The role of the segmentation gene hairy in Tribolium
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Hairy stripes in Tribolium are generated during blastoderm and germ band extension, but a direct role for Tc-h in trunk segmentation was not found. We have studied here several aspects of hairy function and expression in Tribolium, to further elucidate its role. First, we show that there is no functional redundancy with other hairy paralogues in Tribolium. Second, we cloned the hairy orthologue from Tribolium confusum and show that its expression mimics that of Tribolium castaneum, implying that stripe expression should be functional in some way. Third, we show that the dynamics of stripe formation in the growth zone is not compatible with an oscillatory mechanism comparable to the one driving the expression of hairy homologues in vertebrates. Fourth, we use parental RNAi experiments to study Tc-h function and we find that mandible and labium are particularly sensitive to loss of Tc-h, reminiscent of a pair-rule function in the head region. In addition, lack of Tc-h leads to cell death in the gnathal region at later embryonic stages, resulting in a detachment of the head. Cell death patterns are also altered in the midline. Finally, we have analysed the effect of Tc-h knockdown on two of the target genes of hairy in Drosophila, namely fushi tarazu and paired. We find that the trunk expression of Tc-h is required to regulate Tc-ftz, although Tc-ftz is itself also not required for trunk segmentation in Tribolium. Our results imply that there is considerable divergence in hairy function between Tribolium and Drosophila.
KeywordsSegmentation Pair-rule genes Tribolium Short germ embryogenesis
Drosophila hairy (h) forms part of the segmentation cascade at the level of the pair-rule genes (Nüsslein-Volhard and Wieschaus 1980; Ingham 1988). Mutations of h result in the deletion of the posterior part of every odd-numbered segment in the resulting larvae, thus reflecting a classical pair-rule phenotype (Jürgens et al. 1984). h negatively regulates the spatial expression of the pair-rule genes runt (Klingler and Gergen 1993), fushi tarazu (ftz; Carroll et al. 1988; Rushlow et al. 1989; Tsai and Gergen 1995), and paired (prd; Baumgartner and Noll 1990; Gutjahr et al.1993), and was therefore classified as primary pair-rule gene.
Hairy homologues occur also in vertebrates (called her or hes genes) where some of them are involved in the generation of the somites. The somites can be envisaged as the vertebrate analogs to the segments in insects, although segmentation does not proceed in the ectoderm as in insects, but in the mesoderm (Tautz 2004). Analysis of the process of somite formation revealed a fundamentally different regulatory mode for the vertebrate hairy homologues during the generation of these segmental units. Reflecting segmentation in a cellular environment, the process is based on cell signaling factors of the Notch/Delta and other signaling pathways. These regulate oscillating waves of expression of several genes across the growth zone, including some of the her or hes genes (reviewed in Rida et al. 2004; Giudicelli and Lewis 2004).
Intriguingly, a hairy homologue is also expressed during segmentation in the spider Cupiennius salei and functional analysis of the Notch/Delta pathway by RNAi shows strong disruption of segment formation (Stollewerk et al. 2003). This raises the possibility that oscillating expression of hairy homologues may be an ancestral feature of bilateria, and that the Drosophila pattern of direct regulation through transcription factor gradients is highly derived (Tautz 2004). It is therefore of particular interest to study hairy function in short-germband insect embryos, which also undergo a cellular rather than blastodermal mode of segmentation.
Tribolium embryogenesis can be considered as a typical representative of short germ embryogenesis. Tc-h is expressed in pair-rule stripes during blastoderm stage and germband extension (Sommer and Tautz 1993), which would suggest a pair-rule function. However, Choe et al. (2006) found in their functional studies of the pair-rule gene homologues in Tribolium no indication for a direct involvement of Tc-h in trunk segmentation, although they describe some phenotypic effects on head development. Their results have suggested that the pair-rule gene function in Tribolium differs is in many ways from Drosophila. They found that the homologues of even skipped, runt, and odd skipped form a regulatory circuit, regulating each other, as well as target genes, during the extension of the germband. These findings imply that the pair-rule machinery was subject to significant changes during insect evolution.
We have studied here the function of Tc-h in detail. We clarified whether we are indeed dealing with the right homologue and whether its expression pattern is reasonably conserved between more closely related species. We were further interested to study whether the emergence of Tc-h stripes during germband elongation might reflect oscillatory waves of expression. Finally, we wanted to understand its function during segmentation of the head region, as well as its interaction with other pair-rule genes.
Material and methods
Beetle handling and stock keeping
Beetle stocks were essentially kept as described by Berghammer et al. (1999). All experiments were performed using the T. castaneum wild type strain “San Bernadino” and a wild type strain of T. confusum, provided by Dick Beeman, Kansas State University, if not indicated otherwise. Flour was kept at 65°C overnight to prevent parasitic infections.
Cloning of Tribolium confusum hairy homologue
The hairy homologue from T. confusum was cloned by screening a genomic library cloned in Lambda Fix II provided by Sue Brown (Kansas). As probe we used a 383 bp subcloned PCR fragment obtained from T. confusum genomic DNA as template with the primers 5′AAYAARCCNATHATGGARAAR 3′ and 5′YTGNAGRTGYTTNACNGTCAT3′ covering the HLH region. The insert of a positive clone was subcloned as a NotI fragment into a plasmid vector and fully sequenced (Acc. No. EU819553).
Embryo collection and fixation
Eggs were collected from 0–48 h at 30°C to gather all developmental stages before dorsal closure. The embryos were rinsed with tapwater, mildly dechorionated for 1.5 min in 50% bleach and rinsed with tapwater afterwards to remove residual bleach. Fixation was performed in scintillation vials containing 3 ml PEMS (0.1 M Pipes, 2 mM MgSO4, 1 mM EDTA, pH 6.9), 6 ml heptane, and 4% formaldehyde on a shaking platform for 25 min. The water phase was then substituted for 8 ml methanol and the vial vigorously shaken for 30 s, resulting in devitellinization of the embryos by methanol shock. Undevitellinized embryos were mechanically devitellinized by squeezing them through a syringe using a 19 G needle. Embryos were kept at −20°C in methanol for subsequent analysis.
Parental RNAi experiments were performed according to Bucher et al. (2004) with slight modifications. Approximatly 200 female pupae were fixed to microscope slides using double-sided tape (Scotch 665). Pupae were taken off the slides after injection and transferred to “culture vials” containing full grain flour in order to facilitate eclosion. The first eggs were collected approximately 5 days after injection and incubated at 33°C for 4 days to allow full development in order to assess the amount and strength of phenocopies. Eggs were collected every 48 h and fixed for subsequent analysis by in situ hybridization. Once a week, a 24-h collection was allowed to fully develop and cuticle preparations were performed in Hoyer’s medium according to standard procedures (Berghammer et al. 1999) in order to monitor the phenotype/phenocopies over time. Double-stranded RNA was synthesized from PCR templates using the T7 MEGAscript RNAi Kit (Ambion) without additional annealing steps and injected at a concentration of 2 μg/μl in H2O with 10% Phenol red. The injection solution was thoroughly centrifuged at 13,000×g before injection to pellet any particles and reduce clogging of the needle.
In situ hybridization and antibody staining
Whole mount in situ hybridizations were performed according to standard protocols (Tautz and Pfeifle 1989; Klingler and Gergen 1993). Immunological staining was performed as described by MacDonald and Struhl (1986) with slight modifications. For the analysis of LacZ protein distribution in the transgenic lines, an additional signal amplification step using a secondary biotinylated antibody and the Vectastatin ABC HRP KIT (Vector Labs) was introduced to the protocol. For apoptosis detection using the anti cleaved caspase3 antibody (Cell Signalling Technology, Inc.), amplifications steps were omitted and staining was performed using a secondary alkaline phosphatase coupled antibody.
Analysis of hairy paralogues
Comparison to Tribolium confusum
To assess whether the appearance of the Tc-h stripes is conserved in a distantly related Tribolium species, we have cloned the orthologue of Tc-h from Tribolium confusum. It is known that the expression characteristics of her genes can differ substantially between fish species, i.e., these patterns can be subject to regulatory changes (Gajewski et al. 2006).
The overall alignment between the T. castaneum and T. confusum sequences shows a good conservation of the exons and a generally high divergence in non-coding regions, but with conserved blocks that may be related to functional elements (supplementary Fig. S2). This conservation–divergence pattern is comparable to the average patterns of gene comparisons between D. melanogaster and D. virilis, or zebrafish and Medaka and indicates, therefore, a similar evolutionary distance between T. confusum and T. castaneum.
The dynamics of stripe formation
In vertebrates, the stripe formation in the growth zone occurs in cyclic expression waves, emanating from the posterior end. This implies that a given cell goes through several on and off states before it becomes differentiated. Delta–Notch signaling is a major driver of this cyclic expression and this was also found for basal arthropods, such as spiders and millipedes. In Tribolium, we have so far not found any evidence for an involvement of Delta–Notch signaling in segmentation (Aranda 2006; supplementary Fig. S4). However, this would not rule out that cyclic expression occurs, since other signaling mechanisms might substitute Delta–Notch signaling in Tribolium.
Inspection of the expression pattern does not seem to provide clear evidence for an expression wave of hairy across the growth zone. Rather, it appears that one stripe arises after the other, with a more or less fast clearance of the inter-stripe region (see detailed pictures and description in supplementary Fig. S5). In zebrafish, it was possible to show progressive waves via careful timing of fixation times (Holley et al. 2000). However, our attempts to do this also in Tribolium failed, because one can not achieve sufficient synchrony of the egg lays.
Double stainings with the segmental marker engrailed provide a similar picture. engrailed is expressed during formation of the segmental borders and is not expected to be dynamic itself. Figure 3e–i presents the series of stages from the beginning of the formation of the 6th to the 7th engrailed stripe, i.e. the equivalent of the formation of one segment. The distance between the last engrailed stripe and the neighboring hairy stripe does not seem to change during this time. Only the embryo itself elongates and the space for the emergence of the next engrailed stripe is thus formed.
Functional analysis of the gene Tc-hairy
Using RNAi analysis, Choe et al. (2006) found in their study on pair-rule genes in Tribolium that Tc-h does not appear to have a primary function in segmentation of the trunk region. Our results based on parental RNAi (pRNAi—Bucher et al. 2004) confirm this finding in principle, but we find apparently pair-rule-specific functions of Tc-h in the formation of the head segments.
Given the intricate regulation of Tc-h in the growth zone, as well as the conservation of this pattern in T. confusum, it seems surprising that Tc-h does not appear to have a function in the formation of the abdominal segments. Potentially, this could be due to a redundancy with the function of the homologue of deadpan, which is expressed in similar stripes as Tc-h (see above). However, pRNAi experiments with this gene did not yield abdominal segmentation phenotypes, neither in single injections, nor in double injections with Tc-h (data not shown). Hence, the lack of a trunk segmentation phenotype for Tc-h does not appear to be due to a redundancy with another hairy-like gene.
A pair-rule function of Tc-h at blastoderm stage
Depletion of Tc-h leads to induction of apoptosis in anterior segments
Regulation of target genes
The function of hairy in Tribolium remains enigmatic, although our results clarify several points. The apparent lack of function during the trunk segmentation process (Choe et al. 2006) is not due to a redundancy with another hairy homologue in Tribolium. The expression of Tc-h during trunk segmentation could alternatively have been due to an accidental enhancer capture from another pair-rule gene. However, this can now also be ruled out. First, there is no other pair-rule gene homologue close to Tc-h in the genome sequence (the neighboring genes are the “signal recognition particle receptor beta subunit” and the “nicotinic acetylcholine receptor subunit alpha2”) and second, the expression is conserved in T. confusum, which would be very unlikely, if it would have no function.
Interestingly, expression and function of Tc-h during blastoderm is compatible with a pair-rule function. The mandibular and the labial segments correspond to the anterior parts of the first and second Tc-h stripe at blastoderm and they are the ones that are most sensitive to a loss of function of Tc-h. This is in line with a classic pair-rule function and is also not in contrast to the results by Choe et al. (2006) since the formation of the head segments was not specifically addressed in this study. Our analysis of the hunchback function in Tribolium (Marques-Souza et al. 2008) has also suggested that the patterning of the head segments should be seen separately from the trunk segments. This is also a classical conclusion from comparative morphology (reviewed in Tautz 2004). Hence, it seems possible that only the head segment specification is a conserved feature of hairy function between Drosophila and Tribolium. This would imply that the striped expression of hairy, although conserved in other Arthropoda, might have been recruited to the trunk segmentation process only in the lineage leading to Drosophila. We can only speculate about the specific role of the hairy stripe formation in the trunk. Most likely, it is related to some differentiation process in the developing nervous system.
The strong phenotypes in Tc-h knockdowns lead to a loss of adjacent segments in the head and thorax region, i.e., this goes beyond a pair-rule phenotype. This phenotype appears to involve specific cell death in the respective region, although the region that is affected in the strongest cuticle phenotypes is larger than the region where we observe cell death (i.e., it includes part of the thorax). This raises the possibility that Tc-h may have an organizer function in this region, which would determine the fate of surrounding cell groups. It should be noted that the first Tc-h stripe remains expressed much longer than the subsequent stripes, a feature that is also conserved in T. confusum. Interestingly, this expression overlaps with a specific expression of Tc-delta in this region (supplementary Fig. S4) supporting the notion of signaling processes being activated there. In any case, it appears that this possible organizer function occurs subsequently to the segmentation function.
The emergence of the Tc-h stripes in the growth zone is superficially similar to the oscillatory expression of hairy homologues in vertebrates, but none of our experiments supports the notion that there is an expression wave across the growth zone. The fact that the more long-lived lacZ reporter gene shows essentially the same stripe pattern as the wild type expression is probably most telling. If the expression would be a moving wave, the interstripe cells should also express lacZ, i.e., the resolution of the stripes should be blurred. This is not observed, although the expression appears to be somewhat dynamic with respect to the apparent clearance of stripes in the growth zone (Fig. 3). Given that no specific cell-division activity is apparent in this region (supplementary Fig. S6), we have to conclude that cell migration and intercalation are likely to play a role in the generation of this pattern. Similar as in vertebrates, the Tribolium embryo undergoes a convergent extension process during germband growth (compare supplementary Fig. S5). The cellular interactions and movement patterns during convergent extension processes are still not fully understood (see Keller et al. 2008 for a recent discussion), but it appears unlikely that they are involved in the generation of the stripes in the growth zone. Hence, further work will be required to understand this patterning mechanism and the general process of segment formation in short-germband embryos.
This work was supported by the DFG (SFB 572 and SFB 680). H. M. was a fellow of the International Graduate School of Genetics and Functional Genomics in Cologne.
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