MOUSE WHOLE EMBRYO CULTURE PARADIGM OF EAR MORPHOGENESIS

小鼠耳朵形态发生的全胚胎培养范例

• 批准号: 6142028
• 负责人: Donna M Fekete
• 金额: $ 7.34万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6142028
• 关键词: congenital ear disorder; ear; embryo /fetus culture; gene mutation; green fluorescent proteins; histogenesis; laboratory mouse; labyrinth; mammalian embryology; microinjections; technology /technique development; transfection

Efforts to understand the genetic mechanisms that underlie congenital inner ear defects are now focused on both the normal processes of development and on the cascade of events that can arise in response to a single gene mutation. Because the generation of animals with a targeted gene defect has become commonplace in mice, this species has emerged as a powerful model system in which to pursue the relationship between gene expression and morphogenesis. Counteracting the power of mouse genetics is the inaccessibility of the mouse embryo throughout the critical stages of organogenesis, which begins in utero during the second week after fertilization. Because of its inaccessibility, one key experimental approach, fate mapping of an organ primordium, is rarely performed in the mouse embryo. At the present time, it is not known in any species which parts of the otic placode and early otic vesicle give rise to the different parts of the ear. Such information can be obtained by fate mapping, and is critical for interpreting how gene expression domains get converted into patterning information. The ability to superimpose the expression domains of the greater than 40 genes expressed in the ear with a high-resolution fate map of the otocyst could have a major impact on the field of ear development. Furthermore, fate mapping a mouse inner ear that is abnormal due to a known genetic mutation promises to provide insights that are simply not possible by descriptive analysis alone. For example, it may provide information about whether a specific genetic mutation is causing a change in cell fate that can explain the mutant phenotype. The first specific aim is to fate map the mouse otic cup in both the wild-type mouse and in a mouse mutant, kreisler, that develops with gross abnormalities in the inner ear. This will be accomplished by small focal injections of lipophilic carbocyanine dyes directed into the developing ear epithelium of mouse embryos grown in culture. The labelled cells will be mapped to see where they reside after otic vesicle closure (after 24 hours) or otic vesicle morphogenesis (after 48 hours). The second specific aim is to pilot methods to facilitate focal gene transfer into the otocyst of the cultured mouse embryo. This will be accomplished by injection of retrovirus stocks or small numbers of retrovirus-producing cells. The study will be performed with green fluorescent protein as a marker for the purpose of piloting the methods. The long-term goal driving the development of an in vitro paradigm for mouse ear development is that it may lead to intervention strategies (such as virus-mediated gene transfer) designed to rescue the inner ear defects arising from known genetic mutations. If the mouse whole embryo culture paradigm proves successful, its impact is likely to extend far beyond the proposed studies, given that the number of mutant and knockout mice generated as potential models of human deafness genes will continue to rise.

现在，努力理解基于先天性内耳缺陷的遗传机制现在侧重于正常的发育过程和响应单个基因突变的事件级联。 由于具有靶向基因缺陷的动物的产生在小鼠中已变得司空见惯，因此该物种已成为一种强大的模型系统，可以在其中追求基因表达与形态发生之间的关系。 应对小鼠遗传学的能力是在器官发生的整个临界阶段中的小鼠胚胎的无法访问，该阶段在受精后的第二周就在子宫内开始。 由于其无法获取，很少在小鼠胚胎中执行一种关键的实验方法，即器官原始的命运图。 目前，尚不清楚在任何物种中的任何一部分和早期耳囊引起耳朵的不同部位。 这些信息可以通过命运映射获得，对于解释基因表达域如何转化为模式信息至关重要。在耳囊的高分辨率命运图中，在耳朵中表达的大于40个基因的表达域的能力可能会对耳朵发育领域产生重大影响。 此外，由于已知的遗传突变，命运映射了一个异常的小鼠内耳，有望提供洞察力，而仅通过描述性分析，这根本无法实现。 例如，它可以提供有关特定遗传突变是否导致细胞命运变化的信息，该细胞命运可以解释突变表型。 第一个具体的目的是命运野生型小鼠和小鼠突变体Kreisler中的小鼠耳杯，该杯在内耳中以严重的异常发展而发展。 这将通过针对培养物种植的小鼠胚胎发育中的小鼠上皮的亲脂性碳酸染料的小局部注射来实现。 标记的细胞将被映射，以查看它们在耳囊闭合（24小时后）或耳囊形态发生后（48小时后）的居住地。 第二个具体目的是试验方法，以促进局灶性基因转移到培养的小鼠胚胎的耳囊中。 这将通过注入逆转录病毒库存或少量产生逆转录病毒的细胞来实现。该研究将以绿色荧光蛋白作为标记，目的是为了促进方法。 推动小鼠耳朵发育的体外范式发展的长期目标是，它可能导致旨在挽救已知基因突变引起的内耳缺陷的干预策略（例如病毒介导的基因转移）。 如果小鼠的整个胚胎培养范式证明了成功的范围，那么由于人类聋哑基因的潜在模型将继续增加，因此其影响可能远远超出了拟议的研究。