Figure 1
- ID
- ZDB-FIG-230315-10
- Publication
- Krug et al., 2023 - Generation of a transparent killifish line through multiplex CRISPR/Cas9-mediated gene inactivation
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(A–C) Gene expression analysis of mitfa (A), ltk (B), and csf1ra (C) in the skin of male and female wild-type fish via quantitative real-time PCR. The expression of mitfa was only significantly up-regulated in male fish at the age of 6 weeks post-hatching (wph) (p: 0.048). For both sexes, the expression of ltk increased significantly over time (pmale, 2wph: 0.028, pmale, 3wph: 0.026, pmale, 6wph: 0.002; pfemale, 2wph: 0.002, pfemale, 3wph: 0.019, pfemale, 6wph: 0.033), whereas no significant differences were observed for csf1ra. Expression levels were normalized to the expression at 1 wph in the respective sex. Rpl13a was used as housekeeping gene (nmale_1wph = 3, nmale_2wph = 4, nmale_3wph = 4, nmale_6wph = 4; nfemale_1wph = 3, nfemale_2wph = 4, nfemale_3wph = 4, nfemale_6wph = 4). Relative gene expression was calculated using the ΔΔCT method (Pfaffl et al., 2002). Student’s or Welch’s t-tests were computed to determine significant changes in gene expression. Horizontal line represents median. Whiskers show min. to max. values. (D) Phenotypical analysis of F0 embryos revealed a reduction of melanophores in GFP-positive compared to GFP-negative embryos. (E,F) Male (E) and female (F) wild-type N. furzeri. (G–J) Males and females of the F0 generation, display a mosaic loss of body pigmentation. Note that almost completely transparent individuals can be observed in (H–J), allowing a view on inner organs (o: ovary, s: stomach, sb: swim bladder). (J) Microscopic analysis of a female F0 fish with a view on individual eggs within the ovary and blood vessels (cv: cardinal vein, e: egg). (K,K’) Male (K) and female (K’) fish at the age of 38 days post-hatching (dph) with the genotype mitfa-/-,ltk+/-,csf1ra-/- showed a lack of melanophores and xanthophores, whereas iridophores were present. (L,L’) A lack of melanophores and iridophores was observed in male (L) and female (L’) N. furzeri with the genotype mitfa-/-, ltk-/-, csf1ra+/-. Despite a homozygous mutation in ltk (ltk-/-) individual scales with iridophores were detected in fish of both sexes. (M,M’) The presence of homozygous mutations in all the three genes mitfa, ltk, and csf1ra resulted in the loss of body pigmentation in males (M) and females (M’) allowing a view on inner organs.
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