The role of calreticulin (CALR) in vertebrate hematopoiesis

Abstract

Calreticulin (CALR) is a multi-functional protein mainly controlling protein folding and calcium homeostasis in endoplasmic reticulum (ER). Recently, recurrent mutations in CALR have been found in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without Janus kinase 2 (JAK2) or thrombopoietin receptor (MPL) mutations. Previous studies have shown that mutant forms of CALR interacted with MPL to activate JAK2-STAT5 signaling pathway in myeloproliferative neoplasm (MPN). However, the functional role of CALR in normal hematopoiesis remains unknown. In this study, we made use of zebrafish model to examine the unknown roles of Calr in vertebrate hematopoiesis.

A somatic calr loss-of-function zebrafish model was generated by co-injecting two TALEN pairs separately targeting calr exon 1 and exon 5. Western-blot confirmed the reduction of calr protein in whole embryos as well as adult kidney marrow cells. Somatic calr mutations significantly increased the expression of genes associated with myeloid lineages, including pu.1, l-plastin, mpo and mpeg1, while decreased the expression of c-myb (hematopoietic stem cells (HSCs)) and rag1 (lymphocytes) in definitive hematopoiesis as shown by whole mount in situ hybridization (WISH). The myeloid-proliferation phenotype also persisted in adult hematopoiesis as shown by the expansion of myeloid cells in whole kidney marrows (WKMs) examined by flow cytometry. Unlike CALR mutations in MPN, calr loss-of-function did not affect subcellular phospho-stat5 status in the expanded myeloid population, suggests that calr regulates normal definitive hematopoiesis through an alternative pathway. We initially knock-down calr with morpholino (MO), which resulted in decrease in differentiated primitive myeloid cells and increase in definitive HSCs as shown by WISH, and is opposite to the results observed in calr loss-of-function model by double TALEN injection. However, western blot showed that calr protein level increased unexpectedly in calr morphant which might explain the phenotypic discrepancy between calr morphants and somatic mutants.

To model CALR mutations, TALEN was also used to introduce frame-shifting mutations in zebrafish calr exon 9. In particular, a 7-bp deletion mutant, which resulted in a basic C-terminal resembling human CALR type-I and II mutations was generated. Although low fertility and survival rate limited the number of 7-bp mutants available for analysis, two out of four heterozygous F2 7-bp mutants developed tumor-like tissue in the head with infiltration of blastlike hematopoietic cells during early adulthood. Also, the percentage of myeloid cells increased significantly in their kidney marrows. These leukemic and MPN-like phenotypes were not observed in wild-type siblings or 5-bp mutants (resulted in a truncated C-terminal), under-scored the pathological potential of the novel basic C-terminal.

In summary, we demonstrated the previously unknown role of Calr in regulating vertebrate definitive hematopoiesis, in particular, myelopoiesis. Loss-of-function calr mutation resulted in myeloproliferation phenotype during both embryonic and adult hematopoiesis. In addition, by TALEN-mediated mutagenesis targeting calr exon 9, zebrafish mutant carrying frame-shifted calr with a novel basic C-terminal resembling human CALR type-I/II mutations was generated. Preliminary data showed that heterozygous F2 7-bp mutants developed leukemic and MPN-like phenotypes, highlighted the potential of this mutant to model human MPN or leukemic transformation from MPN with CALR mutations.