A quantitative gene network underlying robust epidermal stem cell fate patterning

了解干细胞维持和细胞命运模式的鲁棒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。稳健性定义为在面对内部和外部干扰时对表型变化的抵抗力。发育最健壮的后生动物之一是C。优雅它的整个发育过程是高度定型的，包括单个细胞的发育路径，以至于我们知道每个细胞在其发育过程中在成年线虫中经历的分裂的确切数量和类型。C.线虫在成年后有959个细胞，其中32个对应于侧缝细胞。缝细胞具有干细胞的基本特性。它们能够自我更新并分化为神经元和表皮细胞命运。这些细胞的选择是由一个转录因子网络调节的，其确切的结构还没有很好地解决。 我们将从一个网络开始，专注于三个转录因子参与接缝细胞的维护。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）的特化所必需的，并且其表达在整个发育过程中维持在接缝细胞中。EGL-18也是一种加塔因子，并被假设受ELT-1调节。CEH-16是人Engrailed同源框1/2基因的直系同源物。它被认为在阻止缝细胞融合中起作用，并被认为影响elt-1和egl-18的表达。这三个基因都是发育过程中非常重要的基因。这三个基因中的任何一个的完全敲除都会导致线虫的严重畸形，在elt-1和ceh-16的情况下，这是胚胎致死的。因此，我们将主要研究组织特异性修饰和部分功能丧失突变体。 我们将采取两种主要方法。第一个是更深入地评估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体将在接缝细胞中的分子和表型后果方面进行评价。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，我们的目标是评估我们的三个基因随着时间的推移通过幼虫发育的行为。我们的目标是使用实时成像的转录报告收集详细的信息，这三个基因的行为。此外，使用转录报告可以使我们能够确定转录和降解速率，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的基因和连接来扩展网络。为了这个目的，我们将研究被认为与我们在文献中选择的三个基因有关的基因。此外，我们将通过实验室中的靶向DamID（TaDa）实验整合其他连接，揭示下游目标并确认核心组件之间的相互作用。 总的来说，我们的目标是阐明基因网络中的连接所需的接缝细胞的维护和分裂的机制，并了解网络的动态。我们的目标是提供一个定量的模型，它们的转录因子之间的相互作用，以便有一个更深入的了解的原因背后缝细胞的鲁棒性。

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.