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: 7934077
• 负责人: Shuo Lin
• 金额: $ 7.61万
• 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/7934077
• 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斑马鱼（一种非常适合胚胎发生和遗传研究的模式生物）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，细胞通过细胞迁移以24hpf的速度聚集成一个簇（胰岛）。两个突变体la676（猎豹）和la572 （minime）具有胰岛素阳性细胞的异常分裂形态。任何一个都表现出明显的形态学异常。Mnm突变体也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。这些数据导致了一个假设，即che和mnm突变导致细胞迁移缺陷，而mnm也调节细胞规格或增殖。本研究的目的是利用定位克隆技术鉴定che和mnm基因。在具体目标1中，研究者将重点关注基于简单序列长度多态性确定che和mnm基因的染色体位置；在Specific Aim 2中，通过高分辨率的减数分裂定位来定义包含che和mnm基因的关键区域；在Specific Aim 3中，从BAC文库和跨越che和mnm位点的基因组序列中鉴定克隆。候选基因将通过morpholino反义寡核苷酸敲低检测突变表型，并通过PCR和DNA测序分析基因突变。通过注入候选基因的cDNA或mRNA来挽救突变体表型；在Specific Aim 4中，利用胰岛素：GFP转基因鱼，通过时间圈共聚焦成像详细研究细胞迁移缺陷；在特异性目标5中，检查这些突变体中的细胞增殖和细胞死亡。这些研究将为胰腺内分泌器官发生的遗传调控提供进一步的见解。