Page 56 - 2019_07 resto del Mondo-web

M. Mazzola et al.
Tg(CD41:GFP) zebrafish embryos at 3 days post-fertilization (dpf) or PU.1-stained cells derived from embryos at 3 dpf.
Injections and indomethacin treatment
Injections were carried out on one- to two-cell stage embryos. Morpholinos were injected as described in the Online Supplementary Methods. Human NPM1 and NPMc+ and zebrafish dkk1b mRNA were generated as previously described12 and inject- ed as detailed in the Online Supplementary Methods. Indomethacin (Sigma-Aldrich St. Louis, MO, USA) treatment was administered as described by North et al.26
Confocal imaging
Previously immunostained 3 dpf embryos were equilibrated and mounted in 85% glycerol solution in phosphate-buffered saline and imaged using a TCS-SP2 confocal microscope (Leica), with 40X oil immersion objective, 488 nm argon ion and 405 nm diode lasers. For each sample, single stack images were acquired.
Statistical analyses
For quantitative RT-PCR experiments, data were statistically analyzed applying one-way analysis of variance (ANOVA) or t- tests, defining P≤0.05 (*), P≤0.01 (**), and P≤0.001 (***) as statisti- cally significant values.27 Data were analyzed using the compara- tive ΔΔCt method. Both t- test and standard deviation (SD) values refer to data from triplicate samples. In zebrafish at least three dif- ferent experiments were done for each analysis. For cell counts and phenotypic analyses, statistical analysis was performed by the χ2 test, with Yates correction when needed.
Results
Cohesin expression in subgroups of adult patients with acute myeloid leukemia carrying NPM1 mutations and in a zebrafish model of NPMc+ expression
It was recently reported that somatic mutations in cohesin genes are frequently associated with NPM1 muta- tions,28 but not with other common AML mutations; how- ever, the expression levels of cohesins have never been analyzed. We, therefore, decided to investigate the pres- ence of alterations in the expression of cohesin genes in a cohort of 40 adult AML patients stratified into two homo- geneous molecular subgroups according to the absence or presence of a NPM1 mutation (NPMc+). Each subgroup was analyzed by means of quantitative RT-PCR for the expression of the cohesin genes SMC1A, SMC3, NIPBL and RAD21 (Figure 1A-D). Interestingly, only NIPBL showed a significantly decreased expression in AML patients carrying a NPM1 mutation (Figure 1B). Conversely, when the same patients were divided into subgroups depending on the presence or absence of a FLT3-internal tandem duplication (ITD), none of the cohesin genes analyzed was differently expressed between the two subgroups (Online Supplementary Figure S1).
We also used quantitative RT-PCR to analyze the expression of cohesin genes in a previously generated and well-characterized zebrafish model with human NPMc+ overexpression.12,13 The injection of 100 pg/embryo of NPMc+ transcript led to a significant downregulation of nipblb in the whole embryo at 3 dpf, while the expression of other cohesin genes analyzed was unaltered or even increased (Figure 1E). We performed a stage-dependent analysis to determine at which developmental stage the overexpression of NPMc+ started to downregulate nipblb
expression in the whole embryo. We observed a signifi- cant decrease of nipblb expression starting from 48 hours post-fertilization (hpf), while no differential decreases between controls and NPMc+ mRNA-injected embryos were observed at 24 and 36 hpf (Figure 1F). To further ver- ify that the downregulation of zebrafish nipblb was strictly dependent on the forced expression of NPMc+, we inject- ed different doses of the NPMc+ transcript (25 pg/embryo, 75 pg/embryo and 125 pg/embryo) and found a significant inverse correlation between reduced nipblb expression and increased NPMc+ (Figure 1G). Moreover, western blot studies of embryos at 3 dpf confirmed that the injection of NPMc+ mRNA, at a dose of 100 pg/embryo, caused a reduction of the level of Nipbl protein in comparison to the level in control-injected embryos (Figure 1H).
nipblb loss-of-function and NPMc+ overexpression generate hematopoietic defects in zebrafish embryos
Since NIPBL is downregulated in AML patients in asso- ciation with NPM1 mutations, we wondered whether nip- blb knockdown might generate defects in myeloid cell dif- ferentiation. To investigate this hypothesis, we downreg- ulated nipblb in zebrafish by injecting a nipblb antisense morpholino targeting the ATG region (nipblb-MO), as pre- viously described for the knockdown of the Nipbl protein.29,30 In AML patients, HSC and immature myeloid blasts are increased. Therefore, to screen for myeloid defects in conditions resembling those of AML patients, we analyzed the same cell populations in nipblb-MO injected embryos. Knockdown of nipblb did not lead to significant differences in the HSC, as shown by confocal images of the caudal hematopoietic tissues (CHT) of Tg(CD41:GFP) embryos31 at 3 dpf (Figure 2A,B) and by cytofluorimetric analyses to quantify the CD41:GFPlow HSC cells in comparison to those in controls (Figure 2D,E,G). On the other hand, at the same stage, nipblb-MO injected embryos showed an increase of myeloid progen- itors, positive for the PU.1 antibody (Figure 2H,I; FACS in K,L,N). These observations were also confirmed by means of WISH techniques with the HSC marker cmyb and the myeloid progenitor marker spi1b with relative quantifica- tions of the observed phenotypes, and RT-qPCR analyses (Online Supplementary Figure S2). Moreover, to verify whether the increased myeloid cells were blocked in an undifferentiated state, we used Sudan black staining to visualize mature myeloid cells.24 At 4 dpf we observed a reduction of neutrophils (Figure 2O,P).
It was previously reported that the forced expression of human NPMc+ in zebrafish embryos increased the HSC population at 30-36 hpf.13 To further analyze whether the hematopoietic defects are still present later during hematopoiesis and are extended to myeloid cells, we injected the embryos with NPMc+ mRNA (100 pg/embryo) and analyzed the hematopoietic phenotype. The CD41:GFPlow cells in the CHT of NPMc+-injected embryos were expanded in comparison to those in the controls (Figure 2A,C; FACS in D,F,G), as were PU.1-posi- tive myeloid precursors (Figure 2H,J; FACS in K,M,N). Sudan black staining showed a decrease in mature myeloid cells in NPMc+-injected embryos in comparison to those in controls (Figure 2O,Q). Moreover, the expres- sion levels of cmyb and spi1b, investigated by WISH and quantitative RT-PCR techniques, corroborated our previ- ous findings (Online Supplementary Figure S3).
To confirm the specificity of the hematopoietic defects
1334
haematologica | 2019; 104(7)