Development of the blue cone bipolar cell in the mouse retina

神经元过程如何发展并与它们的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一个杰出的模型系统。 其中一个关键的视觉功能，色觉，需要视网膜神经元的精确布线。 在小鼠视网膜中，存在两种类型的视锥光感受器，即仅表达S-视蛋白的短波长敏感视锥（S-视锥）和其中许多共表达S-视蛋白的长波长敏感视锥（M-视锥）。为了产生颜色对比，来自这两种类型的视锥细胞的信号必须在它们在神经节细胞水平上形成对比之前被分离。 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锥中寻找非常稀疏的S锥的艰巨任务。结果是SCBC形成了一个非常独特的树突状乔木，具有长而微薄的树突，与少数S-视锥细胞接触。 S-锥和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极好模型系统。 在表达由thy 1启动子驱动的Clomeleon（Clm）的转基因小鼠系中标记SCB。两种小鼠模型用于改变视锥细胞的密度和类型。在缺乏甲状腺激素受体2（TR 2）的Thrb 2-/-小鼠中，M-视蛋白表达被消除，并且所有M-视锥都变成S-视锥32。 在S-视蛋白-/-小鼠中，S-视蛋白基因被敲除。我们将两个细胞系交叉，并产生了既没有S-视蛋白表达也没有M-视蛋白表达的双敲除（DKO）。我们发现Thrb 2-/-、S-视蛋白-/-和DKO小鼠中SCB的数量与野生型中的那些相似。在形态学上，Thrb 2-/-和S-视蛋白-/-小鼠中的SCB在树突状分支和视锥接触的数量方面与野生型中的SCB没有区别。 这些小鼠中的SCB似乎靶向特定的真S-视锥，即使视蛋白表达的类型在所有视锥中是相同的。我们的研究结果表明：1）SCBs的树突发育似乎是内在的; 2）S-视锥的身份可能是由S-视蛋白表达以外的因素指定的，我们将在未来调查的假设。 为了比较S锥和M锥的转录组，我们需要分离真正的S锥。 现有的表达由S-视蛋白启动子驱动的EGFP的转基因小鼠系是不可靠的，因为一些S-视蛋白阴性视锥细胞也表达GFP。 这可能是由于用于产生小鼠系的短S-视蛋白启动子区。 由于低百分比的真S-视锥细胞（5%），来自M-视锥细胞的这种污染将对RNAseq是毁灭性的。 因此，我们目前正在使用细菌人工染色体（BAC）重组方法来产生我们自己的小鼠系，以在内源性S-视蛋白基因座处包括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（Venus）的小鼠品系。

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.