1 1Interfakultäres Institut für Zellbiologie, Universität
Tübingen, Abt. Genetik der Tiere, Auf der Morgenstelle 28, 72076
Tübingen, Germany
2 Institut für Genetik, Universität zu Köln, Weyertal 121, 50931
Köln, Germany
* Author for correspondence (e-mail: reinhard.schroeder{at}uni-tuebingen.de)
Accepted 5 November 2003
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SUMMARY |
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Key words: Tribolium, Sp8, Leg elongation, Allometric growth, Serrate
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Introduction |
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The molecular basis of limb development has been studied intensively in
Drosophila. The anlagen of the adult appendages develop during
embryonic and larval stages as flat, set-aside cell nests, the imaginal discs.
Initially, they become subdivided by the signalling proteins Hedgehog (Hh),
Decapentaplegic (Dpp) and Wingless (Wg). Gradients of Dpp and Wg activate its
downstream target genes homothorax (hth) in the proximal,
and Distal-less (Dll) and dachshund (dac)
in the distal part of the leg disc
(Morata, 2001). At present,
the leg gap genes Dll, dac and hth are thought to be
sufficient for the formation of all the adult leg segments in
Drosophila by activating their target genes
(Rauskolb, 2001
). Recently, a
Sp-like gene has been isolated in Drosophila
(D-Sp1) that is expressed in a Dll like fashion in the leg
imaginal discs (Wimmer et al.,
1996
). This raises the question whether possibly more genes are
required early for the process of leg formation. In contrast to
Drosophila, the red flour beetle Tribolium
(Sokoloff, 1972
) and many
hemimetabolous insect species, such as the cricket, the grasshopper and
gryllus (Inoue et al., 2002
;
Jockusch et al., 2000
;
Niwa et al., 2000
;
Panganiban et al., 1994
), show
a more ancestral mode of appendage formation. In these species, the formation
of an appendage starts already early during embryogenesis as a small outgrowth
from the body wall, the limb bud (Brown et
al., 1994
). As development proceeds, the leg continuously
elongates and the leg segments differentiate before hatching. Despite the
morphological differences, a hierarchical subdivision of the appendage anlage
takes place in both species Drosophila and Tribolium
(Prpic et al., 2001
;
Rauskolb, 2001
) and orthologs
genes are required for this process
(Abzhanov et al., 2001
;
Beermann et al., 2001
;
Prpic and Tautz, 2003
;
Prpic et al., 2001
).
We describe the isolation, the expression pattern and the function of the
D-Sp1 ortholog from the beetle Tribolium, termed
T-Sp8. We found that T-Sp8 belongs to the Sp-class of
zinc-finger transcription factors that have been isolated from nematodes,
insects and vertebrates (Kaczynski et al.,
2003). Functionally, Sp-like genes are involved as transcriptional
regulators in the segmentation process, growth control, tissue differentiation
and neoplastic transformation. Members of this protein family share a highly
conserved Cys2-His2 zinc-finger protein motif that has been shown to bind to
GC-rich promotors (Kaczynski et al.,
2003
). We show that T-Sp8, together with its
Drosophila ortholog D-Sp1, can phylogenetically be grouped
close to the vertebrate Sp7 and Sp8 genes. In the embryo,
TSp8 expression was observed in segmental stripes prior to the
formation of the appendages and, like the gene Distal-less
(Beermann et al., 2001
), during
the complete process of limb development. Double staining of T-Sp8
with Dll revealed co-expression of both genes in more distal regions,
and exclusive expression of T-Sp8 in the proximal leg. In the early
limb bud, T-Sp8 was shown to be uniformly expressed. As the limb
elongated, ring-like expression domains developed sequentially. The number of
T-Sp8 expression domains correlated with the final length of the
appendage. We observed a remaining uniform expression in short appendages,
such as the labium, whereas in the antennae and in the legs, two and four
T-Sp8 expression domains, respectively, developed. A knock-down of
T-Sp8 function via RNAi led to a shortening of the appendages from
the head and the thorax. Affected legs retained proximal, medial and distal
values, and therefore TSp8 cannot be designated as a leg gap gene.
Adult beetles that displayed the T-Sp8 RNAi phenotype as larvae, also
had dwarfed legs and shortened antennae. We conclude that T-Sp8 is
required for the differential outgrowth of the body appendages and thus
contributes to shape the insect limb.
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Materials and methods |
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Isolation and cloning of T-Sp8 and T-Serrate
PCR fragments of the Sp family have been obtained using degenerate PCR
primer directed against a part within the zinc-finger region (Sp-f,
5'CAYATHGGN GARMGNCCNTTYMARTG 3'; and Sp-r,
5'TGNRTYTTCATRTGYTTYTTNARR TGRTC 3'). The resulting 176 bp PCR
products were cloned in TA-vectors (Invitrogen) and sequenced. The complete
cDNA sequence was generated with 3' and 5' RACE reactions using
SMART technology (Clontech). The Serrate ortholog has been isolated
from Tribolium using the degenerate PCR primer DL2 and DL2re/DL3re as
described (Stollewerk, 2002).
The PCR products (597 bp) were subcloned and verified by sequencing. The
GenBank Accession Numbers for T-Sp8 and T-Serrate are
AY316682 and AY453651, respectively.
Sequence alignment and phylogenetic analysis
Clustal W alignments were obtained with the LASERGENEDNASTAR® package
and phylogenetically analyzed using TREE-PUZZLE
(Strimmer and von Haeseler,
1996).
The amino acid positions for the alignment shown in Fig. 1B are (GenBank Accession Number/position of amino acids): Tribolium castaneum T-Sp8, AY316682/287-398; Drosophila melanogaster DSp1, AAF46519/333-444; Homo sapiens Sp8-Hs, XP_166519/290-401; Gallus gallus Gg, CAC84905/amino acids 586-694; Mus musculus Sp3Mm, AAC16322/amino acids 540-648; ostMm: NP_569725/263-374; HsSp4, NP_003103/621-730; HsSp1, AAF67726/597-706; UKLF, AB015132/197-302; TIEG1, U21847/amino acids 341-452; TIEG2, AF028008/amino acids 366-477; EZF, U70663/365-470; BTEB2, D14520/112-218; btdDmel, NP_511100/303-414; SP7Hs, NM_152860/366-377; SP5mm, NP_071880/270-379; and SP6, XP_064386/300-413. The amino acid positions for the alignment shown in Fig. 1C are: Tc Sp, pos 27-72; Dm Sp1, 14-59; Hs Sp 8, 7-56; Rn Sp 8, 149-198; Hs Sp 7, 7-53; Mm Sp5, 23-72; Hs Sp4, pos 39-88; Hs Sp 2, 19-66; Hs Sp 1, 52-101.
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|
RNA interference
Double-stranded (ds) RNA corresponding to nucleotide position 1-1325 of the
T-Sp8 gene was prepared and injected at a concentration of 500
ng/µl into pupae or embryos as described
(Bucher et al., 2002;
Schröder, 2003
). The same
dsRNA preparation was used for parental and embryonic RNAi experiments.
Cuticle preparation and in situ hybridisation for analysing embryos, larvae and adults
Larval and adult cuticles were embedded in Hoyer's medium
(Van der Meer, 1977). Adult
beetles were boiled in 10% KOH prior to embedding. In situ hybridisation was
carried out as previously described (Tautz
and Pfeifle, 1989
) using labelled riboprobes
(Klingler and Gergen,
1993
).
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Results |
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T-Sp8 is dynamically expressed in the appendages during the complete process of limb formation
Prior to the formation of the limb buds, T-Sp8 is expressed in
every segment in an embryo undergoing germ band elongation. Like the
Dll ortholog in Tribolium
(Beermann et al., 2001),
T-Sp8 is expressed in the body appendages from the beginning of bud
formation onwards (Fig. 2).
During the successive stages of leg growth, T-Sp8 expression resolves
into two ring domains: one proximal (T-Sp8prox) and one
distal (T-Sp8dist). The distal domain initially also
covers the leg tip (Fig. 2B-D),
but retracts quickly to establish a subterminal ring that persists until the
end of leg growth. During further leg outgrowth, a third, slightly weaker
T-Sp8 expression domain (T-Sp8med) intercalates
between the primary rings (Fig.
2F). At the end of the leg elongation process,
T-Sp8prox has stretched out and a weak fourth ring appears
at its distal boundary (Fig.
2J). T-Sp8/Dll double labelling reveals that
both genes are expressed at the same time in the growing
(Fig. 2K) and in the fully
elongated leg (Fig. 2L).
T-Sp8dist and Dll are partially co-expressed in
the distal part of the leg. The other expression domains of both genes border
each other with no considerable overlap
(Fig. 2L). In the head
appendages of the Tribolium embryo, T-Sp8 expression
characteristically reflects the length and fate of the respective limb.
T-Sp8 is strongly expressed in the antennae, the maxillae and the
labium but only weakly, diffuse and transiently in the labrum and the mandible
(Fig. 2G). As in the leg, an
initially uniform expression pattern develops into ring domains in the
antennae and the telopodite of the maxillae. Until the end of embryogenesis,
these limbs stretch out and develop into `long' appendages. In contrast to the
even longer thoracic legs, no additional T-Sp8 domain develops in the
antennae or maxillae. Rather, the proximal antennal T-Sp8 domain
ceases early (Fig. 2G-I) and
only the distal domain remains strong until the antenna reaches its final
size. The distal domain in the antenna is different to that in the leg in that
it never retracts from the tip, whereas the distal T-Sp8 domain in
the maxilla forms as a subterminal ring
(Fig. 2G). In the `short' head
appendages - the labial palps - uniform T-Sp8 expression remains at
high levels and does not resolve into ring domains. The two labial buds do not
grow out but fuse to build the unpaired labium at the end of embryogenesis.
T-Sp8 is only faintly expressed in the pleuropodia, the appendages of
the first abdominal segment with leg-like character
(Lewis et al., 2000
)
(Fig. 2A-D). Expression in the
pleuropodia ceases at a stage when the thoracic legs reach
70% of their
final length (Fig. 2E). In
contrast to the legs, the pleuropodia do not grow out further and do not
contribute to a cuticular structure of the larva. Instead, these limbs become
internalized later during development and function as a hatching gland.
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Dwarfed SpRNAi legs are composed of proximal, medial and distal positional values
At the cuticle level, the strong T-Sp8 RNAi phenotype shows a
severe shortening of the proximodistal axis. It remains unclear which parts of
the wild-type leg contribute to the T-Sp8RNAi leg.
Therefore we analysed the expression of the leg gap genes dachshund
and Distal-less in the developing embryonic leg. The expression
pattern of these genes in the wild type can be used as marker for proximal
(dachshund, dac) (Prpic et al.,
2001), medial (dachshund)
(Dong et al., 2001
;
Mardon et al., 1994
) and
distal (Distal-less) (Cohen et
al., 1989
; Sunkel and Whittle,
1987
) positional information. A small spot of Dll
expression at the tip of the T-Sp8RNAi leg proves the
presence of the most distal limb fate (Fig.
5B). The proximal (S-spot) and part of the distal (P-region)
dac expression domains in such legs represent proximal and medial
positions along the proximodistal axis
(Fig. 5D)
(Prpic et al., 2001
).
|
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Discussion |
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T-Sp8 is required for appendage elongation and is involved in the regulation of allometric growth
T-Sp8 expression starts early during germ band elongation in a
segmental pattern even before a limb bud is seen. At the beginning of bud
formation, T-Sp8 expression is uniform in all the appendage anlagen
including the mandibles. This suggests an important function of T-Sp8
for setting up the proximodistal (PD) axis rather than secondarily subdividing
the growing appendages like the dachshund (dac) gene that is
expressed for the first time when they are substantially elongated
(Abzhanov et al., 2001;
Inoue et al., 2002
;
Prpic et al., 2001
). As the
limbs start to grow out, segmental expression of T-Sp8 ceases.
During limb elongation, T-Sp8 remains to be expressed until the end of appendage differentiation. The development of the T-Sp8 expression pattern during appendage elongation is different, depending on limb identity. In appendages that do not elongate, such as the labrum and the labium, T-Sp8 remains uniformly expressed. Despite developing a distinct expression domain, T-Sp8 contributes to the outgrowth of these limbs: in T-Sp8 RNAi embryos the region distal to the dac expression domain is lost in the labrum (Fig. 7).
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The differential expression of T-Sp8 together with the short leg
RNAi phenotype suggests that T-Sp8 directs the extent of the
outgrowth of an appendage. In this way, T-Sp8 regulates the length of
the different limb types of an individual in relation to body size. Therefore,
T-Sp8 can be accounted for as a component of a genetic framework
involved in allometric growth (Stern and
Emlen, 1999). Such a limb-specific allometry obviously contributes
to the phenotype of an individual. During evolution, changes in the regulatory
region of genes like T-Sp8 could result in an altered expression
profile (e.g. more rings) and thus lead to changes in limb-length. This
phenotypic change could then lead to a change in the ability of using new
ecological resources and can be seen as a first step in the evolution towards
a new species.
Besides the appendage specific expression, T-Sp8 transcripts can
also be seen in the brain and in cells of the peripheral nervous system. That
animals treated with dsT-Sp8-RNA hatch and even survive to adulthood
can be explained by a redundantly acting Sp-like gene in these cells.
A likely candidate is a buttonhead ortholog that also belongs to the
Sp gene family, but has not been isolated from Tribolium so far. In
Drosophila, D-Sp1 and buttonhead are partially functional
redundant in the nervous system during post-blastodermal stages of
embryogenesis (Wimmer et al.,
1996). Redundancy of D-Sp1 and btd also applies
to the process of leg formation. Only flies without D-Sp1 and
btd function develop a short-leg phenotype
(Estella et al., 2003
). The
fact that such a phenotype has been obtained in Tribolium with double
stranded T-Sp8-RNA alone suggests, that a putative
buttonhead ortholog in the beetle is not involved in leg formation in
parallel to T-Sp8. This function might have evolved recently during
the evolution of the higher diptera.
Regulation of T-Sp8
How the Sp8 orthologs specifically contribute to limb outgrowth in
Drosophila and Tribolium is unclear. Based on the following
observations, Sp8 could be connected to the Notch signalling pathway.
In Drosophila, activated Notch and its ligand Serrate are
expressed like buttonhead and D-Sp1 in rings in the leg
imaginal disc (de Celis et al.,
1998; Estella et al.,
2003
), suggesting that these genes are functionally related.
Furthermore, the impairment of Drosophila Notch function results in
shortened adult legs (de Celis et al.,
1998
), as this is seen in legs lacking both D-Sp1 and
btd function (Estella et al.,
2003
). In Tribolium, the Notch ligand Serrate is
expressed in the same way as T-Sp8 in rings in the fully elongated
larval leg (Fig. 8B). It is
tempting to speculate that the T-Sp8 RNAi phenotype could be due to a
failure of Notch activation because the control of cell proliferation mediated
by Notch signalling has been found in both invertebrates and vertebrates
(Kenyon et al., 2003
;
Rao and Kadesch, 2003
).
Indeed, we find that the number of Serrate rings in T-Sp8
RNAi legs is reduced (Fig.
8C,D) showing that T-Sp8 function is directly or
indirectly required for initiating the Notch signalling pathway.
|
Further targets of Sp-like genes involved in the regulation of
cell proliferation have been described for other members of the Sp-KLF gene
family (Alpy et al., 2003;
Black et al., 2001
).
Indirectly, T-Sp8 could specify groups of cells that specifically
respond to signals like hormones or growth factors by changing their rate of
cell proliferation and/or by cell growth.
Based on the analysis of the RNAi leg phenotype, we suggest that one target
of T-Sp8 is the Distal-less gene. Our hypothesis is based on
the observation, that: (1) the strongest affected
T-Sp8RNAi leg also lacks the most distal structure, the
pretarsal claw that is Distal-less dependent
(Fig. 3E); and (2) all of the
proximal and most parts of the distal Dll expression domain are
missing in slightly weaker T-Sp8 legs
(Fig. 5B). A dramatic
shortening of the limbs therefore might be cause by a failure of
Distal-less activation. However, Distal-less seems not to be
required for T-Sp8 expression, as embryos homozygous mutant for the
strongest Dll allele Sa-8
(Beermann et al., 2001) still
show T-Sp8 expression on their leg stumps (not shown). Hox genes that
control segment identity are among the candidates acting upstream of
T-Sp8 and could lead to the limb specific modulation of
T-Sp8 expression. To test this hypothesis, Hox-binding sites within
the upstream sequences of T-Sp8 have to be identified and transgenes
with altered binding sites have to be tested in beetles and flies.
Does the SpRNAi-leg phenotype reflect the evolutionary ground state of a leg?
The evolutionary ground state of a limb has been proposed by Snodgrass as
an `undivided lobe or tubular outgrowth of the body wall, serving as an aid in
locomotion' (Snodgrass, 1935).
Morphologically, strongly affected T-Sp8RNAi legs fulfill
these requirements. But according to Snodgrass, a leg representing the ground
state is composed of only proximal and distal pattern elements. The
T-Sp8RNAi legs we have shown to include proximal, medial
and distal positional values may therefore reflect the state of a more
advanced ground state. We suggest calling such an appendage the `Ur-limb'. The
evolutionary invention of genes such as Sp, the co-option of an
already existing Sp8-like gene for the process of limb-elongation or
the addition of `ring-elements' to the regulatory region of an already
existing Sp gene required for the process of limb development have
contributed to stretch proximal, medial and distal positions apart from each
other to result in the elongated `modern' leg.
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ACKNOWLEDGMENTS |
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