Overexpression of Calr Mutants Perturbs Developmental Hematopoiesis in Zebrafish Embryos

• Published in: Blood 2016-12, Vol.128 (22), p.4282-4282
• Main Authors: Lim, Ken-Hong, Chang, Yu-Cheng, Chiang, Yi-Hao, Lin, Huan-Chau, Chang, Chiao-Yi, Lin, Ching-Sung, Huang, Ling, Wang, Wei-Ting, Chen, Caleb Gon-Shen, Chou, Wen-Chien, Kuo, Yuan-Yeh
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V128.22.4282.4282

Introduction: Calreticulin (CALR) is a 46-kDa highly conserved, multicompartmental and multifunctional protein. CALR acts as a Ca2+ binding chaperone protein and participates in ensuring proper protein and glycoprotein folding in the endoplasmic reticulum. CALR mutations have been identified in about 30% of JAK2 and MPL unmutated essential thrombocythemia and primary myelofibrosis. Although the expression of CALR mutants resulted in pathogenic thrombocytosis in adult mice, whether CALR mutants may disrupt normal hematopoiesis during early development remains unknown. Here we aimed to evaluate the effects of mutant CALR during embryonic hematopoietic development using the in vivo zebrafish model. Methods: Full-length CALR wild-type,and CALR-del52 and CALR-ins5 mutants cDNAs were subcloned in the pCS2+ vector and a bicistronic pSYC-102 T2A vector, respectively. Capped CALR mRNAs from the above vectors were micro-injected into 1-2 cells stage wild-type AB strain, Tg(cd41:GFP) and Tg(fli1:EGFP) zebrafish embryos, respectively. cd41+ thrombocytes were counted at 3 and 5 days post fertilization (dpf), respectively. Gene expression of hematopoietic lineage-specific and cytokine and cytokine receptor genes were evaluated by quantitative reverse-transcription and real-time polymerase chain reaction (Q-PCR) from 1 to 3 dpf. Morpholino (MO) was used to knock down cytokine receptor genes mpl, epor and csf3r. Results: The expression of CALR proteins from the injection of 100 pg mRNA was confirmed by CALR N-terminal and mutant specific antibodies, respectively. Expression of both CALR-del52 and CALR-ins5 mutant mRNA significantly increased the numbers of hematopoietic stem and progenitor cells in the caudal hematopoietic tissue when compared with CALR-wt mRNA at 3 dpf. No obvious changes in the angiogenesis were visualized in CALR-wt and mutant CALR expressing embryos at 3 dpf in the fli1:EGFP line when compared with uninjected control. Mutant CALR-del52 significantly increased the number of cd41+ thrombocytes at 5 dpf (mean 162.5±4.1 per embryo) when compared to CALR-wt (mean 117.1±3.1 per embryo, P