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Vgll3 is linked to age at maturity in Atlantic salmon (Salmo salar). However, the molecular mechanisms involving Vgll3 in controlling timing of puberty as well as relevant tissue and cell types are currently unknown. Vgll3 and the associated Hippo pathway has been linked to reduced proliferation activity in different tissues. Analysis of gene expression reveals for the first time that vgll3 and several members of the Hippo pathway were down-regulated in salmon testis during onset of puberty and remained repressed in maturing testis. In the gonads, we found expression in Sertoli and granulosa cells in males and females, respectively. We hypothesize that vgll3 negatively regulates Sertoli cell proliferation in testis and therefore acts as an inhibitor of pubertal testis growth. Gonadal expression of vgll3 is located to somatic cells that are in direct contact with germ cells in both sexes, however our results indicate sex-biased regulation of vgll3 during puberty.

Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out dnd, a factor required for germ cell survival in vertebrates. To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer (alb) and the target (dnd) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish.