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Fig. 3 SDF1 inhibits fgf20a and activates Wnt10a expression during fin regeneration.
A: Over-expression of SDF1 at the time of amputation turns off fgf20a expression. The protein SDF1, or BSA as a control, were injected into the fin at the time of amputation. Fins were allowed to regenerate for 48 hours before fgf20a expression was checked by ISH. Dotted lines demarcate amputation plane. Scale bar, 100 μm. B: fgf20a expression is enhanced in medusa mutant. fgf20a as well as wnt10a expression were analyzed by quantitative RT-PCR in sdf1a/medusa mutant and sibling fins at 48 hpa. fgf20a expression is increased 2.2±0.03 fold in sdf1 mutant compared to siblings, while wnt10a expression is reduced 1.5±0.04 fold. Average values (±s.e.m.) from experiments performed in triplicate are presented. C, D: Fins regenerate slower in sdf1/medusa mutant compared to siblings (C) whereas cxcr4b mutant fish regenerates caudal fin with no abnormality (D). Length of the third dorsal and ventral regenerating fin ray of the regenerates were measured in ody-/- mutant fish and siblings (ody+/- and ody+/+) (D) and in medusa/sdf1-/- mutant fish and siblings (sdf1+/+ and sdf1+/-) (C) at 4 and 7 dpa. Precise measures were made in pixels (1 pixel corresponding to 3 μm). The average length of the regenerate allows to calculate the regeneration speed. No significant difference was observed between ody-/- mutant fish and siblings. Two independent experiments were performed. Experiment 1: n = 5 ody-/-, n = 7 siblings. Experiment 2 n = 5 ody-/-, n = 7 siblings (data not shown). Errors bars represent the s.e.m. of the average regenerate length. Fins regenerate slower in sdf1/medusa mutant n = 18 siblings (sdf1+/+ and sdf1+/-), n = 18 medusa/sdf1-/-. Errors bars represent the s.e.m. of the average regenerate length (* p<0.05). E–F: cxcr7 overexpression inhibits blastema formation. Plasmid DNA expressing cxcr7 (pCS2-cxcr7) was injected into the dorsal half fin whereas an empty plasmid (pCS2) was injected into the ventral part of the fin at the time of amputation prior to electroporation. Fins were allowed to regenerate for 48 hours before scoring blastema formation (n = 8). The percent area of dorsal versus ventral regrowth is presented in E and a representative fin in F. Scale bar, 500 μm. Dotted lines demarcate amputation plane. G: cxcr7 overexpression inhibits fgf20 expression. Plasmid DNA expressing cxcr7 (pCS2-cxcr7) or an empty plasmid (pCS2) as control were electroporated into total fin at the time of amputation. Fins were allowed to regenerate for 48 hours before checking fgf20 expression by quantitative PCR. Two independent experiments are presented in G. Experiment 1: n = 5; experiment 2: n = 5.