MAB21L Family in Human Ocular Disease and Development

项目摘要/摘要 缺损是一种先天性节段性眼部缺陷，可影响眼睛的各种结构。 缺损通常与小眼炎、小角膜和/或视网膜脱离有关，并导致 视力受损。我们和另一个小组最近报道了一种与缺陷症相关的新因素， MAB21L2。作为这些研究的一部分，我们产生了斑马鱼mab21l2等位基因序列， 同源基因区的框内插入和错义突变以及观察到的缺陷性和 这些变种人严重的眼部组织紊乱。接下来，对另一个MAB21L基因MAB21L1进行了筛选 人类患者发现了可能的致病等位基因，表明在人类眼病中起着独立的作用。 我们进一步研究了携带移码等位基因的斑马鱼新品系。 Mab21l1基因，并在这些突变体中鉴定出缺陷性和眼前段缺陷。这些研究 揭示MAB21L1/mab21L1和MAB21L2/mab21L2在眼部的基本和保守作用 发展。MAB21L因子编码与线虫MAB-21细胞命运决定相似的蛋白质 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MAB21L 在眼睛发育中的作用。我们的主要假设是MAB21L因子代表调控 通过与PAX6和BMP信号通路相互作用，指导正常眼部模式的蛋白质。 这将通过以下目的进行研究：1)揭示细胞和分子过程 在眼睛发育过程中受MAB21L/mab21l因子的调控；2)揭示MAB21L/mab21L的功能 MAB21L/mab21L蛋白在眼睛发育过程中的作用。第一个目标是探索mab21l2和mab21l1 使用包括延时4D在内的各种方法识别被破坏的细胞过程的突变体 显微技术，与关博士合作。为了深入了解mab21l 将对突变体的眼睛发育、转录组和蛋白质组进行直接因素分析 识别显著偏离正常模式的转录本/蛋白质。确定的因素将是 通过斑马鱼突变体的拯救实验和同源基因的测序进一步评估 在人类病人身上。第二个目标将集中在使用SELEX对MAB21L蛋白进行功能检测 以及mab21l2-FLAG敲入线中的芯片测序。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定义潜在的 机械装置。Mab21l与Pax6因子以及mab21l与Bmp4的相互作用 途径，将使用相应的突变体和报告基因株进行研究。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.