Development of the blue cone bipolar cell in the mouse retina

小鼠视网膜中蓝锥双极细胞的发育

• 批准号: 8737665
• 负责人: Wei Li
• 金额: $ 33.09万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8737665
• 关键词: Accounting; Affect; Bacterial Artificial Chromosomes; Biological Models; Cells; Color; Color Visions; Cues; Dendrites; Development; Face; Foundations; Future; Gene Expression Profile; Genes; Genetic Recombination; Knock-out; Knockout Mice; Label; Light; Mammals; Molecular; Mus; Neurons; Neurosciences; Opsin; Outcome; Population; Primates; Process; Promoter Regions; Proteins; Retina; Retinal Cone; Signal Transduction; Specific qualifier value; Specificity; Synapses; Thyroid Hormone Receptor; Transgenic Mice; Variant; Venus; Vision; Work; clomeleon; density; ganglion cell; interest; mouse model; neuron development; postsynaptic; presynaptic; promoter; retinal neuron

How neuronal processes develop and establish proper wirings with their synaptic partners is one of the fundamental questions of neuroscience. The vertebrate retina is an outstanding model system for studying dendritic development and neuronal connections. One of the critical visual functions, color vision, requires precise wiring of retinal neurons. In the mouse retina, there are two types of cone photoreceptors, the short wavelength sensitive cones (S-cones), which only express S-opsin, and the long wavelength sensitive cones (M-cones), many of which co-express S-opsin. In order to generate color opponency, signals from these two types of cones have to be segregated before they are contrasted at the ganglion cell level. S-cones only account for 2-5% of the total cone population. Thus, the downstream S-cone bipolar cells (SCBCs) face the daunting task of seeking out very sparse S-cones from a majority of M-cones. The outcome is that SCBCs develop a very unique dendritic arbor with long, meager dendrites that contact a handful of S-cones. This distinctive connection between S-cones and SCBCs makes it an excellent model system to study how presynaptic neurons affect the dendritic development and synaptic targeting of postsynaptic neurons. SCBs are labeled in a transgenic mouse line expressing Clomeleon (Clm) driven by thy1 promoter. Two mouse models were used to change the density and type of cones. In Thrb2-/- mice which lack thyroid hormone receptor 2 (TR2), M-opsin expression is abolished and all M-cones are turned into S-cones32. In S-opsin-/- mice, the S-opsin gene is knocked out. We crossed two lines and generated a double knockout (DKO) with neither S- nor M-opsin expression. We found that the numbers of SCBs in Thrb2-/-, S-opsin-/- and DKO mice are similar to those in wildtype. Morphologically, SCBs in Thrb2-/- and S-opsin-/- mice were indistinguishable from those in wildtype in terms of number of dendritic branches and cone contacts. SCBs in these mice appear to target specific true S-cones, even though the type of opsin expression is identical across all cones. Our results suggest that 1) dendritic development of SCBs appears to be intrinsic; 2) S-cone identity may be specified by factors other than S-opsin expression a hypothesis that we will investigate in the future. In order to compare the transcriptome of S- and M-cones, we need to isolate true S-cones. An existing transgenic mouse line that expresses EGFP driven by an S-opsin promoter is not reliable in that some S-opsin negative cones also express GFP. This is likely due to the short S-opsin promoter region used in generating the mouse line. Due to low percentage of true S-cones (5%), this contamination from M-cones will be ruinous for RNAseq. We thus are currently working on generating our own mouse line using the Bacterial Artificial Chromosome (BAC) recombination approach to include longer upstream and downstream regulatory sequences at the endogenous S-opsin gene locus. We are sceening mouse lines that express yellow fluorescent protein YFP (Venus) only in S-opsin expressing cones.

神经元过程如何发育并与其突触伙伴建立适当的连接是神经科学的基本问题之一。脊椎动物视网膜是研究树突发育和神经元连接的杰出模型系统。 色觉是关键视觉功能之一，需要视网膜神经元的精确连接。 在小鼠视网膜中，有两种类型的视锥细胞：短波长敏感视锥细胞（S-视锥细胞），仅表达 S-视蛋白；以及长波长敏感视锥细胞（M-视锥细胞），其中许多共同表达 S-视蛋白。为了产生颜色对比，来自这两种类型视锥细胞的信号必须在神经节细胞水平上进行对比之前进行分离。 S-视锥细胞仅占视锥细胞总数的2-5%。因此，下游的 S 锥双极细胞 (SCBC) 面临着从大多数 M 锥中找出非常稀疏的 S 锥的艰巨任务。结果是，SCBC 形成了非常独特的树突乔木，其长而细小的树突与少数 S 锥体接触。 S 锥体和 SCBC 之间的这种独特联系使其成为研究突触前神经元如何影响突触后神经元的树突发育和突触靶向的优秀模型系统。 SCB 在表达由 thy1 启动子驱动的 Clomeleon (Clm) 的转基因小鼠系中进行标记。使用两种小鼠模型来改变视锥细胞的密度和类型。在缺乏甲状腺激素受体 2 (TR2) 的 Thrb2-/- 小鼠中，M-视蛋白表达被消除，所有 M-视锥细胞转化为 S-视锥细胞32。 在 S-视蛋白-/- 小鼠中，S-视蛋白基因被敲除。我们将两条细胞系杂交，产生了既不表达 S 视蛋白又不表达 M 视蛋白的双敲除 (DKO)。我们发现Thrb2-/-、S-opsin-/-和DKO小鼠中的SCB数量与野生型相似。在形态学上，Thrb2-/-和S-视蛋白-/-小鼠中的SCB在树突分支和视锥接触的数量方面与野生型小鼠中的SCB没有区别。 这些小鼠中的 SCB 似乎针对特定的真正的 S 视锥细胞，尽管所有视锥细胞的视蛋白表达类型都是相同的。我们的结果表明：1）SCB 的树突发育似乎是内在的； 2）S-视锥细胞身份可能由S-视蛋白表达以外的因素指定，这是我们将来将研究的假设。 为了比较 S 锥体和 M 锥体的转录组，我们需要分离真正的 S 锥体。 现有的表达由 S-视蛋白启动子驱动的 EGFP 的转基因小鼠系并不可靠，因为一些 S-视蛋白阴性视锥细胞也表达 GFP。 这可能是由于用于生成小鼠品系的短 S-视蛋白启动子区域所致。 由于真正的 S 锥体的比例较低 (5%)，这种来自 M 锥体的污染对于 RNAseq 来说将是毁灭性的。 因此，我们目前正致力于使用细菌人工染色体 (BAC) 重组方法产生我们自己的小鼠品系，在内源性 S-视蛋白基因座上包含更长的上游和下游调控序列。我们正在筛选仅在表达 S-视蛋白的视锥细胞中表达黄色荧光蛋白 YFP（金星）的小鼠品系。