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First published online 9 November 2005
doi: 10.1242/dev.02154

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Breakdown of abdominal patterning in the Tribolium Krüppel mutant jaws

Alexander C. Cerny^1,*, Gregor Bucher^1,*,, Reinhard Schröder² and Martin Klingler^1,

¹ Institute for Biology, Department Developmental Biology, Friedrich-Alexander-University Erlangen, Staudtstrasse 5, 91058 Erlangen, Germany
² Interfakultäres Institut für Zellbiologie, Universität Tübingen, Abt. Genetik der Tiere, Auf der Morgenstelle 28, 72076 Tübingen, Germany

View larger version (96K): [in a new window]   Fig. 1. Segmental position of the Tc'Kr expression domain. Wild-type embryos stained for Tc'Kr (brown, A-G) and Tc'eve (blue, B-E). As in the remaining figures, all embryos are oriented anterior towards the left. The gap domain of Tc'Kr arises at the posterior pole of the blastoderm (A, posterior pit stage). In early germ rudiments (B,C) Tc'Kr expression extends from the 2nd Tc'eve stripe to beyond the 3rd Tc'eve stripe. When the 2nd (D) and 3rd (E) Tc'eve stripes split into segmental stripes 2a, 2b, 3a and 3b, the Tc'Kr gap domain (brown bar) is demarcated by 2a and 3b, i.e. it covers the three thoracic segments (E). At later stages, secondary expression domains arise in the head and eventually in all segments of the extended germ band (F,G). md mandible; T1 to T3, thoracic segments 1 to 3; A9, 9th abdominal segment.

View larger version (59K): [in a new window]   Fig. 2. jaws is a mutation in the first Zn finger of Tc'Kr. (A) After repeated outcrossing of the jaws mutation (induced in a GA-1 background) with the SB wild-type strain, all adult beetles that were jaws mutant carriers are also heterozygous for polymorphisms at the Tc'Kr locus, indicating close linkage between the mutation and the polymorphism. The polymorphism `SB' represents a Tc'Kr 3'UTR sequence specific to the SB strain that can be detected as a 205 bp RFLP band. The polymorphism `Tiw' is specific to the Tc'Kr copy of the jaws-carrying chromosome and results in a 141 bp RFLP (this polymorphism originally had been identified in the Tiw-1 wild-type strain). Depicted is an agarose gel with 11 samples (of 80 total). As controls, AseI-digested DNA amplified from a SB animal (SB/SB), a Tiw-1 animal (Tiw/Tiw) and an animal that resulted from a cross between Tiw-1 and SB parents (SB/Tiw) are shown. (B) Tc'Kr gene structure; the four Zn fingers are shown as black boxes. (C) Sequence of the first Zn finger of Tc'Kr. In the jaws mutant, the 2nd histidine is altered to a tyrosine.

View larger version (132K): [in a new window]   Fig. 3. Phenotypic series for Tc'Kr: ventral views of first instar larvae (confocal image projections based on cuticle autofluorescence). (A,B) Wild-type first instar larva (A). (B) Enlarged view of the ventral head, with left maxilla and labium outlined in white. (C-E) Tc'Kr RNAi embryos of increasing phenotypic strength. (F) Tc'Krjaws mutant embryo. Appendages resembling maxilla are labelled with arrows, those resembling labial palps with arrowheads. In some embryos, the urogomphi and pygopodes, corresponding to the 9th and 10th abdominal segments, respectively, are also indicated. For detailed phenotypic description see Results. The embryo in F is at a higher magnification than those in A-E. mx maxilla; lb labium; T1 to T3, thoracic segments 1 to 3; A1 to A8, abdominal segments 1 to 8; pp, pygopod; ug, urogomphi.

View larger version (135K): [in a new window]   Fig. 4. Tc'Krjaws is an amorphic mutation, and the homeotic transformations in jaws are epistatic over those in Tc'gt RNAi embryos. (A) Tc'Krjaws mutant embryo in which the defective Tc'Kr mRNA additionally has been depleted by RNAi. The phenotype of this embryo is not stronger than in Tc'Krjaws alone, indicating that the Tc'Krjaws mutant itself fully inactivates the gene. (B) Tc'Krjaws mutant embryo in which the Tc'gt gene has been inactivated by RNAi. The normally present maxilla and labium are fully developed, followed by one ectopic maxillary and one labial segment. Thus, the homeotic effect of Tc'Kr inactivation is epistatic over that observed in Tc'gt RNAi larvae. Because Tc'gt also results in segmentation defects, the 2nd ectopic pair of maxillary and labial segments are incomplete or missing. Arrows indicate maxillary structures; arrowheads labial appendages; pp, pygopode. (C) Phenotype of a Tc'gt RNAi larva. In addition to abdominal segmentation defects, maxillary and labial segments are transformed into thoracic segments.

View larger version (88K): [in a new window]   Fig. 5. Hox gene expression in wild-type, Tc'Krjaws and Tc'gt RNAi embryos. (A-D) Tc'Dfd (purple) and Tc'Ubx (blue) in situ double staining in stage-matched wild-type (A,B) and Tc'Krjaws mutant (C,D) embryos. In the mutant (D), the anterior boundary of the Tc'Ubx domain recedes towards the posterior. Tc'Dfd is expressed in three strong and one weak domain of double segmental periodicity. The two ectopic domains with stronger expression correspond to the ectopic maxillary segments in Tc'Krjaws. (E-J) Tc'Scr (purple) and Tc'Antp (blue) staining of wild-type (E-G) and Tc'Krjaws mutant (H-J) embryos. Tc'Scr is also strongly expressed in two ectopic domains with double-segmental periodicity, corresponding to the two ectopic labial segments (J). In contrast to Tc'Ubx, Tc'Antp expression is not shifted in Tc'Krjaws germbands (H-J). (K,L) Tc'Scr (brown) and Tc'Antp (blue) in Tc'gt RNAi embryos. Tc'Antp expands towards anterior by two segments, which correlates with the fact that the maxillary and labial segments attain thoracic appearance in Tc'gt RNAi embryos. High-level expression of Tc'Scr in the labial segment is repressed in these embryos. Weak Tc'Scr expression in the maxillary segment of older embryos (L) probably corresponds to the weak expression seen in the prothoracic segment in wild type (G).

View larger version (120K): [in a new window]   Fig. 6. The first five Tc'eve stripes are formed normally in Tc'Krjaws. Ventral views of stage-matched wild-type (A-D) and Tc'Krjaws (E-H) embryos. The youngest wild-type (A,B) and all Tc'Krjaws (E-H) embryos have been triply labelled for Tc'eve (brown), Tc'en and Tc'gt mRNA (both blue); in the older wild-type embryos (C,D), only Tc'eve (brown) and Tc'en or Tc'wg (blue) are labelled. Tc'gt expression was used to identify mutant embryos: the posterior giant domain(s) present in wild-type germbands (A,B) are missing in Tc'Krjaws (E,F); black arrowheads indicate the position of these domains. In older Tc'Krjaws embryos, two ectopic Tc'gt domains appear in the first and third thoracic segments (grey arrowheads in F-H). In Tc'Krjaws mutant germbands (E,F), the primary eve stripes 3 and 4, and the secondary segmental stripes of eve2 form in a similar way to wild type (A,B) (stripes eve1a and eve1b are partially obscured by en stripes and the maxillary Tc'gt expression). Later on, eve3 splits into segmental stripes, while eve5 becomes detectable (C,G). The segmental stripes 4a and 4b remain imperfect in Tc'Krjaws, while eve5 forms a sharp anterior boundary (H).

View larger version (110K): [in a new window]   Fig. 7. Segment formation in Tc'Krjaws is not re-established during later stages of development. Embryos are doubly labelled for Tc'eve (brown) and Tc'wg RNA (blue). Numbers relate to canonical Tc'wg stripes, i.e. starting with the mandibular segment. (A-F) Wild-type embryos of increasing developmental age, stage-matched to Tc'Krjaws embryos (G-L; staging is based on the dynamic Tc'wg head expression). Compared with wild type (A-C), Tc'eve expression becomes irregular and prematurely fades in Tc'Krjaws embryos (G-I). Initially, only the seven anteriormost wg stripes (md to A1) are properly formed in Tc'Krjaws. Posterior to A1, several irregular or fragmentary wg stripes form (G-K), which then reorganize such that in more mature embryos (L) normal Tc'wg stripes 8-10 are frequently observed, consistent with the 10 differentiated segments in Tc'Krjaws larvae. No new Tc'eve or Tc'wg activity is evident at later stages, when in wild-type embryos the last Tc'wg stripes form (15 and 16 in our notation, which correspond to abdominal segments A9 and A10) (D-F). A non-segmental Tc'wg domain is present in the growth zone throughout development. This domain represents terminal fates and eventually becomes part of the proctodeum.

View larger version (30K): [in a new window]   Fig. 8. Regulation of Hox genes by Krüppel in Tribolium. The repressor activity of the anterior giant domain delimits the anterior border of Tc'Kr. Tc'Kr in turn acts as general posterior repressor for Tc'Dfd and Tc'Scr. The precise boundaries of these gnathal Hox genes are defined by pair-rule genes. Therefore, ectopic gnathal Hox gene expression in Tc'Krjaws is interrupted with double-segmental periodicity. Tc'Kr also determines the anterior border of Tc'Ubx through activation, whereas the anterior border of Tc'Antp is probably set by Tc'gt directly.