Early (4 -168 hpf) phenotypes of the established cyp1b1-KO zebrafish line. (A–P) The phenotypes were analyzed in F4 zebrafish resulting from inbreeding of young (< six months) F3 siblings. Progenitors’ genotypes (PG) and embryos’ genotypes (EG) are indicated on the left. (A,E,I,M) The difference in egg volume (white dotted line). Yellow dotted line: animal pole. (B,F,J,N) The differences in yolk extension (YE) length and yolk ball’s (YB) largest diameter. Red line: wildtype YE length extrapolated to embryos with different genotypes. No differences were observed in larval development at 48 hpf and 168 hpf. The images are representative of the results observed in at least 10 embryos per experimental group. Scale bars represent 500 µm in 4 hpf, 24 hpf and 168 hpf photographs and 1000 µm in 48 hpf photographs. (Q) Relative egg volume at 4 hpf. The values are expressed as the percentage of the volume of wildtype eggs (n = 20). (R) YE/YB ratio at 24 hpf (n = 3–6). (S) Cyp1b1 mRNA levels in zebrafish at 48 hpf and 168 hpf. Pools of 45 F4 zebrafish at 48 and 168 hpf were used to calculate cyp1b1 mRNA levels by RT-qPCR. The values are expressed as relative expression levels normalized to the wild type. Three independent experiments carried out in triplicate were used for calculation of mean expression values in each sample. Asterisks indicate statistical significance compared to the wild type, p < 0.05 (*), p < 0.001 (***).
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