Mechanisms of Pbx-directed Genetic &Transcriptional Control of Limb Development

描述（由申请人提供）：在脊椎动物中，Hox基因在大多数重要器官的形成中起主要作用。已经提出，允许不同的Hox蛋白调节特定靶基因，从而指示不同身体结构的身份的精致的DNA结合特异性取决于与其他同源异型蛋白的相互作用，这些同源异型蛋白充当Hox辅因子。在过去的十五年中，基于分子和生物化学分析，流行的观点是TALE同源结构域蛋白，其包含由Pbx基因家族编码的产物，作为Hox的辅助辅因子。Pbx 1是果蝇外齿蛋白（exd）的同源物，在果蝇身体的图案形成中具有关键作用。虽然exd是果蝇中唯一的Pbx编码基因，但小鼠有四个这样的基因（Pbx 1 -4）。尽管它们在器官发生和身体和肢体轴的模式中起着至关重要的作用，但Hox调节的分子机制仍然难以捉摸。我们的目标是使用小鼠肢体作为最易处理和建立的系统来描绘是否调控Hox的“共线”表达，一个基本的和神秘的生物现象，是由Pbx。我们已经建立了不同的Pbx基因，类似于Hox基因，在肢体模式和生长中具有重叠的作用。因此，Pbx 1/Pbx 2双纯合（Pbx 1-/-; Pbx 2-/-）胚胎完全缺乏肢体，而Pbx 1-/-; Pbx 2突变体表现出类似于HoxA/D突变体的肢体截短。此外，我们发现Pbx 1/Pbx 2控制肢体间充质中5' HoxA/D表达的起始和空间分布。这些发现证实Pbx蛋白分级控制肢体中的5' HoxA/D基因表达。鉴于这些新的发现，我们的假设提出了一种新的机制，Hox基因的调控，即5' Hox的表达直接控制在转录水平的Pbx在芽间充质的肢体形态发生和数字的形成。我们将在小鼠中使用胚胎学、遗传学和分子方法来验证我们的假设。首先，通过分子生物学方法，我们将确定Pbx 1/2是否通过直接控制HoxD GCR（一个控制HoxD共线表达的基因组区域）来调节5' HoxD转录。然后，我们将测试是否Pbx结合到HoxD GCR转录的功能轴承在细胞培养中的瞬时转染和小鼠中的瞬时转基因实验。此外，通过组织特异性和诱导的遗传消融，使用我们新的Pbx 1条件等位基因（在Pbx 2缺陷的背景下）和可用的Cre系（其中一个在间充质中诱导），我们将解剖Pbx 1/Pbx 2在肢体领域和芽间充质中的空间和时间要求。通过这种方法，我们将确定Hox表达何时首先受到肢芽中Pbx损失的影响。这些研究的完成将定义新的调控网络，管理Hox基因的转录，并将直接有助于人类先天性肢体畸形的理解。广泛地说，考虑到人类HOX基因参与白血病和实体瘤，我们的研究将为人类肿瘤中HOX调控的一般理解提供信息。 公共卫生相关性：Hox蛋白在哺乳动物包括肢体在内的许多重要器官的形成中发挥着重要作用;然而，Hox基因调控的分子机制仍然难以捉摸。拟议的研究将使用小鼠模型，为肢体中Pbx蛋白控制Hox调控提供新的见解。因此，这项工作的广泛影响将是产生有关人类先天性畸形发病机制的新知识，包括那些影响肢体骨骼发育和功能的畸形，发生在大约500例活产婴儿中的1例。

DESCRIPTION (provided by applicant): In vertebrates, Hox genes play major roles in the formation of most vital organs. It has been proposed that the exquisite DNA-binding specificities that allow different Hox proteins to regulate specific target genes, thus instructing the identity of distinct body structures, depend on interactions with other homeoproteins, which act as Hox cofactors. For the last fifteen years, based on molecular and biochemical analyses, the prevailing view has been that TALE homeodomain proteins, which comprise the products encoded by the Pbx gene family, act as ancillary cofactors for Hox. Pbx1 is a homolog of Drosophila extradenticle (exd), which has critical roles in patterning of the fly body. While exd is the sole Pbx-encoding gene in the fly, the mouse has four such genes (Pbx1-4). Despite their paramount roles in organogenesis and patterning of the body and limb axes, the molecular mechanisms of Hox regulation remain elusive. Our objectives are to use the mouse limb as the most tractable and established system to delineate whether regulation of Hox "collinear" expression, a basic and mysterious biological phenomenon, is governed by Pbx. We have established that different Pbx genes, similarly to Hox genes, share overlapping roles in limb patterning and outgrowth. Accordingly, Pbx1/Pbx2 double homozygous (Pbx1-/-;Pbx2-/-) embryos lack limbs altogether, while Pbx1-/-;Pbx2 mutants exhibit limb truncations similar to those of HoxA/D mutants. Additionally, we have found that Pbx1/Pbx2 control the onset and spatial distribution of 5' HoxA/D expression in limb mesenchyme. These findings establish that Pbx proteins hierarchically govern 5' HoxA/D gene expression in the limb. In view of these new findings, our hypothesis proposes a novel mechanism for Hox gene regulation, whereby 5' Hox expression is directly controlled at the transcriptional level by Pbx in the bud mesenchyme for limb morphogenesis and digit formation. We will test our hypothesis using embryologic, genetic and molecular approaches in the mouse. First, by molecular methods, we will determine whether Pbx1/2 regulate 5' HoxD transcription by direct control of the HoxD GCR, a genomic region that governs HoxD collinear expression in the autopod. We will then test whether Pbx binding to the HoxD GCR has functional bearings on transcription by both transient transfections in cell culture and transient transgenesis experiments in the mouse. Moreover, by tissue-specific and inducible genetic ablation, using our new Pbx1 conditional allele (on a Pbx2-deficient background), and available Cre lines (one of which inducible in the mesenchyme), we will dissect Pbx1/Pbx2 spatial and temporal requirements in the limb field and bud mesenchyme. By this approach, we will determine when Hox expression is first affected by Pbx loss in the limb bud. Completion of these studies will define novel regulatory networks that govern transcription of Hox genes and will directly contribute to the understanding of human congenital limb malformations. Broadly, given the involvement of human HOX genes in leukemias and solid tumors, our studies will inform general comprehension of HOX regulation also in human neoplasia. PUBLIC HEALTH RELEVANCE: Hox proteins play essential roles in the formation of many critical organs in mammals, including the limb; however, the molecular mechanisms underlying Hox gene regulation remain elusive. The proposed studies will use mouse models to provide novel insights into the control of Hox regulation by Pbx proteins in the limb. Accordingly, a broad impact of this work will be the generation of new knowledge on the pathogenesis of human congenital malformations, including those that affect limb skeletal development and function, occurring in approximately 1 of 500 human live births.