Molecular mechanisms of embryonic tail development in the self-fertilizing mangrove killifish Kryptolebias marmoratus

Abstract

Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

Developmental defects in ectodermal appendages caused by missense mutation inedaraddgene in thenfrmangrove killifish,Kryptolebias marmoratus

“…Laboratory strains were established by successive self-fertilisation for many generations (over 20 years) leading to the generation of highly isogenic and inbred lines (Tatarenkov et al 2010;Moore et al, 2012). Self-fertilisation and isogenicity makes this animal model extremely unique and useful for generating mutant lines and identifying the mutated gene(s) (Sucar et al 2016;Saud et al, 2021). Firstly, when a mutated F1 fish contains a mutation in the genome, as this fish is a self-fertilising hermaphrodite possessing ovary and testis in the same body, the fish already produces eggs and sperms heterozygous for the same mutation.…”

“…Self-fertilisation and isogenicity makes this animal model extremely unique and useful for generating mutant lines and identifying the mutated gene(s) (Sucar et al . 2016; Saud et al ., 2021). Firstly, when a mutated F1 fish contains a mutation in the genome, as this fish is a self-fertilising hermaphrodite possessing ovary and testis in the same body, the fish already produces eggs and sperms heterozygous for the same mutation.…”

“…Therefore, it is not necessary to separate groups of siblings in individual tanks and screen for carriers of the same mutation from two both sexes. Secondly, due to the isogenicity of the laboratory lines, genetic variations between two complementary chromosomes are highly limited, facilitating instant identification of mutated genes by a single run of sequencing with a small number of mutant, siblings and parental wild type strains, avoiding the need for genetic mapping (Saud et al ., 2021).…”

“…The first ENU mutagenesis applied to the self-fertilising mangrove killifish generated a variety of mutant phenotypes (Moore et al ., 2012, Sucar et al ., 2016). Among these, two lines R109 and R228 were sequenced and identified the mutations in the genes, noto and msgn1 causing the phenotype of the shorttail/R109 and balltail/R228 respectively (Saud et al ., 2021). While maintaining the mutant lines, we identified another mutant phenotype that we named no-fin-ray ( nfr ) that was found from the R228 progenitor lineage.…”

Genetic tools for the study of the mangrove killifish, Kryptolebias marmoratus, an emerging vertebrate model for phenotypic plasticity

“…These attributes hinder the systematic injection and production of morphant, transgenic, or genome-edited strains required to take full advantage of forward genetics. For example, morpholino knockdowns that characterized two mutations impacting Kmar tail development found in a forward screen (Sucar et al, 2016) were conducted with medaka because "it is difficult to obtain many one-cell-stage embryos for … injections" (Saud et al, 2021). Manual coaxing of egg laying, however, can be done for IVF (Nakamura et al, 2008).…”