Abnormalities in 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 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 made significant advances in the development of novel strategies to image living vertebrate embryos (mouse and chick) at subcellular resolution, allowing us to visualize the full interplay of cell and tissue dynamics during limb 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 cytoplasmic extensions that we have termed ?specialized filopodia? present on limb mesenchyme 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. Strikingly, these cytoplasmic extensions create a dense and dynamic communication network in the chick limb bud that can be used as a ?highway? for the directed distribution of key signaling molecules such as Sonic Hedgehog (Shh). We hypothesize that long-range signaling via specialized filopodia constitutes a previously underappreciated mechanism for shaping and patterning limb skeletal development and is likely to reflect a more wide-spread mechanism for delivery of key signaling molecules in space and time.
In Aim1 we will develop and employ state-of-the-art optogenetic molecular motors to manipulate filopodia dynamics in vivo for the first time allowing us to change the orientation, lengths, and dynamics of these cellular extensions to address their functional roles in signaling.
In Aim2 we will develop the first tissue specific mouse transgenes to specifically alter the length and characteristics of specialized filopodia that will enable us to systematically delineate and extend the role of specialized filopodia in multiple tissue types that rely on the precise dissemination of signaling molecules.
In Aim3 we will systematically delineate the cellular mechanisms that are utilized for the direct delivery of critical morphogens, such as Shh, by filopodial extensions underlying control of digit number and patterning. Together, the imaging technology and studies described in this proposal will open a portal into a previously unexplored area: the dynamics of cell-to-cell signaling, visualized at subcellular resolution, leading to the precise delivery and reception of signaling molecules vital for mammalian development.
Abnormalities of the human limb are one of the most common human birth defects, yet little is known as to the cellular changes underlying these congenital malformations. The research proposed in this application will significantly advance the understanding of the underlying causes for these birth defects and help to lay the foundation for developing cellular and pharmacological based approaches for the treatment of congenital limb malformations.
| Fujii, Kotaro; Shi, Zhen; Zhulyn, Olena et al. (2017) Pervasive translational regulation of the cell signalling circuitry underlies mammalian development. Nat Commun 8:14443 |
