Production of Germ Cell-Less Rainbow Trout by dead end Gene Knockout and their Use as Recipients for Germ Cell Transplantation

Fujihara, Ryo; Katayama, Naoto; Sadaie, Sakiko; Miwa, Misako; Matias, Gabriela Angelica Sanchez; Ichida, Kensuke; Fujii, Wataru; Naito, Kunihiko; Hayashi, Makoto; Yoshizaki, Goro

doi:10.1007/s10126-022-10128-w

Cited by 31 publications

(19 citation statements)

References 39 publications

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“…Thus, this gene will likely serve as an effective marker gene for editing in any species of Oncorhynchus . Another example is the dead-end gene ( dnd1) which has been edited in S. salar and O. mykiss with the intent to produce fish with inherited sterility (Fujihara et al, 2022; Güralp et al, 2020; Wargelius et al, 2016). The gene dnd1 , represented by gene family 13,707, is single copy across Oncorhynchus but is double copy in S. salar (Figure 5d).…”

Section: Resultsmentioning

confidence: 99%

A Homology Guide for Pacific Salmon Genus Oncorhynchus resolves patterns of Duplicate Retention, Rediploidization and Local Adaptation Following the Salmonid Specific Whole Genome Duplication Event

Dimos

Phelps

2022

Preprint

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Salmonid fishes have emerged as a tractable model to study whole genome duplications (WGD) as this group has undergone four rounds of WGD, with a significant proportion of the genome yet to rediploidize. The fact that much of modern salmonid genomes retain duplicates from the most recent WGD while other regions have rediploidized creates complications for genetic studies by obscuring homology relationships and the necessity of filtering duplicate regions from many analyses. The difficulty this creates is particularly prominent in Pacific salmonids genus Oncorhynchus who are the focus of intense genetics-based conservation and management efforts owing to the important ecological and cultural role these fish play. To address this gap, we generated a homology guide for six species of Oncorhynchus with available genomes and used this guide to describe patterns of duplicate retention and rediploidization. Overall, we find that retained duplicates comprise over half of modern gene repertoires and that retained duplicate genes are enriched for genes involved in nuclear stability, while rediploidized genes which represent a smaller proportion of genes are heavily enriched in dosage sensitive processes such as mitochondria. Additionally, by reanalyzing published expression data from locally adapted strains of O. mykiss we demonstrate that retained duplicates are more likely to be associated with adaptive divergence than rediploidized genes, highlighting the potential of WGDs to promote adaptation. Finally, we demonstrate the utility of our homology guide by investigating the evolutionary relationship among genes highlighted as playing a role in salmonid life history traits or gene editing targets.

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Section: Resultsmentioning

confidence: 99%

A Homology Guide for Pacific Salmon Genus Oncorhynchus resolves patterns of Duplicate Retention, Rediploidization and Local Adaptation Following the Salmonid Specific Whole Genome Duplication Event

Dimos

Phelps

2022

Preprint

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“…The phenotype of a GCF ovary from salmon piwil1 −/− F1 and dnd crispant females ( Wargelius et al, 2016 ) resembles that of the rainbow trout dnd KO ovary ( Fujihara et al, 2022 ): a thin and dense structure of connective tissue with rare groups of somatic cells that may represent granulosa cells (GCF piwil1 −/− F1 ovaries express aromatase, cyp19a1a , likewise the dnd mutant ovaries; Wargelius et al, 2016 ). The resemblance in phenotype between dnd crispant and piwil1 −/− females may further suggest that germ cells are lost during early ontogenesis in piwil1 −/− salmon embryos, as germ cells are no longer detectable 90 days post fertilization in dnd KO rainbow trout ( Fujihara et al, 2022 ). Future work will have to elucidate if the loss of germ cells in salmon occurred as early as during germ line establishment, as reported for piwil1 KO in medaka ( Li et al, 2012 ).…”

Section: Discussionmentioning

confidence: 99%

The Piwil1 N domain is required for germ cell survival in Atlantic salmon

O²,

Andersson³

et al. 2022

Front. Cell Dev. Biol.

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Genetic introgression of farmed salmon into wild populations can damage the genetic integrity of wild stocks and is therefore considered as an environmental threat. One possible solution is to induce sterility in farmed salmon. We have searched for proteins potentially essential for germline survival in Atlantic salmon. One of these is the argonaute protein Piwil1, known to be required for germ cell survival. To examine Piwil1 function in salmon, we induced indels in the N domain by CRISPR-Cas9. The encoded domain is present in all vertebrate Piwi proteins and has been linked to Tdrd1 protein interaction and PAZ lobe structure. The F0 founder generation of piwil1 crispant males and females displayed a mosaic pattern of piwil1 mutations, exhibiting highly mutated alleles (53%–97%) in their fin gDNA samples. In general, piwil1 crispants carried germ cells, went through puberty and became fertile, although a transient and partial germ cell loss and delays during the spermatogenic process were observed in many male crispants, suggesting that Piwil1 functions during salmon spermatogenesis. By crossing highly mutated F0 founders, we produced F1 fish with a mixture of: loss-of-function alleles (−); functional in frame mutated alleles (+) and wt alleles (+). In F1, all piwil1−/− fish lacked germ cells, while piwil1+/+ siblings showed normal ovaries and testes. Yet, most juvenile F1 piwil1+/−males and females displayed an intermediate phenotype with a higher somatic/germ cell ratio without an increase in germ cell apoptosis, suggestive of a gene dose effect on the number of germ cells and/or insufficient replacement of lost germ cells in heterozygous fish. Interestingly, the two longest in-frame indels in the N domain also ensured germ cell loss. Hence, the loss of 4–6 aa in this region Phe130-Ser136 may result in crucial changes of the protein structure, potentially affecting piRNA binding of the PAZ lobe, and/or affecting the binding of Piwil1 interacting proteins such as Tdrd protein, with critical consequences for the survival of primordial germ cells. In conclusion, we show that loss of piwil1 leads to loss of germ cells in salmon and that part of the N domain of Piwil1 is crucial for its function.

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“…Germ cells are a prerequisite for female development in zebrafish ( Danio rerio ), medaka ( Oryzias latipes ), and mackerel ( Scomber australasicus ) as ablation of PGCs results in phenotypic, though sterile, males only (Kawamura et al, 2020; Kurokawa et al, 2007; Siegfried & Nüsslein‐Volhard, 2008; Slanchev et al, 2005; Weidinger et al, 2003). In comparison, PGC ablation failed to affect the sexual fate of gonadal somatic cells in loach ( Misgurnus anguillicaudatus ), goldfish ( Carassius auratus ), grass puffer (Tak ifugu alboplumbeus ), and salmonids, which could develop as either phenotypic males or females (Fujihara et al, 2022; Fujimoto et al, 2010; Goto et al, 2012; Wargelius et al, 2016; Yoshikawa et al, 2020; Yoshizaki et al, 2016). In zebrafish, removal of germ cells led to incomplete masculinization of the brain despite normal sex steroid levels (Pradhan & Olsson, 2018), while dnd ‐knockout Atlantic salmon ( Salmo salar ) males and females showed basal plasma sex steroid concentrations and low gene expression levels along the brain–pituitary–gonadal (BPG) axis at the seawater stage (Kleppe et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Impact of germ cell ablation on the activation of the brain–pituitary–gonadal axis in precocious Atlantic salmon (Salmo salar L.) males

Tveiten

Karlsen

Thesslund

et al. 2022

Molecular Reproduction Devel

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The germ cells are essential for sexual reproduction by giving rise to the gametes, but the importance of germ cells for gonadal somatic functions varies among vertebrates. The RNA-binding dead end (Dnd) protein is necessary for the specification and migration of primordial germ cells to the future reproductive organs. Here, we ablated the gametes in Atlantic salmon males and females by microinjecting dnd antisense gapmer oligonucleotides at the zygotic stage.Precocious maturation was induced in above 50% of both germ cell-depleted and intact fertile males, but not in females, by exposure to an off-season photoperiod regime. Sterile and fertile males showed similar body growth, but maturing fish tended to be heavier than their immature counterparts. Pituitary fshβ messenger RNA levels strongly increased in maturing sterile and fertile males concomitant with the upregulated expression of Sertoli and Leydig cell markers. Plasma concentrations of 11-ketotestosterone and testosterone in maturing sterile males were significantly higher than the basal levels in immature fish, but lower than those in maturing fertile males. The study demonstrates that germ cells are not a prerequisite for the activation of the brain-pituitary-gonad axis and sex steroidogenesis in Atlantic salmon males, but may be important for the maintenance of gonadal somatic functions.

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Production of Germ Cell-Less Rainbow Trout by dead end Gene Knockout and their Use as Recipients for Germ Cell Transplantation

Cited by 31 publications

References 39 publications

A Homology Guide for Pacific Salmon Genus Oncorhynchus resolves patterns of Duplicate Retention, Rediploidization and Local Adaptation Following the Salmonid Specific Whole Genome Duplication Event

A Homology Guide for Pacific Salmon Genus Oncorhynchus resolves patterns of Duplicate Retention, Rediploidization and Local Adaptation Following the Salmonid Specific Whole Genome Duplication Event

The Piwil1 N domain is required for germ cell survival in Atlantic salmon

Impact of germ cell ablation on the activation of the brain–pituitary–gonadal axis in precocious Atlantic salmon (Salmo salar L.) males

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