The Roles of Lysophosphatidic Acid Receptor 3 in Hematopoiesis of Zebrafish

• Main Authors: Ya-Chi Chang, 張雅琪
• Other Authors: 李心予
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/q86u77

碩士 === 國立臺灣大學 === 生命科學系 === 105 === Hematopoiesis is a dynamic process by which hematopoietic stem cells (HSCs) differentiate into the specific blood cell lineages. Lysophosphatidic acid (LPA) receptors have been shown to be involved in the regulation of hematopoiesis in HSCs and bone marrow microenvironment. In our previous studies, we demonstrated that activation of LPA receptor 3 (LPA3) promotes erythropoietin(EPO)-dependent erythropoiesis in HSCs and zebrafish. Moreover, knockdown of Lpa3 expression by using morpholino (MO) caused erythropoietic defects in zebrafish embryos. However, the pharmacological activation and chemical-dependent gene-depletion of LPA3 may have redundant effects during the development of zebrafish. Therefore, we aim to generate Lpa3 knockout zebrafish to clarify the roles of Lpa3 in hematopoiesis. In this study, we established LPA3 knockout (Lpa3-/-) zebrafish by deleting 2 base pairs in exon 1 using transcription activator-like effector nucleases (TALEN). As expected, the expressions of early hematopoietic markers are delayed in Lpa3-/- embryos within 3 days post fertilization (dpf) by real-time PCR, implying that Lpa3 is involved in the embryonic hematopoiesis in zebrafish. However, 5 dpf Lpa3-/- larvae showed no deficiency in the amount of hemoglobin and locomotor activities by histochemistry staining and high-throughput behavioral studies, respectively. Interestingly, adult Lpa3-/- zebrafish showed smaller whole kidney marrow (WKM) in histological analysis compared to the wild type. Flow cytometry analysis also showed the decreased population of precursors and lymphocytes in adult Lpa3-/- zebrafish, suggesting that Lpa3 may involve in the lymphopoiesis of zebrafish. Taken together, these findings reveal critical roles of Lpa3 in the embryonic development and adult hematopoiesis in zebrafish.