Developmental defects in ectodermal appendages caused by missense mutation in edaradd gene in the nfr mangrove killifish, Kryptolebias marmoratus
The mangrove killifish, Kryptolebias marmoratus, can reproduce with self-fertilisation, offering a unique and useful genetic tool for generation of genetic mutants and quick identification of mutated genes. From an ENU-mutated mangrove killifish line R228, we have isolated a novel mutant line, no-fin-ray/nfr in which homozygous mutant of adult fish fin ray development is largely reduced. Besides the reduction of the fin, the nfr mutant also exhibited other phenotype associated with ectodermal cell lineages including loss of scales, deformation in the gill structure such as decreasing the number of gill filaments, the reduction in the number of jaw teeth, pharyngeal teeth and gill rakers. Illumina RNAseq with 3 embryos each from mutants, siblings and the parental WT strain Hon9 (only 9 embryos as total) identified a mutation in the edaradd in a highly conserved C-terminal death domain. Edaradd is known as a cytoplasmic accessory protein for the Ectodysplasin A (EDA) signalling pathway. To confirm the crucial role of edaradd during fin development, CRISPR RNAs were designed to knock out the gene in another killifish species, Arabian killifish. Indeed, Arabian killifish edaradd crispants showed a potent reduction of the fin development with 100% frequency. Furthermore, EDA crispants also showed identical phenotypes to that of edaradd crispants, confirming the fin defect in the mutants/crispants is caused by the signalling pathway of the EDA in the killifish species.   These data demonstrate a powerful genetic approach using isogenic self-fertilising mangrove killifish as a tool for identifying mutants and their mutation, and revealed the crucial role of edaradd for the first time in the fish fin development and other ectoderm derived epithelial tissues. Overall design: RNAseq was conducted using embryos from no-fin-ray mutants, siblings, and wild type mangrove killifish. Mutated genes in the no-fin-ray mutant were identified.