Mechanisms of repressive interactions between developmental gene regulatory networks

细胞在发育过程中通过逐渐出现不同的特征而获得其独特的身份 转录程序。这些转录程序可以被视为相互作用的动态网络 称为基因调控网络（GRN）的基因。 GRN 因其在以下方面的积极作用而得到了深入研究： 激活赋予细胞特殊特性的基因表达；然而，现在很明显 这些网络的一个同样重要的功能是排除其他潜在的替代性转录 程序。这种抑制性相互作用在决定细胞 无脊椎动物和哺乳动物等多种生物体的身份。它们在以下背景下也很重要 再生医学。通过谱系特异性转录因子 (TF) 对体细胞进行直接重编程 伴随着预先存在的转录程序的全面沉默。尽管发挥着举足轻重的作用 转录网络之间的抑制性相互作用在胚胎细胞命运规范和 体细胞重编程的潜在机制尚不清楚。 我们将利用海胆来解决这个重要问题，海胆是生物多样性研究的一个著名实验模型。 发育机制和 GRN 生物学分析。最具特征的海胆 GRN 之一 是构成骨骼的细胞发育的基础。该网络的一个关键组成部分是 Alx1，它是一个谱系- 特定的 TF 为许多支持骨骼发生的基因提供直接、积极的输入。与其并行的 Alx1 发挥积极作用，抑制潜在的替代转录程序，这些程序通常仅限于 周围的非成骨中胚层 (NSM) 细胞。成骨细胞中 Alx1 功能的扰动结果 NSM GRN 在这些细胞中异位部署，并导致它们采用 NSM 命运。的镇压 因此，Alx1 的 NSM GRN 提供了一个绝佳的机会来揭示转录的机制。 网络之间相互作用，从而确保了独特的细胞身份的出现。 为了剖析这种 GRN 相互作用的机制，我将首先定义关键的空间和 Alx1 抑制 NSM GRN 的时间方面，使用定量方法和空间基因表达 分析来表征干扰 Alx1 功能后发生的基因表达变化（目标 1）。接下来，我 将探索 Alx1 通过使用荧光报告构建体直接抑制 NSM 基因的假设 转基因来剖析这些基因的顺式调控元件（目标 2）。最后，我将检验以下假设： Alx1 控制细胞对 Notch/Delta 信号的自主无反应，这是驱动 NSM 的途径 正常开发期间的规范（目标 3）。这些研究将揭示其机制 Alx1 抑制 NSM GRN。更广泛地说，它们将导致更好地理解之间的相互作用 调节细胞身份的转录网络。

Cells acquire their unique identities during development through the progressive emergence of distinct transcriptional programs. These transcriptional programs can be viewed as dynamic networks of interacting genes known as gene regulatory networks (GRNs). GRNs have been well studied for their positive role in activating the expression of genes that endow cells with their specialized properties; however, it is now apparent that an equally important function of these networks is to exclude other, potentially alternative, transcriptional programs. Repressive interactions of this kind play a widespread, fundamental role in determining cellular identities in organisms as diverse as invertebrates and mammals. They are also important in the context of regenerative medicine. The direct reprogramming of somatic cells by lineage-specific transcription factors (TFs) is accompanied by the comprehensive silencing of pre-existing transcriptional programs. Despite the pivotal role that repressive interactions between transcriptional networks play in both embryonic cell fate specification and somatic cell reprogramming, the underlying mechanisms are poorly understood. We will address this important problem using the sea urchin, a prominent experimental model for the analysis of developmental mechanisms and for GRN biology. One of the best characterized sea urchin GRNs underlies the development of cells that form the skeleton. A key component of this network is Alx1, a lineage- specific TF that provides direct, positive inputs into many genes that support skeletogenesis. In parallel with its positive role, Alx1 represses potential, alternative transcriptional programs that are ordinarily restricted to surrounding non-skeletogenic mesoderm (NSM) cells. Perturbation of Alx1 function in skeletogenic cells results in the ectopic deployment of NSM GRNs in these cells and causes them to adopt NSM fates. The repression of NSM GRNs by Alx1 thus provides an outstanding opportunity to uncover mechanisms by which transcriptional networks interact with one another, thereby ensuring the emergence of unique cellular identities. To dissect the mechanisms underlying this GRN interaction, I will begin by defining key spatial and temporal aspects of NSM GRN repression by Alx1, using both quantitative methods and spatial gene expression analysis to characterize changes in gene expression that occur after perturbing Alx1 function (Aim 1). Next, I will explore the hypothesis that Alx1 directly represses NSM genes by using fluorescent reporter constructs and transgenesis to dissect cis-regulatory elements of these genes (Aim 2). Finally, I will test the hypothesis that Alx1 controls a cell-autonomous unresponsiveness to Notch/Delta signaling, a pathway that drives NSM specification during normal development (Aim 3). These studies will shed light on the mechanisms by which Alx1 represses NSM GRNs. More broadly, they will lead to a better understanding of interactions between transcriptional networks that regulate cell identity.