Molecular Analysis of Hindbrain Formation

描述（由申请人提供）：在胚胎发生过程中，脊椎动物中枢神经系统（CNS）被细分为几个结构。其中之一，后脑，产生小脑和脑干，它们调节基本功能（如姿势和运动）和认知功能（如语言）。后脑缺陷因此与运动控制问题（如共济失调）和认知缺陷（如自闭症）有关。为了更好地理解一般的神经模式，特别是后脑发育，我们最近克隆了几个在后脑发育早期表达的基因，包括斑马鱼的meis3基因。根据我们迄今为止的研究结果，我们假设Meis3通过与Hox和Pbx蛋白形成转录调控复合物，在后脑发育过程中充当Hox辅助因子。具体来说，我们发现Meis蛋白在体外与Pbx和Hox形成三聚体复合物，而Hox蛋白在体内缺乏Pbx和Meis时无法激活转录。然而，仍有几个问题没有得到解答。首先，目前尚不清楚Meis、Pbx和Hox蛋白是否总是一起起作用，还是它们也有独立的功能。为了解决这个问题，我们将首先比较减少Meis， Pbx和Hox功能在体内的影响。这将证明这些蛋白质是否一起起作用（即，降低它们的水平会产生相同的表型），并揭示每种蛋白质在后脑发育过程中的作用。然后，我们将直接讨论这三种蛋白质是否必须通过使用结合缺陷的Pbx结构来相互作用，以挽救Pbx突变的鱼线。第二个问题是为什么Meis， Pbx和Hox蛋白都是Hox靶点转录所必需的。我们发现Meis， Pbx和Hox与转录共调节因子（激活因子和抑制因子）相互作用，我们将测试这些相互作用在体内是否必要。首先，我们将使用不能与共同调节因子相互作用的每种蛋白质的突变形式，并确定这些突变形式是否活跃。其次，我们将在体内干扰共调节功能，并测试这是否会影响Hox靶基因的表达。我们的研究结果将提供有关后脑发育的信息，并且由于Hox蛋白参与了神经发育的许多方面（例如神经管的背侧模式），因此也将广泛适用于神经发育。hox， meis和pbx基因也是参与白血病的原癌基因，通过外推，我们的结果也可能为hox蛋白及其辅助因子的致癌性质提供见解。

DESCRIPTION (provided by applicant): During embryogenesis, the vertebrate central nervous system (CNS) becomes subdivided (patterned) into several structures. One of these, the hindbrain, gives rise to the cerebellum and brainstem, which regulate basic functions (e.g., posture and movement) and cognitive functions (e.g., language). Hindbrain defects are therefore associated with both motor control problems (e.g., ataxias) and cognitive defects (e.g., autism). To better understand neural patterning in general and hindbrain development in particular, we recently cloned several genes expressed early during hindbrain development, including the zebrafish meis3 gene. Based on our results to date, we hypothesize that Meis3 acts as a Hox cofactor during hindbrain development by forming transcription regulatory complexes with Hox and Pbx proteins. Specifically we find that Meis proteins form trimeric complexes with Pbx and Hox in vitro and that Hox proteins are unable to activate transcription in the absence of Pbx and Meis in vivo. However, several questions remain unanswered. First, it is unclear if Meis, Pbx and Hox proteins always function together, or if they also have independent functions. To address this question we will first compare the effects of reducing Meis, Pbx and Hox function in vivo. This will both demonstrate whether the proteins act together (i.e., reducing their levels give the same phenotype) and reveal the role for each protein during hindbrain development. We will then directly address whether the three proteins must interact by using binding-deficient Pbx constructs to rescue a Pbx mutant fish line. A second question is why Meis, Pbx and Hox proteins are all required for transcription of Hox targets. We find that Meis, Pbx and Hox interact with transcriptional co-regulators (activators and repressors) and we will test if these interactions are essential in vivo. First we will use mutant forms of each protein that cannot interact with co-regulators and determine if these mutant forms are active. Second, we will interfere with co-regulator function in vivo and test if this affects expression of Hox target genes. Our results will provide information about hindbrain development and, since Hox proteins are involved in a number of aspects of neural development (e.g., dorsoventral patterning of the neural tube), will also be broadly applicable to neural development. hox, meis and pbx genes are also proto-oncogenes involved in leukemia and, by extrapolation, our results may also provide insights into the oncogenic nature of Hox proteins and their co-factors.