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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. CALRmutations have been identified in about 30% of JAK2and MPLunmutated essential thrombocythemia and primary myelofibrosis. Although the expression of CALRmutants resulted in pathogenic thrombocytosis in adult mice, whether CALRmutants 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 vivozebrafish model.

Introduction: Essential thrombocythemia (ET) is a classic BCL-ABL1-negative chronic myeloproliferative neoplasm (MPN), and is associated with an increased risk of hemorrhagic and thrombotic complications and leukemic transformation. Recently, two research groups discovered a high frequency of somatic calreticulin (CALR) mutations in patients with JAK2V617F/MPL-unmutated ET and primary myelofibrosis. The pattern of most CALRmutations in MPNs is indels in exon 9 causing one base pair reading frameshift. CALRmutations have been found to have important diagnostic and prognostic significances in patients with ET. High-resolution melting analysis (HRMA) is a well-established method for the detection of or screening for mutations. The aims of this study were to screen for CALRmutations and to determine its clinical correlation in a cohort of adult Taiwanese patients with ET, and to test for the feasibility of HRMA as a screening method for the detection of CALRexon 9 mutations.

Loss-of-function mutations in Ten-Eleven-Translocation 2 (TET2) gene have been identified in various human myeloid and lymphoid malignancies. Recently, the TETgene family (TET1, TET2, and TET3) was found to function as DNA methylcytosine dioxygenase that is able to oxidize 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). In Tet2-deficient mouse models, Tet2has been shown to play an important role in regulating self-renewal and differentiation of hematopoietic stem cells. These Tet2-deficient mice would gradually develop a chronic myeloid neoplasm resembling human chronic myelomonocytic leukemia suggesting that TET2may function as a tumor suppressor. In the present study, we investigated the role of tet2in zebrafish early hematopoiesis. During zebrafish early development, the expression of tet1, tet2, and tet3by qRT-PCR can be detected mainly after the segmentation stage (26-somite), with fluctuated expression levels thereafter. Whole-mount in situhybridization revealed that tet2expression was strong over aorta-gonad-mesonephros region at 48 hours post-fertilization (hpf). Morpholino oligonucleotide (MO) knock-down of tet2increased the expression of tet1, tet3, dnmt3aa, gata-1, alpha-Hband fli1a(48 hpf) as well as rag2and lck(4 days post-fertilization), and the expression of spi1band mpodecreased (48 hpf). The expression of primitive hematopoietic stem cell markers scland lmo2, as well as dnmt3ab, beta-Hb, l-plastin, and rag1were unaffected. The levels of 5-mC and 5-hmC measured by ELISA were also decreased after MO knock-down of tet2. The number of gata-1expressing red blood cells increased after tet2MO knock-down as evaluated by flow-cytometry indicating that tet2 deficiency increased erythropoiesis. These preliminary results suggest that tet2might play a role in the epigenetic regulation of zebrafish early hematopoiesis including erythropoiesis. Recently, transcription activator-like effector nuclease (TALEN) has been shown to generate targeted genomic editing in zebrafish. To validate our observation, we therefore utilized customized TALENs pair to generate tet2knock-out zebrafish animal model. We designed a pair of TALENs targeting first exon of tet2and our tet2TALENs were able to generate insertion and/or deletion in targeted region of tet2exon 1 in 25% to 44% zebrafish embryos. We obtained a total of fifteen different tet2mutation genotypes F1 fish, and seven of them were predicted to cause early termination of transcription. The in-cross of these F1 genotypes matched the Mendelian inheritance. The tet2-/-knock-out F2 zebrafish is not embryonic lethal and can grow to sexually mature adult fish. The detailed analysis of tet2-/-knock-out zebrafish early hematopoiesis will be presented at the meeting.