Megakaryocytes and platelets are essential for hemostasis, immune regulation and hematopoietic niche maintenance, while also promoting thrombosis. Comprehensive characterization of late-stage megakaryocyte maturation in mammals using single-cell RNA-sequencing (scRNA-Seq) remains challenging due to the large size of megakaryocytes and the low RNA content of platelets. Although zebrafish thrombocytes are functionally analogous and retain high transcriptional activity at maturity, their transcriptional similarities to mammals have not been systematically defined. To address this, we generated a single-cell transcriptomic atlas spanning the full developmental trajectory of zebrafish thrombocytes, demonstrating that lineage progression is continuous and functionally coordinated. Cross-species comparison with human and mouse datasets defined conserved differentiation stages and gene modules of the megakaryocyte–platelet lineage, indicating evolutionary conservation of lineage development at single-cell resolution. Moreover, we identified a gene set (M3pan-PLT) that marks a platelet-like transcriptional state in zebrafish thrombocytes, supporting their transcriptomic analogy to mammalian platelets. Finally, bulk transcriptomic analyses using these conserved gene signatures demonstrated their ability to capture physiological megakaryocyte states, as well as disease-associated transcriptional alterations in acute megakaryoblastic leukemia. Together, our study defines a conserved and continuous developmental framework of megakaryocytes at single-cell resolution and establishes zebrafish thrombocytes as a transcriptionally conserved model for dissecting late-stage megakaryocyte-platelet biology.
