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着重要作用。有人提出，精致的dna结合特异性，使不同的Hox蛋白调节特定的靶基因，从而指导不同的身体结构的身份，依赖于与其他同型蛋白的相互作用，这些同型蛋白作为Hox辅助因子。在过去的15年里，基于分子和生化分析，流行的观点是TALE同源结构域蛋白，它包含Pbx基因家族编码的产物，作为Hox的辅助辅因子。Pbx1是果蝇外皮层（exd）的同源基因，在果蝇体的模式化中起着关键作用。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（Pbx1-4）。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。我们的目标是利用小鼠肢体作为最易于处理和建立的系统来描述Hox“共线”表达的调节是否由Pbx控制，这是一种基本而神秘的生物现象。我们已经确定了不同的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，Pbx1/Pbx2双纯合子（Pbx1-/-;Pbx2-/-）胚胎完全没有四肢，而Pbx1-/-；Pbx2突变体表现出与HoxA/D突变体相似的肢体截断。此外，我们发现Pbx1/Pbx2控制肢体间质中5' HoxA/D表达的开始和空间分布。这些发现证实了Pbx蛋白在肢体中分层控制5' HoxA/D基因的表达。鉴于这些新发现，我们的假设提出了一种新的Hox基因调控机制，即5' Hox的表达在转录水平上由芽间质中的Pbx直接控制肢体形态发生和手指形成。我们将在小鼠身上使用胚胎学、遗传学和分子方法来验证我们的假设。首先，通过分子方法，我们将确定Pbx1/2是否通过直接控制HoxD GCR来调节5' HoxD的转录，HoxD GCR是一个控制HoxD共线表达的基因组区域。然后，我们将通过细胞培养中的瞬时转染和小鼠的瞬时转基因实验来测试Pbx与HoxD GCR的结合是否对转录具有功能影响。此外，通过组织特异性和可诱导的基因消融，使用我们的新Pbx1条件等位基因（在Pbx2缺乏的背景下）和可用的Cre系（其中一个在间质中可诱导），我们将解剖Pbx1/Pbx2在肢体场和芽间质中的空间和时间需求。通过这种方法，我们将确定Hox表达何时首先受到肢体芽中Pbx丢失的影响。这些研究的完成将定义控制Hox基因转录的新调控网络，并将直接有助于理解人类先天性肢体畸形。从广义上讲，考虑到人类HOX基因在白血病和实体瘤中的参与，我们的研究将有助于对人类肿瘤中HOX调控的一般理解。

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.