Characterization of diverse melanopsin-expressing cells in zebrafish

描述(申请人提供)：动物需要光来使它们的生理和行为活动与光：黑暗周期保持一致。在人类中，这种同步性的破坏可能会导致生理和行为障碍，影响情绪、睡眠-觉醒周期、癌症发展和治疗。除了经典的光感受器、视杆细胞和视锥细胞外，哺乳动物视网膜中的一小部分视网膜神经节细胞本质上是光敏的。这些神经节细胞表达光色素黑素(Opn4)，它负责调节小鼠的昼夜节律和其他离子图像形成光反应。系统发育分析和序列相似性分析表明，脊椎动物进化了两个黑素蛋白组：Opn4x和Opn4m，前者类似于非洲爪哇发现的第一个黑素蛋白，后者类似于哺乳动物蛋白质。哺乳动物只有一个黑素蛋白基因(Opn4m)，其表达仅限于视网膜神经节细胞(RGC)。我已经在斑马鱼中发现了五个与Opn4相关的基因，它们在视网膜中显示出独特的、不重叠的模式。此外，所有这些基因都在发育中的斑马鱼大脑的不同区域瞬时表达。斑马鱼opn4x2基因的表达模式类似于哺乳动物的基因，但也在感光器官松果体的第3酶的少数细胞中表达。本研究的目的是鉴定和探索表达Opn4的细胞在视网膜中的功能。1.测定5种斑马鱼蛋白的吸收光谱和光反应能力；2.利用已知的视网膜神经节细胞opn4x2的视网膜标志物，通过原位杂交和免疫荧光检测3pn4表达细胞的表达模式和同源性；3.利用Tol2转座技术建立稳定的转基因系，以定位opn4x2的神经回路并确定脑靶点；4.选择性地去除表达opn4x2的细胞，并形成行为学分析以评估这些细胞的功能。这些研究将对视网膜Opn4表达细胞在影响视觉和非视觉光反应的电路和功能中的不同作用提供深入的见解。此外，这些研究将进一步加深我们对非哺乳动物脊椎动物中表达Opn4的细胞的视黄醇连接的理解。

DESCRIPTION (provided by applicant): Animals require light to align their physiological and behavioral activities to the light:dark cycle. In humans, disruption of this synchronization can lead to physiological and behavioral disorders that affect mood, sleep- wake cycles, cancer development and treatment. In addition to the classical photoreceptors, rods and cones, a small subset of retinal ganglion cells is intrinsically photosensitive in the mammalian retina. These ganglion sells express the photopigment Melanopsin (Opn4), which is responsible for mediating circadian and other ion-Image forming light responses in mice. Phylogenetic analysis and sequence similarity demonstrate that vertebrates evolved two Melanopsin groups: Opn4x, similar to the first Melanopsin discovered in Xenopus, and Opn4m, similar to the mammalian protein. Mammals have only one melanopsin gene (opn4m) and its expression is restricted to retinal ganglion cells (RGCs). I have identified five opn4-related genes in the zebrafish that show unique and non-overlapping patterns in the retina. In addition, all ofthe genes are expressed transiently in distinct regions ofthe developing zebrafish brain. The zebrafish opn4x2 gene is expressed in a pattern similar to the mammalian gene, but also is expressed in a small number of cells at the 3ase ofthe phototoreceptive organ, the pineal gland. The goal of this study is to identify and explore the unctions of opn4-expressing cells in the retina. 1 aim to 1. Determine the absorption spectra ofthe five zebrafish proteins and capability to respond to light, 2. Determine the expression pattern and identity ofthe 3pn4-expressing cells by in situ hybridization and immunofluorescence with known retinal markers of opn4x2 autative retinal ganglion cells, 3. Generate stable transgenic lines using Tol2 transposition to map the neural circuitry of opn4x2 and identify the brain targets and 4. Ablate the opn4x2-expressing cells selectively and Derform behavioral assays to assess the function of these cells. These studies will provide insights into the diverse roles of retinal opn4-expressing cells in influencing the circuitry and function of visual and non-visual ight responses. In addition these studies will further our understanding of the retinofugal connectivity of opn4-expressing cells in a diurnal, non-mammalian vertebrate.