A quantitative gene network underlying robust epidermal stem cell fate patterning

强大的表皮干细胞命运模式背后的定量基因网络

• 批准号: 2283976
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2283976
• 关键词: quantitative; gene; network; underlying; robust

Understanding the robustness underlying stem cell maintenance and cell fate patterning is a fundamental problem in developmental biology. It has applications in many disciplines from regenerative medicine to cancer treatment. Robustness is defined as resistance to phenotypic change in the face of internal and external perturbations. One of the most developmentally robust metazoan animals is C. elegans. Its whole developmental process is highly stereotyped including the developmental path of individual cells, to the extent that we know the exact number and type of divisions each cell has undergone in an adult nematode over the course of its development. C. elegans have exactly 959 cells upon reaching adulthood, of these 32 correspond to the lateral seam cells. Seam cells have the essential properties of stem cells. They are able to both self-renew and differentiate into neuronal and epidermal cell fates. The choices that these cells make are regulated by a network of transcription factors, the exact architecture of which is not well resolved. We will start from a network focused on the three transcription factors involved in seam cell maintenance. These genes are ceh-16, elt-1 and egl-18. ELT-1 is a GATA-binding transcription factor and is required for the specification of the entire epidermis, including the seam cells and its expression is maintained in seam cells throughout development. EGL-18 is also a GATA factor and has been hypothesised to be regulated by ELT-1. CEH-16 is an ortholog of human Engrailed Homeobox 1/2 genes. It is thought to have a role in preventing seam cell fusion and has been thought to influence both elt-1 and egl-18 expression. All three of these are highly important genes in development. Full knockouts of any of the three results in severe malformations of the nematode, which in the case of elt-1 and ceh-16 are embryonic lethal. Due to this we will be working mostly with tissue-specific modifications and partial loss of function mutants. There are two main approaches that we will take. The first is to evaluate individual connections established by an initial Boolean network in more depth. This approach would require the confirmation of certain behaviours predicted by the Boolean network. Therefore, double and triple mutants of these core genes will be evaluated in terms of their molecular and phenotypic consequences in seam cells. This will allow us to test the regulatory connections between the genes proposed by the model. On top of this, we aim to evaluate the behaviour of our three genes over time through larval development. We aim on using live imaging of transcriptional reporters to collect detailed information on the behaviour of the three genes. Additionally, the use of transcriptional reporters could allow us to identify transcription and degradation rates, which would be essential for more complex modelling of the system's behaviour. Additionally, the second approach is to expand the network through the addition of more genes and connections. For this purpose, we will investigate the genes thought to be connected to our selected three genes in the literature. Additionally, we will integrate additional connections through targeted DamID (TaDa) experiments in our lab revealing downstream targets as well as confirming interactions between core components. Overall, we aim to elucidate the connections in the gene network underlying the mechanisms required for seam cell maintenance and division and understand the network's dynamics. We aim to provide a quantitative model of the interactions between their transcription factors in order to have a more in depth understanding of the causes underlying seam cells robustness.

了解干细胞维持和细胞命运模式的稳健性是发育生物学的一个基本问题。它在从再生医学到癌症治疗的许多学科中都有应用。稳健性定义为面对内部和外部扰动时对表型变化的抵抗力。发育最强大的后生动物之一是秀丽隐杆线虫。它的整个发育过程包括单个细胞的发育路径都是高度定型的，以至于我们知道成体线虫在其发育过程中每个细胞所经历的确切分裂次数和类型。线虫在成年后总共有 959 个细胞，其中 32 个对应于侧缝细胞。 Seam 细胞具有干细胞的基本特性。它们能够自我更新并分化成神经元和表皮细胞。这些细胞做出的选择受到转录因子网络的调节，但其确切的结构尚未得到很好的解决。 我们将从一个专注于缝细胞维护涉及的三种转录因子的网络开始。这些基因是ceh-16、elt-1和egl-18。 ELT-1 是一种 GATA 结合转录因子，是整个表皮（包括接缝细胞）规范所必需的，并且在整个发育过程中接缝细胞中都维持其表达。 EGL-18 也是一种 GATA 因子，假设受 ELT-1 调节。 CEH-16 是人类 Engrailed Homeobox 1/2 基因的直向同源物。它被认为具有防止接缝细胞融合的作用，并且被认为影响 elt-1 和 egl-18 的表达。所有这三个基因在发育过程中都是非常重要的。完全敲除这三种线虫中的任何一种都会导致线虫严重畸形，就 elt-1 和 ceh-16 而言，这种畸形是胚胎致死的。因此，我们将主要研究组织特异性修饰和部分功能丧失的突变体。 我们将采取两种主要方法。第一个是更深入地评估由初始布尔网络建立的个体连接。这种方法需要确认布尔网络预测的某些行为。因此，将根据它们在接缝细胞中的分子和表型后果来评估这些核心基因的双突变体和三突变体。这将使我们能够测试模型提出的基因之间的调控联系。最重要的是，我们的目标是通过幼虫发育来评估我们的三个基因随时间的行为。我们的目标是使用转录报告基因的实时成像来收集有关这三个基因行为的详细信息。此外，转录记者的使用可以让我们识别转录和降解率，这对于更复杂的系统行为建模至关重要。 此外，第二种方法是通过添加更多基因和连接来扩展网络。为此，我们将研究被认为与我们在文献中选择的三个基因相关的基因。此外，我们将在实验室中通过有针对性的 DamID (TaDa) 实验集成额外的连接，揭示下游目标并确认核心组件之间的相互作用。 总体而言，我们的目标是阐明接缝细胞维持和分裂所需机制背后的基因网络中的连接，并了解网络的动态。我们的目标是提供转录因子之间相互作用的定量模型，以便更深入地了解接缝细胞稳健性的原因。