Fig. 3.

Engineered crz1 mutants exhibit maturation delay, dampened growth rate, and posterior regeneration, while food intake is unaffected. (A) Schematized crz1/gnrhl1 locus highlighting the site in the first coding exon (exon 2) selected for targeted mutagenesis and the alignment of wild-type and crz1/gnrhl1 mutant gene sequences (Bottom). Black rectangles represent exons, and yellow sections highlight the binding sites for TALENs. Blue arrows schematize primers used for genotyping, and gray areas correspond to the restriction site around which TALENs were designed. In the DNA sequence alignment, the resulting amino acid sequences of wild-type peptide (blue) and the corresponding aberrant peptide (magenta) are boxed. (B and C) In crz1 mutants (KO), maturation is delayed compared to wild-type siblings. Data are presented as cumulative curves based on the percentage of mature animals over time (weeks) (B), as well as plotting the age at which worms reached maturation (C). Wild types are shown in blue, and mutants are shown in magenta. n ≥ 213. Statistical significance was tested using t test. ****P < 0.0001. (D and E) crz1 mutants (KO) exhibit a reduction in growth rate. (D) Length in mm of 2-mo-old young worms does not differ between crz1^−/− and ^+/+. n ≥ 232. (E) Length of 5-mo-old premature crz1^−/− and ^+/+ worms. Age-matched mutant worms are significantly smaller compared to wild-type controls. n ≥ 75. Statistical significance was tested using t test. ****P < 0.0001. (F and G) Posterior regeneration is dampened in crz1 mutants (KO). (F) Schematic representation of the experimental design. Fifteen segments have been amputated from age-matched ∼50-segment-long premature crz1^−/− and ^+/+ worms, and regenerated segments (red area) have been counted weekly for 4 wk. (G) Mutants regenerate a reduced number of segments per time unit (week) compared to wild types. Data are shown as cumulative number of regenerated segments for all of the 4 wk. n ≥ 48. Statistical significance was tested using t test. *P < 0.05; **P < 0.01. (H–J) Food intake is not affected in crz1 mutants (KO). (H) Representative spinach circle provided in the feeding experiments and representative spinach circles after a 1-d experiment. (Scale bar, 18 mm.) The eaten area was calculated subtracting the residual leaf area (Bottom) from the averaged area of 6 control leaves (Top). (I) Eaten leaf area reported for crz1^−/− and ^+/+ worms in the 2 time points (after 1 and 3 d). n = 12. (J) Food intake rate is not affected in crz1^−/− mutants. Data are reported as average time (days) required to eat completely a single spinach circle. n ≥ 25.