Genetics of Thrombopoiesis - Project Summary/Abstract Despite significant progress in understanding factors involved in megakaryocyte development and differentiation, much remains unknown about the mechanisms through which these factors affect megakaryocytes, particularly during maturation. This represents a critical gap in megakaryocyte biology that requires further investigation. Our previous research has shown that zebrafish thrombocytes express approximately 50% of the genes found in megakaryocytes, although they differ in aspects such as polyploidy. Therefore, zebrafish thrombocyte maturation can serve as a valuable model for studying conserved genes and their roles in megakaryocyte development. In this renewal application, building on the progress from our R15 grant, we will investigate the downstream factors of 10 newly identified genes that affect either young or mature thrombocyte production. Additionally, we have identified 453 protein-coding genes expressed in mature thrombocytes that are also found in megakaryocytes. Assuming around 300 genes are expressed downstream of the 10 novel factors, this brings the total to 753 genes to be examined for their involvement in thrombocyte development. The core hypothesis of this proposal is that common downstream factors exist for the novel genes involved in young thrombocyte production, as well as a distinct pathway for the production of mature thrombocytes. To test this hypothesis, we propose the following three specific aims: Aim 1: Generate CRISPR/Cas9-based thrombocyte-specific knockouts for the 10 novel genes and characterize the resulting zebrafish for thrombocyte types. Aim 2: Perform single-cell RNA sequencing on the young and mature thrombocyte populations from the knockout fish generated in aim 1, where these populations are labeled with RFP and GFP, respectively, identify differentially expressed downstream genes for the 10 novel factors, compared to control fish and search for common genes expressed across these mutants to gain further insight into shared regulatory pathways. Aim 3: Conduct a comprehensive knockdown of the downstream genes identified in aim 2, along with mature thrombocyte-specific genes that are also expressed in megakaryocytes, using the high-throughput piggyback knockdown method. The results of this proposal will lead to the identification and confirmation of common downstream factors for the 10 newly identified genes from our previous R15 grant and uncover novel factors involved in thrombocyte maturation. These findings will serve as a foundation for future grant proposals. Additionally, this project will provide training opportunities for two graduate students, who will also mentor two undergraduate students. We will encourage undergraduate students to contribute to the publication of research papers as part of their training.
