Defining the p63 regulatory network driving periderm development in the mouse

细胞如何在不同的细胞状态之间转换是一个基本的生物学问题。在这种情况下，简单的外胚层通过一系列动态的细胞状态来产生自我补充的多层表皮，以保护有机体免受脱水、机械创伤和微生物的入侵。第一次层结事件导致形成一层扁平的周皮细胞。周皮发育始于尾部和四肢，然后扩展到躯干和面部，因此胚胎在胚胎第14天就被覆盖；因此，周皮发育的所有阶段都存在于单个E11.5胚胎中。周皮起着胚胎屏障的作用，在发育过程中防止上皮之间的病理性粘连。周皮形成的失败是几个出生缺陷的基础，这些缺陷的特征是皮肤在主要关节、唇裂和/或腭裂、手指和/或脚趾的融合以及生殖器畸形。虽然我们已经证明转录因子DeltaNp63Alpha(DNp63A)是周皮发育的关键调节因子，但控制这一根本重要的细胞层的调控网络的特征很差；这是理解表皮发育中的重大事件和解剖疾病病因的障碍。我们组建了一个具有发育生物学、基因调控和计算生物学专业知识的研究团队，目的是描绘驱动细胞从外胚层剥离到形成外胚层的调控相互作用。将致力于两个目标：1.以单细胞(Sc)分辨率预测驱动小鼠周皮形成的调控网络：我们将通过对E11.5野生型小鼠的皮肤进行scRNA-seq分析来定义发育中的周皮的转录组，在E11.5野生型小鼠的周皮发育的所有阶段都存在。聚类算法将被用来识别细胞的亚群体，并将分析它们的“标记基因”的表达，以将群体重建为组织背景。我们将通过计算来推断发育轨迹，以便我们研究发育过程中簇的关系。为了识别调控元件，我们将进行scATAC-seq分析，并推断转录因子及其靶基因的相互作用。我们将把开放的调控区域与他们的目标基因联系起来，并使用scATAC-seq数据通过转录因子结合基序的差异浓缩来推断潜在的上游调控因子。驱动周皮发育的调控网络的进化将通过计算进行分析。交付成果：我们将在单细胞分辨率下预测驱动周皮发育的调控网络，从而使我们能够制定关于DNp63A调控级联的假说。解剖转录因子DNp63A下游的分子级联：我们将使用目标1中产生的数据来扩展DNp63A调控网络。我们将对E11.5 TP63基因缺失小鼠的皮肤进行scRNA-seq和scATAC-seq分析，并进行计算分析，以调查预测的调控网络是如何受到干扰的。推断的调控相互作用将得到实验验证。DNp63A的直接转录靶点将通过对E11.5皮肤的芯片-qPCR分析来确定。随后，我们将芯片-qPCR分析扩展到网络监管的第二层。为了分析转录因子是作为激活因子还是作为抑制因子，我们将进行荧光素酶报告分析和Dead Cas9介导的CRISPR激活/抑制研究。成果：我们将在单细胞分辨率下验证驱动周皮发育的DNp63A调控网络。数据将提供对发育过程中细胞状态转变的机械性见解，并将对确定一系列先天性疾病的发病机制产生潜在影响。

How cells transition between different cellular states is a fundamental biological question. In this context, the simple ectoderm passes through a dynamic series of cell states to produce a self-replenishing, multi-layered epidermis that protects the organism from dehydration, mechanical trauma, and microbial invasion. The first stratification event results in the formation of a layer of flattened periderm cells. Periderm development commences on the tail and limbs before spreading over the torso and face so that the embryo is covered by embryonic day (E)14; all stages of periderm development are therefore present in individual E11.5 embryos.Periderm functions as an embryonic barrier that prevents pathological adhesion between epithelia during development. Failure of periderm formation underlies several birth defects that are characterised by webbing of the skin across the major joints, cleft lip and/or palate, fusion of the fingers and/or toes, and genital malformations.While we have demonstrated that the transcription factor DeltaNp63Alpha (DNp63A) is a key regulator of periderm development, the regulatory networks controlling this fundamentally important cell layer are poorly characterised; this is a barrier to understanding a major event in epidermal development and in dissecting disease pathogenesis.We have assembled a team of investigators with expertise in developmental biology, gene regulation, and computational biology with the aim of delineating the regulatory interactions that drive cells to delaminate from the ectoderm to form periderm. Two objectives will be addressed:1. Predicting the regulatory networks driving mouse periderm formation at single-cell (sc) resolution: We will define the transcriptome of the developing periderm by performing scRNA-seq analysis of skin dissected from E11.5 wild-type mice in which all stages of periderm development are present. Clustering algorithms will be used to identify sub-populations of cells and expression of their 'marker genes' will be analysed to reconstruct the populations into a tissue context. Developmental trajectories will be inferred computationally allowing us to study the relationships of the clusters during development.To identify regulatory elements, we will perform scATAC-seq analysis and infer the interactions of transcription factors and their target genes. We will link open regulatory regions to their target genes and use the scATAC-seq data to infer potential upstream regulators through differential enrichment of transcription factor binding motifs. Evolution of the regulatory networks driving periderm development will be analysed computationally.Deliverables: We will predict the regulatory networks driving periderm development at single-cell resolution allowing us to formulate hypotheses about the DNp63A regulatory cascade.2. Dissecting the molecular cascade downstream of the transcription factor DNp63A: We will use the data generated in Objective 1 to extend the DNp63A regulatory network. We will perform scRNA-seq and scATAC-seq analyses of skin dissected from E11.5 Tp63-null mice and perform computational analyses to investigate how the predicted regulatory networks are perturbed.The inferred regulatory interactions will be verified experimentally. Direct transcriptional targets of DNp63A will be confirmed by ChIP-qPCR analysis of E11.5 skin. Subsequently, we will extend the ChIP-qPCR analyses to the second layer of network regulation. To analyse whether the transcription factors act as activators or repressors, we will perform luciferase reporter assays and deadCas9-mediated CRISPR activation/inhibition studies.Deliverables: We will validate the DNp63A regulatory network driving periderm development at single-cell resolution.The data will provide mechanistic insights into cell state transitions during development and will have potential impact in determining the pathogenesis of a series of congenital disorders.