MAB21L Family in Human Ocular Disease and Development

项目总结/摘要 眼缺损是一种先天性的节段性眼缺损，可影响眼的各种结构。 缺损通常与小眼球、小角膜和/或视网膜脱离相关，并导致 视力障碍我们和另一个小组最近报道了一种与缺损相关的新因素， MAB21L2。作为这些研究的一部分，我们产生了携带移码的斑马鱼mab 21 l2等位基因系列， 在orthotropic基因区域中的框内插入缺失和错义突变，并观察到缺损， 严重的眼部紊乱接下来，在大肠杆菌中筛选另一个MAB 21 L基因MAB 21 L1， 人类患者鉴定了可能的致病等位基因，表明在人类眼病中的独立作用。 我们对这一发现进行了进一步的研究，发现了携带移码等位基因的其他斑马鱼品系， MAB 21 L1基因，并确定了这些突变体中的缺损和眼前节缺陷。这些研究 揭示了MAB 21 L1/mab 21 l1和MAB 21 L2/mab 21 l2在眼内分泌系统中的重要和保守作用。 发展MAB 21 L因子编码与C. elegans mAb-21细胞命运决定 然而，这些蛋白质的功能在很大程度上是未知的。在这个项目中，我们将揭示MAB 21 L 在眼部发育中的作用。我们的主要假设是MAB 21 L因子代表了调节性的 这些蛋白质通过与PAX 6和BMP信号通路相互作用，指导正常的眼睛图案。 本研究的主要目的是：1）揭示细胞和分子过程 2）揭示MAB 21 L/mab 21 l因子在眼发育过程中的作用， MAB 21 L/mab 21 l蛋白在眼发育中的作用第一个目标将探索mab 21 l2和mab 21 l1 突变体，以确定破坏细胞过程使用各种方法，包括时间推移4D 与关博士合作的显微镜。为了深入了解mab 21 l 将对突变体进行转录组和蛋白质组分析， 鉴定与正常模式有显著偏差的转录物/蛋白质。确定的因素将是 通过在斑马鱼突变体中的拯救实验以及正向同源基因的测序进一步评估 在人类病人身上。第二个目标将集中于使用SELEX对MAB 21 L蛋白进行功能检查 和在mab 2112-FLAG敲入系中进行ChIP测序。所获得的数据将与 转录组/蛋白质组数据，以揭示分子变化的层次结构，并确定潜在的 机制等mab 21 l与pax 6因子以及mab 21 l与bmp 4之间的相互作用 将使用相应的突变体和报告细胞系来检查信号通路。概述的实验将 揭示脊椎动物眼睛发育和人类疾病的新机制。

PROJECT SUMMARY/ABSTRACT Coloboma is a congenital segmental ocular defect which can affect various structures of the eye. Coloboma is often associated with microphthalmia, microcornea, and/or retinal detachment and leads to visual impairment. We and another group recently reported a novel factor associated with coloboma, MAB21L2. As part of these studies, we generated a zebrafish mab21l2 allelic series carrying frameshift, in-frame indel and missense mutations in the orthologous gene region and observed coloboma and severe ocular disorganization in these mutants. Next, screening of another MAB21L gene, MAB21L1, in human patients identified likely pathogenic alleles indicating an independent role in human ocular disease. We followed up this finding with the development of additional zebrafish lines carrying frameshift alleles in the mab21l1 gene and identified coloboma and anterior segment defects in these mutants. These studies revealed essential and conserved roles for MAB21L1/mab21l1 and MAB21L2/mab21l2 in ocular development. The MAB21L factors encode proteins similar to C. elegans mab-21 cell fate-determining factor; however, the function of these proteins is largely unknown. In this project, we will reveal MAB21L function in ocular development. Our primary hypothesis is that MAB21L factors represent regulatory proteins that, through interaction with PAX6 and the BMP signaling pathway, direct normal eye patterning. This will be investigated through the following aims: 1) to uncover the cellular and molecular processes controlled by MAB21L/mab21l factors during eye development and 2) to reveal the function of MAB21L/mab21l proteins during eye development. The first aim will explore mab21l2 and mab21l1 mutants to identify disrupted cellular processes using various methods including time-lapse 4D microscopy in collaboration with Dr. Kwan. To gain an insight into the mechanisms by which mab21l factors direct ocular development, transcriptome and proteome analyses of mutants will be performed to identify transcripts/proteins with significant deviation from normal patterns. The identified factors will be further evaluated by rescue experiments in zebrafish mutants as well as sequencing of orthologous genes in human patients. The second aim will focus on functional examination of MAB21L proteins using SELEX and ChIP sequencing in a mab21l2-FLAG knock-in line. The obtained data will be analyzed together with transcriptome/proteome data to reveal the hierarchy of molecular changes and define the underlying mechanisms. The interaction between mab21l and pax6 factors, as well as mab21l and the bmp4 pathway, will be examined using corresponding mutant and reporter lines. The outlined experiments will reveal novel mechanisms of vertebrate ocular development and human disease.