Abstract
ABSTRACTPolyamines are small poly-cations essential for all cellular life. The main polyamines present in metazoans are putrescine, spermidine and spermine. Their exact functions are still largely unclear; however, they are involved in a wide variety of processes affecting cell growth, proliferation, apoptosis and aging. Here we identify idefix, a mutation in the zebrafish gene encoding the enzyme spermidine synthase, leading to a severe reduction in spermidine levels as shown by capillary electrophoresis-mass spectrometry. We show that spermidine, but not spermine, is essential for early development, organogenesis and colour pattern formation. Whereas in other vertebrates spermidine deficiency leads to very early embryonic lethality, maternally provided spermidine synthase in zebrafish is sufficient to rescue the early developmental defects. This allows us to uncouple them from events occurring later during colour patterning. Factors involved in the cellular interactions essential for colour patterning, likely targets for spermidine, are the gap junction components Cx41.8, Cx39.4, and Kir7.1, an inwardly rectifying potassium channel, all known to be regulated by polyamines. Thus, zebrafish provide a vertebrate model to study the in vivo effects of polyamines.
Highlights
One of the most prominent features of adult zebrafish is their pigmentation; the fish display a stereotypical pattern of horizontal dark and light stripes on their flanks and in the anal and caudal fins
Gap junctions allow the molecular and electrical coupling between neighbouring cells via the transfer of small molecules and ions (Kumar and Gilula, 1996). Both leo and luc are required in melanophores and xanthophores (Irion et al, 2014a; Maderspacher and Nüsslein-Volhard, 2003) and it has been suggested that they form heteromeric gap junctions allowing cell-cell communications necessary for normal pattern formation
It was shown that the N-terminus of connexin41.8, which is affected in leo mutants, contains a putative polyamine-binding motif, ExxxE, necessary for the correct pigment pattern formation in vivo (Watanabe et al, 2012); the same motif is present in Cx39.4, which is encoded by luc (Irion et al, 2014a)
Summary
One of the most prominent features of adult zebrafish is their pigmentation; the fish display a stereotypical pattern of horizontal dark and light stripes on their flanks and in the anal and caudal fins. In all three cases we found that the ide mutant chromatophores can restore the wild-type pattern in the chimeric animals and generate stripes of normal width (Fig. 3) This demonstrates that the gene function of ide is not required in pigment cells, but rather influences their behaviour indirectly. Sequencing of all predicted coding sequences in the entire region and of both miRNA genes revealed only one significant difference between wild-type fish and ide mutants, a T to A transversion in the second exon of the gene coding for the enzyme spermidine synthase (srm) This mutation leads to a premature termination codon (TTG to TAG) in the resulting mRNA, and is predicted to give rise to a truncated protein of only 52 amino acids (Leu to Stop) instead of 289 residues (Fig. 4B). Low quantities of both spermine and spermidine are still detectable, indicating uptake of at least spermidine e.g. via food or from the water
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