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

基本信息

批准号：
10164722
负责人：
Kimberly Lynn Cooper
金额：
$ 37.86万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
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/10164722
关键词：
ATAC-seq Address Adult Animals Arm Bones Biochemical Biology Candidate Disease Gene Cartilage Chick Embryo 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

项目摘要

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.

每个骨骼元件的长度在开发和演变过程中独立变化将具有相似大小软骨的胚胎骨骼转化为一系列成人形式和功能。许多基因的功能突变的丧失产生的骨骼成比例矮小的骨骼，这表明一个常见的骨骼所有长骨的伸长需要“工具包”。然而，不太了解的是在每个生长板上建立特定速率和延伸持续时间的机制，共同确定成年肢体骨骼比例。定义骨骼比例的基因是什么？是差异由模块化增强子控制的生长，这些增强子在局部调节所有生长共有基因的表达板和/或仅在生长板子集中起作用的基因？我们的实验室可以回答有关脊椎动物如何如何使用两个独特的物种获取形式和功能：实验室小鼠和较小的埃及人杰博亚。在最近的老鼠亲戚中，Jerboa具有最大不同的后肢非常长的脚，但它的前肢与鼠标相似。这些相似性和差异耦合具有高基因组序列同源性可以鉴定局部控制的遗传机制骨骼生长速率。小鼠和Jerboa前肢和后肢元素的RNA-seq分析表明，直系同源基因的10％与内部和之间的相对增长率相关。物种。其中包括40个基因，有强烈的证据证明这两个物种都有增强子模块化。目标1意志实施比较ATAC-SEQ和鼠标转基因以识别和在功能上测试模块化增强器在小鼠和Jerboa基因组中。我们预测，这40个基因中的一些受半径/尺度控制物种和通过功能差异的不同meta骨增强剂之间保守的增强剂在Jerboa中，允许Jerboa Hindb比例未耦合。我们的表达数据还提供了一个宝贵的机会，以填补我们对调节所有脊椎动物的肢体骨骼生长和比例的基因。我们以前表明IGF1 小鼠需要信号传导，以使生长板的肥厚软骨细胞尺寸差异拉长尽管IGF1在整个生物体和器官的生长中都有良好的作用，但尚不清楚该途径如何在局部调节以调节差异生长。在AIM 2中，我们将在生化测试假设升高的蛋白酶表达在快速伸长的骨骼元件中裂解IGF结合蛋白质因此使生物活性IGF1蛋白释放信号传导以加速生长。虽然其他六个高优先级预计候选基因也将是骨骼生长的关键调节因子，尚未归因于生长板功能。 AIM 3将在鸡胚胎中实施强大的过表达方法进行测试这些基因中的每一个都足以加速或抑制肢体生长速率的假设。