Leveraging comparative genomics to elucidate the genetic determinants of limb skeletal proportion

利用比较基因组学阐明肢体骨骼比例的遗传决定因素

• 批准号: 9762600
• 负责人: Kimberly Lynn Cooper
• 金额: $ 47.25万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762600
• 关键词: ATAC-seq; Address; Adult; Animals; Arm Bones; Biochemical; Biology; Candidate Disease Gene; Cartilage; Chickens; Chiroptera; Chondrocytes; Chondrogenesis; Cleaved cell; Complex; Coupled; Data; Data Set; Development; Digit structure; Dipodidae; Distal; Dolphins; Dwarfism; Elements; Embryo; Enhancers; Epiphysial cartilage; Evolution; Fingers; Forelimb; Gene Expression; Gene Transfer Techniques; Genes; Genetic; Genetic Determinism; Genome; Growth; Hindlimb; Human; IGF1 gene; IGFBP5 gene; In Vitro; Insulin-Like Growth-Factor-Binding Proteins; Knowledge; Laboratories; Laboratory mice; Leg Bones; Length; Limb structure; Link; Location; Metatarsal bone structure; Modeling; Mus; Organ; Organism; Orthologous Gene; Pathway interactions; Peptide Hydrolases; Phalanx of hand; Phylogenetic Analysis; Positioning Attribute; Public Health; Radial; Regulatory Element; Research; Resistance; Role; Sequence Homology; Shapes; Signal Pathway; Signal Transduction; Signaling Protein; Skeletal Development; Skeleton; Structure; Testing; Tissues; Toes; Transgenic Mice; Vertebrates; Whole Organism; Wing; Work; bone; comparative; comparative genomics; dexterity; differential expression; foot; gene function; grasp; in vivo; inhibitor/antagonist; long bone; loss of function mutation; mutant; new growth; organ growth; overexpression; predictive modeling; skeletal; transcriptome sequencing; ulna

The length of each skeletal element changes independently during development and evolution to transform an embryonic skeleton with similar sized cartilages into a diverse array of adult forms and functions. Loss of function mutations of many genes produce proportionately dwarfed skeletons that suggest a common “toolkit” is required for elongation of all of the long bones. Far less well understood, however, are the mechanisms that establish the specific rate and duration of elongation at each growth plate, which together determine adult limb skeletal proportion. What are the genes that define skeletal proportion? Is differential growth controlled by modular enhancers that locally tune expression of genes common to all growth plates and/or by genes that function only in subsets of growth plates? Our laboratory is positioned to answer these profoundly important questions about how vertebrate limbs acquire form and function using two uniquely suitable species: the laboratory mouse and the lesser Egyptian jerboa. Among the nearest mouse relatives, the jerboa has the most extremely different hindlimbs with extraordinarily long feet, but its forelimbs are similar to the mouse. These similarities and differences coupled with high genome sequence homology enable the identification of genetic mechanisms that locally control skeletal growth rate. RNA-Seq analysis of mouse and jerboa forelimb and hindlimb elements revealed that 10% of orthologous genes are differentially expressed correlating with relative growth rates within and between species. These include 40 genes with strong evidence for enhancer modularity in both species. Aim 1 will implement comparative ATAC-Seq and mouse transgenesis to identify and functionally test modular enhancers in the mouse and jerboa genomes. We predict that some of these 40 genes are controlled by radius/ulna enhancers that are conserved between species and by distinct metatarsal enhancers that functionally diverged in jerboa and allowed the uncoupled evolution of jerboa hindlimb proportion. Our expression data also provides a valuable opportunity to fill critical gaps in our understanding of the genes that regulate limb skeletal growth and proportion in all vertebrates. We previously showed that IGF1 signaling is required in mice for hypertrophic chondrocyte size differences in growth plates that elongate at different rates. Although IGF1 has a well-established role in whole organism and organ growth, it is unclear how the pathway is locally regulated to modulate differential growth. In Aim 2, we will biochemically test the hypothesis that elevated protease expression in rapidly elongating skeletal elements cleaves IGF binding proteins thus freeing bioactive IGF1 protein for signaling to accelerate growth. Although six other high priority candidate genes are also expected to be critical regulators of skeletal growth, they have not yet been attributed growth plate functions. Aim 3 will implement a powerful overexpression approach in chicken embryos to test the hypothesis that each of these genes is sufficient to accelerate or inhibit limb growth rate.

每个骨骼元素的长度在发育和进化过程中独立变化 将具有相似大小软骨的胚胎骨骼转化为各种成人形式和功能。 许多基因的功能丧失突变会产生比例矮小的骨骼，这表明存在一种常见的现象 延长所有长骨需要“工具包”。然而，人们不太了解的是 建立每个生长板的特定伸长率和持续时间的机制，这些机制一起 确定成人肢体骨骼比例。决定骨骼比例的基因是什么？是微分的 生长由模块化增强子控制，局部调节所有生长共有的基因表达 板和/或仅在生长板子集中起作用的基因？ 我们的实验室致力于回答这些极其重要的问题，即脊椎动物的四肢如何 使用两种独特合适的物种获得形态和功能：实验室小鼠和小埃及小鼠 跳鼠。在老鼠的近亲中，跳鼠的后肢差异最大 脚特别长，但前肢却与老鼠相似。这些相似点和不同点耦合在一起 具有高基因组序列同源性，能够识别局部控制的遗传机制 骨骼生长速度。对小鼠和跳鼠前肢和后肢元件的 RNA-Seq 分析表明 10% 的直系同源基因存在差异表达，与组内和组间的相对生长率相关 物种。其中包括 40 个基因，这些基因在两个物种中均具有增强子模块化的有力证据。目标1将 实施比较 ATAC-Seq 和小鼠转基因来识别和功能测试模块化增强子 在小鼠和跳鼠基因组中。我们预测这 40 个基因中的一些是由桡骨/尺骨控制的 物种间保守的增强子以及功能上不同的不同跖骨增强子 在跳鼠中，并允许跳鼠后肢比例的非耦合进化。 我们的表达数据还提供了一个宝贵的机会来填补我们理解的关键空白 调节所有脊椎动物肢体骨骼生长和比例的基因。我们之前表明IGF1 小鼠生长板中肥大软骨细胞尺寸差异需要信号传导，这些差异在 不同的费率。尽管 IGF1 在整个生物体和器官生长中具有明确的作用，但尚不清楚 如何局部调节该途径以调节差异生长。在目标 2 中，我们将进行生化测试 假设快速伸长的骨骼元件中蛋白酶表达升高会裂解 IGF 结合 从而释放具有生物活性的 IGF1 蛋白，用于发出加速生长的信号。尽管其他六个高度优先 候选基因也有望成为骨骼生长的关键调节因子，但尚未被归因 生长板功能。 Aim 3 将在鸡胚胎中实施强大的过表达方法进行测试 假设这些基因中的每一个都足以加速或抑制肢体生长速度。