PSCAL在视网膜感光细胞形成中的功能和作用机理研究

The retinal photoreceptor layer is an important component of the eye, its abnormality or degradation will cause eye defects or even ocular diseases. However, the molecular mechanisms underlying photoreceptor formation remain to be fully elucidated. .The ability to explore the genetic and embryonic accessibility of zebrafish in combination with a behavioral assessment of visual function has made zebrafish a popular model in vivo to study visual system. Very recently, we have characterized two novel PSCA-like genes PSCALa and PSCALb, which are both Ly-6 superfamily members in zebrafish (Danio rerio), and encode GPI-anchored membrane proteins. Whole-mount in situ hybridization showed that PSCAL are specifically expressed in the retinal photoreceptor cell layer. Moreover, knockdown of PSCALa leads to abnormal retinal photoreceptor, and the expression of rhodopsin and four photoreceptor opsins (blue, green, red, UV) are significantly downregulated. We also found that knockdown of PSCALa in transgenic Bm3-GFP fish line affects the visual function. In addition, PSCALa knockout induces double cone mislocalization. Knockdown and knockout of PSCALb is currently under way. All these preliminary results suggest that PSCALa may be required for the formation and function of retinal photoreceptor. However, its precise role and mode of action remain enigmatic, and demand detailed study. .The aim of this proposal is thus to explore the function and mechanisms of PSCAL gene/protein in photoreceptor formation. First, we will analyze the implication of PSCAL in the formation of retinal photoreceptor by morpholino-mediated gene knockdown and TALENs-mediated gene knockout. We will then examine the visual function in mutants by behavioral experiments. We also plan to test whether PSCAL is involved in regulating cell adhesion; to identify putative protein partners which interact with PSCAL; to analyze the biochemical and functional interaction between PSCAL and its interaction partners. Eventually, these experiments will elucidate the mechanism of PSCAL in the formation and function of retinal photoreceptor. This will also provide novel insights into the gene regulatory network underlying the retinal photoreceptor formation.

视网膜感光细胞层对视觉功能至关重要，探索其发育机制是视觉系统研究的热点。我们发现斑马鱼Ly6家族基因PSCAL (PSCA Like-a和PSCA Like-b)在视网膜感光细胞特异表达，基因敲降和敲除时感光细胞异常，视网膜双视锥细胞定位错误，排列混乱，提示其在感光细胞中起重要作用，但其作用机制尚不明确。我们拟：确定PSCAL的蛋白定位；通过基因敲降和敲除研究PSCAL对感光细胞及视觉功能的影响；通过细胞转染方法确定PSCAL是否促进细胞粘附；通过酵母双杂交筛选可能与PSCAL相互作用的蛋白，重点关注其中是否有在视网膜感光细胞表达的受体或离子通道蛋白等，并研究在视网膜发育中的功能；研究PSCAL与其相互作用蛋白的共定位、调控机制以及二者功能互作对视网膜感光细胞的影响。在上述基础上，阐明PSCAL的作用机制，构建视网膜感光细胞发育的调控网络模型，并为早期眼睛发育缺陷的分子机制提供参考。

视网膜感光细胞层对视觉功能至关重要，探索其发育机制是视觉系统研究的热点。我们发现斑马鱼Ly6家族基因PSCAL(lypge)在视网膜感光细胞特异表达，敲降和敲除时感光细胞异常，视网膜双视锥细胞定位错误，排列混乱，但其具体功能和作用机制尚不明确。我们发现斑马鱼中有两个在视网膜感光细胞表达的Ly6家族基因，二者的表达模式有差异， 其中pscal/lypge 在眼睛感光细胞和松果体特异表达，其编码的蛋白是GPI锚定的含LU功能域的膜蛋白。Lypge呈现节律性表达，其启动子含有多个 Otx5的结合位点，表达受Otx5的正调控，是Otx5的靶基因。发现pscal/lypge基因敲降不影响视网膜的背腹模式形成，但会影响视网膜感光细胞发育，导致眼睛变小；利用Talens方法构建pacal/lypge基因敲除的斑马鱼品系，发现一月龄斑马鱼突变体的视网膜视杆细胞未受影响，但视锥细胞排列不规则，出现明显的退化，并且GNAT2在OS上的表达明显减少，且会错误的定位。lypge敲除导致endothelin-2表达量的升高，除此，视投射神经的突出明显变少，视力受到影响。且随发育进行，视网膜感光细胞退化会加重。据此，认为Lypge在成体的视网膜感光细胞正常发育中起重要作用。明确了Pscal/lypge 调控视网膜感光细胞发育的机制，发现Lypge并不影响细胞的增殖，体外细胞实验表明Lypge与细胞粘附无关；lypge的敲降会导致细胞凋亡，从而导致视网膜感光细胞发育异常。测定lypge敲除后胚胎的转录组，发现C2H2型锌指蛋白转录因子的表达变化较大。除此，研究还发现NFELb通过调控神经元的凋亡从而影响早期神经系统发生。本项目的研究阐明Lypge的功能和作用机制，完善了视网膜感光细胞发育的调控网络模型，并为早期眼睛发育缺陷的分子机制提供参考。