Dead-End (dnd) Gene Cloning and Gonad-Specific Expression Pattern in Starry Flounder (Platichthys stellatus)

Abstract

dnd is a germline-specific maternal RNA expressed in various vertebrate classes, which encodes an RNA-binding protein that is essential for PGC migration. The purpose of this study is fundamental research about starry flounder dnd gene for germ cell marker development. In this study, we cloned and analyzed the expression levels of Platichthys stellatus dead end (psdnd) in various tissues and embryonic stages. The psdnd gene was isolated from starry flounder ovaries, cloned into a pGEM-t vector, and sequenced. Full-length of psdnd cDNA was 1495 bp long, encoding 395 amino acids. psdnd expression levels were investigated by real-time polymerase chain reaction (qRT-PCR) in various tissues and embryo developmental stages. psdnd transcripts were detected in the testes and ovaries, but not in somatic tissues. Embryonic psdnd expression levels were higher during early embryo development stages than during late embryogenesis; psdnd expression was highest at the 1 cell stage, then gradually decreased throughout the subsequent developmental stages. The spatial expression pattern was analyzed by whole-mount in situ hybridization (WISH). The psdnd transcripts migration pattern was similar with zebrafish (Danio rerio). Our results suggest that psdnd may function as a germ cell-specific marker.

Characterization of ddx4 and dnd Homologs in Snakeskin Gourami (Trichopodus pectoralis) and Their Expression Levels during Larval Development and in Gonads of Males and Females

“…Ontogenetic variations in the level of ddx4 mRNA in several fish species have demonstrated that maternal transcripts of ddx4 persist, and their amounts have been shown to be relatively high at the early developmental stage and diluted in later embryogenesis [ 18 , 20 , 51 , 52 ]. A similar ontogenetic expression pattern of dnd during embryo development has also been revealed in a number of fish [ 9 , 11 , 15 , 19 , 21 , 29 , 65 , 66 ]. The present study determined the expression levels of ddx4 and dnd1 , which have different patterns, from hatching through to early larval development at 35 dph (1 cm).…”

“…Similar specific expressions of ddx4 mRNA in the gonads have been demonstrated in rainbow trout [ 50 ], medaka [ 51 ], tilapia [ 30 ], gibel carp [ 52 ], rice field eel [ 53 ], Pacific bluefin tuna [ 54 ], carp [ 55 ], catfish [ 18 ], European seabass [ 56 ], rare minnow [ 57 ], Atlantic cod [ 58 ], Korean rockfish [ 59 ], Solea senegalensis [ 60 ], toadfish [ 61 ], Japanese flounder [ 20 ], brown-marbled grouper [ 31 ], striped catfish [ 7 ], and Mekong giant catfish [ 62 ]. Coincidentally, the specific expression of dnd1 in testes and ovaries was revealed in zebrafish [ 15 ], medaka [ 29 ], rare minnow [ 63 ], gibel carp [ 13 ], tambaqui [ 64 ], Atlantic salmon [ 65 ], and starry flounder [ 21 ]. In addition, in situ hybridization using antisense ddx4 and dnd1 probes demonstrated that ddx4 and dnd1 mRNAs were observed in germ cells at various stages, and strong expression was observed in immature germ cells in testes, including spermatogonia, PSC, and SSC, and in ovarian cells such as oogonia and primary oocytes.…”

“…Several germ cell markers have been investigated and used in reproductive biological and biotechnological studies on vertebrates, including DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 ( ddx4 ; previously known as vasa ), microRNA-mediated repression inhibitor 1 ( dnd1; previously known as dnd ), nanos protein ( nanos3 ; previously known as nanos ), piwi-like RNA-mediated gene silencing 1 ( piwil1 ; previously known as piwi ), sry-box transcription factor sox-3 ( sox3 ), and deleted in azoospermia-like ( dazl ) [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ]. Specifically, ddx4 and dnd have been extensively used as DNA markers for germline and gonad development, sex determination, and sex differentiation in fish [ 9 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. It has been reported that ddx4 is responsible for a wide variety of functions, such as RNA binding, RNA splicing, RNA editing, ATP binding, and hydrolysis [ 22 , 23 , 24 , 25 ].…”

“…It prevents germ-cell-specific RNA degradation via the inhibition of miRNA function by binding to the 3′-untranslated regions (3′UTRs) of germ-cell-specific RNAs [ 28 ]. Both ddx4 and dnd have been identified and their functions related to gonadal development and/or gametogenesis in several fish, including medaka ( Oryzias latipes ) [ 29 ], turbot ( Scophthalmus maximus ) [ 19 ], olive flounder ( Paralichthys olivaceus ) [ 9 ], gibel carp ( Carassius gibelio ) [ 13 ], starry flounder ( Platichthys stellatus ) [ 21 ], tilapia ( Oreochromis niloticus ) [ 30 ], brown-marbled grouper ( Epinephelus fuscoguttatus ) [ 31 ], striped catfish ( Pangasianodon hypophthalmus ) [ 7 ], and black rockfish ( Sebastes schlegelii) [ 8 ].…”

Spinyhead Croaker Germ Cells Gene dnd Visualizes Primordial Germ Cells in Medaka

“…In mice, dnd is only detectable in testis [ 5 ], whereas it is restricted to the ovary in frog [ 6 ]. Dnd occurs in germ cells of both sexes in medaka [ 8 ], zebrafish ( Danio rerio ) [ 4 ], carp ( Carassius gibelio ) [ 10 ], turbot ( Scophthalmus maximus ) [ 13 ] and starry flounder ( Platichthys stellatus ) [ 14 ].…”