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M. Mazzola et al.
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Figure 6. Co-injection of subcritical doses of nipblb-MO and NPMc+, which singularly do not have effects, indicates cooperation between NPMc+ and nipblb in myeloid differentiation. (A-D) The number of green fluorescent protein (GFP)+ cells in caudal hematopoietic tissue (CHT) was increased when embryos at 3 dpf were co-injected with subcritical doses of both nipblb-MO and NPMc+ mRNA (D) in comparison to a single injection of a subcritical dose of NPMc+ mRNA (B) or nipblb- MO (C). The numbers of GFP+ cells in the CHT following the single injections were comparable to those in the controls (A). (E) Quantification of GFP+ cells in the CHT. (F-I) Whole mount in situ hybridization analyses of the myeloid precursor marker spi1b. spi1b expression was increased in embryos co-injected with subcritical doses of both nipblb-MO and NPMc+ mRNA (I) in comparison to the expression in controls and embryos injected with a subcritical dose of either NPMc+ (sub NPMc+) (G) or nipblb-MO (sub nipblb-MO) (H). (J) Quantification of the embryos presenting increased spi1b expression. Images were processed as described in the Online Supplementary Methods. The scale bar represents 100 μm. **P<0.01 and ***P<0.001. ctrl: control; MO: morpholino.
been investigated in clinical trials26 and was able to rescue the hematopoietic phenotype in our study, could be attractive candidates for the development of new treat- ments for NPMc+ AML patients.
Further analyses are necessary to investigate the mech- anisms through which NPMc+ interacts with NIPBL and how they regulate the canonical Wnt pathway and the hematopoietic phenotype. One hypothesis raised by pre- vious research and our analyses is the involvement of other members of the cohesin complex such as Rad21, which is negatively regulated by Nipbl in both murine fibroblasts47 and zebrafish (see Figure 1F). Alternatively, the aberrant cytoplasmic dislocation of mutant NPM1 might alter the normal regulation of NIPBL expression in the nucleus, similarly to what happens in the important PU.1-NPM1-mediated regulation in myeloid precursors.48 Large scale transcriptomic and/or proteomic analyses will be useful to unravel the mechanism and, to this purpose, the use of the zebrafish platform with cohesin knock- down might be a suitable model system.
In conclusion, our study correlates NIPBL with NPM1 mutations in adult AML patients for the first time and demonstrates their interplay in myeloid cell differentiation in zebrafish through involvement of the canonical Wnt pathway. The results obtained will foster the identifica- tion of new potential targets for the treatment of sub- groups of AML.
Acknowledgments
We thank R Monteiro, (University of Birmingham), for the Tg(CD41:GFP/kdrl:dsRED) zebrafish line, N. Bolli, (University of Milan) for useful discussion of the data, the cytometry desk staff (IFOM, Milan) for technical help in FACS experiments, MC Crosti of the INGM FACS facility for sorting experiments and M Spreafico and M Cafora (University of Milan) for their priceless support in experimental procedures. This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) (MFAG#18714). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.
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