The Roles of LPA2 during Erythrocyte and Megakaryocyte Differentiation in Zebrafish

• Main Authors: Shih-Hung Lin, 林士閎
• Other Authors: Hsinyu Lee
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/91946306718005941980

碩士 === 國立臺灣大學 === 生命科學系 === 102 === Lysophosphatidic acid (LPA) is a small lysophospholipid which regulates many cell behaviors, such as cell proliferation, migration, and survival. LPA binds to a family of G-protein-coupled receptor, LPA receptor 1-6, and activates its downstream pathways. Hematopoiesis is a developmental process that hematopoietic stem cells (HSCs) differentiate into all types of blood cells, including erythrocytes, lymphocytes, and megakaryocytes. In our previous study, we demonstrated that activation of LPA3 could enhance erythropoiesis processes. However, the roles of other LPA receptors in hematopoiesis remain unclear. Since erythrocyte and megakaryocyte share the common progenitor cell, we attempted to investigate the role of LPA receptors in megakaryopoiesis processes. To clarify the role of LPA2 in erythrocyte and megakaryocyte differentiation in zebrafish, we first injected anti-sense morpholino oligonucleotide of LPA2 into wild-type zebrafish embryos at one-cell-stage. O-dianisidine staining for hemoglobin and real-time PCR were used to determine the expression of erythrocyte and mRNA expression of erythropoietic markers, Hbae1 and GATA1, or megakaryocyte marker, CD41, respectively. Moreover, we also injected LPA2 morpholino into embryos of Tg(eGFP:CD41) transgenic line. Our results demonstrated that knockdown of LPA2 enhanced the staining at CHT and mRNA levels of Hbea1, GATA1, and CD41. In addition, the number of megakaryocyte in Tg(eGFP:CD41) zebrafish embryos was also increased. Furthermore, we performed pharmacological experiment to confirm these results by incubating embryos with the LPA2 agonist GRI977143 and RP-239. Results from staining of o-dianisidine and real-time PCR of Hbea1, and CD41 showed that both protein and mRNA levels were suppressed by LPA2 agonists. In conclusion, our results suggested that activation of LPA2 inhibit differentiation of erythrocyte and megakaryocyte in zebrafish.