Fig. S5

Additional Evidence that Dkk1b-Producing ET Inhibit Regeneration

(A) Quantification of regenerate lengths after distal amputations of 10-20% of pectoral fins. n = 18. Data are mean ± s.d. ‘N.S.’: not significant. (B) Quantification of regenerate length after proximal amputation of 50% of pectoral fins. n = 18. Data are mean ± s.d.*P < 0.001 by one-way ANOVA with Bonferroni’s posttest.(D-E) Additional confocal images of dkk1b:EGFP in amputated anterior regions of male pectoral fins. (D) dkk1b:EGFP+ET cells are located over and/or dorsally adjacent to the amputation place. Note that there are only few BrdU+ proliferating cells (red) near the amputation plane. (E) dkk1b:EGFP expression in dysmorphic 4 dpa regenerates. Proliferation appears to be directed away from dkk1b:EGFP+ET. Green: EGFP immunofluorescence; red: BrdU immunofluorescence; blue: DAPI. Scale bars = 50 μm. (F) Increased expression of dkk1b in the anterior regions of 2 dpa male pectoral fin tissue compared with females, as assessed by quantitative PCR (n = 3). Error bars indicate standard deviation. Data are normalized to actb2 (beta-actin2). AU = arbitraty units. *P < 0.05 by two tailed Student’s t-test. (G) axin2 expression at 2 dpa in female (Left) and male (Right) pectoral fins, as assessed by in situ hybridization. Note that axin2 (violet) is clearly detectable in the female regenerate, but weak in the male sample. Arrowheads indicate amputation plane. amputations through the ET field, but not after amputation distal to the ET field. Note that growth rates during fin regeneration are position-dependent, with distal regenerates growing slower than proximal regenerates (Lee et al., 2005). (C) Adult females treated with vehicle or Eth for 14 days prior to one day of washout had specific defects in anterior regeneration that mirrored male defects. n = 16. Data are mean ± s.d.; *P < 0.001 by one-way ANOVA with Bonferroni’s posttest.