MOUSE WHOLE EMBRYO CULTURE PARADIGM OF EAR MORPHOGENESIS

努力了解先天性内耳缺陷的遗传机制，现在集中在两个正常的发展过程和事件的级联反应，可以出现在一个单一的基因突变。 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，因此该物种已经成为一个强大的模型系统，在其中追求基因表达和形态发生之间的关系。 与小鼠遗传学的力量相抵消的是小鼠胚胎在整个器官形成的关键阶段中的不可接近性，该器官形成在受精后的第二周开始于子宫内。 由于其不可及性，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，耳基板和早期耳泡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作图获得，并且对于解释基因表达结构域如何转化为模式信息至关重要。用耳囊的高分辨率命运图来确定耳中表达的超过40个基因的表达域的能力可能对耳发育领域产生重大影响。 此外，由于已知的基因突变而异常的小鼠内耳的命运映射有望提供仅通过描述性分析根本不可能的见解。 例如，它可以提供有关特定基因突变是否引起细胞命运变化的信息，从而可以解释突变表型。 第一个具体的目标是对野生型小鼠和一种突变型小鼠（kreisler）的小鼠耳杯进行命运定位，这种突变型小鼠的内耳出现了明显的异常。 这将通过向培养中生长的小鼠胚胎的发育中的耳上皮细胞中直接注射亲脂性碳菁染料来实现。 标记的细胞将被定位，以观察它们在耳泡闭合（24小时后）或耳泡形态发生（48小时后）后的位置。 第二个具体目标是试验方法，以促进重点基因转移到培养的小鼠胚胎的耳囊。 这将通过注射逆转录病毒储备液或少量逆转录病毒产生细胞来实现。本研究将使用绿色荧光蛋白作为标记物进行，目的是对方法进行中试。 推动小鼠耳发育体外模式发展的长期目标是，它可能导致旨在挽救由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产生的突变和敲除小鼠的数量将继续增加。

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.