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First published online 14 January 2004
doi: 10.1242/dev.00974

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The Sp8 zinc-finger transcription factor is involved in allometric growth of the limbs in the beetle Tribolium castaneum

¹ 1Interfakultäres Institut für Zellbiologie, Universität Tübingen, Abt. Genetik der Tiere, Auf der Morgenstelle 28, 72076 Tübingen, Germany
² Institut für Genetik, Universität zu Köln, Weyertal 121, 50931 Köln, Germany

View larger version (79K): [in a new window]   Fig. 1. Sequence alignment of Sp-like proteins. (A) Comparison of the Sp8 orthologs from Tribolium and Drosophila. Serines and threonines are only moderately enriched and glutamine rich regions are not present in the deduced T-Sp8 protein. These regions are present in the Drosophila ortholog and are thought to be targets for post-translational modification (serines/threonines) or to be essential for transcriptional activation (glutamines) (Bouwman and Philipsen, 2002). (B) Alignment of the Btd-box and the adjacent zinc-finger region of different species; the cysteines (C) and histidines (H) structuring the zinc fingers are marked in green in A. (C) A comparison of the N-terminal Sp-box reveals a closer relationship of the insect sequences to Sp8 than to Sp7. The Sp-box is suggested to function in regulating the proteolysis of Sp factors or alternatively as a motif for interaction with a repressor (Bouwman and Philipsen, 2002).| indicates identical amino acid positions, - refers to gaps. Hs, Homo sapiens; Gg, Gallus gallus; Mm, Mus musculus; Rn, Rattus norvegicus.

View larger version (24K): [in a new window]   Fig. 6. Phylogenetic analysis of zinc-finger proteins that belong to the Sp class. A Tree-Puzzle analysis based on a Clustal W alignment places the T-Sp8 gene (boxed) together with its ortholog from Drosophila melanogaster (D-Sp1) close to the Sp7/osterix and the Sp8 (Bouwman and Philipsen, 2002) genes from vertebrates (Clustal W alignment; btd-Dm: buttonhead/Drosophila melanogaster was used as outgroup).

View larger version (99K): [in a new window]   Fig. 2. T-Sp8 expression in wild-type Tribolium embryos. Successively older stages are shown from A-F. (A) T-Sp8 is expressed in segmental stripes in the complete embryo prior to limb bud formation. Only the thoracic segments (T1-3) and abdominal segment 1 (A1) are shown. (B) T-Sp8 is uniformly expressed in the young limb bud (arrowhead indicates the pleuropodium, the appendage of abdominal segment 1). (C) T-Sp8 resolves into two rings but is still seen at the limb tip (arrowhead indicates the pleuropodia). (D) T-Sp8 begins to retract from the limb tip (arrowhead indicates the pleuropodia). (E) The process of leg elongation continues. No T-Sp8 expression is detectable in the pleuropodia (arrowhead). (F) A third, slightly weaker T-Sp8 domain (arrow) intercalates between the primary rings. (G) T-Sp8 expression in the head (same embryo as in D). Two ring-shaped expression domains can be seen in the antennae (arrowheads) and the outer branch of the maxillae, the telopodit (arrows). T-Sp8 expression is strong in the labial buds (Lb) and diffuse in the labrum (Lr). One small distal positioned T-Sp8 spot is visible in the mandibles (Md) that presumably marks a sensory organ. (H) T-Sp8 expression in the head (same embryo as in E). Strong T-Sp8 expression is seen at the distal tip of the antennae (arrowhead) whereas in the rest of this limb, T-Sp8 expression is diffuse. (I) T-Sp8 expression in the head (same embryo as in F). In the antennae, only the distal tip shows T-Sp8 expression (arrowhead). In the maxillae the proximal T-Sp8 domain is still visible (arrow). (J) The three rings of T-Sp8 expression are now fully developed (solid arrows) and a fourth ring appears (outlined arrow). The staining in the pleuropodia (arrowhead) is due to a known artefact. (K,L) Double labelling of T-Sp8 (in blue) and Distal-less (Dll, in red) expression reveals that the proximal T-Sp8 ring lays outside of the distal Dll expression domain (arrowhead indicates the pleuropodia in J).

View larger version (131K): [in a new window]   Fig. 3. Cuticle preparations of wild-type and T-Sp8 RNAi larval legs. (A-E) The joint between coxa and trochanter is easily identifiable by its heavy cuticularized structure (arrow). + indicates the presence of two bristles of different length (`mother and father') that are indicative for the coxa. The arrowhead indicates the tip of the pretarsal claw. (A) The leg of a wild-type first instar larvae is subdivided into the coxa (Cx), the trochanter (Tr), the femur (Fe), the tibiotarsus (Tita) and the pretarsal claw (Ptc). (B) Cuticle of a T Sp8RNAi leg: weak phenotype. The coxa (Cx) is not affected; the segment distal to the coxa appears to be of mixed character. (C) Cuticle of a T-Sp8RNAi leg: strong phenotype. The coxa is shorter than in the wild type, where the joint has developed partially. (D) Cuticle of a T-Sp8RNAi leg: very strong phenotype; only part of the pretarsal claw present (arrowhead). (E) Cuticle of a T-Sp8RNAi leg: very strong phenotype, no pretarsal claw present. Note that the coxa is also proximally shortened (C-E). (F) Cuticle of a larval head (wild-type) Lb: labium, arrowheads indicate the palps of the labium, the arrow indicates the telopodite (t) of the maxilla (Mx); e, endite of the maxilla. Ant, antenna; so, sense organ of the antennae; m, middle part; a, arista. (G) Effect of T-Sp8 RNAi on the head appendages. The asterisk marks the affected limbs: the distal palps of the labium are missing, the telopodite of the maxilla is strongly reduced. In weaker affected embryos, a reduction of the length has been observed (not shown). In the antenna, the middle segment is strongly reduced in size. No effect was seen in the mandibles. Only half of the head is shown in F,G.

View larger version (112K): [in a new window]   Fig. 4. Cuticle preparations of wild-type and T-Sp8 RNAi adult legs and antennae (proximal points to the left, distal to the right). (A) The adult leg is composed of the same leg segments as the embryo except that the tibia (Ti) and the tarsal segments (Ta) are distinct. (B) T-Sp8 RNAi weak phenotype. The leg lacks the tarsus. The pretarsal claw (Ptc) together with the club shaped tibia-like structure (arrowhead) is fused to a shortened femur. (C) T-Sp8 RNAi strong leg phenotype. Only the pretarsal claw (Ptc) is connected with a joint to the otherwise jointless leg stump. (D) In the wild-type antenna, six segments (dots) form the middle region between pedicel (Pd) and the club (Cl). (E) Adult T-Sp8 RNAi antennae frequently show a reduction in the number of middle segments. In this example, four segments (dots) can be seen. Cx, coxa; Tr, trochanter; Fe, femur.

View larger version (126K): [in a new window]   Fig. 5. Distal-less and dachshund expression in wild-type- and in TSp8 RNAi embryonic legs. The pleuropodium (pl) is nonspecifically stained in A and C. (A) Wild-type embryo at the end of the segmentation process. The long outstretched legs show Distal-less expression as a proximal `ring' (arrowhead) and a distal positioned `sock' (arrow). (B) In very strongly affected T-Sp8RNAi legs, Distalless expression can be seen at the tip of the dramatically shortened leg (arrow). At the base of such a leg a dot of Distal-less expression (arrowhead) represents the rest of the `ring' domain. (C) The dachshund gene is expressed in three domains in wild-type embryonic legs: as a spot (S) near the body wall, weakly in a proximal (P) and stronger in a distal (D) domain. (D) All three domains appear to be present in T-Sp8RNAi legs. The distal domain (D) is reduced in width. The distal leg tip as in the wild type is free of dachshund expression. The embryos shown in B and D most probably represents the very strong phenotype shown in Fig. 3D.

View larger version (62K): [in a new window]   Fig. 7. dachshund expression in the labrum of wild-type and TSp8RNAi embryos. (A) In the wild type, the dac expression domain (arrow) is seen in a middle position of the labrum. (B) In T-Sp8 RNAi embryos the distal part of the labrum is lost and the dac domain (arrow) comes to lie at its anterior margin.

View larger version (58K): [in a new window]   Fig. 8. T-Serrate expression in wild-type and T-Sp8 RNAi legs. The rings mark the positions of the leg joints. 0, body wall/coxa; 1, coxa/trochanter; 2, trochanter/femur; 3, femur/tibiotarsus; 4, tibiotarsus/pretarsal claw. (A) T-Sp8 expression in wild-type legs (same embryo as in Fig. 2J). No T-Sp8 expression is seen in the region of the body-wall/coxa joint. (B) T-Serrate expression in wild-type legs. (C) T-Serrate expression (*) in T-Sp8-RNAi legs (weak RNAi effect). (D) T-Serrate expression (*) in T-Sp8-RNAi legs (strong RNAi effect).

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