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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Dev Genes Evol. Author manuscript; available in PMC 2011 November 2.
Published in final edited form as:
PMCID: PMC3206738
NIHMSID: NIHMS48841

Tribolium Wnts: evidence for a larger repertoire in insects with overlapping expression patterns that suggest multiple redundant functions in embryogenesis

Abstract

wingless (wg)/Wnt family genes encode secreted glycoproteins that function as signaling molecules in the development of vertebrates as well as invertebrates. In a survey of Wnt family genes in the newly sequenced Tribolium genome, we found a total of nine Wnt genes. In addition to wg or Wnt1, Tribolium contains orthologs of the vertebrate Wnt5–7 and 9–11 genes. As in Drosophila, Wnt1, 6 and 10 are clustered in the genome. Comparative genomics indicates that Wnt9 is also a conserved member of this cluster in several insects for which genome sequence is available. One of the Tribolium Wnt genes appears to be a member of the WntA family, members of which have been identified in Anopheles and other invertebrates, but not in Drosophila or vertebrates. Careful phylogenetic examination suggests an Apis Wnt gene, previously identified as a Wnt4 homolog, is also a member of the WntA family. The ninth Tribolium Wnt gene is related to the diverged Drosophila WntD gene, both of which phylogenetically group with Wnt8 genes. Some of the Tribolium Wnt genes display multiple overlapping expression patterns, suggesting that they may be functionally redundant in segmentation, brain, appendage and hindgut development. In contrast, the unique expression patterns of Wnt5, Wnt7 and Wnt11 in developing appendages likely indicate novel functions.

Introduction

During embryogenesis, all insects reach a phylotypic stage when all the future segments are present, although there are often species-specific differences in the events leading to this stage. In the long-germ insect Drosophila melanogaster, segmentation of the embryo occurs almost simultaneously during the blastoderm stage. In contrast, in short-germ insects such as Tribolium castaneum, most segments are added sequentially from the posterior growth zone. These different mechanisms of segmentation might require diverse sets of genes, some of which are likely to be evolutionarily conserved among insects and possible all arthropods.

The Wnt gene family encodes secreted signalling molecules that control cell fate specification, proliferation, polarity and movements during animal development. Each Wnt protein has one or more sites for N-linked glycosylation and up to 24 cysteines, the positions of which are highly conserved. Most of the deduced proteins are about 350 to 380 amino acids in length, with over 100 conserved residues scattered across the entire sequence (Nusse and Varmus 1992).

Phylogenetic comparison between Wnt family genes from diverse animal phyla including chordates, echinoderms, insects, cnidarians and bilaterians have identified 12 phylogenetically conserved Wnt subfamilies (Prud’homme et al. 2002; Kusserow et al. 2005). In addition, orphan Wnt genes have been found in Drosophila (Ganguly et al. 2005), nematodes and humans (Kusserow et al. 2005). These studies, combined with the availability of the full sequence of the nematode Caenorhabditis elegans and human genomes, plus the insects genomes including that of the fly Drosophila melanogaster, the mosquito Anopheles gambiae and the honey bee Apis melifera, allow comparison with the set of genes present in other organisms, including the newly sequenced Tribolium genome (The Tribolium genome consortium).

Within these genomes, Wnt genes are generally dispersed, however a cluster of three genes was found to be highly conserved when comparing Wnt genes from human and Drosophila (Nusse 2001). In Drosophila, wingless (wg), Wnt6 and Wnt10 are immediately adjacent to each other and are transcribed in the same orientation, with no recognizable genes between them. On human chromosome 2q35, Wnt6 and the adjacent gene Wnt10A, are transcribed in the same direction, while Wnt1 (the ortholog of Drosophila wg) and Wnt10B, immediately adjacent to each other on chromosome 12q13, are transcribed of opposite strands. Since Wnt10A and Wnt10B are thought to be the result of a recent duplication, these observations suggest that there was a common ancestral cluster of Wnt genes containing Wnt1, Wnt6 and Wnt10 (Nusse 2001, Sullivan et al. 2007).

In insects, the expression pattern and segment polarity function of the canonical wg gene are widely conserved, but little is known about other Wnt molecules. In Drosophila, wg gene expression in the presumptive head region and in a caudal ring is detected in the early blastoderm (Baker, 1988). The expression of the wg ortholog in Tribolium (Tcwg/Wnt1) is first seen during the blastoderm stage at the posterior pole of the egg. Shortly thereafter stripes appear in the presumptive head lobes (Nagy and Carroll 1994). Later, wg is expressed in both insects in segmental stripes. In Drosophila, the stripes are one cell wide and appear simultaneously in response to the activity of pair-rule genes (Baker 1988; Ingham & Hidalgo 1993). In Tribolium, these stripes develop in a sequential order, as the germ band elongates (Nagy and Carroll 1994). The first Tcwg stripe appears in the presumptive mandibular segment, followed sequentially by stripes in the other gnathal segments, three thoracic segments and ten abdominal segments. Additional expression domains in the developing head appear on an independent schedule (Nagy and Carroll 1994). Although the expression dynamics of Tcwg expression differ from Drosophila, the timing and position of segmental wg stripes are consistent with their regulation by pair-rule genes (Choe et al. 2006). In contrast to this detailed analysis of Tcwg, there are no data concerning the extent to which other Wnt family genes are conserved in Tribolium or how they might be expressed. However, in Drosophila, studies on Wnt genes have implicated Wg signalling in several developmental processes in different life stages (Wnt5: Fradkin et al. 1995; Wnt6, 10: Janson et al, 2001; Wnt7: Kozopas et al. 1998; Wnt9: Cohen et al. 2002; WntD: Gordon et al. 2005, Ganguly et al. 2005).

Here we describe nine Wnt family genes in the Tribolium genome, four of which are located in a highly conserved cluster. We also analyse the expression pattern of the newly identified Wnt genes in Tribolium and discuss possible differences and/or redundancies in gene function among the Wnt gene family members.

Materils and Methods

Isolation of Wnt genes from Tribolium castaneum and in situ hybridization

Tribolium Wnt genes were identified by BLAST analysis of the genome. Candidate genes were amplified from cDNA, cloned into TOPO4 vector (Invitrogen) and sequenced to confirm their identity. 400–600-bp fragments where used as template for digoxigenin-labeled RNA probes. Tribolium eggs were collected and whole-mount in situ hybridization was performed as previously described (Klingler and Gergen 1993; Tautz and Pfeifle 1989; Brown et al. 1997).

Nomenclature

The insect Wnt genes are named to match previous nomenclature for vertebrates and other metazoans.

Alignment and phylogenetic analysis of Wnt gene sequences

Wnt protein sequences were downloaded from NCBI (htpp://www.ncbi.nlm.nih.gov/). ClustalW within the MEGA 3.0 software was used to generate a multiple alignment of the protein sequences, which was subsequently manually improved by considering only the conserved regions of the proteins. Neighbour joining analysis in MEGA 3.0 was performed using a bootstrap value of 1000.

Results

Wnt family genes in Tribolium

We found a total of nine Wnt genes in the Tribolium genome. Alignment of the predicted Tribolium proteins with other insect and human Wnt proteins was used to identify conserved blocks of amino acid sequence for phylogenetic analysis. The neighbour-joining algorithm in the MEGA phylogenetic software package identified Tribolium homologs of most of the human Wnts (Fig. 1a). In addition to an ortholog encoding wg or Wnt1, Tribolium contains obvious orthologs of vertebrate Wnt5–7 and 9–11. Similar to other sequenced insect genomes (Prud’homme et al. 2002), orthologs of Wnt2, 3 or 4 were not found in the Tribolium genome (Fig. 1).

Fig. 1Fig. 1
a Neighbour-joining phylogenetic tree of Wnt gene family. Analysis in MEGA 3.0 was performed using a bootstrap value of 1000. Tribolium Wnt8/D is clustered with Drosophila WntD and other Wnt8 family members. WntA from Tribolium, Apis and Anopheles also ...

Among insects with completed genomes examined to date, Tribolium appears to contain the largest complement of Wnt genes (Fig. 1). Compared to the nine Wnt genes in Tribolium, seven have been identified in honeybee (Apis), seven in Drosophila and six in Anopheles (Fig. 1b).However, the quality of each genome project may influence the number of Wnt genes identified. Orthologs of wg/Wnt1, Wnt5, 6 and 10 have been identified in all of these insects. A Wnt11 ortholog is found in Apis and Tribolium, but not in Anopheles or Drosophila. Of these insects, Anopheles is the only one that appears to be lacking a Wnt7 ortholog, while Apis is the only one that appears to be lacking a Wnt9 gene.

Drosophila and Tribolium each contain an additional Wnt gene, that appear to be related to one another (Fig. 1a). The Drosophila gene, WntD is required for proper development of the mesectoderm (Ganguly et al. 2005; Gordon et al. 2005). In phylogenetic anlaysis, the length of the Drosophila WntD branch suggests that this gene is diverging more rapidly than its Tribolium counterpart. In addition, these insect WntD genes phylogenetically group in a larger clade with Wnt8 subfamily genes, consistent with the previous analysis of Drosphila WntD (Ganguly et al. 2005; Gordon et al. 2005) and supporting the hypothesis that these are divergent members of the Wnt8 subfamily.

The ninth Tribolium Wnt gene has no obvious Drosophila ortholog. Surprisingly, it groups with both Anopheles WntA and Apis Wnt4 (Fig. 1a); two Wnt genes that have not been previously grouped together. This group also includes WntA genes from polycheate and cnideria (Fig. 1a), suggesting that these three insect Wnt genes are most likely WntA subfamily members. The presence of WntA genes in Anopheles, Apis and Tribolium suggests that this Wnt gene family, although not represented in Drosophila, is conserved in insects. and perhaps among arthropods. Indeed, a spider Wnt gene has been grouped with WntA family genes in a previous analysis (Prud’homme et al. 2002). Similar results were obtained using the same multiple sequence alignment in tree puzzle (Strimmer and von Haeseler, 1996), which uses a maximum likelihood method to derive the phylogenetic tree (data not shown).

A conserved Wnt gene cluster

Most of the Tribolium Wnt genes are dispersed throughout the genome. However, four genes, including wg/Wnt1, Wnt6, Wnt9 and Wnt10, are closely linked on LG 5 (Fig. 2).They are all clustered within a 52 kb region that contains only one other unrelated gene, which encodes CDP diglyceride synthetase. Wnt9 is located 5′ of Wnt1, while Wnt6 and Wnt10 are located 3’ of Wnt1.Wg and Wnt10 are transcribed off one strand while Wnt6 and Wnt9 are transcribed off the opposite strand (Fig. 2).

Fig. 2
Insect Wnt gene clusters. The relative size, order and orientation of Wnt genes, including Wnt9, Wnt1/wg, Wnt6 and Wnt10 are compared. Non-Wnt genes found in the Tribolium and Drosophila clusters are also shown The Drosophila non-Wnt gene is not annotated, ...

A similar Wnt gene cluster exists in other insects (Nusse 2001; Dearden et al. 2006). In Drosophila, Wnt9 (initially identified as DWnt4, which groups with Wnt9 family members in phylogenetic analysis (Prud’homme et al. 2002; Fig. 1a), is located 5′ of wg with a single gene (CG13785) between them. This Wnt cluster is contained within 120kb (Fig. 2) on chromosome 2L. The non-Wnt genes in the Drosophila and Tribolium clusters are not related to one another. In Apis, Wnt1, Wnt6 and Wnt10 are also clustered (Dearden et al. 2006) within an 80kb interval (Fig. 2). Here the cluster is composed of only three Wnt genes, since Wnt9 appears to be absent from the Apis genome. This Wnt gene cluster seems to be highly conserved (Sullivan et al. 2007), thus loss of Wnt9 may reflect the draft status of the genome sequence rather than a true gene loss. In Anopheles, Wnt1, 6 and 10 are clustered in a genomic interval spanning 100kb. Wnt9 is located more distantly 5′ of Wnt1, such that all four genes are located within 150kb and no other genes have been predicted within this interval (Fig. 2). In Aedes aegypti, the gene order is conserved (Wnt1-Wnt6-Wnt10), but they do not seem to be clustered as they span an interval of 420 kb (NCBI, Aedes aegytpi mapviewer).

The orientation of genes within these insect Wnt clusters differs (Fig. 2), suggesting that lineage specific reorganisation of the Wnt cluster has occurred during evolution. The orientation of Wnt genes 1, 6 and 10 are the same in Tribolium, Anopheles and Apis. In Drosophila, the orientation of Wnt6 is reversed relative to the other two genes. The orientation of Wnt9 relative to the other three genes is identical in all the insects for which Wnt9 orthologs have been identified. These results suggest an ancestral insect cluster existed in which Wnt9 and Wnt6 are transcribed off one strand while wg/Wnt1 and Wnt10 are transcribed off the other. Despite some degree of rearrangement, the overall structure of this cluster has been conserved arguing for a functional constraint on Wnt-cluster maintenance (Nusse, 2001; Sullivan et al. 2007).

Wnt expression patterns

To analyze the possible function of different Wnt genes during embryogenesis, we assayed the expression patterns of the nine Tribolium Wnt genes by in situ hybridization during embryogenesis (Fig. 3 and and4).4). The early blastoderm stage embryos contain head and tail primordia, as well as the presumptive gnathal and thoracic segments, while the late germband embryos also contain nine abdominal segments, sequentially added from the posterior growth zone. We followed the expression of the newly identified Wnt genes through these embryonic stages, comparing them with the expression of wg (Figs. 3 and and44).

Fig. 3
Only three Tribolium Wnt genes show expression in blastoderm embryos (whole mount in situ hybridization). Anterior pole points to the left. a wingless / Wnt 1. b Wnt 8/D. c Wnt A.
Fig. 4
Expression of Tribolium Wnt genes in germband embryos (whole mount in situ hybridization). Ventral views, anterior to the left

wg/Wnt1 is expressed in Tribolium during embryogenesis at the blastoderm and germband stages (Nagy and Carroll 1994 and Fig. 3a, 4a–c). Initially, wg is expressed in a domain at the posterior end of the embryo (Fig. 3a), and then laterally in each head lobe (Nagy and Carroll, 1994). In the germ rudiment, wg is expressed at the anterior dorsal rim of the head lobes and posteriorly in a subterminal position that marks the developing hindgut and leaves the posterior pole free of expression (Fig. 4a). During axis elongation, segmental stripes of wg expression appear one at a time, anterior to the growth zone (Fig. 4b), and extend ventrally into the appendages as they develop (Fig. 4c). Later, wg expression appears in patches along the lateral edge of the embryo in each segment (Fig. 4c), as well as in the labrum and faintly in the stomodeum while the expression domains expand in the developing brain (Fig. 4c).

Similar to wg, Wnt5 is expressed during germband elongation in the lateral head lobes, in segmental stripes and in the posterior growth zone (Fig. 4d, e). Wnt5 expression also extends ventrally into the developing limbs (Fig. 4f).In contrast to wg, the domains of Wnt5 expression in the head lobes are broader (compare Fig. 4b and e) and the segmental stripes cross the midline (compare Fig. 4e, f with b, c). The domain of Wnt5 expression in the posterior growth zone is also broader than that of wg and includes the posterior tip of the growth zone (compare Fig. 4e with b), but fades after germband elongation (Fig. 4f). Interestingly, Wnt5 is expressed in the tips of the developing limbs. Both wg and Wnt5 are expressed in the labrum of older embryos (Fig. 4c, f).

As the germband begins to elongate, Wnt6 is expressed similar to wg (albeit much fainter) in lateral domains of the head lobes, segmental stripes, and a subterminal region of the posterior growth zone (Fig. 4g).Wnt6 is also expressed along the ventral edge of the developing limbs and in segmental patches along the lateral edges of the elongating germband similar to wg (Fig. 4g–h). In the abdomen, faint spots of expression in the CNS appear to overlap with wg expression (compare 4h with c). Unlike wg, Wnt6 is expressed in the anlagen of the Malpighian tubules (arrowhead in Fig. 4h). During germband retraction, Wnt6 expression appears in the anlagen of the fore- and hindgut, as well as in the precursors of the tracheal openings (Fig. 4i).

Wnt7 expression is first visible during germband elongation in segmental stripes that resemble wg stripes (Fig. 4j). As they develop, these stripes do not cross the ventral midline, similar to wg. However, later they extend into the dorsal mesoderm of the developing limbs, where wg is not expressed (Fig4l).Wnt7 is expressed in only a few cells of the developing brain (Fig. 4k, l), some of which may be outside the wg domain.

Wnt8/D is one of the two other Wnt genes expressed in the blastoderm embryo (Fig. 3b), where it is expressed in a small domain at the posterior pole. As the germ rudiment condenses and elongates, Wnt8/D expression resolves into spots on either side of the ventral mesoderm in the posterior growth zone that are connected by a faint subterminal u-shaped line of cells (Fig. 4m). During germband elongation, the posterior domains of wg and Wnt8/D appear to partially overlap. Unlike wg, expression of Wnt8/D fades completely after germband elongation (data not shown).

Unlike wg, Wnt9 is not expressed during early stages of embryonic development. In the retracted germband, Wnt9 marks cells in the fore- and hindgut at the border between the ectodermal and endodermal derivatives of the gut (Fig. 4n–p).

Wnt10 expression also resembles that of wg in many aspects. It first appears in a single stripe that marks the mandibular segment (Fig. 4q). During germband elongation, the segmental stripes of Wnt10 do not cross the ventral midline, but ventrally extend into the developing appendages (Fig. 4r, s). Unlike wg, these stripes are not uniform but appear as dots. Three prominent neurons in each hemisphere of the brain that express Wnt10 may also fall within the wg domains (Fig. 4r).

Wnt11 expression domains have little in common with those of wg. During early germband extension, Wnt11 is expressed in cells of the serosa (Fig. 4t). A little later, Wnt11 appears in the distal tips of newly formed gnathal and thoracic appendages, with exception of the mandibles (Fig. 4u). At this stage, Wnt11 expression is also observed faintly at the border of the dorsal ectoderm (Fig. 4u). As the limbs develop, Wnt11 is expressed in a ring at midlength and in a ventral patch slightly proximal to the distal tip, whereas in the maxilla and labium no such refinement is seen, and the mandibles remain free of expression (Fig. 4v). Wnt11 is also expressed in a few cells of the antennae and strongly in the anal plate (Fig. 4v).

Similar to wg and Wnt8/D (although fainter), expression of WntA first appears at the posterior end of the embryo during the blastoderm stage (Fig. 3c). As the germ rudiment condenses, additional WntA expression appears in a mandibular stripe and in lateral head lobe domains (Fig. 4w). Soon after the germband begins to elongate, WntA is expressed in segmental stripes that initially resemble wg stripes, but quickly resolve into small domains on either side of the ventral midline (Fig. 4x). Head- and appendage specific WntA expression is also observed during later stages (Fig. 4x, y), however expression in the posterior growth zone fades during germband elongation (compare Fig. 4w and 4x) expression in the hindgut anlagen appears after the germband is fully extended (Fig. 4y). In the limbs, WntA expression extends proximally from the distal tips (Fig. 4y). WntA is also expressed in segmentally reiterated patches along the lateral edge of the embryo and in the labrum.

Discussion

Phylogenetic characterization of insect Wnt genes

Tribolium has the largest complement of Wnt genes among insects for which genome sequence data is available (Fig. 1b). We identified nine Wnt genes in Tribolium while seven have been identified in Apis and Drosophila, and six in Anopheles. The higher complexity of this important signalling gene family in the beetle Tribolium compared to the more derived dipteran insect Drosophila reflects specific gene losses in the lineage leading to Drosophila since the divergence of these groups appr. 250 million years ago (xx). Eleven Wnt family members have been found in phylogenetically basal cnidarians (Kusserow et al. 2005).Drosophila has representatives of seven Wnt gene subfamilies (Wnt1, 5–7, 9, and 10) including a diverged Wnt8 gene (WntD). Although the complement of Wnt genes reported for Anopheles is smaller, (Wnt7, 11 and D appear to be missing) this dipteran possesses a WntA family member, suggesting that the common ancestor of diptera contained at least eight Wnt genes. When compared to the other insects (Fig. 1b), Apis and Tribolium share the presence of Wnt11, which is absent from the dipteran species. However, Wnt9, which is present in the other three species, appears to be missing in Apis. All together, these data indicate that at least nine of the 12 different Wnt genes known so far from other studies (Kusserow et al. 2005) are represented in insects in different combinations. Thus, with its large repertoire of Wnt genes, Tribolium is the only insect to date to include representatives of all the known insect Wnt genes.

An insect Wnt gene cluster

Despite species-specific Wnt gene losses, all insects examined to date, including Tribolium, contain the evolutionarily conserved cluster of Wnt1, Wnt6 and Wnt10. In addition, most insects for which genome data are available have a fourth gene (Wnt9), which also clusters with Wnt1-Wnt6-Wnt10. The Wnt9 gene is located 5′ of Wnt1 and the four-gene cluster spans 70–150 kb, depending on the insect species. We also found evidence for a cluster of Wnt genes in the genome of Bombyx mori. In this Lepidopteran insect, Wnt1, Wnt6 and Wnt10 are located within 60 kb of one another in the same order as in other insects (NCBI BLAST results, data not shown). The Wnt9 ortholog is also located 5′ of Wnt1, resulting in a four-gene cluster of 99 kb, supporting our suggestion that a four-gene Wnt cluster may be ancestral to insects. It is interesting to note that in the current assembly version of the Bombyx genome, all four genes are oriented in the same direction. The conservation of this Wnt cluster during evolution implies that these genes may be co-ordinately regulated.

Wnt gene synexpression groups

The nine Tribolium Wnt genes display distinct expression patterns. However, overlapping tissue-specific expression patterns suggests that Wnt genes may be functionally redundant in some aspects of embryonic development. In the discussion below we classify these genes into synexpression groups that share partially overlapping expression domains in the head, segments, growth zone and/or limbs. However, the intensity and developmental profile of each gene exhibits differences that are often subtle, but in some cases more dramatic, suggesting they are also involved in gene-specific functions.

Expression in the embryonic head /brain

wg expression in the developing head lobes of the differentiated blastoderm, is soon followed by expression of Wnt5, Wnt6 and WntA as the germband begins to elongate. The wedge-shaped domains of Wnt5 are broader and more intense than those of the other genes, while the domains of Wnt6 and WntA are fainter. Wnt7 and Wnt10 are also expressed in the head, although they appear later and are restricted to a few neurons in the developing brain.wg and WntA are both expressed in the labrum as it develops.

Segmental aspects of Wnt gene expression

In addition to wg/Wnt1, Wnt5, 6, 7, 10 and A are expressed segmentally during germband elongation. While the segmental stripes of Wnt6 are very faint and fade early during axis elongation, wg/Wnt1, Wnt5 and Wnt10 show much stronger expression that lasts until axis elongation has ended. On the other hand, Wnt7 is not strongly expressed in every abdominal segment until late in germband extension.

Wnt gene expression in the growth zone

Various Wnt genes are expressed in the posterior region of the embryo, in the anlagen of the hindgut or more broadly in the growth zone including wg/Wnt1, Wnt5, Wnt6, Wnt8/D, Wnt9 and WntA. Although very faintly expressed, Wnt6 resembles wg/Wnt1 in that its early expression occupies a subterminal position. In contrast, early germband expression of Wnt5 and WntA covers the posterior tip of the embryo. While posterior expression of Wnt5 and Wnt8/D fade during germband retraction, continued expression of wg and Wnt6 in the anlagen of the hindgut is joined by expression of Wnt9. Posterior WntA expression is the most dynamic, fading from the growth zone during germband elongation, but reappearing in the hindgut during retraction. These expression patterns suggest these genes may function in germband extension as well as growth and differentiation of the integument.

Limb specific expression of Wnt genes

wg/Wnt1 is strongly expressed ventrally along the proximo-distal axis of the growing limb buds in a domain shared by Wnt5, 6, and 10. In contrast, expression of Wnt7 in the leg dorsal mesoderm suggests a distinct function. In young stages Wnt5 11 and A are each expressed in the limb buds and resolve into distinct patterns as the limbs mature. Wnt5 expression continues in a small cap at the tip of the limb in addition to expression along the ventral edge. Wnt11 expression fades at the tip but develops into a ring at midlength and slightly more distal as a ventral patch. WntA expression extends more proximal than Wnt5, but is excluded from the distal tip. A more intense ring of WntA expression appears at midlength, similar to Wnt11. These complex dynamics suggest Wnt signalling may be required in the development of several different limb features.

Wnt gene expression in the blastoderm

In addition to the genes expressed in overlapping domains during germband elongation and retraction, we can consider another group of Wnt genes that are expressed at the blastoderm stage. This group includes wg/Wnt1, Wnt8/D and WntA. They are all expressed in the posterior region of the embryo. The expression of Wnt8/D is largely complementary to that of wg/Wnt1, suggesting they may be only partially redundant in function. Expression of wg/Wnt1 and Wnt8/D quickly resolves into specific domains in the germ rudiment suggesting more specialized functions, while expression of WntA remains broad.

Genes in the Wnt cluster belong to similar synexpression groups, supporting the hypothesis that these genes may be co-regulated. Wnt1, 6 and 10 display overlapping expression patterns in the head, limbs and segments, while Wnt1, 6 and 9 are expressed in the developing gut. In Drosophila, Wnt6 and 10 are not expressed segmentally, but are expressed in the gut (Janson et al. 2001). DWnt9 has been implicated in the regulation of cell movement in the developing ovaries, but is not required for embryonic development (Cohen et al. 2002). Most Drosophila larvae lacking Wnt9 activity appear outwardly normal, but die as first or second instars. Our analysis of Wnt9 in Tribolium suggests a role in gut development, which, if conserved, might help explain the larval lethal phenotype observed in Drosophila.

The expression patterns of other Wnt genes do not appear to be largely conserved between Drosophila and Tribolium, suggesting they may be required for different functions in each insect. Wnt5 is expressed in the labrum and the primordial of the thoracic limbs of both insects. Later it is expressed strongly in the CNS in Drosophila (Fradkin et al. 1995), but not Tribolium. During embryogensis, Wnt7 is expressed segmentally in both insects, but in flies it is expressed in dorsolateral and ventral patches (Russell et al. 1992), while in beetles it is expressed in segmental stripes. In Drosophila, Wnt8/D is expressed at both poles of the egg at the blastoderm stage, while in Tribolium it is only expressed at the posterior end. In Drosophila it is expressed continuously in the ventral mesectoderm, and is required for proper mesoderm formation (Ganguly et al. 2005). In Tribolium it continues to be expressed at the posterior end of the growth zone during germ-band elongation.

Wg signalling has been implicated in germband extension from the posterior growth zone in Gryllus bimaculatus, and Oncopeltus fasciatus, but wg/Wnt1 itself does not appear to be the ligand (Miyawaki et al. 2004; Angelini and Kaufman 2005). As described above, the expression of several Wnt genes in Tribolium appears to overlap in the posterior growth zone (Wnt1, 5, 6, A and D), providing several ligand candidates to activate the wg signalling pathway involved in germband extension. Careful investigation of single and multiple RNAi experiments will be required to sort out the functions of the Tribolium Wnt genes in this domain.

The overlapping expression patterns of Tribolium Wnt genes suggest they participate in similar functions, perhaps redundantly. On the other hand, minor differences observed in the temporal and spatial expression of each gene within a synexpression group (eg. varied expression in the growth zone) might imply functional discrepancies. Functional analysis of each Tribolium Wnt gene is required to determine the relationship between expression patterns and functions. Furthermore, it may be necessary to analyze multiple genes simultaneously to elucidate genes that function redundantly.

By accessing the genome sequence we have completed a comprehensive analysis of the Wnt gene family in Tribolium. We found a larger complement of Wnt genes in Trioblium, which was used to provide a framework for a comparative genomic analysis of Wnt genes in insects. Our results suggest that the insect repertoire of Wnt genes includes nine of the more broadly conserved Wnt gene subfamilies:wg/Wnt1, 5–7, 9–11, A and a highly diverged Wnt8/D.

Acknowledgments

The work of R. Bolognesi, L. Farzana and S. Brown is supported by NIH grant HD29594. A. Beermann and R. Schröder thank R. Reuter for continuous support, T. Mader for technical assistance and the German Research Council (DFG) for funding (SCHR 4435/3-1/3/2).

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