaws homeotic phenotype can be explained by defective Hox gene regulation, and they suggest inhibition of Tc'Dfd and Tc'Scr by Tc'Kr, whereas Tc'Ubx positively depends on Kr activity. In addition, the double-segmental appearance of ectopic gnathal Hox expression domains suggests that the Hox genes Tc'Dfd and Tc'Scr also are under strict pair-rule control.
Function of Tc'Kr in regulating segmentation genes
Previous work has already revealed that the pattern of the segment-polarity
gene engrailed (Tc'en) and the pair-rule genes
Tc'eve and Tc'runt are altered in
Tc'Krjaws (Sulston and
Anderson, 1998). We repeated and extended this work in order to
relate the defects observed with what we now know about the spatial expression
of the gene that is inactivated in this mutant.
We first attempted to identify which stripes of Tc'eve exactly are affected by the Tc'Krjaws mutation. To distinguish pair-rule stripes arising in the growing germ band, we performed double staining with segment polarity genes, and to identify Tc'Krjaws mutant embryos at stages before morphological differences to wild types become evident, Tc'giant was included as an additional marker in some experiments (in Tc'Krjaws embryos, the posterior domain of Tc'gt is absent, while an additional stripe of expression appears; A.C., unpublished). We find that the first three Tc'eve stripes arise and split into segmental stripes in Tc'Krjaws mutant embryos exactly as in wild type (Fig. 6A-C,E-G). In addition, a stripe of eve4 is formed in the growth zone as a distinct band with sharp boundaries. Although this stripe arises just posterior to the Tc'Kr domain, Tc'Kr apparently has no role in defining its anterior boundary. However, segmentation defects become evident at subsequent stages: while eve4 does split into segmental stripes 4a and 4b, these segmental stripes (particularly eve4b) appear somewhat irregular. The anterior boundary of eve5 also forms perfectly in Tc'Krjaws (Fig. 6G,H), very similar to wild type. However, this stripe never progresses into segmental stripes 5a and 5b (Fig. 7A-C); instead, its expression becomes irregular in shape and then decreases in strength and fades away (Fig. 7G-I). The defects of Tc'eve patterning observed in Tc'Krjaws differ strongly from the situation in Drosophila gap gene mutants, where negative regulation of stripe-specific elements results in widened stripes.
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Discussion |
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Regulation of homeotic genes by Tc'Kr
The most obvious difference between the phenotypes of Krüppel
in Tribolium and Drosophila are the homeotic transformations
in Tc'Krjaws and Tc'Kr RNAi larvae that are not
evident in Dm'Kr mutants. Such transformations are not entirely
unexpected given that in Drosophila the expression boundaries of Hox
genes are also set by gap genes, including Dm'Kr. However, in
Drosophila gap mutants all segments that would be transformed because
of misregulation of homeotic genes usually also suffer segmentation defects
and fail to develop. By contrast, Tribolium segment primordia
anterior of, and within, the Krüppel expression domain do
differentiate, such that homeotic transformations can manifest themselves in
the differentiated larva.
The expression of homeotic genes in Tc'Krjaws embryos
is consistent with the morphological transformations observed
(Fig. 3F,
Fig. 5). Our results with
Tc'Dfd, Tc'Scr, Tc'Antp and Tc'Ubx confirm and extend
earlier findings for Tc'pb and Tc'UBX/Tc'ABD-A expression
(Sulston and Anderson, 1998).
Notably, the complementary double-segmental expression of Dfd and
Scr in Tc'Krjaws embryos explains the phenotype
of alternating maxillary and labial segments. As summarized in
Fig. 8, these expression
patterns indicate that the posterior limit of Tc'Dfd and
Tc'Scr domains is set through inhibition by Tc'Kr. In this
respect, Tc'Kr fulfils a function similar to Drosophila gap
genes.
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In addition to gap gene input, Drosophila Hox genes also receive input from pair-rule genes. The near-pair-rule pattern of Tc'Dfd and Tc'Scr in Tc'Krjaws embryos reveals an important role of pair-rule genes also in defining Tribolium Hox domain boundaries. It seems likely that regulation of Tc'Dfd and Tc'Scr by pair-rule genes is responsible for the precision of their expression boundaries in wild-type Tribolium embryos, while input from gap genes defines the broad region were a particular Hox gene can become active (Fig. 8).
Tc'Kr does not function as a canonical gap gene during segmentation
In Drosophila, Krüppel is expressed in a bell-shaped profile
centered over the primordia of segments T2 to A3
(Gaul and Jäckle, 1987;
Myasnikova et al., 2001
). In
the Tribolium blastoderm, only one such gradient is present as the
Tc'Kr domain covers the posterior pole
(Sommer and Tautz, 1993
). When
the germ rudiment has formed, the Tc'Kr domain retracts from the
posterior end and forms a distinct domain overlapping the three thoracic
segment primordia (Fig. 1). At
this stage, therefore, the Tc'Kr domain covers more anterior segment
primordia (and more anterior pair-rule stripes) than does its
Drosophila counterpart.
Both boundaries of the Dm'Kr expression domain have been shown to
serve as short-range gradients that provide positional information to define
the margins of pair-rule stripes (Klingler
et al., 1996; Langeland et
al., 1994
; Small et al.,
1991
). A similar function should have been expected at least for
the anterior boundary of Tc'Kr, which already forms during the
syncytial blastoderm. However, although this anterior boundary evidently is
used for limiting gnathal Hox gene expression (Figs
5,
8), it is not required for the
formation of gnathal or thoracic segments. The segment polarity genes
Tc'en and Tc'wg are expressed normally in all segments up to
the first abdominal segment (Figs
6,
7). In addition, the first four
pair-rule stripes of Tc'eve show little or no change compared with
wild type (Fig. 6). The same is
the case for the first four stripes of Tc'hairy and Tc'runt
(data not shown). Severe deviations from the wild-type pattern only become
apparent beginning with the 5th Tc'eve stripe. These data clearly
show that the Krüppel domain in Tribolium has no
significant role in generating those primordia that arise within the reach of
its blastoderm expression domain.
|
Compared with the classical gap phenotype of Dm'Kr mutants, the
segmental defects in Tc'Krjaws are shifted towards
posterior. Based on its larval phenotype, Tc'Krjaws has
been described as a gap gene, in that most abdominal segments are deleted
while gnathal and thoracic segments, as well as the most posterior abdominal
segments (A9 and A10), remain intact
(Sulston and Anderson, 1996).
However, when analysing pair-rule and segment-polarity expression, we did not
observe resumption of stripe formation posterior of a defect zone (Figs
6,
7) as is observed for the
mutation krusty, for example
(Maderspacher et al., 1998
).
In contrast to the earlier report, we interpret the progression of the
en/wg pattern in Tc'Krjaws embryos as reflecting
a breakdown of segmentation, not a temporal gap in the sequence of abdominal
segment additions. While the 9th and 10th abdominal segments usually are
present in Tc'Krjaws mutant and Tc'Kr RNAi larvae
and give rise to urogomphi and pygopods, we conclude from the time series in
Fig. 7 that these structures
actually derive from the fragmentary stripes formed immediately after the
anterior seven unaffected stripes have been generated. This implies that the
remnants of middle-abdominal segments later on differentiate as posterior
abdominal segments in Tc'Krjaws mutant embryos. To explain
the specification of earlier formed segments as A9 and A10, we speculate that
after completion of germ band growth, a signal emanates from the posterior
terminalia and instructs the next two segments to fuse with the telson and to
form urogomphi and pygopods. In addition, non-segmental terminal structures
are present in Tc'Krjaws embryos. These primordia are
known to arise early in the blastoderm, posterior of the growth zone proper
(reviewed by Anderson, 1972
).
One marker for terminal structures is the posterior terminal domain of
Tc'wg (Nagy and Carroll,
1994
), which is formed and maintained in
Tc'Krjaws embryos similar to wild type
(Fig. 7). In addition, the
cuticle lining of the hindgut is present in mutant larvae (e.g.
Fig. 4A).
The role of Krüppel in short germ insects
As the growth zone is a patterning environment very different from the
syncytial blastoderm, it was expected that segmentation genes in short germ
embryos would play similar roles as in Drosophila during early
stages, while abdominal segmentation was predicted to be fundamentally
different. It is surprising that knock-down of several short germ gap gene
homologues, i.e. Tc'gt (Bucher and
Klingler, 2004), Tc'Kr, Gb'hb
(Mito et al., 2005
) and
Of'hb (Liu and Kaufman,
2004a
), results mainly in homeotic transformations in those
segments that form during the blastoderm. This also pertains to Tc'hb
(Schröder, 2003
), where
homeotic transformations occur in addition to segmentation defects (A.C. and
R.S. unpublished). That so many of these gap gene homologues do not seem to
have strong roles in the formation of anterior segments raises the possibility
that the original role of gap genes early during arthropod evolution may have
been to regulate Hox genes, but not to directly regulate pair-rule genes (G.
Bucher, PhD thesis, Ludwig-Maximilians-Universität, München, 2002)
(Liu and Kaufman, 2004a
). In
Tribolium, however, some blastoderm pair-rule stripes are affected by
gap gene orthologues other than Kr (A.C.C. and M.K., in preparation),
and there is good evidence for stripe-specific elements driving at least the
first two Tc'hairy stripes
(Eckert et al., 2004
).
Our results for Tc'Kr deviate from those obtained for
Krüppel in Oncopeltus fasciatus
(Liu and Kaufman, 2004b). In
this short-germ insect, knock-down of Kr also results in
mis-expression of Hox genes, although the effects are more limited as only one
ectopic Of'Dfd domain is detected. Interestingly, expression of
Of'en in such embryos seems to indicate a clear gap phenotype, i.e.
perfect segmental stripes reappear posterior to a region of segmental
disruption. Incomplete inactivation of Of'Kr could be responsible for
this difference; we note, however, that weak Tc'Kr RNAi situations do
not result in obvious gap phenotypes (see Fig. S2 in the supplementary
material). Rather, in such embryos the segmentation process simply breaks down
somewhat later than in Tc'Krjaws, i.e. the additional
segments present in weak Tc'Kr RNAi embryos appear to represent
anterior abdominal rather than posterior (post-gap) abdominal segments.
Oncopeltus is sometimes denoted an intermediate-germ insect, because
a few more segments are formed already in the blastoderm than, for example, in
Tribolium. It will be interesting to see if the `next posterior' gap
gene in Oncopeltus will also display a `gap' phenotype, and to find
out whether pair-rule gene expression in Of'Kr RNAi embryos indicates
a role in the regulation of specific stripes boundaries.
If our interpretation is correct that Tc'Kr does not directly specify pair-rule stripes during abdomen formation, what could its function be in this process? All abdominal cells derive from progenitors that expressed Tc'Kr at the blastoderm stage. Therefore, regulation of later-acting abdominal expression domains (e.g. the posterior domains of Tc'gt and Tc'hb), may depend on Tc'Kr activity in the blastoderm, rather than on its activity at later stages when its domain forms a distinct posterior boundary. In this way, the long-ranging action of Tc'Kr could be explained through a temporal persistence rather than a spatial diffusion mechanism. Later acting genes depending on Tc'Kr activity then could have a role in regulating pair-rule genes.
However, the discovery that a segmentation clock appears to pattern lower
arthropods (Chipman et al.,
2004; Stollewerk et al.,
2003
) raises the issue of when in the evolutionary line leading to
the diptera this clock was replaced by the hierarchical mode of
Drosophila segmentation. Although at present no evidence is available
for a segmentation clock functioning in Tribolium, it is conceivable
that a modified clock is installed at the posterior end of the blastoderm
embryo. Tc'Kr could have a role in initiation of this clock
machinery. Alternatively, it could be required for its continued function.
Because the number of abdominal segments is constant in insects, some type of
counting principle would be required to stop the clock once the last segment
has formed. Such a counting mechanism could be provided, for example, by a
series of abdominal `gap gene' activities (including the posterior domains of
Tc'gt and Tc'hb), the last of which would shut off the
clock. In this view, abdominal `gap genes' would have a permissive rather than
a positionally instructive function during abdominal segmentation of short
germ embryos.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/24/5353/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Institute for Zoology, Anthropology and Developmental
Biology, Department of Developmental Biology, Georg August Universität,
Justus-von-Liebig-Weg-11, 37077 Göttingen, Germany
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