Louisiana State University, Department of Biological Sciences, Baton Rouge, LA 70803, USA Department of Biological Sciences, 202 Life Sciences Building, Louisiana State University, Baton Rouge, LA 70803, USA
* Author for correspondence (e-mail: broger2{at}lsu.edu)
Accepted 8 June 2004
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SUMMARY |
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Key words: Oncopeltus fasciatus, tiptop, Antennapedia, Leg development, Ground state, RNA-interference
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Introduction |
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The primary model organism for insect appendage formation is the dipteran
D. melanogaster, in which regulators of leg development have been
well characterized. In D. melanogaster, combinations of selector gene
activity control the segmentation and differentiation of leg segments
(Fig. 7) to produce coxa,
trochanter, femur, tibia, tarsi, and pretarsus (proximal to distal). In brief,
extradenticle (exd) and teashirt (tsh) are
required for proximal development
(Abu-Shaar and Mann, 1998;
Erkner et al., 1999
;
Wu and Cohen, 2000
);
Distalless (Dll) is required for distal development
(Cohen et al., 1989
), and
dachshund (dac) is required for medial development
(Mardon et al., 1994
). While
these genes are required for elaboration of the axis of the leg and for normal
leg development, the primary modifiers of ground state development are thought
to be the Hox genes and homothorax (hth). These latter genes
act as switches that choose among the various appendage types
(Casares and Mann, 2001
;
Kaufman et al., 1990
; Struhl,
1982
,
1981
).
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Here we present the functional analyses of two selector genes (tiptop, Antp) that control leg development in the milkweed bug Oncopeltus fasciatus (Hemiptera). We show that, in O. fasciatus, tiptop is a selector gene that is required for the segmentation of the distal leg and is also required to switch appendage development from antenna to leg. We also show that, while the role of Antp in the segmentation of the medial leg is conserved between D. melanogaster and O. fasciatus, its role in specifying leg versus antennal development is not. The implication is that, in O. fasciatus, it is tiptop and not Antp which represses hth activity in the leg. We discuss the significance of the regulatory variation in the leg specification mechanism on the evolution of insects and arthropods.
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Materials and methods |
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Cloning of tiptop used the forward primer GTNTGGYTNGGNAARGG (VWLGKG) and the reverse primer TTRTCRCANACYTTRCA (CKVCDK). Cloning of Antp used the forward primer GAYTAYCCNTGGATGMGN (DYPWMR) and the reverse primer CKNCKRTTYTGRAACCA (WFQNRR). Cloning of Ubx used the forward primer GARYTNGARAARGARTTY (ELEKEF) and the reverse primer CKNCKRTTYTGRAACCA (WFQNRR).
RNA-interference (RNA-I) was performed generally as described in Hughes and
Kaufman (Hughes and Kaufman,
2000). In brief, sense and antisense single-stranded (ss)RNAs were
generated from cDNA clones by MEGASCRIPT (Ambion), extracted with
phenol-chloroform, ethanol precipitated, and resuspended in injection buffer
(0.1 mM NaPO4, 5 mM KCl, pH 6.8). The double-stranded (ds)RNA was
produced by combining complementary ssRNAs in roughly equimolar ratios,
heating at 95°C (1 min), and allowing the heat block to cool to room
temperature. Production of dsRNA was verified by briefly treating samples of
both ssRNA and dsRNA with weak solutions of RNAse A, and separating the
products in 2% agarose gels stained with ethidium bromide. Eggs were attached
to glass slides with acid-free and xylene-free glue. The dsRNAs were injected
into embryos (<4-8 h old) either anteriorly or posteriorly at
concentrations ranging from 4-10 g/l of injection buffer. Viable eggs were
internally pressurized; thus, actual injection volumes were minimal and higher
transformation efficiencies were generated when dsRNA concentrations were at
least 8 g/l. Eggs were kept in humidified, glass petri dishes until at least
one day after the control (i.e. non-stabbed) eggs hatched; any non-hatched
embryos were then dissected to check for RNA-I induced abnormalities.
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Results |
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From O. fasciatus embryos, we cloned partial cDNAs that represent
a homolog of the D. melanogaster gene tiptop
(Fig. 1). tiptop is a
member of the tsh-family that is typified by zinc-finger motifs and is
presumed to be a transcription factor
(Andrew et al., 1994;
Röder et al., 1992
)
(GenBank Accession Number, AF219383). Also, while present in the D.
melanogaster genome, this gene was identified solely on the basis of
molecular data. No mutations have been reported in tiptop, and its
developmental function has not been previously reported for any arthropod.
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Injection of tiptop dsRNA produces specific defects
To gain insight into the function of several O. fasciatus genes on
the development of the larval cuticle, gene activity was reduced using RNA-I.
RNA-I was performed by injecting in vitro synthesized dsRNA corresponding to a
specific cDNA into precellular embryos. The developmental effects of injecting
specific dsRNAs were compared to control groups consisting of: (1) eggs
collected and prepared but not injected, and (2) group of eggs which are
injected with buffer or mock transcription reactions. Developmental defects
detected in group 1 would reveal any natural variation in development or
defects in development caused by manipulation of the eggs. Nearly all of these
eggs hatch with normal phenotypes (data not shown), except for some
unfertilized eggs which are easily recognized and are not included in the
other experimental regimes. The developmental defects observed in group 2
reveal the effects of damaging the eggs by injection and the effects of
transcription reagents that may remain in the injection mix. Common defects in
this group include randomly positioned deletions which we attribute to partial
losses of the embryo from the egg during injection. Specific defects caused by
the injection of a particular dsRNA are defects which are observed in repeated
injections and are not observed in the control populations. These specific
defects are presumed to be the phenocopies caused by reductions in gene
activity. The collective defects caused by injection of dsRNA from a
particular gene are usually different from those caused by dsRNA from other
genes, suggesting a reduction in activity of a specific gene.
The results of RNA-I analysis for tiptop and several other genes are summarized in Table 1. The table shows that the percentage of developing animals showing a specific defect is reasonably high (approximately 25-40%) and that the specific defect associated with reducing the activity of each gene is unique.
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The partial homeotic transformation of legs to antennae in the strong phenocopies suggests that tiptop also acts as a selector gene that can switch appendage development from antenna to leg. The persistence of leg identity in the proximal segments of the leg suggest that more than one selector gene is required to establish the identity of the entire appendage.
Antp does not act as a switch between antennal and leg development in O. fasciatus
We considered the Hox genes to be candidates for genes with leg identity
specification functions because the activity of Hox genes can establish leg
versus antennal identity in D. melanogaster, and because the leg
morphology of the strong tiptop phenocopy was similar to that of
D. melanogaster with reduced Hox function. We examined the role of
Hox genes in specifying leg development in O. fasciatus embryos using
RNA-I. Our analysis of Hox genes in O. fasciatus suggests several
significant differences in their activity compared to their activity in D.
melanogaster.
In O. fasciatus, the moderate Antp phenocopy is typified by a distortion or a fusion of the medial segments (M*; produced by the fusion of femur and tibia, in Fig. 6A). Here the fusion, rather than loss of segments is more apparent. There are femur-like sense organs on the proximal edge of the segment and (on first thoracic legs) a tibial comb on the distal edge. The legs of the strong Antp phenocopy are characterized by a normal coxa and trochanter as well as by a third segment (DM*; produced by the fusion of the medial and distal segments) that is tipped with normal, tiptop-dependent pretarsi (Fig. 6B, Table 1). All three pairs of legs are affected equally.
In O. fasciatus, reducing the activities of both Antp and tiptop results in legs composed of normal proximal segments and a third segment (DM*; femur+tibia+tarsi) that has an antennal identity (Fig. 6D, Table 1). Reducing the activity for two other Hox genes that control leg development in D. melanogaster, Sex combs reduced (Scr) and Ultrabithorax (Ubx), produces little or no effect on leg morphology (Table 1).
We interpret our results to mean that Scr and Ubx are not
specifiers of leg development in O. fasciatus. Also, Antp
activity is required for the development of distal and medial segments, but
not for the proximal leg or pretarsi. Further, in contrast to D.
melanogaster, where loss of Hox (Antp, Scr, Ubx) function
produces dramatic transformations of leg to antenna
(Struhl, 1982;
Struhl, 1981
), we detected no
transformation toward antennae of Antp-phenocopy legs. Finally, in
O. fasciatus, reducing the activities of both Antp and
tiptop simultaneously had a strictly additive effect. Thus, there is
no evidence for a genetic interaction between tiptop and
Antp.
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Discussion |
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Expression of tiptop mRNA over the distal thoracic appendage
during early stages of embryonic development is consistent with its activity
in the leg. Modulation of the leg expression pattern in later stages seems
consistent with tiptop's role in segmentation. However, the loss of
expression in the distal region of the leg suggests that the effect of
tiptop on distal identity results from a transient and indirect
activity. It may be that all the defects in the tiptop phenocopies
result from misregulation of a single gene such as hth.
Alternatively, the segmentation and distal specification function of
tiptop may result from a more direct role in stabilizing or
modulating distally required genes such as Dll or EGFR
(Campbell, 2002;
Galindo et al., 2002
).
The restriction of tiptop activity to the leg is somewhat
surprising given its broad mRNA expression pattern and the conservation of
this pattern to T. domestica. This conserved mRNA expression pattern
may simply be unnecessarily broad, or some tiptop activity may be
redundant, or the pattern may reflect a requirement for undetected (e.g. not
cuticular) activities of tiptop. Such activities are suggested by the
lethal effect seen in strong tiptop phenocopies. It is also possible
that tiptop is regulated post-transcriptionally, perhaps by a
mechanism similar to the one for tsh in D. melanogaster
(Erkner et al., 1999;
Waltzer et al., 2001
), and
that the distribution of gene activity is more restricted than the mRNA
distribution.
A comparison of the tsh-like genes of insects suggests a recent duplication in the lineage leading to Drosophila
As illustrated in Fig. 1,
tsh-family genes have been cloned by different methods from at least
five insect species and, with the exception of D. melanogaster tsh,
all appear to have greater similarity to tiptop. Through extensive
PCR on genomic DNA and cDNA, we recovered both tiptop and
tsh from D. melanogaster, but we only recovered only a
single gene from O. fasciatus or other insects. Also, a gene tree
(not shown) constructed using PAUP*
(Swofford, 1996) for the
tsh-family genes shows that the two D. melanogaster genes
cluster together. The gene tree and the absence of a tsh gene in the
other insects surveyed suggest that the two tsh-family genes in D.
melanogaster result from a recent duplication of an ancestral gene. We
call this ancestral gene tiptop because of its greater similarity to
that gene and not to imply a closer evolutionary relationship of the ancestral
gene to either the D. melanogaster tsh or tiptop.
tsh has a variety of developmental functions in the cuticle of the
Drosophila larva and adult. Specifically, tsh is thought to
specify trunk identity in the larva through interactions with Hox genes
(Röder et al., 1992), is
required for the formation of proximal regions of appendages in adults
(Abu-Shaar and Mann, 1998
;
Erkner et al., 1999
;
Wu and Cohen, 2000
), and plays
a role in restricting the development of the adult eye
(Bessa et al., 2002
;
Singh et al., 2002
). There is
little similarity between any of these activities of the Drosophila
tsh gene and O. fasciatus tiptop. Thus, these activities appear
to have been acquired by the tsh-family relatively recently.
Significant to the discussion here is that the function tsh has in
the formation of the proximal region of the leg in D. melanogaster
cannot be provided by tsh in O. fasciatus because the gene
is not present. These roles may be provided by other proximally expressed
genes such as hth and exd.
Variation in the genetic mechanism of leg specification among insects
A model of the leg specification mechanisms in D. melanogaster and
the proposed differences from O. fasciatus are shown in
Fig. 7. In contrast to D.
melanogaster, where loss of Hox (Antp, Scr, Ubx) function
produces dramatic transformations of leg to antenna
(Struhl, 1981;
Struhl, 1982
), we detected no
transformation toward antennae of Antp-phenocopy legs that could be
interpreted as an expansion of hth activity. Thus, although it is
possible that some residual Antp function remains in our animals, we
suggest that it is tiptop and not Antp that represses the
activity of hth (or other antennal specifier) in the O.
fasciatus leg. A role for Antp in the segmentation of the distal
region is absent in D. melanogaster while its role in medial
segmentation is conserved between the two insects. Also, given that neither
tiptop nor the Hox genes act as specifiers of proximal identity (leg
vs. antenna) in the leg specification mechanism of milkweed bugs, additional
undetermined genes are implicated. This further distances the mechanism of
appendage specification in milkweed bugs from the relatively simple two-gene
(Hox, hth) system evident in D. melanogaster.
A tiptop-like activity is also evident in D.
melanogaster. This is illustrated most convincingly by the persistent
pretarsi formed on legs that are otherwise transformed to antennae in the
absence of Antp activity (Struhl,
1981; Struhl,
1982
). Also, the leg-like appendage (composed primarily of tarsi
and pretarsi) produced by hth Antp null clones in D.
melanogaster (Casares and Mann,
2001
) is what might be predicted if an independent
tiptop-like activity for distal segmentation and specification
remained active in these appendages. Genetic analysis of Drosophila
tiptop has not revealed a role in distal specification or segmentation of
the adult leg (Laurent Fasano, personal communication). However, due to the
technical difficulties of determining the role embryonic gene activities have
in adult structures in D. melanogaster, it has not been possible to
rule out that embryonic activities of either tiptop or tsh
affect the adult leg. Thus, it remains a possibility that a
tiptop-like activity could be provided by tiptop or
tsh, as well as by other genes in D. melanogaster.
Interestingly, the defects induced by reduced Antp activity in
O. fasciatus are in striking contrast to the transformations of
mouthparts to antennae seen when Scr and Dfd activity are
reduced (Table 1)
(Hughes and Kaufman, 2000).
These latter transformations have been used as evidence for a universal
mechanism of Hox specification of insect appendages
(Hughes and Kaufman, 2000
;
Casares and Mann, 2001
).
However, in O. fasciatus, Scr and Dfd apparently repress the
activity of antennal specifierin gnathal appendages while Antp does
not repress this activity in thoracic appendages. Thus, in O.
fasciatus, two mechanisms (Hox-dependent and a Hox-independent) exist for
specifying the identity of appendages. Additional factors (including
tiptop) must mediate the differences in the active mechanisms in
these tagma.
It should be noted that, while reduced Scr and Ubx
activity cause segmental transformations of identity (labial and A1,
respectively), the absence of such a transformation by reduced Antp
is not unexpected. The primary and most frequent transformation caused by
loss-of-function (hypomorphic and null) Antp alleles of D.
melanogaster is of the second thoracic segment (T2) toward the first
thoracic segment (T1). This is frequently seen as the formation of a
T1-specific structure, the denticle beard, in T2
(Wakimoto and Kaufman, 1981).
In our analysis, T1 and T2 of the first instar milkweed bug are homomorphic
(except for the Scr-dependent leg comb)
(Hughes and Kaufman, 2000
).
Therefore, a T1 to T2 transformation in milkweed bugs, as might be expected
for reduced Antp function, would be undetected at the level of the
cuticle. Other cuticle defects produced by loss-of-function Antp
alleles in D. melanogaster include disruptions in the denticle band
pattern, the formation of sclerotized tissue, and occasional rudimentary
denticle beards in T3. We did not observe comparable defects in our
investigations with O. fasciatus. However, their apparent absence may
be due to a low frequency of these defects, insufficient knowledge of the
detail of the O. fasciatus cuticle, or residual Antp
activity remaining in our experimental animals.
Evolution of the genetic mechanism for leg specification
Our discovery of significant variation in the mechanism of leg
specification can be added to a growing number of examples that suggest there
is greater regulatory diversity in the mechanisms underlying development than
could have been suspected from the high conservation of the regulatory genes
themselves or the processes they regulate. In this case, both O.
fasciatus and D. melanogaster form legs of similar composition,
and thus the processes of segmentation and specification are conserved.
However, the regulatory mechanisms governing these processes are not
conserved. This regulatory variation suggests how developmental mechanisms can
evolve (using regulatory changes, gene duplication, and divergence) without
compromising the organism. It is this kind of genetic variation that can
provide significant raw material for morphological evolution.
A path of evolutionary change, and possible transitional states, in the leg specification mechanism can be inferred by assuming that the ancestral state of insect leg development is better represented by O. fasciatus than by D. melanogaster. This assumption is reasonable, given that O. fasciatus appears to have retained the ancestral states of embryonic leg formation and two tiptop-related characters (i.e. genome content of tsh- family members and expression pattern). Combined with its more basal position on the phylogenetic tree, O. fasciatus seems more likely than D. melanogaster to have conserved the ancestral mechanisms of leg formation.
Therefore, we can describe the genetic changes required for the O. fasciatus mechanism of leg development to evolve into that of D. melanogaster. First, Antp acquired the ability to repress the antennal specifier (hth) in the distal leg and lost its role in distal segmentation. These changes might have been relatively simple. A mechanism for Hox genes (e.g. Scr and Dfd) to repress the antennal specifier already existed and the segmentation functions of Antp might be partially provided by tiptop. Second, the change in Antp function relaxed the constraints on tiptop, thereby allowing its function (including hth repression) to diverge. Finally, duplication and further divergence of the ancestral tiptop gene produced the tsh and tiptop genes of D. melanogaster.
Genetic evolution of leg specification from a ground state
The variation we have detected in the genetic control of leg development,
and specifically that the segmentation and specification activities of
independent genes evolve independently, impacts genetic models of the ground
state appendage and the evolution of its variations. By definition, the ground
state appendage is formed in the absence of all appendage-modifying selector
genes. In our proposed model (Fig.
7), the activities of at least three genes (Antp, hth,
and a tiptop-like activity) in D. melanogaster, and perhaps
additional genes in O. fasciatus, need to be eliminated to recreate
the ground state.
At this time, such a recreation has not been accomplished in any insect and the morphology of a recreated ground state appendage is difficult to predict. Significantly, even when formed in the absence of identity specifier activity, an appendage of an extant insect might have the identity of a ground state appendage, but it is unlikely to have the morphology of an ancestral ground state appendage because the segmentation and identity-specifying activities of selector genes evolve independently.
Although a recreated ground state that is leg-like or antenna-like would suggest that insect antennae are evolutionary modifications of primitive leg-like structures (or vice versa), one that has neither identity would suggest that both antennae and legs could be independent modifications of a more basic appendage type. Based on the requirement for segmentation activities in all appendage types, as well as the presence of distinct cephalic and thoracic appendages among all arthropods, we suggest a model in which the cephalic and thoracic appendages diverged from a segmented, but undifferentiated ground state. This divergence appears to predate the evolution of insects and it may be shared by all modern arthropods. In our example, the segmentation activities of the appendage selector genes evolved first, and the identity functions of the selector genes evolved later. Thus, in this example, the variable types of appendages would have evolved only after multisegmented, but relatively undifferentiated appendages were present.
Only further genetic investigations of additional genes in a greater variety of arthropod species will clarify the genetic evolution of appendage diversification. These investigations could verify the existence of a common appendage ground state and help identify the ancestral specifiers of appendage segmentation and identity in arthropods.
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ACKNOWLEDGMENTS |
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