Platelets are important players in primary hemostasis. They are produced in copious numbers as the megakaryocyte maturation. Thrombocytes also mature and undergo changes in transcriptional machinery similar to megakaryocytes. Thus zebrafish thrombocyte maturation could be easily studied along with the power of genetic manipulations available in zebrafish. The information gained from this study should shed light on megakaryocyte maturation and enhance our understanding platelet production and thus, this proposal is important and has clinical value. In our preliminary studies, we developed a transgenic line that has both young and mature thrombocytes that are differentially labeled by RFP and GFP. We are thus poised with identifying the genes that are differentially expressed in these two populations of thrombocytes by RNA sequencing. Knockdown of these differentially expressed genes should determine the genes whose loss of function results in a change in the ratio between young and mature thrombocytes. Thus the major hypothesis in this investigation is that there are unidentified factors that control the maturation of thrombocytes and identifying these factors may enhance our understanding of platelet biogenesis. Recently, we have developed a novel piggyback knockdown method for knockdown of genes with unprecedented ease and efficiency in adult zebrafish. In our preliminary results using this method, knockdowns were performed on 3000 genes in less than six month period giving hope that genome-wide knockdowns for thrombocyte specific gene are an achievable goal in this proposal. However, there is still room for improving the efficiency of this method to meet the demands of this proposal to understand the mechanism of thrombocyte maturation. To verify the efficacy of the method and to identify novel genes involved in thrombocyte maturation two specific aims are proposed.
Aim 1 will be to optimize conditions for the piggyback knockdown method to efficiently knockdown genes and then test the efficiency of knockdowns in multiple tissues utilizing genes that are expressed in a tissue-specific manner. The goal of Aim 2 is to comprehensively perform genome-wide knockdowns of thrombocyte specific genes to understand thrombocyte maturation. The results from these aims will identify hitherto unidentified factor(s) that regulate thrombocyte development. Such identification may lead to novel drugs to treat disorders such as thrombocytopenia. Furthermore, the technology developed here will have a large utility to study other physiological pathway.
Megakaryocytes increase in their size and produce platelets by a process known as maturation. The mechanism of megakaryocyte maturation is poorly understood. Using zebrafish thrombocyte maturation as a model, we propose to identify genes involved in this maturation process by developing a cost- effective method.
