Understanding organ identity in flowers at the level of single cells

多细胞生物中的细胞通常包含相同的遗传信息，但在形式和功能上却表现出惊人的差异。在植物中，大多数发育过程发生在胚胎后，从分生组织中的干细胞池开始。花过渡后，拟南芥茎尖分生组织产生花分生组织，分生组织分化为萼片、花瓣、雄蕊和心皮四种器官。过去的研究已经确定了控制器官规格和分化的主要转录因子，但它们的细胞类型特异性功能仍然知之甚少。在这个项目中，我们将把单细胞和细胞类型特异性基因组学技术与靶向启动子分析结合起来。目的是为花的细胞分化建立一个分子框架，包括主要细胞类型的表征，它们的发育轨迹和细胞类型特异性调控的表征。更具体地说，我们将在野生型和同型突变花的单细胞水平上测量mRNA的表达。为了便于细胞类型的分类，我们将在特定的gfp标记的细胞群中额外使用mRNA-seq。单细胞RNA-seq数据将允许预测新的细胞类型特异性标记基因，这些基因将在植物中使用专门的报告基因分析进行分析。在一种补充方法中，我们将使用单细胞ATAC-seq分析顺式调控区域的活性。我们将生成野生型和同型突变系的scATAC-seq数据集，并通过分类细胞群体的ATAC-seq数据来补充数据。因此，我们将获得可以直接与单细胞RNA-seq数据进行比较和整合的数据集，从而可以识别控制细胞类型特异性基因表达模式的调控元件组合。然后可以在植物中利用报告基因分析发育中的花分生组织和花来验证这些结果。总之，这里提出的研究计划将把我们对花卉器官规格的理解提升到一个新的水平，因为我们将能够解剖同源转录因子的细胞和器官特异性活性，并预测相应的调控模块。

Cells in multicellular organisms typically contain the same genetic information, yet display an amazing variation in form and functions. In plants, most developmental processes occur post-embryonically, starting from pools of stem cells that are located in meristems. After floral transition, the Arabidopsis shoot apical meristem produces floral meristems that differentiate into four types of organ: sepals, petals, stamens and carpels. Past research has identified major transcription factors controlling organ specification and differentiation, yet their cell-type specific functions are still poorly understood. In this project, we will combine single cell and cell-type specific genomics techniques with targeted promoter analyses. The aim is to develop a molecular framework for cellular differentiation in flowers, including the characterization of major cell types, their developmental trajectories and the characterization of cell-type specific regulons. More specifically, we will measure mRNA expression at the level of single cells in wildtype and homeotic mutant flowers. To facilitate the classification of cell types, we will additionally employ mRNA-seq in specific GFP-labelled cell populations. The single cell RNA-seq data will allow the prediction of novel cell-type specific marker genes, which will be analyzed using dedicated reporter gene analyses in planta. In a complementary approach, we will analyze the activity of cis-regulatory regions using single cell ATAC-seq. We will generate scATAC-seq datasets for wildtype and homeotic mutant lines, and complement the data by ATAC-seq data from sorted cell populations. Thereby, we will obtain datasets that can be directly compared and integrated with the data from single cell RNA-seq, allowing the identification of combinations of regulatory elements that control cell-type specific gene expression patterns. These can then be validated in planta using reporter gene analysis in developing floral meristems and flowers. Together, the research plan proposed here will bring our understanding of organ specification in flowers to a new level, because we will be able to dissect cell- and organ-specific activities of homeotic transcription factors and predict the corresponding regulatory modules.