The role of the segmentation gene hairy in Tribolium

Abstract

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.

Fig. 1

Phylogenetic comparison of hairy-like sequences. Neighbor-joining distance tree using full amino acid sequences of all genes with an HLH domain and a C-terminal WRPW motif from Drosophila (Dm-), Tribolium (Tc-) and mouse (Mm-). The hey gene homologues from these species serve as outgroup. Bootstrap values higher than 60% (out of 1,000 runs) are shown at the branches. Accession numbers are added to the gene names where these are not unequivocal

Fig. 2

Comparison of hairy expression between T. confusum (left) and T. castaneum (right). Equivalent stages are compared to each other. The largest difference occurs at the early germ band stage (a versus e), where stripes are more distinct and more persistent

Fig. 3

Dynamics of hairy stripe formation in the growth zone. a–c In situ hybridization with the Tribolium hairy probe, d–i double staining with an engrailed antibody (brown). a to c shows three apparently successive stages during hairy stripe #5 formation. The embryos are aligned with respect to the anterior borders of stripes #2 and #3 (grey lines). d–i Successive stages during the formation of engrailed stripe 7, with an enlargement of the relevant section of the growth zone. Ordering of the stages is based on the appearance of the engrailed staining and the appearance of the enlargement of the growth zone

Fig. 4

a–e Expression pattern of the Tc-h-lacZ reporter gene construct KN8.8 (Eckert et al. 2004), as determined by in situ hybridization with the lacZ probe. The construct drives the expression of eight stripes, faithfully mimicking the wild type expression pattern. f–j Show an antibody staining for LacZ in the respective reporter line. Note that both the lacZ in situ (a–e) as well as the antibody staining (f–j) show formation of precise stripes (black arrows) with no staining in the inter-stripe region (white arrows), i.e., no fusions are detected as expected in the case of an expression wave k–o. Wild type Tc-h staining in comparable stages. Comparison of the expression in stripe #2 and #3 in b, g, and l shows higher stability of the lacZ transcripts and protein compared to the endogeneous hairy transcripts

Fig. 5

Cuticle preparations of Tc-h pRNAi knockdown embryos. a Wild type cuticle for comparison, lateral view. b–e Phenotypic series of cuticles, lateral view. In b, the distance between the antennae and the maxillae is strongly reduced, suggesting that the mandibular segment is missing (see also magnification in i). c–e Show successively stronger phenotypes were the entire head region is missing (c) and thoracic segments are affected (d–e). f, g Show magnifications of the head region in wild type (f) and intermediate Tc-h phenotypes (g) in a ventral view. In g, no mandibular structures are visible and the labium is strongly reduced. h–k Magnifications of the head region of a wild type (h) and three Tc-h pRNAi cuticles (i–k) in a lateral view. i Shows a magnification of the phenotype in b, note the distance between the antennae and the maxilla which lie directly adjacent in contrast to the wild type situation (h). In j, the labrum and the mandibles are absent whereas in k the labrum is present, but an antenna is formed only on one side, the maxillae are both present. Frequencies of phenotypes obtained with 2 μg/μl Tc-h dsRNA (b) 45%; (i–k) 29%; (c) 7%; (d) 3%; (e) 2%; 16% wild type, n = 114

Fig. 6

Analysis of the Tc-h knockdown phenotype during development. a–e Tc-gsb expression in wild type. Tc-gsb is expressed in the posterior part of the specified segments and serves as segmental marker. The positions of the mandibular and labial segments are marked with white arrows in a and b. f–j Tc-gsb expression in embryos depleted for Tc-h by pRNAi. The embryos depicted in f and g show disruption of Tc-gsb expression in the mandibular and labial segments (white arrows, compare to a and b). In older stages, the stripe corresponding to the maxillary segment is also affected (h–j), suggesting a secondary loss of this stripe, since a loss at early stages was never observed. Furthermore, head development appears to be significantly disturbed (compare i–j with d and e). Note the asymmetries of the phenotype in the embryos in h and j which we observed frequently

Fig. 7

Effects of Tc-h knockdown on apoptosis during development. Antibody staining against cleaved caspase3 (as apoptosis marker) in wild type (a and b, e and h) and in Tc-h pRNAi (c and d, f, g, and i). In wild type embryos apoptosis is only detected in a few cells in young stages (a). In older stages, apoptotic cells are found in the headlobes, the ventral midline and a few dispersed cells (b). Tc-h depleted embryos show patches of staining in the gnathal region as well as the first thoracic segment (c–d, f–g). Furthermore, disruption of the segments fusing the headlobes to the trunk region is seen, probably resulting in the detachment of all anterior segments during further development (f–g, black arrows). Note that no stained cells are detected along the ventral midline (h and i). Magnifications of the posterior segments of the embryos depicted in c and d. The midline appears wider and deeper in Tc-h knockdown embryos (black arrow) and large cells are found lining the border to the lateral regions of the embryo (white arrow, compare h and i). Segment identities abbreviated as follows: mandible, (md); maxilla. (mx); labium, (lb); T1–T3, thoracic segments

Fig. 8

Effects of Tc-h knockdown on Tc-ftz expression. a–e Wild type expression of Tc-ftz and (f–j) in Tc-h pRNAi knockdowns. Tc-ftz is expressed in a pair-rule-like fashion complementary to Tc-h. Expression is detected in the maxillary segment of young germbands (a); thereafter, seven additional stripes appear sequentially, de novo near the tip of the expanding germband (b–e). In later stages, a second expression appears in the developing nervous system (e). In Tc-h knockouts, the anterior border of the first stripe seems unaffected (f) while the remaining stripes are formed, but appear to fuse with ongoing development (g–j). Interestingly, expression in the anterior region does not cease, as seen for the wild type expression. Instead, the expression persists throughout the segmentation process

Fig. 9

Effects of Tc-h knockdown on Tc-prd expression. a–f, m Wild type expression of Tc-prd and (g–l, n) in Tc-h pRNAi knockdowns. Expression of Tc-prd starts as a circumferential stripe in the blastoderm in the maxillary segment, which is followed by a second stripe of expression in the segment corresponding to T1 before the formation of the germ rudiment (a–b). The following stripes appear de novo at the anterior border of the growth zone (c–f). Shortly after the appearance of the stripes, they split into a segmental expression pattern. At the end of segmentation Tc-prd is strongly expressed in the mandible and the maxilla (m). In Tc-h knockdown embryos, the first Tc-prd stripe appears normally in the blastoderm (g), whereas the second stripe is formed directly adjacent to the first (h), suggesting a loss of the intermediate labial segment. During further development, these stripes do not split as seen in wild type. Instead, a weaker stripe is seen in the region of the presumptive labial segment (compare i and c, white arrow). In some cases, a loss of expression in the maxillary segment is observed during further germband growth (j, black arrow). The remaining stripes appear unaffected (k–l). m–n Show magnifications of the head region in wild type (m) and Tc-h depleted embryo (n). Expression in the mandible is not detected and no mandibular nor labial structures are formed (compare n and m)