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

在发育和进化过程中，每个骨骼元素的长度独立变化 将具有相似大小软骨的胚胎骨骼转变为一系列不同的成人形式和功能。 许多基因的功能突变丢失会产生比例矮小的骨骼，这表明 所有长骨的拉长都需要“工具包”。然而，远不为人所知的是， 在每个生长板上建立特定的伸长率和持续时间的机制，这些共同 测定成人四肢骨骼比例。决定骨骼比例的基因是什么？是不同的 生长受模块化增强剂控制，局部调节所有生长共同基因的表达 和/或只在生长板的亚群中起作用的基因？ 我们的实验室能够回答这些关于脊椎动物四肢如何 使用两个独特的合适物种获得形式和功能：实验室小鼠和小埃及鼠 跳鼠。在最接近的老鼠近亲中，跳鼠的后肢差异最大， 脚非常长，但它的前肢类似于老鼠。这些相似之处和不同之处 具有很高的基因组序列同源性使得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成分的RNA-Seq分析表明 10%的同源基因差异表达与内部和之间的相对生长速度相关 物种。其中包括40个基因，这些基因在两个物种中都有很强的增强子模块化证据。目标1将 实施比较ATAC-Seq和小鼠转基因，以识别和功能测试模块增强剂 在老鼠和跳鼠的基因组中。我们预测，这40个基因中的一些基因受尺骨/桡骨控制。 在物种之间保守的增强剂，以及在功能上分化的不同的跖骨增强剂 在跳鼠和允许跳鼠后肢比例的非耦合进化。 我们的表情数据也提供了一个宝贵的机会来填补我们对 调节所有脊椎动物肢体骨骼生长和比例的基因。我们之前已经证明了IGF1 小鼠生长板中肥大的软骨细胞大小差异需要信号传递 不同的价格。尽管IGF1在整个生物体和器官生长中的作用已经得到证实，但它还不清楚 该途径是如何在局部调节以调节差异生长的。在目标2中，我们将对 快速伸长的骨骼元素中蛋白水解酶的表达增加会导致IGF结合断裂的假说 从而释放具有生物活性的IGF1蛋白，以传递加速生长的信号。尽管其他六个高优先级 候选基因也被认为是骨骼生长的关键调节因素，但它们尚未被归类 生长板功能正常。Aim 3将在鸡胚胎中实施一种强大的过表达方法来测试 假说认为这些基因中的每一个都足以加速或抑制肢体生长速度。

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.