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.

项目概要/摘要 缺损是一种先天性节段性眼部缺陷，可影响眼睛的各个结构。 缺损通常与小眼症、小角膜和/或视网膜脱离有关，并导致 视力障碍。我们和另一个小组最近报告了一种与缺损相关的新因素， MAB21L2。作为这些研究的一部分，我们生成了携带移码的斑马鱼 mab21l2 等位基因系列， 直系同源基因区域中的框内插入缺失和错义突变以及观察到的缺损和 这些突变体的眼部严重紊乱。接下来，筛选另一个 MAB21L 基因 MAB21L1， 人类患者鉴定出可能的致病等位基因，表明其在人类眼部疾病中具有独立作用。 我们根据这一发现开发了携带移码等位基因的其他斑马鱼品系 mab21l1 基因并鉴定出这些突变体中的缺损和眼前节缺陷。这些研究 揭示了 MAB21L1/mab21l1 和 MAB21L2/mab21l2 在眼病中的重要和保守作用 发展。 MAB21L 因子编码与线虫 mab-21 细胞命运决定相似的蛋白质 因素;然而，这些蛋白质的功能很大程度上未知。在这个项目中，我们将展示MAB21L 在眼部发育中发挥作用。我们的主要假设是 MAB21L 因子代表监管 通过与 PAX6 和 BMP 信号通路相互作用，指导正常眼睛模式的蛋白质。 这将通过以下目标进行研究：1）揭示细胞和分子过程 在眼睛发育过程中受 MAB21L/mab21l 因子控制，2) 揭示 眼睛发育过程中的 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 与关博士合作进行显微镜检查。为了深入了解 mab21l 的机制 直接眼部发育的因素、突变体的转录组和蛋白质组分析将被进行 识别与正常模式有显着偏差的转录本/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。第二个目标将侧重于使用 SELEX 对 MAB21L 蛋白进行功能检查 以及 mab21l2-FLAG 敲入系中的 ChIP 测序。获得的数据将与 转录组/蛋白质组数据揭示分子变化的层次结构并定义潜在的变化 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系进行检查。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。