Characterizing the R8 cell subtype fate in Drosophila

描述(申请人提供)：为了获得真彩色视觉，动物必须有能力比较不同吸收波长的光感受器的输出。果蝇的复眼由大约800个被称为小眼的重复单位组成，每个小眼又细分为20个专门的细胞。其中八个细胞是吸收光线所需的光感受器。六个外部光感受器(R1-R6)表达广谱视紫红质Rh1，是运动检测所必需的。这些细胞与人类的杆状细胞相似。果蝇眼睛内的两个光感受器(R7和R8)与人类视锥细胞相似，是色觉所必需的，位于相同的视路中，R7位于近端R8细胞的顶部。它们含有不同的视紫红质感光色素，它们的输出在视叶的髓质部分进行了比较，每个视叶都在那里投射轴突。在果蝇的视网膜上随机分布着两类不同的小眼：在70%的小眼(黄色(Y)小眼)中，R7含有UV-Rh4，而R8含有绿色-Rh6，它们专门用于区分较长波长的光。在其余30%的苍白(P)小眼中，R7含有对紫外线敏感的Rh3，而R8含有蓝色Rh5，这使得这些小眼很好地适应于区分短波长的光。我们的实验室最近发现，在位于远处的R7细胞中，当无刺转录因子被激活时，随机选择成为y或小眼。这发生在化蛹期间70%的R7中，指定它们为yr7。剩余的R7在默认情况下成为PR7，随后指示它们的底层R8成为PR8。在这里，我提出了三个目标，旨在揭示R8亚型规范所需的信号机制，该机制导致Rh5在PR8中的稳健表达，Rh6在YR8中的稳健表达。我们实验室最近的工作表明，在R7启动的DPP信号通路中，通过激活素受体Babo(在R8中)在建立R8命运的非规范途径中发挥了作用。然后，BABO可能会与疣/融化的双稳环相互作用，以维持这一命运。我将研究整个DPP和Activin信号通路，在这个命运决定中，这两个通路都没有被充分研究过是否有额外或多余的作用。由于亚型规范可能涉及其他因素，我还将对3000多个已知和预测的膜相关分子和信号分子进行基于RNAi的筛选，并使用RNAseq技术对p和y R7和R8细胞进行亚型表达谱分析。这项工作将扩大我们对视网膜模式和细胞命运建立的知识，特别是在随机决定之后。它还将增加我们对河马肿瘤抑制通路以及DPP和Activin信号通路的了解，所有这些信号通路在这里都以非规范的方式使用。好了！

DESCRIPTION (provided by applicant): In order to achieve true color vision, animals must have the ability to compare the outputs of photoreceptors with different wavelengths of absorption. The compound eye of Drosophila is made up of approximately 800 repeating units called ommatidia and each ommatidia is sub-divided into 20 specialized cells. Eight of these cells are photoreceptors required for absorbing light. The six outer photoreceptors (R1-R6) express the wide- spectrum rhodopsin Rh1 and are required for motion detection. These are similar to human rod cells. The two inner photoreceptors in the Drosophila eye (R7 and R8), which are similar to human cone cells, are required for color vision and lie in the same optic path where R7 sits distally on top of the proximal R8 cell. They contain distinct rhodopsin photopigments and their outputs are compared in the medulla part of the optic lobe where each projects their axons. Two distinct classes of ommatidia are stochastically distributed throughout the fly retina: In 70% of ommatidia (yellow (y) ommatidia), R7 contains UV-Rh4 while R8 contains green-Rh6, specializing them to discriminate longer wavelengths of light. In the remaining 30% pale (p) ommatidia, R7 contains UV-sensitive Rh3 while R8 contains blue-Rh5 making these ommatidia well adapted to discriminate among short wavelengths of light. Our lab has recently found that the stochastic choice to become a y or p ommatidium is made in the distally located R7 cell, when the transcription factor Spineless is activated. This occurs in 70% of R7 during pupation, specifying them as yR7. The remaining R7 become pR7 by default and subsequently instruct their underlying R8 to become pR8. Here I propose three aims designed to uncover the signaling mechanism required for R8 subtype specification, which leads to robust expression of Rh5 in pR8 and Rh6 in yR8. Recent work in our lab has suggested a role for Dpp signaling, initiated in R7, through the Activin receptor, Babo (in R8) in a non-canonical pathway to establish R8 fate. Babo may then interact with the warts/melted bi-stable loop to maintain this fate. I will examine the entire Dpp and Activin signaling pathways, neither of which have been fully examined for additional or redundant roles in this fate decision. As there are likely additional factors involved in subtype specification, I will also conduct an RNAi-based screen of over 3000 known and predicted membrane- associated and signaling molecules as well as perform subtype expression profiling of p and y R7 and R8 cells using RNAseq techniques. This work will expand our knowledge of retinal patterning and cell fate establishment, specifically following a stochastic decision. It will also add to our knowledge of the Hippo tumor suppressor pathway as well as Dpp and Activin signaling pathways, all of which are utilized in non-canonical ways here. !