MOUSE WHOLE EMBRYO CULTURE PARADIGM OF EAR MORPHOGENESIS

了解先天性内耳缺陷背后的遗传机制的努力现在既集中在正常的发育过程上，也集中在单个基因突变可能引发的一连串事件上。由于具有靶向基因缺陷的动物的产生在小鼠中已经变得司空见惯，这个物种已经成为探索基因表达和形态发生之间关系的一个强大的模式系统。抵消小鼠遗传学力量的是，小鼠胚胎在器官发生的关键阶段都无法获得，这一阶段始于受精后第二周的子宫内。由于它的不可访问性，一个关键的实验方法，器官原基的命运映射，很少在小鼠胚胎中进行。目前，还不知道在任何物种中，耳道胎盘和早期耳囊的哪些部分产生了耳朵的不同部分。这些信息可以通过命运图谱获得，对于解释基因表达结构域如何转化为模式信息至关重要。将耳朵中表达的40多个基因的表达结构域与高分辨率的耳囊命运图叠加的能力可能会对耳朵发育领域产生重大影响。此外，命运映射由于已知基因突变而异常的小鼠内耳有望提供仅通过描述性分析根本不可能提供的见解。例如，它可能提供关于特定基因突变是否导致细胞命运改变的信息，这可以解释突变的表型。第一个明确的目标是在野生型小鼠和内耳发育严重异常的突变小鼠克莱斯勒身上绘制小鼠耳杯的命运图。这将通过向培养中生长的小鼠胚胎发育中的耳朵上皮直接注射少量亲脂性碳菁染料来完成。标记的细胞将在耳泡关闭(24小时后)或耳泡形态形成(48小时后)后被映射到它们所在的位置。第二个具体目标是试验促进将局部基因转移到培养的小鼠胚胎耳囊中的方法。这将通过注射逆转录病毒库存或少量逆转录病毒产生细胞来实现。这项研究将以绿色荧光蛋白作为标记物，用于试验这些方法。推动小鼠耳朵体外发育范例发展的长期目标是，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(如病毒介导的基因转移)。如果小鼠全胚胎培养模式被证明是成功的，其影响可能远远超出拟议的研究，因为作为人类耳聋基因潜在模型而产生的突变和敲除小鼠的数量将继续上升。

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.