Cloning and Characterization of Zebrafish Endocrine Pancreas Mutants

描述(申请人提供)：本申请的长期目标是开发基于细胞的替代疗法的策略，通过了解斑马鱼调节细胞规格的分子机制来治疗糖尿病，斑马鱼是一种非常适合胚胎发育和遗传学研究的模式生物。研究人员进行了化学诱变筛选，以确定控制斑马鱼胰腺细胞形成的各个方面的基因。在发育中的斑马鱼胰腺中，细胞在24hpf时通过细胞迁移聚集成簇(胰岛)。两个突变体la676(猎豹)和la572(Mini)具有胰岛素阳性细胞的异常分裂形态。任何一个都显示出明显的形态异常。MNM突变体也表现出胰岛素表达减少。肝脏和肠道的发育在这两个突变体中都没有受到影响。这些数据导致了一种假设，即CHE和MNM突变导致细胞迁移缺陷，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。在特定目标1中，研究者将专注于基于简单序列长度多态性来确定ChE和MNM基因的染色体位置；在特定目标2中，通过高分辨率减数分裂定位来确定包含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基因座的基因组重叠群中鉴定克隆。候选基因将通过吗啡反义寡核苷酸敲除的方式进行检测，以复制突变，并通过聚合酶链式反应和DNA测序分析基因突变。通过注射候选基因的c DNA或m RNA来挽救突变的表型；在特定的目标4中，利用胰岛素：GFP转基因FISH的时间圈共聚焦成像详细研究细胞迁移缺陷；在特定的目标5中，检测这些突变的细胞增殖和细胞死亡。这些研究将对内分泌胰腺器官发生的遗传调控提供进一步的见解。 项目简介：该应用程序建议克隆两个在内分泌细胞发育过程中调节细胞迁移的基因，以形成胰岛。胰岛的正确形成对细胞功能至关重要，以确保饥饿期间胰岛素分泌量低，食物摄取后胰岛素分泌量充足。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。

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.