PDF(Pubmed)
Abstract：
As a member of the fibronectin leucine-rich transmembrane (flrt) gene family, fibronectin leucine-rich transmembrane 2 (flrt2) is strongly expressed in a subset of sclerotome cells, and the resultant protein interacts with FGFR1 in the FGF signaling pathway during development. Studies on flrt2 have focused mainly on its roles in the brain, heart and chondrogenesis. However, reports on its expression and function in the zebrafish retina are lacking. Here, we detected the high expression of flrt2 in zebrafish retina using in situ hybridization technique and developed an flrt2-knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. Quantitative real-time PCR was used to measure the expression levels of flrt2, which results in an approximately 60% mRNA reduction. The flrt2-KO zebrafish eyes\' altered morphological, cellular, and molecular events were identified using BrdU labeling, TUNEL assay, immunofluorescent staining, fluorescent dye injection and RNA sequencing. Abnormal eye development, known as microphthalmia, was found in flrt2-KO larvae, and the retinal progenitor cells exhibited increased apoptosis, perhaps owing to the combined effects of crx, neurod4, atoh7, and pcdh8 downregulation and Casp3a and Caspbl upregulation. In contrast, the retinal neural development, as well as retinal progenitor cell differentiation and proliferation, were not affected by the flrt2 deletion. Thus, flrt2 appears to play important roles in retinal development and function, which may provide the basis for further investigations into the molecular mechanisms of retinal development and evolution.
摘要：
作为flrt基因家族的一员,纤维结合蛋白富含亮氨酸的跨膜2（flrt2）在巩膜组细胞的亚群中强烈表达，并且所得蛋白在发育过程中与FGF信号通路中的FGFR1相互作用。对flrt2的研究主要集中在其在大脑中的作用，心脏和软骨形成。然而,关于其在斑马鱼视网膜中的表达和功能的报道尚缺乏。这里,我们使用原位杂交技术检测到flrt2在斑马鱼视网膜中的高表达，并使用CRISPR/Cas9基因组编辑开发了flrt2敲除（KO）斑马鱼细胞系。定量实时PCR用于测量flrt2的表达水平,其导致约60%的mRNA减少。flrt2-KO斑马鱼的眼睛改变了形态，细胞，使用BrdU标记鉴定分子事件，TUNEL检测,免疫荧光染色，荧光染料注射和RNA测序。眼睛发育异常,被称为小眼症，在flrt2-KO幼虫中发现,视网膜祖细胞表现出增加的凋亡，也许是由于crx的综合作用,neurod4、atoh7和pcdh8下调，Casp3a和Caspbl上调。相比之下,视网膜神经发育，以及视网膜祖细胞的分化和增殖，不受flrt2缺失的影响。因此，flrt2似乎在视网膜发育和功能中起重要作用，这可能为进一步研究视网膜发育和进化的分子机制提供基础。
