Genetic control of limb development by Pbx

Pbx 对肢体发育的遗传控制

• 批准号: 7888810
• 负责人: Licia Selleri
• 金额: $ 35万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-10 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7888810
• 关键词: Ablation; Acute Myelocytic Leukemia; Acute leukemia; Affect; Alleles; Anterior; Basic Science; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Breast; Cell Culture Techniques; Cell Line; Cells; Chromosomal translocation; Complex; Comprehension; Congenital Abnormality; Congenital Disorders; Congenital Heart Defects; DNA Binding; Defect; Development; Developmental Process; Diabetes Mellitus; Digit structure; Disease; Distal; Drosophila genus; EMSA; Ectopic Expression; Embryo; Event; Exhibits; Fatal Outcome; Forelimb; Funding; Gene Expression; Gene Expression Regulation; Gene Family; Gene Mutation; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic Crosses; Genetic Transcription; Genital system; Genomics; Glioma; HOXA9 gene; Hand; Hindlimb; Homeobox Genes; Homeodomain Proteins; Homologous Gene; Human; Human Genetics; Knowledge; Light; Limb Bud; Limb Development; Limb structure; Malignant neoplasm of lung; Malignant neoplasm of ovary; Mediating; Medical; Mesenchyme; Modeling; Molecular; Molecular Genetics; Morphogenesis; Mus; Mutation; NUP98 gene; Names; Neoplastic Cell Transformation; Neuroblastoma; Nucleic Acid Regulatory Sequences; Organogenesis; Pathogenesis; Pathway interactions; Pattern; Play; Process; Proteins; Public Health; Regulation; Reporting; Research; Role; Skeletal Development; Skeleton; Spatial Distribution; Specificity; Structure; Syndrome; System; Testing; Tissues; Transcriptional Regulation; Transfection; Ursidae Family; Work; base; cancer type; chromatin immunoprecipitation; cofactor; craniofacial; fly; foot; gene function; homeodomain; in vivo; insight; malformation; mutant; novel; programs; public health relevance; research study; rib bone structure; social; transcription factor

DESCRIPTION (provided by applicant): Hox homeodomain transcription factors play essential roles in vertebrate limb patterning and morphogenesis, but the mechanisms of Hox regulation remain elusive at the molecular level. The Pbx1 TALE homeodomain protein is a homolog of Drosophila extradenticle (Exd), which has critical roles in patterning of the fly body. While the fly has only one TALE-encoding gene, Exd, the mouse has four Pbx genes (Pbx1-4). Based on molecular and biochemical analyses, for the last fifteen years the prevailing view has been that Pbx/Exd primarily helps Hox proteins execute their developmental programs. Hence, Pbx proteins have been named as "Hox cofactors". However, it has been unclear whether Pbx proteins exert their roles more broadly than Hox ancillary factors in vivo. Our objectives are to: 1) use the limb as a tractable system to delineate genetic and molecular networks controlled by TALE proteins in developmental programs; 2) establish whether regulation of Hox "collinear" expression in the limb, an elegant but mysterious developmental phenomenon, is mediated by Pbx TALE homeodomain proteins. Using the mouse as a system, we have established that different Pbx genes share partially overlapping roles in various developmental processes, including limb patterning. Accordingly, Pbx1/Pbx2 double homozygous (Pbx1-/-;Pbx2-/-) embryos lack limbs altogether, while Pbx1-/-;Pbx2 mutants exhibit dramatic limb truncations. Most significantly, we have uncovered that during limb morphogenesis Pbx1/Pbx2 control the onset and spatial distribution of 5' Hox expression in the autopod. Thus, Pbx proteins hierarchically govern Hox gene expression in this system. In view of these unanticipated findings, our current working hypothesis proposes that Pbx homeoproteins do not function exclusively as Hox ancillary factors in the limb, but that they control Hox genes and possibly regulate 5' HoxD expression at the transcriptional level in the limb bud. We will test our hypothesis in the mouse using genetic and molecular approaches. First, 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 is inducible in the mesenchyme), we will dissect Pbx1/Pbx2 spatial and temporal requirements in limb field and bud mesenchyme. We will then determine when Hox expression is first affected by Pbx1/2 loss in the mesenchyme. By molecular approaches, we will subsequently determine whether Pbx1/2 regulate 5' HoxD transcription by direct control of the HoxD GCR, a genomic region that controls HoxD collinear expression in the autopod. Furthermore, we will 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 vivo in the mouse. Finally, we will analyze Pbx1/Pbx3 phenotypic interactions in mouse forelimb (FL) development and assess Pbx1/Pbx3 control of early Hox expression in the FL field. We will specifically focus on FL patterning since Pbx3, which is not expressed in hindlimbs, acts as a FL-specific marker. Completion of these studies will shed light on Pbx- controlled programs in limb development and establish novel regulatory networks that govern transcription of Hox genes, key architects of the body plan. Also, this work will bear directly on our understanding of human genetic limb malformations. More broadly, given the involvement of HOX genes in human chromosomal translocations that perturb HOXD13 or HOXA9 expression resulting in acute leukemia, our studies will inform general comprehension of Hox gene function also in other contexts, as human neoplastic transformation. PUBLIC HEALTH RELEVANCE: Homeodomain proteins control patterning and morphogenesis of the appendicular skeleton within genetic and molecular networks that are poorly understood. We have established critical genetic roles for the TALE-class of homeoproteins Pbx1 and Pbx2 in limb development. The studies proposed in this application will provide novel insight into the control of HoxD gene expression by Pbx in the mesenchyme of the distal limb, as well as strengthen our new model wherein Pbx do not act solely as Hox ancillary factors in limb development, but govern Hox gene expression. Furthermore, the proposed experiments will shed light on the unique and partially overlapping contributions of Pbx1/Pbx2 in limb bud mesenchyme in skeletal development. Finally, the planned studies will establish as yet unknown roles for Pbx3, a forelimb-specific marker, in patterning anterior and proximal forelimb bud mesenchyme together with Pbx1, as well as uncover novel molecular networks that are perturbed when Pbx1/Pbx3 are concomitantly lost. Direct involvement of Hox genes in human congenital malformations has been reported as a result of genetic mutations. Accordingly, a broader impact of this work will be the generation of new knowledge on the pathogenesis of human congenital disorders, including those that affect limb skeletal development and function. Indeed, mutations in 5' HOX genes result in severe limb abnormalities, including synpolydactyly, monodactyly, club foot malformation, hand-foot-genital syndrome, and brachydactyly, among others. Additionally, common forms of human acute myelogenous leukemias with fatal outcome involve chromosomal translocations that join HOXD13 or HOXA9 to NUP98, thereby resulting in perturbed and ectopic expression of the HOX gene. Lastly, perturbed expression of HOX genes in humans has been reported in several types of cancers, including lung cancers, gliomas, neuroblastomas, and breast and ovarian cancers. While this proposal focuses on Pbx functions in the limb and potential regulatory roles of Pbx on Hox transcription in this system, perturbation of Pbx-ruled developmental processes in the mouse leads to a variety of other abnormalities (in addition to craniofacial, axial, rib cage, limb, and girdle defects) that closely model a broad range of human congenital diseases, including congenital heart defects, diabetes mellitus, congenital asplenia. Therefore, it is of high social and medical relevance to conduct basic research that will establish as yet unknown Pbx-controlled networks in organogenesis as well as novel mechanisms for Hox transcriptional regulation and collinear expression. Deeper comprehension of these basic processes in the limb will be applicable to other contexts within the developing embryo and ultimately aid our understanding of the pathogenesis of human congenital diseases as well as neoplastic transformation.

描述（申请人提供）：Hox同源域转录因子在脊椎动物肢体模式和形态发生中发挥重要作用，但Hox调控机制在分子水平上仍不明确。Pbx1 TALE同源结构域蛋白是果蝇外皮层（Exd）的同源物，在果蝇体的模式化中起着关键作用。苍蝇只有一个编码tale的基因Exd，而老鼠有四个Pbx基因（Pbx1-4）。基于分子和生化分析，在过去的15年里，流行的观点是Pbx/Exd主要帮助Hox蛋白执行其发育程序。因此，Pbx蛋白被命名为“Hox辅助因子”。然而，Pbx蛋白在体内是否比Hox辅助因子发挥更广泛的作用尚不清楚。我们的目标是：1)利用肢体作为一个可处理的系统来描述发育程序中由TALE蛋白控制的遗传和分子网络；2)确定肢体中Hox“共线”表达的调控是否由Pbx TALE同源结构域蛋白介导，这是一个优雅而神秘的发育现象。利用小鼠作为一个系统，我们已经确定了不同的Pbx基因在各种发育过程中共享部分重叠的作用，包括肢体模式。因此，Pbx1/Pbx2双纯合子（Pbx1-/-;Pbx2-/-）胚胎完全没有四肢，而Pbx1-/-；Pbx2突变体表现出显著的肢体截断。最重要的是，我们发现在肢体形态发生过程中，Pbx1/Pbx2控制着自细胞中5' Hox表达的开始和空间分布。因此，Pbx蛋白在这个系统中分层次地控制Hox基因的表达。鉴于这些出乎意料的发现，我们目前的工作假设提出Pbx同源蛋白在肢体中并不仅仅作为Hox辅助因子，而是控制Hox基因，并可能在肢体芽的转录水平上调节5' HoxD的表达。我们将用遗传和分子方法在老鼠身上验证我们的假设。首先，通过组织特异性和可诱导的基因消融，利用我们的新Pbx1条件等位基因（在Pbx2缺乏的背景下）和现有的Cre系（其中一个在间质中可诱导），我们将解剖Pbx1/Pbx2在肢体野和芽间质中的时空需求。然后我们将确定什么时候Hox的表达首先受到间质中Pbx1/2缺失的影响。通过分子方法，我们随后将确定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。此外，我们将通过细胞培养中的瞬时转染和小鼠体内的瞬时转基因实验来测试Pbx与HoxD GCR的结合是否对转录有功能影响。最后，我们将分析Pbx1/Pbx3在小鼠前肢（FL）发育中的表型相互作用，并评估Pbx1/Pbx3对前肢早期Hox表达的控制。我们将特别关注FL模式，因为Pbx3在后肢中不表达，它是FL特异性标记物。这些研究的完成将揭示肢体发育中Pbx控制的程序，并建立新的调控网络，控制Hox基因的转录，Hox基因是身体计划的关键建筑师。此外，这项工作将直接影响我们对人类遗传性肢体畸形的理解。更广泛地说，考虑到HOX基因参与人类染色体易位，扰乱HOXD13或HOXA9的表达，导致急性白血病，我们的研究将为在其他情况下（如人类肿瘤转化）对HOX基因功能的普遍理解提供信息。