Page 83 - Haematologica Vol. 109 - July 2024

ARTICLE - Targeting TNF/IL-17/MAPK in hE2A-PBX1 zebrafish H. Luo et al.
(Student t tests, mean ± SEM; *P<0.05, **P<0.01, ****P<0.0001) (C) WISH of myb expressions in Tg(hsp70:E2A-PBX1-EGFP) (right panel) were higher than siblings (left panel) at 5 dpf. (C’) Statistical analysis of the myb+ signals in panel (C). The black asterisks indicate statistical difference (Student t tests, mean ± SEM; *P<0.05). N/N: number of zebrafish larvae showing representative phenotype/total number of zebrafish larvae examined.
the number of myeloid cells was significantly increased (Figure 1E, E’), indicating that transient induction of hE2A- PBX1 expression may disrupt the hematopoiesis in zebrafish. Additionally, survival analysis of Tg(hsp70:E2A-PBX1-EGFP) fish revealed a significant increase in mortality within 15 days following the induction of hE2A-PBX1 expression, in comparison to their siblings (Figure 1F).
hE2A-PBX1 induces abnormal expansion of myeloid cells in zebrafish larvae
In order to further explore the effect of hE2A-PBX1 expression on the development of different hematopoietic lineages, we detected the marker genes of each hematopoietic lin- eage in Tg(hsp70:E2A-PBX1-EGFP) transgenic zebrafish after heat shock by whole mount in situ hybridization (WISH) and specific staining. The results showed a significant increase in myeloid cells (l-plastin: a myeloid gene that marks granulocytes and macrophages, also known as lcp1) (Figure 2A, A’), which was consistent with the hemogram changes in zebrafish larvae (Figure 1E). Further assays of myeloid terminally differentiated granulocytes and mac- rophages revealed a significant increase in the number of immature granulocytes (cebp1), mature granulocytes (lyz, mpx, Sudan Black B [SB]) and macrophages (mfap4) in both 3 dpf and 5 dpf (Figure 2B, B’; Online Supplementary S2A, A’). As myeloid cells originate from HSPC;34 we aimed to investigate whether the increased number of myeloid cells was caused by the tendency of HSPC toward the myeloid lineage or if there was an overall increase in HSPC leading to increased differentiation into various lineages? In order to address this question, we examined the changes of HSPC (myb), lymphocytes (rag1, rag2), and erythrocytes (be1) in Tg(hsp70:E2A-PBX1-EGFP) after heat shock. The results showed that myb+ HSPC increased at 5 dpf (Figure 2C, C’), while the number of rag1+ and rag2+ lymphocytes decreased significantly (Online Supplementary Figure S2B, B’), and be1+ erythrocytes showed no significant change (Online Supplementary Figure S2C, C’). These data suggest that hE2A-PBX1 expression may lead to myeloid expansion in zebrafish by increasing HSPC with myeloid differentiation potential.
In addition, it has been reported that the expression of PBX1 maintains cell proliferation,21 while the E2A protein promotes cell differentiation and has anti-proliferation effect.35 Therefore, we investigated whether hE2A-PBX1 in- fluences the proliferation of myeloid and lymphocytes in Tg(hsp70:E2A-PBX1-EGFP). The proliferation and apoptosis of myeloid/lymphocytes in Tg(hsp70:E2A-PBX1-EGFP) fish were examined after heat shock by co-staining with BrdU/
TUNEL and Lcp1/Rag2, respectively. The results showed a significant increase in the proportion of proliferating cells among Lcp1+ myeloid cells in 5 dpf Tg(hsp70:E2A-PBX1- EGFP) compared to the siblings (Figure 3A, 3A’), while the proportion of apoptotic cells showed a significant decrease (Figure 3B, B’). In contrast, Rag2+ lymphocytes showed a significant decrease in proliferation (Online Supplementary Figure S3A, A’), with no significant change in apoptotic cells (Online Supplementary Figure S3B, B’). These data suggest that expression of hE2A-PBX1 leads to abnormal myeloid expansion by increasing the myeloid differentiation potential of HSPC on the one hand and inducing myeloid hyperplasia on the other hand.
In order to investigate the potential relationship between myeloid expansion and early mortality in hE2A-PBX1 ze- brafish, we treated heat-shocked hE2A-PBX1 fish with drugs capable of reducing myeloid proliferation, namely cytarabine36-38 and flavopiridol.39,40 The results showed that, compared to the DMSO-treated control group, both cytar- abine and flavopiridol treatments significantly reduce the population of SB+ myeloid cells in hE2A-PBX1 fish (Online Supplementary Figure S3C, C’), and increase their survival rate (Online Supplementary Figure S3D). This suggests that myeloid cell proliferation may be one of the contributing factors to the elevated mortality in hE2A-PBX1 zebrafish.
Induction hE2A-PBX1 expression in adult zebrafish leads to an acute myeloid leukemia-like phenotype
In order to analyze whether induced expression of hE2A-PBX1 leads to blood tumors in adult zebrafish, we administered 1-month continuous heat shock to 3-month-old adult fish and collected peripheral blood (PB) cells and kidney marrow (KM) blood cells for Giemsa staining to detect hematological changes. Although 1 month of hE2A-PBX1 induction did not alter the hemogram in PB, it resulted in a significant increase in myeloid cells in KM (Online Supplementary Figure S4A, A’). We then extended the heat shock time to 3 months to determine whether the degree of myeloid expansion induced by hE2A-PBX1 is related to the induction time. The results showed that the longer expression of hE2A- PBX1 not only further exacerbated the myeloid expansion and lymphoid reduction in KM, but also resulted in mild myeloid hyperplasia in PB (Online Supplementary Figure S4B, B’). Since myeloid leukemia is commonly associated with the patient’s age, we induced 3-month continuous expression of hE2A-PBX1 in 1-year-old zebrafish. We found that the AML-like phenotype was more pronounced in aged zebrafish after 3 months of hE2A-PBX1 induced compared to young zebrafish. In addition to the significant increase
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