A key difference between a stem cell and a tumor cell is the ability to regulate proliferation. In most cases, however, it is not known how proliferation is regulated in stem cell populations in vivo. Progenitor cells are stem-like cells defined by their ability to remain undifferentiated until cued to accept a terminal fate. During the early stages of vertebrate embryogenesis, a population of mesodermal progenitor cells resides at the most posterior end of the body in a region called the tailbud. Cells progressively leave the progenitor population of the tailbud and differentiate, producing blocks of mesoderm called somites that will become the muscle and vertebrae of the adult body. How proliferation in the mesodermal progenitors is controlled and coordinated with differentiation is poorly understood. I propose to investigate regulation of proliferation in the mesodermal progenitors and their progeny by using the specific advantages of the zebrafish system including ease of making transgenic lines, optical clarity of the embryos, and the ability to transplant transgenic cells into nontransgenic embryos. This proposal is aimed (1) to determine if the mesodermal progenitors and their descendants are dividing or quiescent in living embryos;(2) to test the hypothesis that Fibroblast growth factor (Fgf) and/or Wnt signaling pathways regulate proliferation in mesodermal progenitors;and (3) to determine how proliferation during somite formation affects somite number, somite size, and the differentiation and morphogenesis of mesodermal cells. Using a modified version of a recently developed technique, Fluorescent Ubiquitin Cell Cycle Indicators (Fucci), I have created a new transgenic zebrafish reporter line, and propose to use the new line to identify when the mesodermal progenitors and their progeny divide. I will also determine where in the cell cycle non-dividing cells are held. To identify if the Fgf or Wnt signaling pathways regulate the rate of proliferation in the mesodermal progenitors, I will block each pathway using a heat shock-inducible, cell autonomous pathway inhibitor followed by analysis of proliferation in transplanted cells using Fucci. Finally, to determine how proliferation affects somite formation, I will increase the rate of proliferation during somite formation with a new zebrafish transgenic line expressing a heat-shock inducible, cell cycle checkpoint regulator. I will quantify the number and size of somites formed in this new transgenic line, and analyze cell fates in transplanted cells by gene expression analysis. These studies are designed to take advantage of the zebrafish system to identify mechanisms regulating growth control throughout the highly conserved process of body formation and elucidate global mechanisms of growth control in a stem cell-like progenitor population.
The research I have proposed has a high degree of relevance to both stem cell biology and cancer biology. A clear understanding of how proliferation changes and what factors regulate proliferation as cells differentiate in vivo will provide essential knowledge as to how vertebrate stem cells function. Because unregulated proliferation in stem cell populations results in tumor formation, these results will also have important implications for cancer biology.
|Bouldin, Cortney M; Kimelman, David (2014) Cdc25 and the importance of G2 control: insights from developmental biology. Cell Cycle 13:2165-71|
|Bouldin, Cortney M; Snelson, Corey D; Farr 3rd, Gist H et al. (2014) Restricted expression of cdc25a in the tailbud is essential for formation of the zebrafish posterior body. Genes Dev 28:384-95|