Genomic Biology of Limb and Gonad Development in the Spanish Mole (Talpa occidentalis)

在这项研究中，我们建议使用鼹鼠（Talpa oceanentalis）作为一个模型来研究进化如何在特定的发育过程中运作，以及这是如何反映在物种基因组的特定变化。对这些适应性变化背后的遗传原因的识别可以提供关于进化如何运作的基本原则，并可以提供对人类疾病分子机制的见解。鼹鼠已经完全适应了它们的觅食生活方式。在这个进化过程中，他们发展出一些特殊的特征，包括前肢和后肢的多趾。此外，雌性痣是真正的雌雄同体，即它们具有功能性卵巢和睾丸。我们的目标是解开这些独特特征的基因组起源。在我们的初步工作中，我们成功地编译了西班牙鼹鼠的完整基因组序列，从而为未来的分子工作提供了框架。组装2Gb大基因组并与其他物种进行比较。我们发现了感兴趣的区域，如Cyp 19 a1的三重序列，Cyp 19 a1是一种参与类固醇途径的基因。此外，我们对性腺组织进行了RNA-seq，比较了雄性睾丸和雌性性腺的睾丸和卵巢部分。我们的数据表明，类固醇途径的变化可能参与了痣的性逆转。我们的第一个目标是通过PacBio测序技术改进我们的鼹鼠基因组数据，该技术提供了超长的读取长度，简化和改进了基因组组装。我们将通过识别与其他物种相比的同线性断裂来分析基因组的结构变异。另外两种没有显示卵睾表型的鼹鼠物种将进行基因组测序，并与Talpa occurentalis进行比较。为了确定参与基因调控的区域，将在肢体和性腺组织中创建组蛋白修饰的全基因组图谱。在我们的第二个目标中，我们将研究鼹鼠趾发育的生物学。我们将研究多指（趾）畸形的发育机制以及候选通路如hedgehog和Hox在这一过程中的参与。为此，我们将在鼹鼠胚胎中进行原位杂交。对鉴定的错误表达基因的调控域和组蛋白标记的分布进行基因组学分析将揭示可能的表型相关差异。将在选定的基因座进行染色体构象捕获（4C）实验，并与小鼠进行比较。将在一组多指（趾）畸形患者中检测确定的基因组变化。在第三个目标中，我们想解开鼹鼠雌性性别逆转的基因组起源。我们将在其他阶段使用RNA-seq来鉴定可能负责雌性鼹鼠卵睾丸的候选靶基因。基因组和组蛋白标记的比较将有助于确定调控元件和进化的变化，与卵精巢表型。将在性发育障碍患者队列中测试确定的变化。

In this study we propose to use the mole (Talpa occidentalis) as a model to study how evolution operates in particular developmental processes and how this is reflected by specific changes in the species genome. The identification of the genetic causes underlying these adaptations can provide basic principles about how evolution operates and can provide insights into the molecular mechanisms of human disease. Moles have perfectly adapted to their fossorial lifestyle. In this evolutionary process they developed some exceptional features including polydactyly of the fore- and hindlimbs. Furthermore, female moles are true hermaphrodites, i.e. they have functioning ovaries and testes. We aim at unravelling the genomic origin of these unique features. In our preliminary work we managed to compile a complete genome sequence of the Spanish mole thereby providing the frame work for any future molecular work. The 2 Gb large genome was assembled and compared to other species. We found regions of interest such as a triplication of Cyp19a1, a gene invovled in the steroid pathway. In addition, we performed RNA-seq of gonadal tissue comparing male testis and the testicular and ovarian part of the female gonad. Our data indicate that changes in the steroid pathway may be involved in sex reversal in moles. Our first aim is to improve our mole genome data by PacBio sequencing a technology that provides extra-long read lengths that simplify and improve genome assembly. We will analyze the genome for structural variations by identifying breaks in synteny in comparison to other species. Two other mole species that do not show the ovotestis phenotype will be genome sequenced and compared to Talpa occidentalis. To identify regions involved in gene regulation, genome wide maps of histone modifications will be created in limb and gonadal tissue. In our second aim we will investigate the biology of digit development in the mole. We will study the developmental mechanisms of polydactyly and the involvement of candidate pathways such as hedgehog and Hox in this process. For this purpose we will perform in situ hybridizations in mole embryos. Genomic analysis of regulatory domains of identified misexpressed genes and the distribution of histone marks will reveal possible phenotype related differences. Chromosome conformation capture (4C) experiments will be performed at selected loci and compared to the mouse. Identified genomic changes will be tested in a cohort of patients with polydactyly. In the third aim we want to unravel the genomic origin of female sex reversal in moles. We will use RNA-seq at additional stages to identify candidate target genes that could be responsible for the ovotestes in female moles. Comparison of genomes and histone marks will help to identify regulatory elements and evolutionary changes that are related to the ovotestis phenotype. Identified changes will be tested in a cohort of patients with disorders of sexual development.