Galicia Carlos A, Sukeena Joshua M, Stenkamp Deborah L, Fuerst Peter G
University of Idaho, Department of Biological Sciences, Moscow, ID.
University of Washington School of Medicine, WWAMI Medical Education Program, Moscow, ID.
Mol Vis. 2018 Jul 19;24:443-458. eCollection 2018.
The differential adhesion hypothesis states that a cell adhesion code provides cues that direct the specificity of nervous system development. The Down syndrome cell adhesion molecule (DSCAM) and sidekick (SDK) proteins belong to the immunoglobulin superfamily of cell adhesion molecules (CAMs) and provide both attractive and repulsive cues that help to organize the nervous system during development, according to the differential adhesion hypothesis. The zebrafish genome is enriched in and genes, making the zebrafish an excellent model system to further test this hypothesis. The goal of this study is to describe the phylogenetic relationships of the paralogous CAM genes and their spatial expression and co-expression patterns in the embryonic zebrafish retina.
Exon-intron structures, karyotypic locations, genomic context, and amino acid sequences of the zebrafish CAM genes (, , , , , , and ) were obtained from the Ensembl genome database. The Prosite and SMART programs were used to determine the number and identity of protein domains for each CAM gene. The randomized axelerated maximum likelihood (RaxML) program was used to perform a phylogenetic analysis of the zebrafish CAM genes and orthologs in other vertebrates. A synteny analysis of regions surrounding zebrafish CAM paralogs was performed. Digoxigenin (dig)-labeled cRNA probes for each CAM gene were generated to perform in situ hybridization of retinal cryosections from zebrafish embryos and larvae. Dual in situ hybridization of retinal cryosections from zebrafish larvae was performed with dig- and fluorescein-labeled cRNA probes.
We found the studied zebrafish CAM genes encode similar protein domain structures as their corresponding orthologs in mammals and possess similar intron-exon organizations. CAM paralogs were located on different chromosomes. Phylogenetic and synteny analyses provided support for zebrafish and paralogs having originated during the teleost genome duplication. We found that and are co-expressed in the ganglion cell layer (GCL) and the basal portion of the inner nuclear layer (INL), with weak expression in the photoreceptor-containing outer nuclear layer (ONL). Of the genes, only was strongly expressed in ONL. and were co-expressed in the GCL and the basal portion of the INL. and also showed co-expression in the GCL and basal portion of the INL. All genes were expressed in the ciliary marginal zone (CMZ). Dual in situ hybridizations revealed alternating patterns of co-expression and exclusive expression for the and paralogs in cells of the GCL and the INL. The same alternating pattern was observed between and paralogs and between and paralogs. The expression of was observed in the INL and the GCL, with some cells in the basal portion of the INL showing co-expression of and .
These findings suggest that zebrafish and paralogs were likely the result of the teleost whole genome duplication and that all CAM duplicates show some differential expression patterns. We also demonstrate that the comparative expression patterns of CAM genes in the zebrafish are distinct from the exclusive expression patterns observed in chick retina, in which retinal ganglion cells express one of the four chick or genes only. The patterns in zebrafish are more similar to those of mice, in which co-expression of and genes is observed. These findings provide the groundwork for future functional analysis of the roles of the CAM paralogs in zebrafish.
差异黏附假说指出，细胞黏附密码提供了指导神经系统发育特异性的线索。唐氏综合征细胞黏附分子（DSCAM）和Sidekick（SDK）蛋白属于细胞黏附分子（CAM）的免疫球蛋白超家族，根据差异黏附假说，它们在发育过程中提供吸引和排斥线索，有助于构建神经系统。斑马鱼基因组中富含 和 基因，这使得斑马鱼成为进一步验证该假说的优秀模型系统。本研究的目的是描述胚胎斑马鱼视网膜中同源CAM基因的系统发育关系及其空间表达和共表达模式。
从Ensembl基因组数据库中获取斑马鱼CAM基因（ 、 、 、 、 、 和 ）的外显子-内含子结构、核型定位、基因组背景和氨基酸序列数据。使用Prosite和SMART程序确定每个CAM基因的蛋白质结构域数量和特征。采用随机加速最大似然法（RaxML）程序对斑马鱼CAM基因及其在其他脊椎动物中的直系同源基因进行系统发育分析。对斑马鱼CAM旁系同源基因周围区域进行共线性分析。制备每个CAM基因的地高辛（dig）标记的cRNA探针，用于对斑马鱼胚胎和幼体视网膜冰冻切片进行原位杂交。使用dig标记和荧光素标记的cRNA探针，对斑马鱼幼体视网膜冰冻切片进行双重原位杂交。
我们发现，所研究的斑马鱼CAM基因编码的蛋白质结构域结构与其在哺乳动物中的相应直系同源基因相似，并且具有相似的内含子-外显子组织。CAM旁系同源基因位于不同的染色体上。系统发育和共线性分析支持斑马鱼 和 旁系同源基因起源于硬骨鱼基因组复制。我们发现 和 在神经节细胞层（GCL）和内核层（INL）的基部共表达，在含有光感受器的外核层（ONL）中表达较弱。在 基因中，只有 在ONL中强烈表达。 和 在GCL和INL的基部共表达。 和 在GCL和INL的基部也显示出共表达。所有 基因均在睫状边缘区（CMZ）表达。双重原位杂交揭示了GCL和INL细胞中 和 旁系同源基因共表达和排他性表达的交替模式。在 和 旁系同源基因之间以及 和 旁系同源基因之间观察到相同的交替模式。在INL和GCL中观察到 的表达，INL基部的一些细胞显示 和 的共表达。
这些发现表明，斑马鱼 和 旁系同源基因可能是硬骨鱼全基因组复制的结果，并且所有CAM重复基因都表现出一些差异表达模式。我们还证明，斑马鱼中CAM基因比较表达模式与在鸡视网膜中观察到的排他性表达模式不同，在鸡视网膜中，视网膜神经节细胞仅表达四个鸡 或 基因之一。斑马鱼中的模式与小鼠中的模式更相似，在小鼠中观察到 和 基因的共表达。这些发现为未来对斑马鱼中CAM旁系同源基因作用的功能分析奠定了基础。
