Abstract
Definitive haematopoiesis occurs during the lifetime of an individual, which continuously replenishes all blood and immune cells. During embryonic development, haematopoietic stem cell (HSC) formation is tightly controlled by growth factors, signalling molecules and transcription factors. But little is known about roles of the cytochrome P450 (CYP) 2 family member in the haematopoiesis. Here we report characterization and functional studies of Cyp2aa9, a novel zebrafish Cyp2 family member. And demonstrate that the cyp2aa9 is required for the HSC formation and homeostasis. Knockdown of cyp2aa9 by antisense morpholino oligos resulted the definitive HSC development is defective and the Wnt/β-catenin activity becomes reduced. The impaired HSC formation caused by cyp2aa9 morpholino can be rescued by administration of PGE2 through the cAMP/PKA pathway. Furthermore, the in vivo PGE2 level decreases in the cyp2aa9 morphants, and none of the PGE2 precursors is able to rescue phenotypes in the Cyp2aa9-deficient embryos. Taken together, these data indicate that Cyp2aa9 is functional in the step of PGE2 synthesis from PGH2, thus promoting Wnt activation and definitive HSC development.
Highlights
Definitive haematopoiesis occurs during the lifetime of an individual, which continuously replenishes all blood and immune cells
Cyp2aa9MO led to reduced number of mitotic active cells at 36 hpf in the AGM as shown by pH3 antibody staining, which could be rescued by prostaglandin E2 (PGE2). (e) Quantification of pH3-positive cells in the AGM (n = 6, mean ±SD). (f–i) cyp2aa9MO led to increases in the number of apoptotic cells at 36 hpf in the AGM as shown by TUNEL assay, which could be rescued by PGE2. (j) Quantification of the number of apoptotic cells in the AGM (n = 6, mean ±SD). *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test
Our work found reduced Wnt activity and PGE2 synthesis in the cyp2aa[9] morphants, suggesting a model that Cyp2aa[9] acts upstream of PGE2 signalling to mediate the full effects of Wnt activation in haematopoietic stem cell (HSC) development
Summary
Definitive haematopoiesis occurs during the lifetime of an individual, which continuously replenishes all blood and immune cells. The in vivo PGE2 level decreases in the cyp2aa[9] morphants, and none of the PGE2 precursors is able to rescue phenotypes in the Cyp2aa9-deficient embryos Taken together, these data indicate that Cyp2aa[9] is functional in the step of PGE2 synthesis from PGH2, promoting Wnt activation and definitive HSC development. HSCs first emerge from the VDA at 28 hours post fertilization (hpf)[6,7] These HSCs migrate to the caudal haematopoietic tissue (CHT) from two days post fertilization (dpf) on[8,9]. Following the phospholipase-mediated release from phospholipids of the cell membrane, AA is sequentially converted to prostaglandin precursors G2 (PGG2) and H2 (PGH2) by cyclooxygenases Cox1/214,15 These precursors are used to synthesize numerous prostanoids, including prostaglandin E2 (PGE2). Roles of CYP2s in the embryonic development remain largely unknown
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