Congenital brain disorders and many central nervous system cancers are in part caused by the misregulation of neural stem cells during embryonic and fetal development. In all vertebrates radial glial cells serve as the neural stem cell during these early periods of development. The brain is built through the proper regulation of radial glia division rates and differentiation of their progeny and alterations to radial glial development is suggested to be one of the primary modules of brain evolution. Understanding what the mechanisms are that regulate radial glial development will have broad implications to nervous system construction, disease states, tumorogenesis, and potentially the ability to unlock regenerative capabilities to treat CNS trauma and degenerative diseases. The fundamental process underling the ability of radial glial cells to build the nervous system and respond to disease or trauma is cell division, yet much still remains to be learned about the molecular mechanism that regulate radial glial division and differentiation. We study the developing zebrafish spinal cord as a simple model of neurogenesis, in which the role of radial glial cells and their progeny can be investigated at the tissue, cellular, and genetic levels. Previously during an insertional mutagenesis screen we identified a class of genes required for proper radial glial development in the spinal cord. With my first NIH R15 award, we investigated the role of kif11 as an intrinsic regulator of radial glial division during neural tube development. We showed Kif11 was necessary for radial glial progression through mitosis, which revealed reductions in specific neuronal and glial lineages dependent upon radial glia for their derivation. For this proposed competitive renewal we will focus on wnt5b, which causes increases to the number of mitotic radial glia like kif11 mutants but functions as a potential external regulator of radial glial cell proliferation. I present preliminary data that lends support for the central hypothesis that Wnt5b functions as a negative regulator of canonical Wnt/?-Catenin signaling, which results in alterations to the proliferation rate of radial glial cells that further impacts neurogenesis during development of the spinal cord. We will use a variety of combinatorial loss and gain of function approaches of both Wnt5b and Wnt/?-Catenin signaling to (1) characterize how the cell cycle in radial glia is influenced by wnt5b, (2) test which cells in the neural stem cll niche require wnt5b function, and (3) determine whether Wnt5b functions through Wnt/?-Catenin signaling to regulate radial glial division and differentiation. This investigation is particularly innovative for several reasons. We will take full advantage of the power of zebrafish genetics to manipulate the function of multiple genes with temporal control, visualize specific stem and post-mitotic cell populations in the live developing embryo, and detect changes in pathway response at the cellular level. We have uniquely adapted the use of Geographic Information Systems to apply spatial statistics to visual data sets for the highest level of objective quantification. Lastly, analysis of Wnt5b regulation of radial glial cell division will b guided by our mathematical modeling to shed light on the parameters of cell cycle control most related to changes in Wnt5b function. The comprehensive nature of this work ensures its broad impact upon the field of developmental biology. From the study of live radial glial cell behaviors during development to the molecular dissection of Wnt signaling pathways in the regulation of radial glial cell cycle control, there promises to be far reaching advances to our understanding of neural stem cells in both development and disease. One of the most important impacts will be to the education of students across the hierarchy of academia, which includes the direct participation of graduate students, the foundational contributions of the undergraduate research scientists in my lab, and the hundreds of primary and secondary education students that directly benefit yearly from my Student Scientists outreach program. Ultimately it is the fully committed integration of research and education, which will yield productive contributions to our knowledge of stem cell development in ways that impact a wide range of human health related needs while inspiring and training the next generation of problem solving scientists and medical professionals.
The proposed research investigates how neural stem cells are precisely regulated to maintain a balance between cell proliferation and differentiation. This work promises to provide new insight into how errors in neural stem cell regulation might lead to congenital disorders and brain tumors, but also identify the mechanisms that enable the unique ability of lower vertebrates to regenerate neural tissues. We take full advantage of the accessibility, cellular clarity, and powerful genetic tools of the zebrafish model system to determine the role of the signaling protein Wnt5b on cell proliferation and neural differentiation during embryonic spinal cord development.
