Molecular Analysis of Hindbrain Development

描述（由申请人提供）：尾侧后脑分成4-7形染色体是由旁系1组（PG1） Hox蛋白调控的，但目前尚不清楚PG1 Hox蛋白如何激活它们的靶基因，以及这些基因随后如何相互作用产生不同的形染色体。我们的初步结果表明，Hoxb1b（斑马鱼PG1 Hox蛋白）与Pbx蛋白和组蛋白去乙酰化酶（hdac）形成抑制复合物，并且需要Meis蛋白将Pbx:Hoxb1b复合物转化为转录激活因子。我们也证实了Hoxb1b调控r4（如hoxb1a）和r5/r6（如val、vhnf1）中几个基因的表达，但尚不清楚这些基因是如何起作用的，很可能存在其他Hoxb1b靶基因。我们假设Meis蛋白取代Pbx:Hoxb1b复合体中的hdac来激活一些靶基因的转录，这些靶基因反过来调节r4-r7形成所需的一系列转录因子。我们的第一个目标是描述hoxb1b调控r4-r6形成的途径。特别是，我们将确定vhnf1， val和hoxb1a的作用顺序以及它们的表达域如何被细化。我们还将探讨在r5/r6中发现的几个新基因的功能。我们的第二个目标是确定Meis和Pbx辅助因子在Hoxb1b功能调节中的作用。我们将探索Meis蛋白取代Pbx中hdac的机制，并在体内测试Hoxb1b靶基因激活过程中Meis是否以类似的方式取代hdac。我们的实验之所以重要，有几个原因。首先，后脑产生了几个基本结构——感觉神经节和支运动神经元，以及脊椎动物头部的骨头、软骨和肌肉。发育中的后脑对各种因素（如环境毒素、传染性病原体和遗传条件）的干扰很敏感，这些因素会导致一系列出生缺陷——运动控制问题，如共济失调，认知缺陷，如自闭症和颅面缺陷。此外，Hox蛋白和Hox辅助因子调节神经发育的其他方面（如神经管的背腹侧模式）和胚胎发生的其他方面（如造血）。Hox， meis和PBX基因也是参与白血病的原癌基因。因此，对Hox功能的更好理解将适用于广泛的生物过程和人类疾病状况。胚胎后脑发育出许多基本结构——神经系统的感觉神经节和支运动神经元，以及头部的骨骼、软骨和肌肉。发育中的后脑对各种因素（如环境毒素、传染性病原体和遗传条件）的干扰很敏感，这些因素会导致一系列出生缺陷——运动控制问题，如共济失调，认知缺陷，如自闭症和颅面缺陷。此外，本提案中研究的基因调节神经发育的其他方面（例如神经管的背腹侧模式）和胚胎发生的其他方面（例如造血）。这些基因也是与白血病有关的原癌基因。因此，我们提出的实验结果将适用于广泛的生物过程和人类疾病状况。

DESCRIPTION (provided by applicant): Segmentation of the caudal hindbrain into rhombomeres (r) 4-7 is regulated by paralog group 1 (PG1) Hox proteins, but it is unclear how PG1 Hox proteins activate their target genes and how these genes subsequently interact to produce distinct rhombomeres. Our preliminary results indicate that Hoxb1b (a zebrafish PG1 Hox protein) forms repressive complexes with Pbx proteins and histone deacetylases (HDACs) and that Meis proteins are required to convert Pbx:Hoxb1b complexes into transcriptional activators. We have also demonstrated that Hoxb1b regulates the expression of several genes in r4 (e.g. hoxb1a) and r5/r6 (e.g. val, vhnf1), but it is unclear how these genes act and it is likely that additional hoxb1b target genes exist. We hypothesize that Meis proteins displace HDACs from Pbx:Hoxb1b complexes to activate transcription of a few target genes that in turn regulate a cascade of transcription factors required for formation of r4-r7. Our first aim is to delineate Hoxb1b-regulated pathways that control formation of r4-r6. In particular, we will establish in which order vhnf1, val and hoxb1a act and how their expression domains become refined. We will also explore the function of several novel genes we have identified in r5/r6. Our second aim is to determine the role of Meis and Pbx cofactors in modulation of Hoxb1b function. We will explore the mechanism whereby Meis proteins displace HDACs from Pbx and we will test whether Meis acts in a similar manner to displace HDACs during activation of Hoxb1b target genes in vivo. Our experiments are important for several reasons. First, the hindbrain gives rise to several essential structures - sensory ganglia and branchiomotor neurons, as well as bone, cartilage and muscle of the vertebrate head. The developing hindbrain is sensitive to disruptions by a variety of factors (e.g. environmental toxins, infectious agents and genetic conditions) that give rise to a range of birth defects - motor control problems such as ataxia, cognitive defects such as autism and craniofacial defects. In addition, Hox proteins and Hox cofactors regulate other aspects of neural development (e.g. dorsoventral patterning of the neural tube), and other aspects of embryogenesis (e.g. hematopoiesis). hox, meis and pbx genes are also proto-oncogenes involved in leukemia. A better understanding of Hox function will therefore be applicable to a broad set of biological processes and human disease conditions. The embryonic hindbrain gives rise to many essential structures - sensory ganglia and branchiomotor neurons of the nervous system, as well as bone, cartilage and muscle of the head. The developing hindbrain is sensitive to disruptions by a variety of factors (e.g. environmental toxins, infectious agents and genetic conditions) that give rise to a range of birth defects - motor control problems such as ataxia, cognitive defects such as autism and craniofacial defects. In addition, the genes studied in this proposal regulate other aspects of neural development (e.g. dorsoventral patterning of the neural tube), and other aspects of embryogenesis (e.g. hematopoiesis). These genes are also proto-oncogenes involved in leukemia. The results from our proposed experiments will therefore be applicable to a broad set of biological processes and human disease conditions.