fgf20 Is Essential for Initiating Zebrafish Fin Regeneration

doi:10.1126/science.1117637

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Science 23 December 2005:
Vol. 310. no. 5756, pp. 1957 - 1960
DOI: 10.1126/science.1117637

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fgf20 Is Essential for Initiating Zebrafish Fin Regeneration

Geoffrey G. Whitehead, Shinji Makino, Ching-Ling Lien and Mark T. Keating^*

Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Department of Cardiology, Children's Hospital, Boston, MA 02115, USA.

Fig. 1. dob, a mutant with early fin regeneration block. Regenerating wild-type (wt) and dob fins at 33°C following amputation. Arrows mark amputation plane. At 1 dpa, both wt and dob have covered the amputation with epithelium. By 2 dpa, wt fins have regenerated beyond the amputation plane, whereas dob mutants have not. At 10 dpa, wt has undergone full reconstitution, but dob fails to regenerate. Scale bars, 400 µm. [View Larger Version of this Image (93K GIF file)]

Fig. 2. dob fails to initiate fin regeneration and does not form a blastema. Dashed line or arrow marks amputation plane. (A) Hematoxylin-stained sections of caudal fin regenerates. At 6 and 12 hpa, the regeneration epithelium in dob is thicker than wild type (wt). At 18 hpa, disorganization of mesenchyme beneath the regeneration epithelium is apparent in wt fins but was not observed in dob. At 36 hpa, a blastema is seen in wt regenerates, yet dob lacks blastema. b, blastema; emb, epithelial-mesenchymal boundary. (B) lef1 and sparc mark the basal epidermal layer of the regeneration epithelium in wt. At 24 hpa, lef1 and sparc are absent from the basal epithelial layer in dob. No msxb expression is apparent in dob. Violet stain, indicated by red arrow, marks gene expression. (C) 24-hpa fin regenerates stained for BrdU (green) and 4',6'-diamidino-2-phenylindole (blue). White dashed line marks epithelial-mesenchymal boundary. During blastema formation, mutants had reduced mesenchymal and epithelial proliferation (n = 19). Graph displays the indices of proliferation in mesenchyme and epithelium of wt and dob regenerates. **, P « 0.01; *, P < 0.05. Scale bars, 50 µm (A), 100 µm [(B) and (C)]. [View Larger Version of this Image (99K GIF file)]

Fig. 3. fgf20a Y148S missense mutation causes dob. (A) Genetic map of dob on chromosome 1. Refined linkage analysis mapped dob to the 0.2 cM region between bef7 and tof24. The only gene between flanking recombinant markers is fgf20a. Numbers above linear map quantify recombination events between dob and linked markers from 2027 meioses. Bef7, fe12, mt254, tof24, and et6 are polymorphic genetic markers between AB and SJD strains identified by random DNA amplification and sequencing within the dob critical region. (B) DNA sequence chromatograms of wild-type, dob^+/–, and dob^–/– fish. A443C transversion leads to Y148S amino acid substitution. (C) Tyr-148 is conserved across vertebrates and among most zebrafish Fgfs. (D) Fgf secondary ß-trefoil structure. Tyr-148 is located in the ß9 strand of Fgf20a. (E) Phenotypic classes and frequencies (%) obtained after injection of wild-type fgf20a, Y148S fgf20a, wild-type fgf3, or Y148C fgf3 mRNAs (10 ng/µl) into wild-type embryos. Y148S fgf20a had no effect on the embryo, suggesting loss of function. Wt, normal; p1, head reduction, loss of tail; p2, lysis. [View Larger Version of this Image (42K GIF file)]

Fig. 4. fgf20a expression localizes to epithelial-mesenchymal boundary during initiation of fin regeneration. (A) Whole-mount in situ hybridization and sections showing fgf20a expression. During initiation of fin regeneration (6 to 12 hpa), fgf20a expression is localized to mesenchymal cells directly underneath the regeneration epithelium. During early blastema formation (18 hpa), fgf20a is confined to the blastema. (B) During blastema formation (24 hpa), fgf20a and msxb colocalize in blastema cells. fgf20a and msxb expression domains overlap at 72 hpa, when fgf20a is concentrated at the distal tip of msxb-positive distal blastema. Violet stain indicated by red arrow marks expression. Scale bars, 100 µm. [View Larger Version of this Image (96K GIF file)]