Germline Specific Expression of a vasa Homologue Gene in the Viviparous Fish Black Rockfish (Sebastes schlegelii) and Functional Analysis of the vasa 3′ Untranslated Region

Abstract

Germ cells play a key role in gonad development. As precursors, primordial germ cells (PGCs) are particularly important for germline formation. However, the origination and migration patterns of PGCs are poorly studied in marine fish, especially for viviparous economic species. The vasa gene has been widely used as a germ cell marker to identify a germline because vasa RNA is a component of germ plasm. In this study, we described the expression pattern of black rockfish (Sebastes schlegelii) vasa (Ssvas) in gonadal formation and development by in situ hybridization. The results showed that Ssvas failed in localization at the cleavage furrows until the late gastrula stage, when PGCs appeared and migrated to the genital ridge and formed elongated gonadal primordia at 10 days after birth. This study firstly revealed the PGCs origination and migration characteristics in viviparous marine fish. Furthermore, we microinjected chimeric mRNA containing EGFP and the 3 untranslated region (3 UTR) of Ssvas into zebrafish (Danio rerio) and marine medaka (Oryzias melastigma) fertilized eggs for tracing PGCs. We found that, although Sebastes schlegelii lacked early localization, similar to red seabream (Pagrus major) and marine medaka, only the 3 UTR of Ssvas vasa 3 UTR of black rockfish was able to label both zebrafish and marine medaka PGCs. In comparison with other three Euteleostei species, besides some basal motifs, black rockfish had three specific motifs of M10, M12, and M19 just presented in zebrafish, which might play an important role in labeling zebrafish PGCs. These results will promote germ cell manipulation technology development and facilitate artificial reproduction regulation in aquaculture.

Primordial Germ Cell Development in the Poeciliid, Gambusia holbrooki, Reveals Shared Features Between Lecithotrophs and Matrotrophs

“…In gambusia, two each of the four clusters merge at gastrula (Ga10) and appear as a cluster each on either side of the body axis, while in zebrafish this rearrangement is postponed to 10 somites. In medaka ( Herpin et al, 2007 ) and black rockfish ( Zhou et al, 2020 ), the Gp markers are dispersed throughout the cytoplasm of blastomeres (stages 7 and 12) until gastrula where the nanos -positive cells are evident at stage 16, forming two clusters in either side of the body. In mice, PGC specification is first evident in egg cylinder stage (E6.25) and the PGCs remain in a single cluster until their colonisation ( Saitou and Yamaji, 2010 ).…”

“…The position of PGCs determines their migration start point which is defined in relation to cell collectivity and body patterning during blastula and gastrula stages, respectively ( Raz, 2002 ). In zebrafish ( Kimmel et al, 1995 ), medaka ( Iwamatsu, 2004 ), and viviparous black rockfish ( Zhou et al, 2020 ), prominent embryonic features ( e.g., sphere, yolk cell doming, multiple stages of epiboly) during blastula stage pose broad somatic environment for cell motility. While in poeciliids including G. holbrooki ( Tavolga and Rugh, 1947 ; Tavolga, 1949 ; Mousavi and Patil, 2021 ), the blastodisc is extremely thin and flat and the growing cells sink inwards into the emerging yolk cavity.…”

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

“…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 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 ].…”

Conserved germ plasm characteristics across theDanioandDevariolineages

“…In these fish species, germ plasm appears to exhibit a conserved pattern of aggregation in the forming furrows for the first several cell cycles. However, there is also evidence of germ plasm heterogeneity among fish, as demonstrated by the lack of furrow localization of maternally inherited vasa/ddx4 RNA in early medaka and seabream embryos (Herpin et al, 2007; Shinomiya et al, 2000; Zhou et al, 2020).…”