Genetic and Genomic Approaches to Study Hair Cell Regeneration in Zebrafish

研究斑马鱼毛细胞再生的遗传和基因组方法

• 批准号: 8601557
• 负责人: Martine Behra
• 金额: $ 2.13万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8601557
• 关键词: Address; Adult; Aging; Animals; Antibodies; Auditory; Behavior; Birth; Candidate Disease Gene; Cell Cycle; Cell Line; Cells; Cellular Structures; Centrosome; Chromatin; Co-Immunoprecipitations; Coupled; Cytoskeleton; Data; Disease; Embryo; Event; Family; Fibroblasts; Fishes; Gene Expression Profile; Genes; Genetic; Genomics; Hair Cells; Hela Cells; Homologous Gene; Human; Image; Immunoprecipitation; In Situ Hybridization; In Vitro; Instruction; Labyrinth; Lamin Type A; Lamins; Larva; Mammals; Mass Spectrum Analysis; Mechanoreceptors; Microtubule Depolymerization; Microtubules; Mitosis; Molecular; Monitor; Mutate; Mutation; Natural regeneration; Nature; Neomycin; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Lamina; Nuclear Outer Membrane; Nuclear Pore Complex; Oligonucleotides; Organ; Patients; Peripheral; Phenotype; Process; Progeria; Proteins; Proteomics; Reverse Transcriptase Polymerase Chain Reaction; Sensory; Small Interfering RNA; Sorting - Cell Movement; Stem cells; Supporting Cell; Syndrome; Techniques; Testing; Therapeutic; Time; Transgenic Organisms; Tubulin; Work; Zebrafish; base; cell type; deafness; follow-up; gene conservation; hair cell regeneration; in vivo; insight; lateral line; liquid chromatography mass spectrometry; mutant; novel; novel therapeutics; overexpression; prevent; regenerative; research study; segregation; time use; tool

Damage or loss of the mechanoreceptors (hair cells) in the inner ear, are the leading cause for human deafness, because both are irreversible after birth. Unlike mammals, fish replace damaged hair cells throughout adulthood. This regenerative power is observed in the inner ear, but also in an evolutionary and physiologically related organ the lateral line. This superficial sensory organ offers a valuable tool to study in vivo the dynamic events of regeneration in time and in the context of a whole animal. Very few genes involved in regeneration have been identified so far, hampering progress towards restoring hair cells in humans. To address this particular point, the long-term objectives of this application are to elucidate the molecular events underlying the regeneration of hair cells in the lateral line. As an entry point, we are using a zebrafish mutant line (phoenix), which we have characterized. The mutated gene is novel and mutant larvae have a clear reduction in regenerated hair cells in the lateral line. The phoenix gene is expressed in supporting cells, which have been previously implicated as progenitor cells. They are not entering the cell cycle in the absence of phoenix. The first specific aim is to decipher the function of phoenix. To do so, we combine a cytological approach in fish cell lines with an expression screen in mutant larvae of previously known or newly discovered molecular players in the regeneration process. The second specific aim is to find partners of phoenix. We use genomic and a proteomic approaches in the wild type and phoenix mutant context. We are characterizing the transcriptome of each cell type of the lateral line and assess their changes during regeneration. The proteomic approach is based on co-immunoprecipitation in fish cells lines using an antibody against phoenix. In the third specific aim, we will pursue preliminary data showing that, phoenix localization is altered in human fibroblasts of Hutchinson-Gilford Progeria Syndrome patients, who are aging prematurely. Using immunoprecipitation coupled with mass spectroscopy, we will isolate and sequence the human homolog of phoenix. This work will provide insight into the genetics of regeneration of hair cells, opening up new therapeutic avenues.

内耳中的机械感受器（毛细胞）的损伤或损失是人类听力损失的主要原因。 耳聋，因为两者在出生后都是不可逆的。与哺乳动物不同，鱼类会取代受损的毛细胞 整个成年期。这种再生能力不仅在内耳中观察到，而且在进化和 生理相关器官侧线。这个表面感觉器官提供了一个有价值的工具来研究 在整个动物的背景下，在时间上再生的动态事件。很少的基因 目前为止，已经确定了参与再生的细胞，阻碍了恢复毛细胞的进展， 人类为了解决这一特定问题，本申请的长期目标是阐明本发明的实施例。 侧线毛细胞再生的分子事件。作为入口点，我们使用 斑马鱼突变系（凤凰），这是我们的特点。突变的基因是新的和突变的幼虫 侧线再生毛细胞明显减少。凤凰基因表达于 支持细胞，其先前被认为是祖细胞。他们不能进牢房 在没有凤凰的情况下循环。第一个具体目标是破译凤凰的功能。为此，我们 联合收割机将鱼类细胞系中的细胞学方法与先前 已知的或新发现的分子在再生过程中的作用。第二个具体目标是找到 凤凰的伙伴我们在野生型和凤凰突变体中使用基因组和蛋白质组学方法 上下文我们正在描述侧线的每种细胞类型的转录组，并评估它们的表达。 再生过程中的变化。蛋白质组学方法是基于鱼类细胞系的免疫共沉淀 用的是针对凤凰的抗体在第三个具体目标中，我们将寻求初步数据，表明， 在Hutchinson-Gilford早衰综合征患者的人成纤维细胞中，凤凰的定位发生了改变， 过早衰老使用免疫沉淀结合质谱，我们将分离和 人类凤凰的同源基因。这项工作将提供深入了解再生的遗传学， 毛细胞，开辟了新的治疗途径。

项目成果

Phoenix is required for mechanosensory hair cell regeneration in the zebrafish lateral line.

• DOI: 10.1371/journal.pgen.1000455
• 发表时间: 2009-04
• 期刊: PLoS genetics
• 影响因子: 4.5
• 作者: Behra M;Bradsher J;Sougrat R;Gallardo V;Allende ML;Burgess SM
• 通讯作者: Burgess SM

Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line.

• DOI: 10.1186/1471-213x-12-6
• 发表时间: 2012-01-24
• 期刊: BMC developmental biology
• 作者: Behra M;Gallardo VE;Bradsher J;Torrado A;Elkahloun A;Idol J;Sheehy J;Zonies S;Xu L;Shaw KM;Satou C;Higashijima S;Weinstein BM;Burgess SM
• 通讯作者: Burgess SM

Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish.

• DOI: 10.1186/1471-213x-10-120
• 发表时间: 2010-12-13
• 期刊: BMC developmental biology
• 作者: Gallardo VE;Liang J;Behra M;Elkahloun A;Villablanca EJ;Russo V;Allende ML;Burgess SM
• 通讯作者: Burgess SM