The Genomik Origin of Hedgehog Spines

进化过程导致了对环境挑战最多样化的适应。这种适应是如何进化的，以及它们是如何在基因组中编码的，仍然是生物学中最大的谜团之一。在这个提议中，我们将研究一个有趣的进化现象，刺猬的刺的发展，特别是四趾刺猬（Atelerix albiventris），我们有一个繁殖群体。我们的基因组分析将与小鼠和刺猬的功能分析相结合。该项目由五个部分组成。(1)我们的第一个目标是将我们已经制作的全染色体刺猬基因组与小鼠和人类基因组进行比较，以(i)寻找基因和基因家族的损失和获得，（ii）检测synsyny的断裂，包括拷贝数变化（缺失，重复），以及（iii）确定拓扑相关结构域（TAD）受到影响的候选基因组区域。然后，我们将在其他刺猬和相关物种（鼹鼠和鼩鼱）的基因组中验证这些修饰的存在，以确定那些特定于刺猬谱系的修饰。(2)我们将对来自刺猬和小鼠胚胎背侧和腹侧皮肤的单细胞（sc）数据进行两个阶段的表达分析：(a)皮肤未分化时和(b)毛/棘前体基板形成时。根据这些数据，我们将生成小鼠和刺猬胚胎在皮肤中的表达图谱。同时，我们将使用ChIP-seq和ATAC-seq鉴定发育中的皮肤中的活性增强子和启动子区域。(3)在综合数据分析中，我们将合并目标1和目标2的结果，以确定增强子活性的变化和/或基因组重排在协同断裂处，从而改变刺猬背皮肤中差异表达基因的调控景观。将在hedgehog基因样本中进行比较——前置基因位点与基因位点皮肤、背侧与腹侧皮肤——以及与小鼠表达谱进行比较。(4)我们将对刺猬和小鼠胚胎进行全贴装原位杂交，确定每个候选基因的准确表达位置和时间，并比较两个物种。一旦确定了每个候选基因的表达时间，我们将在刺猬和小鼠离体皮肤培养中进行功能测试。(5)最后，利用CRISVar技术，我们将能够以一种再现刺猬特有的基因组重排的方式操纵小鼠基因组。我们还将首次利用iGONAD方法在刺猬中建立转基因，目的是至少部分逆转导致刺出现的变化。这些目标的综合结果将使我们能够阐明刺猬刺的基因组起源，并促进我们对新形态特征出现的进化机制的理解。

Evolutionary processes have resulted in the most diverse adaptions to environmental challenges. How suchadaptations evolve and how they are encoded in the genome remains one of the great mysteries in biology. In this proposal, we will study a fascinating phenomenon in evolution, the development of spines in hedgehogs, in particular the four-toed hedgehog (Atelerix albiventris) for which we have a breeding colony. Our genomic analyses will be coupled with functional analyses, both on mice and hedgehogs. The project consists of five parts. (1) Our first aim is to compare the full-chromosome hedgehog genome we already produced to that of mouse and human to (i) search for loss and gain of genes and gene families, (ii) detect breaks of synteny, including copy number variations (deletions, duplications), and (iii) identify candidate genomic regions where Topologically Associating Domains (TAD) have been affected. We will then verify for the presence of these modifications in genomes of other hedgehogs and related species (mole and shrew) in order to identify those that are specific to the hedgehog lineage. (2) We will perform expression analysis from single cell (sc) data from dorsal and ventral skin of hedgehog and mouse embryos at two stages: (a) when the skin is undifferentiated and (b) when placodes, the precursor of hair/spines, form. From these data, we will generate an expression map of the mouse and hedgehog embryonic in skin. In parallel, we will identify active enhancer and promoter regions in the developing skin using ChIP-seq for chromatin modification and ATAC-seq. (3) In an integrated data analysis, we will merge the results from Aims 1 and 2 to identify changes in enhancer activity and/or genomic rearrangements at synteny breaks resulting in altered regulatory landscapes of genes differentially expressed in hedgehog dorsal skin. Comparisons will be performed within the hedgehog samples - pre-placode vs. placode skin and dorsal vs. ventral skin - as well by comparing with the mouse expression profiles. (4) We will carry out whole-mount in situ hybridizations on hedgehog and mouse embryos to determine the exact position and timing of expression of each candidate gene and compare the two species. Once the timing of expression of each candidate gene has been defined, we will proceed to functional testing in hedgehog and mouse ex-vivo skin cultures. (5) Finally, using CRISVar technology, we will be able to manipulate the mouse genome in a way that it recapitulates hedgehog-specific genomic rearrangements. We will also establish transgenesis in hedgehogs for the first time sing the iGONAD approach aiming to at least partially reverse the changes that led to the appearance of spines. The combined results of these aims will allow us to elucidate the genomic origin of hedgehog spines and advance our understanding of the evolutionary mechanisms responsible for the appearance of novel morphological characters.