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First published online 17 March 2004
doi: 10.1242/dev.01073

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Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function

Gregor Bucher and Martin Klingler^*,

Department for Biology II, Ludwig-Maximilian-University Munich, Luisenstraße 14, 80333 Munich, Germany

View larger version (35K): [in a new window]   Fig. 1. Sequence of the Tribolium Giant ortholog. (A) Alignment of Tribolium and Drosophila Giant using Clustal W. Identical and similar amino acids are highlighted in black and grey, respectively. Longer sequence stretches without homology were omitted from the Dm'Giant sequence (indicated by dots, the number of omitted amino acids is indicated). The DNA-binding leucine zipper (large box) is 63% identical and 78% similar between both insects. The binding domain of the co-repressor CtBP (small box) is highly conserved, as are a number of additional motifs of unknown function. These conserved motifs upstream of the leucine zipper substantiate the orthology of Tc'Giant and Dm'Giant, as they are not present in other leucine zipper genes. Note that Dm'Giant is twice as large as the predicted beetle protein. (B) Alignment of the leucine zippers of Tc'Giant, Dm'Giant, Dm'CG4575 and Hs'HLF, the Human hepatic leukemia factor. Dashes indicate sequence identity. The position of the three nested PCR primers used for isolation of Tc'giant is given below.

View larger version (123K): [in a new window]   Fig. 2. Expression of Tc'giant in successive developmental stages. (A-D,F-K) Embryos are oriented anterior towards the left; (E) embryo viewed from the posterior pole. Blastoderm stages (A-E) are oriented dorsal side upwards. Germ band embryos (F-K) were dissected from the yolk and are shown in ventral view. (A-C) After initial ubiquitous maternal expression, Tc'giant forms an anterior domain comprising brain and gnathal segments, but excluding the anteriorly located extra-embryonic serosa. (C,D,F) In the maxilla, expression becomes stronger (black arrowhead) before anterior expression condenses (G-K) in a complex pattern in the brain. (D,E) A second domain arises de novo at the posterior pole of the late blastoderm and later splits in two domains that cover T3 and A2 (white arrowheads). See text for further details.

View larger version (125K): [in a new window]   Fig. 3. Double in situ hybridization with Tc'giant in brown and Tc'eve (A-D), Tc'hairy (E) and Tc'Krüppel (F-H) in blue. eve stripes are numbered, with a/b, indicating the secondary segmental stripes derived from the respective double-segmental primary stripe. (A-D) The Tc'giant stripes in maxilla, T3 and A2 coincide roughly with the first, third and fourth Tc'eve stripes, respectively, and mature in the same relation to each other: early Tc'eve stripes are shifted slightly anterior to the giant stripes. As the pattern matures, the stripes successively coincide (see stripe 3 in B and C). (E) Tc'hairy is expressed in a frame roughly complementary to Tc'eve. Its posterior expression borders initially overlap the Tc'giant stripes but the overlap fades with time and the borders eventually abut each other. The anterior borders of the hairy stripes always remain separated from Tc'giant stripes. (F) Tc'Krüppel appears at the posterior pole of the blastoderm within giant free tissue (compare with C in Fig. 2). (G,H) The posterior Tc'giant domain-arises right within the Tc'Krüppel domain and the genes remain co-expressed in the third thoracic segment (T3 in H). Strong mutual repression as described for the Drosophila orthologs seems unlikely for this region. In addition, this staining shows that Tc'Krüppel is not expressed posteriorly to T3.

View larger version (116K): [in a new window]   Fig. 4. Effect of RNAi gene knock-down on first instar larval cuticles. All larvae are shown anterior towards the left. (A) Wild-type larva with three leg-bearing thoracic segments (T1-T3) and eight abdominal segments. Two additional abdominal segments (A9 and A10) are fused to the telson and bear the urogomphi (u) and pygopods (p). In this lateral view, the mandibles (md) and the labium (lb) cannot be seen because they are covered by the maxilla (mx). (B-D) In all RNAi embryos, maxilla and labium are transformed to T1 and T2, respectively. Intriguingly, the thorax is shifted coordinately such that the mandibula is followed by segments displaying T1, T2 and T3 identity. The following two thoracic segments also have T3 identity. (B) In weak phenocopies, the transformation is not accompanied by segmentation defects. (C) Most giant phenocopies also display segmentation defects. In this specimen, five thoracic and four residual abdominal segments are formed, and the urogomphy are missing. Together, five segments are deleted. Because abdominal segments have identical cuticle pattern, it is not possible to determine which segments are missing. Often, the penultimate pair of legs is less well patterned or homeotically specified than the most posterior one (white arrowhead). (D) In this strong phenocopy, nine segments are deleted. Three thoracic segments are left and the presence of a pair of stomata (white arrowhead) indicates the presence of one abdominal segment. Even in such severely disturbed larvae, the terminal pygopods are usually present. (E,F) The gnathal transformation in a ventral view: antenna (at), labrum (lr) and mandibles (md) are not affected, but maxilla (mx) and labium (lb) are completely transformed to thorax. (F) Schematic representation of E; transformed maxillary appendages highlighted in grey. (G,H) In a few larvae, the transformation of the maxillary segment was not complete. Here, the lower appendage is transformed to leg, while the other appendage adopts an intermediate identity. Note that partial transformations are rarely observed in the maxillary but never in the labial segment. In no case were thoracic identities shifted only one segment towards anterior.

View larger version (15K): [in a new window]   Fig. 6. The distribution of cuticular segmentation defects for different concentrations of dsRNA (A-D) and morpholino oligos (E) are shown. Given is the absolute number of cuticles that lacked a certain number of segments. Only individuals that had been injected with an effective dose of dsRNA/morpholino were counted (as judged by the presence of anterior transformations). The dsRNA concentrations ranged within several orders of magnitude (2000 ng/µl in A to 7.5 ng/µl in D). Nevertheless, the observed dose effect was relatively mild (compare A through D). The proportion of injected embryos that developed cuticles decreased with dsRNA concentration: 20% with 2000 and 750 versus 50% with 75 and 7.5 ng/µl, respectively. Additionally, the proportion of cuticles that produced a phenotype increased with higher concentrations of dsRNA: 75% with 2000 and 750 versus 50% with 75 and 7.5 ng/µl, respectively. Injection of low amounts of the lowest concentration resulted in 80% wild-type cuticles, suggesting that the minimal requirement for dsRNA was approached with 7.5 ng/µl. (E) Although morpholino oligos inhibit gene function by a different mechanism and are chemically distinct, a similar range of deletions was observed.

View larger version (137K): [in a new window]   Fig. 5. In situ detection of engrailed and maxillopedia (mxp, ortholog of Dm'probiscipedia) in wild type (A,I) and in embryos depleted for Tc'giant by RNAi (B-H,J). Black arrowheads in A-H indicate the labial segment. The proctodeum (p) indicates completion of segmentation in D-H. (A) Wild-type germ band shortly before formation of the last (tenth) abdominal engrailed stripe. (B-H) In all germ bands analyzed, the first three segments were unaffected, suggesting, that in the head Tc'giant has a homeotic function. The T1 stripe was often disturbed or deleted in young embryos (stars in B and C), leading to an enlarged segment. By the end of segmentation, no defects are evident in the anterior thorax (D-H), suggesting that the embryo corrects for these early patterning defects. In some cases, the superfluous cells became assigned to the appendages that then appeared enlarged (white arrowheads in D and the close-up E). In cuticles, enlarged appendages were not observed, suggesting further correction. Segmentation is disturbed in a variable pattern in the region between T1 and A9. In germ bands with proctodeum formed (p), the number of deleted segments can be determined (D, 7; F and G, 8; H,4). (I) The Hox gene maxillopedia (red) is expressed in the appendages of the maxillary and labial segments (arrowheads). (J) In Tc'giant RNAi embryos, this expression is reduced or absent (arrowheads), confirming that Tc'giant knock down interferes with proper Hox gene regulation.

View larger version (31K): [in a new window]   Fig. 7. The expression domains of Drosophila and Tribolium gap genes. Head, thorax and abdomen are separated by vertical bars. (A) Fate map and gap gene expression in the Drosophila blastoderm. (B,C) Late blastoderm and early germ band stages in Tribolium (the photos to the right illustrate the stages represented by B and C). (B) In the Tribolium blastoderm only head and thoracic segments are specified. The posterior pole comprises the growth and patterning zone (growth z) and probably includes terminal cells (not shown). Extra-embryonic tissue has been omitted for simplicity. (C) During germ band growth, the posterior giant domain splits into stripes located in segments T3 and A2, far anterior of the segment primordia covered by Dm'giant (A). Although anterior expression domains of gap gene orthologs appear largely conserved (compare anterior domains of tailless, giant and hunchback), the posterior gap domains in Tribolium are shifted relative to the segment primordia. See discussion for further details.