Molecular Mechanisms Underlying Cytoneme Formation by Sonic Hedgehog-Producing Cells

摘要 正常的生理发育依赖于通过分泌的形态原进行细胞间的交流。这些 强大的信号分子在短距离和远距离以时间和组织特有的方式发出信号 以确定细胞命运和组织模式。Sonic Hedgehog(SHH)，一种研究得很好的脊椎动物 形态原在包括神经管在内的各种发育过程中发挥着不可或缺的作用 图案化和肢体发育。该通路的缺陷会导致发育畸形，而 异常激活与发育性疾病和恶变有关。 分泌SHH的细胞延伸以肌动蛋白为基础的细小突起，将SHH运送到接收细胞。这些 特化的丝状伪足因其线状外观而被称为“细胞素”。细胞素是 建议在严密调节形态原浓度梯度方面发挥关键作用 发展。然而，人们对细胞素形成的分子机制知之甚少。 这在很大程度上是因为用传统的固定方法很难研究细微的结构。 为了能够在体外进行机制研究，我们的实验室开发了一种改进的电子显微镜固定剂(MEM- FIX)为共聚焦分析保留细胞质完整性的方案。使用MEM-FIX，以及一个 结合先进的成像和生化技术，我们的实验室发现 Shh可促进培养的小鼠成纤维细胞的细胞素启动。此外，我们还确定了一个 对SHH辅助受体和黏附蛋白的需求，细胞黏附相关，向下 受癌基因(CDON)和CDON兄弟(BOC)调控，以及调度(DISP) SHH介导的细胞线形成和稳定性中的部署受体。这些发现以及 确定SHH、DISP和BOC之间的相互作用，以及DISP和CDON指向 细胞素-启动信号发生在SHH结合到这些跨膜蛋白的下游。 然而，启动细胞线生长的特定相互作用和细胞内信号通路 都还没有确定。我提议的项目是专注于确定SHH激活的信号 发生是由于它与驱动SHH中细胞线粒起始的BOC和DISP的关联- 生产细胞。我的项目的第一个目标是阐明特定的细胞表面相互作用 DISP、BOC、CDON和SHH向细胞内细胞骨架调节因子发出信号。第二个方面 我的项目是阐明直接促进肌动蛋白核化和 对SHH反应的聚合反应。为了解决这些悬而未决的问题，我结合了有针对性的 基因和生化技术与先进的成像方法，有条不紊地询问 信号级联导致SHH介导的细胞素形成的诱导。我将在以下文件中确认调查结果 通过使用我们最近开发的方案，促进了对小鼠胚胎中细胞素的成像。

ABSTRACT Proper physiological development relies on cell-to-cell communication via secreted morphogens. These potent signaling molecules signal at both short and long range in temporal- and tissue-specific manners to determine cell fate and pattern tissues. Sonic Hedgehog (SHH), a well-studied vertebrate morphogen, plays an integral role in a variety of developmental processes including neural tube patterning and limb development. Defects in the pathway result in developmental malformations, while aberrant activation has been associated with developmental disease and malignant transformation. SHH-producing cells extend thin, actin-based projections to transport SHH to receiving cells. These specialized filopodia are termed “cytonemes” due to their thread-like appearance. Cytonemes are proposed to play a key role in tightly regulating morphogen concentration gradients during development. However, little is known about the molecular mechanisms driving cytoneme formation. This is mostly due to the difficulty to study the delicate structures using conventional fixation methods. To permit mechanistic studies in vitro, our lab developed a modified electron microscopy fixative (MEM- fix) protocol that preserves cytoneme integrity for confocal analysis. Using MEM-fix, along with a combination of advanced imaging and biochemical techniques, our lab has found that expression of SHH can promote cytoneme initiation in cultured murine fibroblasts. Furthermore, we identified a requirement for the SHH coreceptors and adhesion proteins, Cell adhesion-associated, Down- regulated by Oncogenes (CDON) and Brother of CDON (BOC), along with Dispatched (DISP) deployment receptor in SHH-mediated cytoneme formation and stability. These findings along with identification of interactions between SHH, DISP, and BOC as well as DISP and CDON point towards cytoneme-initiating signaling occurring downstream of SHH binding to these transmembrane proteins. However, the specific interactions and intracellular signaling pathways that initiate cytoneme outgrowth have yet to be identified. My proposed project is focused on determining the SHH-activated signals occurring in response to its association with BOC and DISP that drive cytoneme initiation in SHH- producing cells. The first goal of my project is to elucidate the specific cell surface interactions between DISP, BOC, CDON, and SHH that signal to intracellular cytoskeletal regulators. The second aspect of my project is to elucidate the regulatory molecules that directly promote actin nucleation and polymerization in response to SHH. To address these outstanding questions, I am combining targeted genetic and biochemical techniques with advanced imaging approaches to methodically interrogate the signal cascade resulting in SHH-mediated induction of cytoneme formation. I will confirm findings in vivo by using our recently developed protocol facilitating imaging of cytonemes in mouse embryos.