MAB21L Family in Human Ocular Disease and Development

MAB21L 家族在人类眼部疾病和发育中的作用

• 批准号: 9247511
• 负责人: Elena V Semina
• 金额: $ 38.46万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9247511
• 关键词: Adult; Affect; Alleles; Anterior; Behavior; Caenorhabditis elegans; Cell physiology; Cells; ChIP-seq; Choroid; Ciliary Body; Collaborations; Coloboma; DNA; Data; Defect; Development; Disease; Electron Microscopy; Embryo; Eye; Eye Development; Eye diseases; Family; Generations; Genes; Histologic; Human; Iris; Knock-in; Knowledge; Lead; Mediating; Methods; Microphthalmos; Microscopy; Missense Mutation; Molecular; Morphology; Mutation; Optic Nerve; Orthologous Gene; Paper; Pathogenicity; Pathway interactions; Patients; Pattern; Phenotype; Process; Proteins; Proteome; Publications; RNA Binding; RNA-Binding Proteins; Reporter; Reporting; Retinal; Retinal Detachment; Role; Sequence Analysis; Series; Signal Pathway; Signal Transduction; Site; Structure; Time; Transcript; Vertebrates; Visual impairment; Work; Zebrafish; experimental study; follow-up; genetic regulatory protein; human disease; improved; insertion/deletion mutation; insight; lens; light microscopy; loss of function; member; mutant; novel; protein function; screening; segregation; time use; transcriptome; young adult

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.

项目摘要/摘要 Coloboma是一种先天性节段性的眼部缺陷，可以影响眼睛的各种结构。 古骨通常与微心心脏，微孔和/或视网膜脱离有关，并导致 视觉障碍。我们和另一个小组最近报道了与山地骨相关的新因素， MAB21L2。作为这些研究的一部分，我们生成了一个携带移码的斑马鱼MAB21L2等位基因系列， 直系同源基因区域中的框架内和错义突变，并观察到了coloboma和 这些突变体中严重的眼部混乱。接下来，筛选另一个MAB21L基因MAB21L1，在 人类患者确定了可能的致病等位基因，表明在人眼疾病中起着独立的作用。 我们跟进了这一发现，并开发了其他斑马鱼线，并带有移架等位基因 MAB21L1基因并鉴定出这些突变体中的coloboma和前部缺陷。这些研究 揭示了Ocular中MAB21L1/MAB21L1和MAB21L2/MAB21L2的必要和保守作用 发展。 MAB21L因子编码类似于秀丽隐杆线虫MAB-21细胞命运确定的蛋白质 因素;但是，这些蛋白质的功能在很大程度上是未知的。在这个项目中，我们将透露MAB21L 眼部发育的功能。我们的主要假设是MAB21L因子代表调节 通过与PAX6和BMP信号通路相互作用的蛋白质直接正常眼睛图案。 这将通过以下目的进行研究：1）揭示细胞和分子过程 在眼睛发育过程中由MAB21L/MAB21L因子控制和2） 眼睛发育过程中MAB21L/MAB21L蛋白。第一个目标将探索MAB21L2和MAB21L1 突变体使用各种方法（包括延时4D）识别破坏的细胞过程 显微镜与Kwan博士合作。洞悉MAB21L的机制 将对突变体的直接眼发育，转录组和蛋白质组分析进行。 识别与正常模式显着偏差的转录本/蛋白质。确定的因素将是 通过在斑马鱼突变体中的救援实验以及直系同源基因的测序进一步评估 在人类患者中。第二个目标将重点介绍使用SELEX的MAB21L蛋白的功能检查 并在MAB21L2-FLAG敲入线中进行芯片测序。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基础 机制。 MAB21L和PAX6因子以及MAB21L和BMP4之间的相互作用 途径将使用相应的突变体和报告基线进行检查。概述的实验将 揭示了脊椎动物眼发育和人类疾病的新型机制。