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Fig. 3
Effects of early developmental exposure to cortisol on adult tailfin regeneration. (A) Examples of morphologically normal, slightly defective and defective tailfin regeneration. (B) Quantification of combined data on regeneration morphology from three different experimental replicates from a single parental stock. Statistical significance was determined by a Chi-squared test (d.f.=1, comparing the total normal versus the total defective and slightly defective). Subsequent experiments using a newly imported parental stock did not show any overt morphological defects (possibly owing to the high polymorphism of the wild-type AB strain); however they did manifest the effects shown in panels C-F. (C) Quantification of neutrophils and macrophages in regenerating fin tissue (dpa, days post-amputation), respectively measured by fluorescence intensity of fins from mpx:GFP and mpeg1:YFP transgenic fish derived from untreated control embryos (-) or embryos treated with 1µM cortisol (+). In one experiment counts of individual cells at a single time point confirmed that the differences in fluorescence intensity reflect differences in cell number. The bars represent the grand means±s.e.m. of measurements from three experimental replicates of the neutrophil counts, and two experimental replicates of the macrophage counts (a third macrophage experiment which did not show significant differences was not included, as it did not include the same time points). For the neutrophils the number of fish in each replicate ranged from 9-18, with overall totals of 41 control and 37 treated. For the macrophages the number of fish in each replicate ranged from 9-11, with overall totals of 21 control and 18 treated. Statistical significance **P=0.0017; *P=0.01; †P=0.028; ‡P=0.0226, by a two tailed t-test. (D) Scatter plots of average expression level and fold-change in expression for each gene in tailfin regenerates at 2dpa compared to 0dpa in 4.5-month-old control (untreated) fish and fish derived from cortisol-treated embryos. The red dots indicate genes with significant differential expression (P<0.05), the numbers of which (over- and under-expressed) are also shown in red. (E) Scatter plot of average expression level and fold-change in expression, showing interaction of cortisol treatment and time (0-2dpa). Red dots indicate genes with significant differential expression (P<0.05), the numbers of which (over- and under-expressed) are also shown in red. (F) RNA-seq time course measurements of seven of the most significantly differentially expressed genes from the analysis shown in E; lines show the averages of three biological replicates, each of which are indicated by individual data points for both cortisol treatment (+, light gray) and untreated control (-, dark gray) groups. (G) qRT-PCR time course measurements of four of the genes shown in (F), during tailfin regeneration in 10month old adults from the same cohort shown in D-F.