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

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多种成人形式和功能。 许多基因的功能丧失突变产生比例不相称的侏儒骨骼，这表明 所有长骨的延长都需要“工具箱”。然而，远不为人所知的是， 在每个生长板上建立特定的伸长速率和持续时间的机制， 确定成人肢体骨骼比例。决定骨骼比例的基因是什么？是差分 由模块化增强子控制的生长，该模块化增强子局部调节所有生长共同的基因表达 板和/或基因的功能，只有在子集的生长板？ 我们的实验室定位于回答这些极其重要的问题， 获得形式和功能使用两个独特的合适的物种：实验室小鼠和较小的埃及 跳鼠。在最近的老鼠亲戚中，跳鼠的后肢最不同， 它的脚特别长，但它的前肢与老鼠相似。这些相似之处和差异结合在一起 具有高基因组序列同源性，能够鉴定局部控制的遗传机制， 骨骼生长率小鼠和跳鼠前肢和后肢元件的RNA-Seq分析显示， 10%的正向基因的差异表达与内部和之间的相对生长率相关 物种这些包括40个基因，在两个物种中具有增强子模块性的强有力证据。目标1将 实施比较ATAC-Seq和小鼠转基因以鉴定和功能测试模块增强子 在老鼠和跳鼠的基因组中。我们预测，这40个基因中的一些是由桡骨/尺骨控制的， 增强子是物种之间的保守和不同的跖骨增强子，功能分歧 并允许跳鼠后肢比例的解耦进化。 我们的表达数据还提供了一个宝贵的机会，以填补我们对基因表达的理解中的关键空白。 在所有脊椎动物中调节四肢骨骼生长和比例的基因。我们之前的研究表明，IGF 1 在小鼠中，生长板中的肥大软骨细胞大小差异需要信号传导， 不同的利率。虽然IGF 1在整个生物体和器官生长中的作用已经得到了很好的证实，但目前还不清楚 该途径如何被局部调节以调节差异生长。在目标2中，我们将对 假设在快速延长的骨骼元件中蛋白酶表达升高会切割IGF结合 蛋白质，从而释放生物活性IGF 1蛋白，用于信号传导以加速生长。虽然其他六个高优先级 候选基因也被认为是骨骼生长的关键调节因子，但它们还没有被归因于 生长板功能。AIM 3将在鸡胚中实施一种强大的过表达方法， 假设这些基因中的每一个都足以加速或抑制肢体的生长速度。

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.