Engrailed genes and cerebellum morphology, spatial gene expression and circuitry

描述（由申请人提供）：小脑（Cb）除了在熟练的运动表现中起关键作用外，还参与认知和社会功能。 因此，Cb与包括自闭症在内的许多使人衰弱的发育疾病有关。一个调节Cb发育并与自闭症有关的基因是enrailed 2（EN 2），这是基于人类研究以及发现En 2缺失小鼠不仅在运动控制方面存在缺陷，而且在社会行为和认知方面也存在缺陷。在我们开始了解Cb的高级功能之前，我们必须对调节Cb发育的基本细胞和遗传过程有更多的了解。我们的方法是使用两个EN同源框转录因子作为分子切入点来研究Cb的发展，因为我们发现En 1/2条件突变体在Cb形态，分子模式和传入电路中存在缺陷。现在，我们将把我们的研究方向转向区分由En 1/2调控的细胞过程，并确定对这些过程至关重要的EN 2靶基因，这些基因可能是复杂行为疾病的易感基因座。我们将重点放在颗粒神经元（GN），包括输入到Cb和产生输出的小脑深核（DCN）的主要接受者。DCN神经元在自闭症患者中持续减少，这可能是某些行为的主要原因，也反映了Cb回路中其他地方的缺陷。我们将应用一种多方面的方法，该方法结合了小鼠中的新型遗传技术，以研究GN和DCN投射神经元的正常和突变行为，包括使用我们的MASTR技术的嵌合突变体分析和一种新方法，以精确靶向EN 2对GN和DCN的过表达，以测试EN 2改变分化的充分性。然后，我们将应用这两种突变的方法，作为一种不同的方法来研究Cb形态发生和细胞增殖/分化的GN的活成像。我们还将解决的问题，是否反馈回路确保正确的比例的细胞类型是通过研究En 1/2和音刺猬（SHH）途径之间的相互作用。最后，为了确定EN 2在大脑中的第一个直接靶点，我们正在设计表达EN 2标记形式的新小鼠品系。目标1.采用条件遗传学方法研究En 1/2对生长中的GN和DCN投射神经元的细胞行为的调控，并在体内和体外用活体成像技术表征细胞行为。目标2.使用比较微阵列分析和ChIP-seq鉴定GN前体和DCN投射神经元中EN 1/2的关键靶基因。

DESCRIPTION (provided by applicant): The cerebellum (Cb) is implicated in contributing to cognitive and social functions, in addition to having a critical role in skilled motor performance. Accordingly, the Cb is associated with many debilitating developmental diseases including autism. One gene that regulates development of the Cb and has been implicated in autism is engrailed 2 (EN2), based on human studies and the finding that En2 null mice not only have deficits in motor control, but also in social behaviors and cognition. Before we can begin to understand higher order functions of the Cb, we must gain more insight into the basic cellular and genetic processes that regulate Cb development. Our approach is to use the two EN homeobox transcription factors as molecular entry points to study Cb development, as we discovered that En1/2 conditional mutants have defects in Cb morphology, molecular patterning and afferent circuitry. We will now direct our studies towards distinguishing the cellular processes regulated by En1/2 and identifying EN2 target genes critical for these processes that could be susceptibility loci for complex behavioral diseases. We will focus on the granule neurons (GNs) that comprise the main recipients of input to the Cb and the deep cerebellar nuclei (DCN) that generate the output. DCN neurons are consistently reduced in autistic patients, which could be a primary cause of some behaviors and also reflect defects elsewhere in the Cb circuit. We will apply a multi-facetted approach that combines novel genetic techniques in mice to study normal and mutant behaviors of GNs and DCN projection neurons, including a mosaic mutant analysis using our MASTR technique and a new method to precisely target over- expression of EN2 to GNs and the DCN to test sufficiency of EN2 to alter differentiation. We will then apply both mutant approaches to live imaging of GNs as a different approach to study Cb morphogenesis and cell proliferation/differentiation. We will also address the question of whether feed back loops ensure the correct proportion of cell types is produced by studying the interaction between En1/2 and the sonic hedgehog (SHH) pathway. Finally, in order to identify the first direct targets of EN2 in the brain, we are engineering new mouse strains expressing a tagged form of EN2. Aim 1. Study the cellular behaviors regulated by En1/2 in developing GNs and DCN projection neurons using conditional genetics and characterizing cellular behaviors in vivo, and in vitro with live imaging. Aim 2. Identify critical target genes o EN1/2 in GN precursors and DCN projection neurons using comparative microarray analysis and ChIP-seq.