Vertebrate cytonemes in cell signaling and skeletal morphogenesis

描述（由申请人提供）：人类肢体骨骼发育异常是最常见的人类出生缺陷之一，但对这些先天性畸形背后的细胞变化知之甚少。许多关于脊椎动物模式生物肢体骨骼发育的研究已经探索了一组相对较小的模式基因的功能。然而，对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，我们的知识还存在许多空白。深入了解这一过程的一个主要限制在于，软骨形态发生在历史上一直是在固定和染色的标本中以静态和不频繁的间隔进行检查的。为了帮助我们弥补这些知识上的空白，我们开发了一种独特的活细胞成像方法，用于软骨形成，动态地观察肢体间充质祖细胞形成间充质凝聚，随后分化成软骨。这种方法已经确定了新的细胞事件，这些事件对软骨模板的形成至关重要，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。我们还在开发新的策略方面取得了进展，以单细胞分辨率成像活的完整脊椎动物胚胎（小鼠和小鸡），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部相互作用，这在以前是不可能解决的。利用这种成像技术，我们发现了一种以前未被识别的细胞间通讯模式，这种模式通过一种新的细胞素样细胞质延伸发生，我们将其称为脊椎动物细胞素（v-细胞素），存在于肢体间质上，它似乎指导软骨的形成。这种细胞质延伸，延长了许多细胞直径的长度，只能在活的而不是固定的组织中看到，因此以前从未在体内的间充质祖细胞中观察到过。我们的初步研究结果表明，细胞质扩展网络连接信号中心和交通信号组件在脊椎动物肢体芽指导软骨模板的形成。在目的1中，我们将在体内确定发育中的肢体芽内v细胞素的形态和方向。这些研究还将通过关键配体Shh和Fgf(s)的v细胞素功能的直接测试来补充，这些配体是肢体软骨模板的图案。在目的2中，我们将验证Shh通过间充质细胞上的v细胞素运动作为远程细胞信号传导机制的假设。在aims中，我们将系统地描述Shh信号通路的组成部分，这些组成部分定位并利用v细胞素作为远程信号传导的机制。本提案中描述的成像技术和研究将为以前未开发的领域打开一个门户：细胞间信号传导的动力学，在单个细胞水平上可视化，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.