Cloning and Characterization of Zebrafish Endocrine Pancreas Mutants

斑马鱼内分泌胰腺突变体的克隆和表征

• 批准号: 7713116
• 负责人: Shuo Lin
• 金额: $ 7.7万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-18 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7713116
• 关键词: Affect; Animal Model; Bromodeoxyuridine; Candidate Disease Gene; Cell Aggregation; Cell Communication; Cell Death; Cell Proliferation; Cell Survival; Cell physiology; Cells; Cheetahs; Chemicals; Chromosomes; Cloning; Complementary DNA; DNA Sequence; DNA analysis; Data; Defect; Development; Diabetes Mellitus; Embryo; Endocrine; Ensure; Exhibits; Fishes; Food; Gene Mutation; Genes; Genetic; Genetic Polymorphism; Genetic Screening; Genomic Library; Genomics; Goals; Image; Insulin; Intestines; Islets of Langerhans; Length; Liver; Location; Maps; Meiosis; Messenger RNA; Molecular; Morphology; Mutagenesis; Mutation; Organogenesis; Pancreas; Phenocopy; Phenotype; Process; Regulation; Replacement Therapy; Research Personnel; Resolution; Siblings; Starvation; TdT-Mediated dUTP Nick End Labeling Assay; Techniques; Testing; Time; Transgenic Organisms; YAC Clone; Zebrafish; base; cell motility; design; insight; insulin secretion; islet; mutant; positional cloning; simple sequence length polymorphism; uptake; zebrafish genome

DESCRIPTION (Provided by Applicant): The long-term goal of this application is to develop strategies for ¿ cell based replacement therapy to treat diabetes by understanding the molecular mechanisms regulating ¿ cell specification using zebrafish, a model organism well suited for embryogenic and genetic studies. The investigator conducted a chemical-induced mutagenesis screen to identify the genes that control various aspects of pancreatic ¿ cell formation in zebrafish. In the developing zebrafish pancreas, ¿ cells aggregate into a cluster (islet) at 24 hpf through cell migration. Two mutants, la676 (cheetah) and la572 (minime), have abnormally split morphology of insulin positive cells. Either one exhibits any visible morphological abnormalities. Mnm mutants also show reduced insulin expression. Liver and intestine development is not affected in either mutant. These data have led to the hypothesis that the che and mnm mutations cause cell migration defects and mnm also regulates ¿ cell specification or proliferation. The objective of this proposed study is to identify the che and mnm genes by positional cloning technique. The investigator will focus on, in Specific Aim 1, determine the chromosomal locations of the che and mnm genes based on simple sequence length polymorphism; in Specific Aim 2, define the critical regions harboring the che and mnm genes by high-resolution meiotic mapping; in Specific Aim 3, identify clones from BAC library and genomic contigs that span the che and mnm loci. The candidate genes will be tested by knockdown using morpholino antisense oligos to phenocopy the mutations and analyze the gene mutations by PCR and DNA sequencing. The mutant phenotypes will be rescued by injecting the cDNA or mRNA of candidate genes; in Specific Aim 4, study cell migration defects in detail by time-laps confocal imaging using insulin:GFP transgenic fish; and in Specific Aim 5, examine cell proliferation and cell death in these mutants. These studies will provide further insights into the genetic regulation of endocrine pancreas organogenesis. PROJECT NARRATIVE: This application proposes to clone two genes that regulate cell migration during endocrine ¿ cell development to form islet. Correct formation of islet is critical for ¿ cell function to ensure low amount of insulin secretion during starvation and sufficient amount of insulin secretion after food uptake. These studies will provide invaluable insights on the molecular mechanisms regulating endocrine islet formation.

描述（由申请人提供）：本申请的长期目标是通过了解使用斑马鱼（一种非常适合胚胎发生和遗传学研究的模型生物体）调节细胞规格的分子机制，开发基于细胞的替代疗法治疗糖尿病的策略。 研究人员进行了化学诱导诱变筛选，以确定控制斑马鱼胰腺细胞形成各个方面的基因。 在发育中的斑马鱼胰腺中，细胞在 24 hpf 时通过细胞迁移聚集成簇（胰岛）。 两种突变体，la676（猎豹）和 la572（迷你），具有异常分裂的胰岛素阳性细胞形态。 任何一种都表现出任何可见的形态异常。 Mnm 突变体还表现出胰岛素表达降低。 两种突变体的肝脏和肠道发育均不受影响。 这些数据导致了这样的假设：che 和 mnm 突变导致细胞迁移缺陷，并且 mnm 还调节细胞特化或增殖。 本研究的目的是通过定位克隆技术鉴定 che 和 mnm 基因。 具体目标1中，研究者将重点根据简单的序列长度多态性确定che和mnm基因的染色体位置；在具体目标 2 中，通过高分辨率减数分裂图谱定义含有 che 和 mnm 基因的关键区域；在特定目标 3 中，从 BAC 文库和跨越 che 和 mnm 位点的基因组重叠群中识别克隆。 将使用吗啉代反义寡核苷酸通过敲低来测试候选基因，以对突变进行表型复制，并通过 PCR 和 DNA 测序来分析基因突变。 通过注射候选基因的cDNA或mRNA来拯救突变表型；在具体目标 4 中，使用胰岛素：GFP 转基因鱼通过延时共聚焦成像详细研究细胞迁移缺陷；在具体目标 5 中，检查这些突变体的细胞增殖和细胞死亡。 这些研究将为内分泌胰腺器官发生的遗传调控提供进一步的见解。 项目叙述：本申请提出克隆两个在内分泌和细胞发育形成胰岛过程中调节细胞迁移的基因。 胰岛的正确形成对于细胞功能至关重要，以确保饥饿期间少量的胰岛素分泌和食物摄取后充足的胰岛素分泌。 这些研究将为调节内分泌胰岛形成的分子机制提供宝贵的见解。