Piggyback knockdown screening of unique genes of zebrafish young thrombocytes identifies eight novel genes in thrombopoiesis

Platelet production, or thrombopoiesis, is a critical process involving the differentiation of hematopoietic stem cells into megakaryocytes, which release platelets into circulation. This study employed a comprehensive screening approach through a piggyback knockdown strategy targeting 394 protein-encoding genes expressed explicitly in young thrombocytes. This approach led us to identify eight candidate genes associated with thrombopoiesis, including spi1b, a transcription factor that potentially regulates thrombocyte development. The sequencing of spi1b mutant progeny harboring a termination codon after Arg254 within the conserved ETS transcription factor domain confirmed the lethality of homozygous mutations, highlighting the essential role of Spi1b in embryonic development. Comparative analysis revealed homology between zebrafish Spi1b and human SPI1, suggesting evolutionary conservation of thrombopoiesis regulatory mechanisms. Additionally, analysis of spi1b knockdown zebrafish and the mutant demonstrated increased bleeding, further emphasizing the importance of spi1b in maintaining hemostasis. Our study provides novel insights into the regulatory networks governing thrombopoiesis and identifies Spi1b as a critical regulator of young thrombocyte development in zebrafish. Further investigations into the functional roles of identified genes in thrombocyte biology may elucidate mechanisms underlying thrombopoiesis and inform therapeutic strategies for bleeding disorders.