Characterization of diverse melanopsin-expressing cells in zebrafish

描述（由申请人提供）：动物需要光来使其生理和行为活动与光：暗循环相一致。在人类中，这种同步的中断可能导致影响情绪，睡眠-觉醒周期，癌症发展和治疗的生理和行为障碍。除了经典的光感受器、视杆细胞和视锥细胞之外，视网膜神经节细胞的一小部分在哺乳动物视网膜中具有固有的光敏性。这些神经节细胞表达黑视素（Opn 4），它负责介导小鼠的昼夜节律和其他离子成像光反应。系统发育分析和序列相似性表明，脊椎动物进化出两个黑视蛋白组：Opn 4x，类似于在非洲爪蟾中发现的第一个黑视蛋白，和Opn 4 m，类似于哺乳动物蛋白。哺乳动物只有一个黑视素基因（opn 4 m），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opn 4相关基因，它们在视网膜中显示出独特的非重叠模式。此外，所有的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opn 4x 2基因以类似于哺乳动物基因的模式表达，但也在光感受器官松果体的少数细胞中表达。本研究的目的是鉴定和探讨opn 4表达细胞在视网膜中的功能。1瞄准1测定斑马鱼五种蛋白质的吸收光谱和对光的响应能力; 2.用已知的opn 4x 2自主视网膜神经节细胞的视网膜标记物通过原位杂交和免疫荧光确定3 pn 4表达细胞的表达模式和身份。使用Tol 2转座产生稳定的转基因株系，以映射opn 4x 2的神经回路并鉴定脑靶点。选择性地扩增表达opn 4x 2的细胞，并进行行为测定以评估这些细胞的功能。这些研究将为深入了解视网膜opn 4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帮助。此外，这些研究将进一步我们的理解，在一个昼夜，非哺乳类脊椎动物的opn 4表达细胞的离视网膜连接。

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.