Spermidine, but not spermine, is essential for pigment pattern formation in zebrafish

Frohnhöfer, H.G., Geiger-Rudolph, S., Pattky, M., Meixner, M., Huhn, C., Maischein, H.M., Geisler, R., Gehring, I., Maderspacher, F., Nüsslein-Volhard, C., Irion, U.
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idefix mutants show defects in pigment pattern formation. Wild-type (A,A′) zebrafish show a stereotypic pattern of horizontal dark and light stripes on their flanks and on the anal and caudal fins. In ide mutants (B,B′) the light stripe areas are expanded, there are fewer and less regular dark stripes, which frequently show interruptions. The striped pattern in the anal and caudal fins is also disrupted in ide mutants. Double mutants of ide with leo (C-D′), luc (E-F′) or obe heterozygous (G-H′) and homozygous (I-J′) show a superimposition of both phenotypes. The light stripe areas are expanded in all cases. Scale bars: 5mm J, 1mm in J′.

The idefix phenotype first becomes visible during metamorphosis. (A,B) Wild-type and ide mutant zebrafish at stage SP, 9.5mm standard length. In the mutants the boundaries between the first light stripe and the developing dark stripes are less regular than in wild type. (C-D′) At stage J++, 16mm standard length, the ide phenotype is fully visible. The light stripe area is wider than in wild type and only two dark stripes develop. The xanthophore densities in the light stripes are similar in wild type and ide mutants (C′,D′). Scale bars: 1mm.

idefix mutant chromatophores contribute to a wild-type pattern in chimeric animals. Chimeric animals derived from blastomere transplantations of ide mutant cells into (A) pfeffer (n=8), (B) rose (n=6) and (C) nacre (n=4) hosts. In all three cases the normally striped wild-type pattern is restored in the regions of donor-derived xanthophores (A), iridophores (B) or melanophores (C), indicated by the brackets. Scale bar: 1mm.

A premature stop codon in spermidine synthase is responsible for the idefix phenotype. (A) The ide mutation, t26743, was placed between the two z-markers z3199 and z14967 by mapping of meiotic recombinants. The region contains 14 annotated protein coding genes and two microRNA genes. (B) idet26743mutants carry a mutation in exon 2 of srm. The gene structure is shown in the top panel, the bottom panel shows the sequence chromatograms of wild-type and homozygous mutant fish. The mutated nucleotide is marked with an asterisk. (C) An alignment of the amino acid sequences of spermidine synthases from zebrafish (Danio rerio), humans (Homo sapiens) and E.coli. Positions of conserved amino acid residues important for substrate binding and catalytic activity are highlighted in red. The position of the mutation in idet26743 (Leu53 to Stop) is underlaid in red. (D) An F0 fish is shown, which was obtained after CRISPR-mediated knockout of srm. (E) The sequence chromatograms from the fish in D. The induced mutations lead to a deterioration of the signal quality in both directions at the same position. (F) A homozygous mutant F2 fish carries an 80bp deletion and shows the typical ide mutant phenotype. (G) The biosynthetic pathway for the generation of the polyamines putrescine, spermidine and spermine from ornithine and S-adenosyl-methionin-amine. Scale bars: 5mm.

idefix mutants show a maternal-effect lethal phenotype. Wild-type embryos and larvae (A-C) and maternal-zygotic idefix mutants (D-I, ide-/- mat. & zyg.) derived from an incross of two homozygous fish are shown at 4h post fertilization (hpf), 26hpf and 3days post fertilization (dpf). A variable proportion of the mutants show morphological defects, which become apparent at different developmental stages; examples were selected to illustrate the most severe phenotypes. Scale bars: 0.5mm.

Observed In:
Stage Range: Sphere to Days 7-13

Development of the exocrine pancreas is defective in ide mutants. Expression of trypsin in 72hpf embryos visualized by in situ hybridization. (A) heterozygous (ide+/-) and (B) homozygous (ide-/- zyg. only, maternal contribution present) mutants derived from heterozygous parents show no defects (n=40). Whereas (C) homozygous mutants derived from an incross of homozygous fish, i.e. zygotic mutants with no maternal contribution (ide-/- mat. & zyg.), show a severe reduction in the expression domain, indicating defects in the development of the exocrine pancreas (n=24). Scale bar: 0.1mm.

A knockout mutation in spermine synthase has no phenotypic consequences. (A) Chromatograms of wild-type, homozygous and heterozygous mutants showing that the CRISPR knockout of sms resulted in an 8bp insertion. (B) Homozygous fish are viable and fertile and show no phenotype. Scale bar: 5mm.

Knockdown Reagent:
Observed In:
Stage: Adult

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