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First published online 21 July 2004
doi: 10.1242/dev.01267

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Signalling by the FGFR-like tyrosine kinase, Kringelchen, is essential for bud detachment in Hydra vulgaris

¹ Philipps University Marburg, FB 17, Morphology and Evolution of Invertebrates, Karl von Frisch Strasse 8, 35032 Marburg, Germany
² INSERM Unité 119, 27 boulevard Lei Roure, 13009 Marseille, France

View larger version (49K): [in a new window]   Fig. 4. Effects of head and foot regeneration induced in the parent on bud development and kringelchen expression. (A-G) Head regeneration and bud development [bud stages according to Otto and Campbell (Otto and Campbell, 1977)]. (A) Competition of both processes results in failure of bud detachment if regeneration is induced early in budding (n=100). (B-G) Expression pattern of kringelchen if the parent head is removed immediately above a stage 3 bud: (B) 10 hours, (C) 26 hours, (D) 22 hours, (E) 31 hours, (F) 36 hours, (G) 57 hours. (H-L) Effect of foot regeneration on bud development. (H) Detachment of the young polyp is slightly accelerated (n=100 each). (I-L) Expression pattern of kringelchen after foot removal immediately below a stage 3 bud, evaluation about 24-25 hours after cutting. The typical ring is present and broader than in normal buds.

View larger version (109K): [in a new window]   Fig. 3. Expression pattern of kringelchen in buds as detected by in situ hybridization with a digoxigenin-labelled antisense probe. Staging of buds is according to Otto and Campbell (Otto and Campbell, 1977). (A,B) Stage 1 (ectoderm thickening and early evagination); (C) stage 3 (about 5 hours after first signs of evagination); (D) stage 4; (E-H) stage 5 (the tentacle in G,H belongs to the parent); (I-K) stage 7 (tentacle buds form); (L) stage 10 (shortly before detachment). (M-O) Budding region of the parent polyp after detachment: (M) immediately after detachment, (N) 30 minutes afterwards, (O) 60 minutes afterwards. (P) Semi-thin section of a stage 5 bud. (Q) Close-up of a stage 2 bud tip, in which about 60 ectodermal cells are distinguishable. (R,S) Single cell preparations of tissue derived from the budding region with kringelchen-positive epithelial cells. (R) Phase contrast imaging, allowing the evaluation of non labelled cell types; (S) a group of epithelial cells from the budding region shown by light microscopy.

View larger version (198K): [in a new window]   Fig. 1. Sequence features of Kringelchen (GenBank Accession Number, AY193769). (A) Proposed structure and full-length sequence: features and their corresponding amino acid positions are given. The partial sequence of splice leader B is underlined. The amino acid sequence contains a signal peptide (boxed and italic), a transmembrane domain (boxed and bold) and an intracellular kinase domain (boxed only). Ig-like loop I and Ig-like loop II are boxed and shaded, and the sequence between Tyr 250 and Phe324 (corresponding to Ig-like loop III) is shaded only. The highly conserved cysteines, which form the Ig-like loops I and II are boxed and shaded. SH2 and SH3 domain-binding consensuses are bold and underlined, bold tyrosines correspond to crucial autophosphorylated tyrosines in vertebrate FGFR. (B,C) Alignment of the Hydra sequence corresponding to the first, second and third putative Ig-like loops with vertebrate and invertebrate FGFR. (B) Ig-like loop I (D1), (C) Ig-like loop II (D2; upper part of the figure) and Ig-like loop III (D3; lower part of the figure). a, acidic amino acid; b, basic amino acid; r, aromatic amino acid. (D) Intracellular (kinase) domain. Underlined is a potential SH2-binding site; (auto)phosphorylation sites (identified in vertebrate FGFR) are marked with +. Shortcuts and Accession Number for the used sequences are: Hy, Hydra vulgaris Kringelchen (AY193769); Ce, C. elegans Egl 15 (AAC46934); Bl and Hl, Drosophila Breathless (Q09147) and Heartless (Q07407); F1-F4, human FGFR 1-4 (P11362, P21802, P22607, P22455).

View larger version (30K): [in a new window]   Fig. 2. Southern blot and phylogenetic tree. (A) Southern blot probed with the full-length kringelchen cDNA. Genomic DNA (10 µg) was digested to completion with HindIII (lane 1), NcoI (lane 2), EcoRV (lane 3) and XhoI (lane 4). HindIII, NcoI and EcoRV cut once within the coding sequence (positions 2076, 1383 and 101 of the full-length cDNA, respectively); XhoI does not cut. The size was determined using a /HindIII digested DNA size marker. (B) Phylogeny of class III, IV, V and XII receptor tyrosine kinases (Grassot et al., 2003). Kinase domain sequences from RTK Class III (CSFR/PDGFR), IV (FGFR), V (VEGFR) and XII (Ret) were used for phylogeny inference (distance method). Class IX (Tie) sequences were used as outgroup. Node with bootstrap values over 50 are indicated (500 bootstrap replicates). Dm, Drosophila melanogaster; Hs, Homo sapiens; Hro, Halocynthia roretzi; Cin, Ciona intestinalis; btl, Drosophila breathless FGFR; htl, Drosophila heartless FGFR. Kringelchen branches at the base of the FGFR tyrosine kinase superfamily.

View larger version (35K): [in a new window]   Fig. 5. Inhibition of bud detachment and kringelchen expression after treatment with either the FGFR inhibitor SU5402 or phosphorothioated oligonucleotides. (A) Treatment scheme for SU5402. Buds were treated for 24 hours with SU5402 and examined at stages 3, 5 and 7. Asterisk indicates when in situ hybridization was carried out. (B) Scheme of the increasingly severe abnormalities obtained after treatment with SU5402. (C) Effects of SU5402 treatment on the characteristics of the animals at three different stages after treatment. The most severe abnormality predominated at earlier stages after treatment with either SU5402 or phosphorothioate antisense oligonucleotides. (D-F) In situ hybridization of the kringelchen antisense RNA probe to Hydra with increasingly severe abnormalities produced by SU5402 treatment. (G) Effects of electroporation of PT-antisense oligo 1 in stage 3 buds. Sixty polyps were electroporated, the 39 survivors evaluated. (H) In situ hybridization of kringelchen RNA probe to a typical abnormally elongated bud obtained by the antisense oligonucleotide treatment. Evaluation of the abnormalities and in situ hybridization were carried out 7 days after the end of treatment.

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