Tracking GnRH neurons in vivo following CRISPR knock out of Six6 in Atlantic salmon

Abstract

Gonadotropin releasing hormone (GnRH) is a conserved neuroendocrine decapeptide, crucial for the onset of puberty and for the functioning of the hypothalamic-pituitary-gonadal axis (HPG). The homeodomain protein sine oculis related homeobox 6 (Six6) is necessary for GnRH neuronal development in mammals. We wanted to investigate the role of Six6 in GnRH neuronal development in the Atlantic salmon (Salmo salar), since the effects of Six6 during embryogenesis in Atlantic salmon is little studied. I sought to induce knockout of the Six6 gene in vivo in salmon embryos during the one cell stage using Clustered-Regularly-Interspaced-Short-Palindromic-Repeats (CRISPR) Cas9 and perform immunohistochemistry with GnRH specific antibodies on the knocked-out embryos as they developed to track changes in GnRH neuronal development. While the immunohistochemistry procedure failed to produce successful immunostaining of GnRH neurons, I did manage to knockout Six6 with a very high efficiency, helping establish a CRISPR protocol that can be used to knockout Six6 in Atlantic salmon.