Rainy Nir, Etzion Talya, Alon Shahar, Pomeranz Adi, Nisgav Yael, Livnat Tami, Bach Michael, Gerstner Cecilia D, Baehr Wolfgang, Gothilf Yoav, Stiebel-Kalish Hadas
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Israel.
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Israel; Sagol School of Neuroscience Tel Aviv University, Israel.
Biochem Biophys Res Commun. 2016 May 13;473(4):1211-1217. doi: 10.1016/j.bbrc.2016.04.041. Epub 2016 Apr 11.
UNC119 proteins are involved in G protein trafficking in mouse retinal photoreceptors and Caenorhabditis elegans olfactory neurons. An Unc119 null allele is associated with cone-rod dystrophy in mouse, but the mechanism leading to disease is not understood. We studied the role of Unc119 paralogs and Arl3l2 in zebrafish vision and retinal organization resulting from unc119c and arl3l2 knockdown.
Zebrafish unc119c was amplified by PCR from retina and pineal gland cDNA. Its expression pattern in the eye and pineal gland was determined by whole-mount in-situ hybridization. unc119c and arl3l2 were knocked down using morpholino-modified oligonucleotides (MO). Their visual function was assessed with a quantitative optomotor assay on 6 days post-fertilization larvae. Retinal morphology was analyzed using immunohistochemistry with anti-cone arrestin (zpr-1) and anti-cone transducin-α (GNAT2) antibodies.
The zebrafish genome contains four genes encoding unc119 paralogs located on different chromosomes. The exon/intron arrangements of these genes are identical. Three Unc119 paralogs are expressed in the zebrafish retina, termed Unc119a-c. Based on sequence similarity, Unc119a and Unc119b are orthologs of mammalian UNC119a and UNC119b, respectively. A third, Unc119c, is unique and not present in mammals. Whole mount in-situ hybridization revealed that unc119a and unc119b RNA are ubiquitously expressed in the CNS, and unc119c is specifically expressed in photoreceptive tissues (pineal gland and retina). A Unc119 interactant, Arl3l2 also localizes to the pineal gland and the retina. As measured by the optomotor response, unc119c and arl3l2 knockdown resulted in significantly lower vision compared to wild-type zebrafish larvae and control morpholino (MO). Immunohistological analysis with anti-cone transducin and anti-cone arrestin (zpr-1) indicates that knockdown of unc119c leads to photoreceptor degeneration mostly affecting cones.
Our results suggest that Unc119c is the only Unc119 paralog that is highly specific to the retina in zebrafish. Unc119c and Arl3l2 proteins are important for the function of cones.
UNC119蛋白参与小鼠视网膜光感受器和秀丽隐杆线虫嗅觉神经元中的G蛋白运输。UNC119无效等位基因与小鼠的视锥-视杆营养不良相关，但导致疾病的机制尚不清楚。我们研究了Unc119旁系同源物和Arl3l2在斑马鱼视觉以及由unc119c和arl3l2敲低导致的视网膜组织中的作用。
通过PCR从视网膜和松果体cDNA中扩增斑马鱼unc119c。通过整体原位杂交确定其在眼睛和松果体中的表达模式。使用吗啉代修饰的寡核苷酸（MO）敲低unc119c和arl3l2。在受精后6天的幼虫上通过定量视动反应试验评估它们的视觉功能。使用抗视锥抑制蛋白（zpr-1）和抗视锥转导蛋白-α（GNAT2）抗体的免疫组织化学分析视网膜形态。
斑马鱼基因组包含四个编码位于不同染色体上的Unc119旁系同源物的基因。这些基因的外显子/内含子排列相同。三个Unc119旁系同源物在斑马鱼视网膜中表达，称为Unc119a - c。基于序列相似性，Unc119a和Unc119b分别是哺乳动物UNC119a和UNC119b的直系同源物。第三个，Unc119c是独特的，在哺乳动物中不存在。整体原位杂交显示unc119a和unc119b RNA在中枢神经系统中普遍表达，而unc119c在感光组织（松果体和视网膜）中特异性表达。一种Unc119相互作用蛋白，Arl3l2也定位于松果体和视网膜。通过视动反应测量，与野生型斑马鱼幼虫和对照吗啉代（MO）相比unc119c和arl3l2敲低导致视力显著降低。用抗视锥转导蛋白和抗视锥抑制蛋白（zpr-1）进行的免疫组织学分析表明unc119c敲低导致主要影响视锥细胞的光感受器退化。
我们的结果表明Unc119c是斑马鱼视网膜中唯一高度特异性存在的Unc119旁系同源物。Unc119c和Arl3l2蛋白对视锥细胞的功能很重要。
