Overexpression of Calr Mutants Perturbs Developmental Hematopoiesis in Zebrafish Embryos
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 
Published: October 2019

Abstract
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<0.001), mutant CALR-ins5 (mean 128.3±6.1 per embryo, P<0.001) and uninjected control (mean 136.7±3.0 per embryo, P<0.001), respectively.
At 5 dpf, the number of cd41+ thrombocytes significantly decreased upon mpl MO knockdown (mean 43.6±4.9 per embryo) when compared to the control MO group (mean 123.5±5.9 per embryo, P<0.001) and the mutant CALR-del52 group (P<0.001). Importantly, co-injection of CALR-del52 mutant mRNA (mean 73.7±5.1 per embryo) can only partially reverse the knockdown effect of mpl MO. In contrast, the numbers of cd41+ thrombocytes did not decrease significantly upon epor MO or csf3r MO knocked-down compared with the control MO group. When CALR-del52 mutant mRNA was co-injected with epor or csf3r MOs, the numbers of cd41+ thrombocytes were comparable to those of CALR-del52-injected embryos.
In Q-PCR experiments, the expression of cmyb, runx1 and scl significantly increased in only CALR-ins5 mutant group at 3 dpf when compared to 2 dpf. The expression of αeHb and βeHb significantly decreased in both CALR-del52 and CALR-ins5 mutant groups at 2 dpf but the expression of gata1 remained unchange. The effects of CALR-del52 and CALR-ins5 mutant groups on the expression of cytokine and cytokine receptor genes included the upregulation of mpl at 1 dpf followed by progressive downregulation at 2 dpf and 3 dpf but relatively stable expression of tpo and epo during early development. In the group of genes related to thrombopoiesis, the expression of emilin1a and nbeal2 was significantly downregulated in CALR-del52 group at 3 dpf when compared to 2 dpf.
Conclusions: In this study, we showed that the expression of mutant CALR causes thrombocytosis through an mpl-dependent mechanism and perturbs developmental hematopoiesis in zebrafish embryos.

Disclosures
No relevant conflicts of interest to declare.