Hang Chong Yee, Moriya Shogo, Ogawa Satoshi, Parhar Ishwar S
Brain Research Institute, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, Selangor, Malaysia.
PLoS One. 2016 Oct 28;11(10):e0165535. doi: 10.1371/journal.pone.0165535. eCollection 2016.
Non-rod non-cone photopigments in the eyes and the brain can directly mediate non-visual functions of light in non-mammals. This was supported by our recent findings on vertebrate ancient long (VAL)-opsin photopigments encoded by the val-opsinA (valopa) and val-opsinB (valopb) genes in zebrafish. However, the physiological functions of valop isoforms remain unknown. Here, we generated valop-mutant zebrafish using CRISPR/Cas genome editing, and examined the phenotypes of loss-of-function mutants. F0 mosaic mutations and germline transmission were confirmed via targeted insertions and/or deletions in the valopa or valopb gene in F1 mutants. Based on in silico analysis, frameshift mutations converted VAL-opsin proteins to non-functional truncated forms with pre-mature stop codons. Most F1 eggs or embryos from F0 female valopa/b mutants showed either no or only partial chorion elevation, and the eggs or embryos died within 26 hour-post-fertilization. However, most F1 embryos from F0 male valopa mutant developed but hatched late compared to wild-type embryos, which hatched at 4 day-post-fertilization. Late-hatched F1 offspring included wild-type and mutants, indicating the parental effects of valop knockout. This study shows valop gene knockout affects chorion formation and embryonic hatching in the zebrafish.
眼睛和大脑中的非视杆非视锥光色素可直接介导非哺乳动物中光的非视觉功能。我们最近在斑马鱼中对由val-视蛋白A（valopa）和val-视蛋白B（valopb）基因编码的脊椎动物古老长链（VAL）-视蛋白光色素的研究结果支持了这一点。然而，valop同工型的生理功能仍然未知。在这里，我们使用CRISPR/Cas基因组编辑技术生成了valop突变斑马鱼，并检查了功能丧失突变体的表型。通过F1突变体中valopa或valopb基因的靶向插入和/或缺失，确认了F0镶嵌突变和种系传递。基于计算机分析，移码突变将VAL-视蛋白转化为带有过早终止密码子的无功能截短形式。来自F0雌性valopa/b突变体的大多数F1卵或胚胎要么没有绒毛膜隆起，要么只有部分绒毛膜隆起，并且这些卵或胚胎在受精后26小时内死亡。然而，来自F0雄性valopa突变体的大多数F1胚胎能够发育，但与在受精后4天孵化的野生型胚胎相比孵化较晚。孵化较晚的F1后代包括野生型和突变体，这表明valop基因敲除存在亲代效应。这项研究表明，valop基因敲除会影响斑马鱼的绒毛膜形成和胚胎孵化。
