Vertebrate cytonemes in cell signaling and skeletal morphogenesis

描述(申请人提供)：人类四肢骨骼发育异常是人类最常见的先天缺陷之一，但对这些先天畸形背后的细胞变化知之甚少。在脊椎动物模式生物中，许多关于肢体骨骼发育的研究都探索了相对较小的一组模式基因的功能。然而，关于这些基因的活性如何控制导致正确数量、形状和大小的软骨元素形成的动态细胞事件，我们的知识中有许多空白。深入了解这一过程的一个主要限制在于，在历史上，软骨形态发生在固定和染色的标本中以静态和罕见的间隔进行检查。为了帮助我们弥合这些知识上的差距，我们开发了一种独特的软骨形成的活细胞成像方法，以动态显示肢体间充质前体细胞形成间充质凝聚，然后分化为软骨。这种方法已经确定了形成软骨模板所必需的新的细胞事件，该模板由在人类肢体和软骨发育不良综合征中发现的突变的关键分子调节因子控制。我们还在开发以单细胞分辨率成像活的完整脊椎动物胚胎(小鼠和小鸡)的新策略方面取得了进展，使我们能够可视化软骨发育过程中细胞和组织动力学的全面相互作用，这是以前无法解决的。利用这一成像技术，我们已经确定了一种以前未知的细胞间通讯模式，这种模式通过新的细胞素样细胞质延伸发生，我们称之为脊椎动物细胞素(v-Cytonemes)，存在于肢体间充质中，似乎指导软骨的形成。这种胞质延伸延伸了许多细胞直径，只能在活的组织中看到，而不能在固定的组织中看到，因此以前从未在体内的间充质祖细胞上观察到过。我们的初步发现表明，一个细胞质延伸网络连接了脊椎动物肢芽中的信号中心和交通信号组件，以指导软骨模板的形成。在Aim1中，我们将在体内确定发育中的肢芽中v-细胞素的景观和方向。这些研究还将得到关键配体Shh和FGFs(S)对v-细胞素功能的直接测试，它们构成肢体软骨模板。在AIM2中，我们将测试Shh通过存在于间充质细胞上的v-细胞素移动作为远程细胞信号机制的假设。在Aim3中，我们将系统地描述Shh信号通路的组件，这些组件定位并使用v-Cytonemes作为长距离信号传递的机制。这项提案中描述的成像技术和研究将打开进入一个以前未被探索的领域的门户：在单个细胞水平上可视化的细胞到细胞信号的动力学，导致软骨模板的形成。

DESCRIPTION (provided by applicant): Abnormalities in human limb skeletal development are one of the most common human birth defects, yet little is known as to the cellular changes underlying these congenital malformations. Many of the studies on limb skeletal development in vertebrate model organisms have explored the function of a relatively small set of patterning genes. However, there are many gaps in our knowledge as to how the activity of these genes control the dynamic cellular events that lead to the formation of cartilage elements of the correct number, shape, and size. A major limitation to a deeper understanding of this process resides in the fact that cartilage morphogenesis has historically been examined in fixed and stained specimens at static and infrequent intervals. To help close these gaps in our knowledge, we have developed a unique live cell imaging approach for cartilage formation to dynamically visualize limb mesenchymal progenitor cells as they form mesenchymal condensations that subsequently differentiate into cartilage. This approach has identified novel cellular events that are critically required for the formation of a cartilage template, controlled by key molecular regulators found mutated in human limb and chondrodysplasia syndromes. We have also made advances in the development of novel strategies to image living intact vertebrate embryos (mouse and chick) at a single cells resolution, allowing us to visualize the full interplay of cell and tissue dynamics during cartilage development that has not been previously possible to resolve. Utilizing this imaging technology, we have identified a previously unrecognized mode of cell-to-cell communication that occurs via novel cytoneme-like cytoplasmic extensions that we have termed vertebrate cytonemes (v-cytonemes) present on limb mesenchyme, which appear to direct cartilage formation. Such cytoplasmic extensions, extending many cell diameters in length, can only be visualized in living but not fixed tissue and have thereby never been previously observed on mesenchymal progenitor cells in vivo. Our preliminary findings suggest that a network of cytoplasmic extensions connect signaling centers and traffic signaling components in the vertebrate limb bud to direct the formation of a cartilage template. In Aim1 we wil determine the landscape and orientation of v- cytonemes within the developing limb bud in-vivo. These studies will also be complemented by direct tests of v-cytoneme function by key ligands, Shh and Fgf(s), which pattern the limb cartilage template. In Aim2 we wil test the hypothesis that Shh movement through v-cytonemes present on mesenchymal cells acts as a mechanism for long-range cell signaling. In Aim3 we will systematically delineate the components of the Shh signaling pathway that localize and employ v-cytonemes as a mechanism for long range signaling. The imaging technology and studies described in this proposal wil open a portal into a previously unexplored area: the dynamics of cell-to-cell signaling, visualized at a single cell level, leading to the formation of a cartilage template.