Combining genetic and modelling approaches to understanding vascular development in Arabidopsis

背景：在高等植物中，胚后组织来源于多能干细胞群，这些多能干细胞群存在于被称为分生组织的壁龛中。分生组织由中央组织细胞和相邻的分裂干细胞组成，这些细胞为新器官提供细胞。负责植物纵向生长的分生组织位于根和茎尖。径向扩张也通过分生组织中的分裂发生，称为形成层和原形成层，它们存在于维管组织中。植物维管组织由木质部和韧皮部两种输导组织组成。木质部是水分运输所必需的，而韧皮部主要是养分运输所必需的。木质部细胞是陆地生物量的重要组成部分，木质部细胞是木本植物组织的重要组成部分，是重要的可再生能源和生物材料。木质部和韧皮部都是由维管分生组织（形成层和原形成层）的细胞分裂产生的。影响植物维管发育的几个方面的一个主要组分是信号传导模块，其特征在于在韧皮部中表达的称为TDIF的肽配体及其同源受体PXY，其在原形成层中表达。这些因素的非细胞自主相互作用导致特别复杂的输出。这种配体-受体对被认为调节原形成层中细胞分裂的速率，将木质部身份排除在分裂区之外，并且还通过影响细胞分裂的方向来控制维管组织。TDIF通过与许多其他信号传导组分和转录调节因子的相互作用来影响这些输出。虽然已经成功地识别了调节PXY信号输出的各个方面的目标和相互作用物，但仍然不清楚这些复杂的输出是如何整合的。目的：为了了解PXY信号的三个输出（即维管组织，细胞分裂和从原形成层中排除木质部身份）是如何整合的，我们的目标是生成定量数据，这些数据将用于制作模拟PXY信号的逻辑网络模型。我们的模型将有助于产生的假设，将使用遗传学和分子biology.Methodology测试：学生将首先结合联合收割机使用组织学，显微镜和成像方法与计算分析（如细胞分析软件），以确定精确的数量木质部韧皮部和原形成层细胞存在于花序发育过程中的维管组织。这些分析将与学生使用定量RT-PCR生成的基因表达数据相结合。将使用野生型拟南芥和pxy突变体，以及其中TDIF已被去除的品系。TDIF来源于3个基因，我们与Zack Nimchuk（北卡罗来纳州大学）合作，他使用CRISPR/Cas9产生了一个三重突变体，以完全消除配体。该数据将用于构建系统的数学/计算模型。该模型将采用空间分布的逻辑网络的形式，由学生在共同监督人Natasha Savage（利物浦大学）的监督下生成。这个模型将被用来产生可检验的假设，随后的实验将以模型数据为指导。我们设想模型预测将通过操纵植物中的基因表达来测试。例如，如果模型预测组分的负反馈对于维持系统至关重要，则我们将使用对这种反馈不敏感的异源和/或诱导型启动子操纵表达，并评估血管发育的后果。其他操作可用于测试各种新出现的特性，包括形态发生梯度、细胞分裂速率等。活动时间表：0-12月：生成拟南芥维管组织的生长数据和基因表达数据。

Background: In higher plants, post-embryonic tissues are derived from pluripotent stem cell populations present in niches referred to as meristems. Meristems are composed of central organizing cells with adjacent dividing stem cells that provide cells for new organs. Meristems that are responsible for length-wise plant growth are located at the root and shoot apices. Radial expansion also occurs via divisions in meristematic tissues, referred to as the cambium and procambium, which are present in the vascular tissue. Plant vascular tissue is constituted of two conductive tissues, the xylem and phloem. Xylem is required for water transport, and phloem is required primarily for nutrient transport. A significant proportion of terrestrial biomass is constituted of xylem cells that make up woody plant tissue, which represents a large renewable resource of energy and biomaterials.Both xylem and phloem are derived from cell divisions in vascular meristems known as the cambium and procambium. One major component that influences several aspects of plant vascular development, is a signalling module characterised by a peptide ligand called TDIF that is expressed in the phloem, and its cognate receptor, PXY which is procambium expressed. The non-cell autonomous interaction of these factors results in particularly complex outputs. This ligand-receptor pair is thought to regulate the rate of cell division in the procambium, exclude xylem identity from the division zone and also control vascular organization by influencing the orientation of cell division. TDIF influences these outputs via interactions with a number of other signalling components and transcriptional regulators. While the identification of targets and interactors that regulate various aspects of PXY signalling outputs has been successful, it remains unclear as to how these complex outputs are integrated. Aims: In order to understand how the three outputs of PXY signalling (i.e. vascular organisation, cell division and exclusion of xylem identity from the procambium) are integrated, we aim to generate quantitative data that will be used to make a logical network model that simulates PXY signalling. Our model will assist in generating hypotheses that will be tested using genetics and molecular biology.Methodology: The student will initially combine the use of histology, microscopy and imaging methodologies with computational analysis (such as cell profiler software) to determine precise numbers of xylem phloem and procambium cells present in vascular tissue during inflorescence development. These analyses will be combined with gene expression data that the student will generate using quantitative RT-PCR. Wild-type Arabidopsis and pxy mutants will be used, as will lines where TDIF has been removed. TDIF is derived from 3 genes, and we have collaborated with Zack Nimchuk (University of North Carolina) who has generated a triple mutant using CRISPR/Cas9 to entirely eliminate the ligand. This data will be used build a mathematical/computational model of the system. The model will take the form of a spatially distributed logical network, generated by the student under the supervision of co-supervisor Natasha Savage (University of Liverpool). This model will then be used to produce testable hypotheses.Subsequent experiments will be guided by modelling data. We envisage model predictions will be tested by manipulating gene expression in planta. For example, if the model predicts that negative feedback of components is critical for maintaining the system, we will manipulate expression using heterologous and/or inducible promoters that are not susceptible to such feedback and assessing the consequences for vascular development. Other manipulations could be used to test a variety of emergent properties including morphogen gradients, rates of cell division etc.Timetable of Activities:Month 0-12: Generate growth data and gene expression data for Arabidopsis vascular tissu