A quantitative gene network underlying robust epidermal stem cell fate patterning

理解干细胞维持和细胞命运模式背后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。稳健性被定义为在面对内部和外部扰动时对表型变化的抵抗力。秀丽线虫是发育最健壮的后生动物之一。它的整个发育过程是高度刻板的，包括单个细胞的发育路径，只要我们知道每个细胞在成虫发育过程中经历的分裂的确切数量和类型。线虫成年后正好有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。它们既能自我更新，又能分化为神经元和表皮细胞的命运。这些细胞做出的选择受转录因子网络的调控，其确切的结构尚未很好地解决。我们将从一个专注于参与Seam细胞维持的三个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，是包括Seam细胞在内的整个表皮发育所必需的，其表达在整个发育过程中维持在Seam细胞中。EGL-18也是一个GATA因子，并被假设为受ELT-1调节。CEH-16是人类嵌入的Homeobox 1/2基因的同源基因。它被认为在阻止Seam细胞融合方面具有作用，并被认为影响ELT-1和EGL-18的表达。这三个基因在发育过程中都非常重要。完全敲除这三种基因中的任何一种都会导致线虫的严重畸形，在ELT-1和CEH-16的情况下，这种畸形是胚胎致命的。由于这一点，我们将主要致力于组织特异性修饰和部分功能突变。我们将采取两种主要方法。第一种方法是更深入地评估由初始布尔网络建立的各个连接。这种方法需要确认布尔网络预测的某些行为。因此，这些核心基因的双重和三重突变将根据它们在Seam细胞中的分子和表型结果进行评估。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的发育来评估我们的三个基因随着时间的推移的行为。我们的目标是使用转录记者的实时成像来收集关于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确定转录和降解率，这对于更复杂的系统行为建模是必不可少的。此外，第二种方法是通过增加更多的基因和连接来扩大网络。为此，我们将调查被认为与我们在文献中选择的三个基因有关的基因。此外，我们将通过我们实验室中的目标DAMID(TADA)实验整合更多的连接，揭示下游目标以及确认核心组件之间的相互作用。总体而言，我们的目标是阐明基因网络中与Seam细胞维持和分裂所需机制的联系，并了解该网络的动态。我们的目标是提供一个转录因子之间相互作用的定量模型，以便更深入地了解Seam细胞健壮性的原因。

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.