Specific somatic mutations in hematopoietic stem and progenitor cells (HSPCs) provide a proliferative advantage, leading to clonal hematopoiesis and the development of myeloproliferative disorders (MPDs) and acute myeloid leukemia (AML). Studies of patients with TET2 or DNMT3A mutations show that hematopoiesis can be clonal without pathology, suggesting that clonal expansion may be a prerequisite for disease development. It is unclear which combination of these mutations are necessary or sufficient to promote clonal dominance or whether the order of the mutational events are critical for disease progression. The zebrafish has emerged as an excellent model for hematopoietic malignancies, notably acute lymphoblastic leukemias. Models of MPDs and AML show low penetrance with no overt AML, although it is not clear in these models whether a non-pathological clonal dominance is established. It would be interesting to evaluate clonality of normal HSCs as genetic perturbations occur in the premalignant state. Viral clonal marking would be helpful, but the relatively small number of HSCs present in the zebrafish kidney marrow require a less complex clonal labeling system. To investigate HSPCs at the clonal level, we have optimized the Brainbow system for use in zebrafish hematopoiesis. This system extends clonal analysis to the erythrocyte lineage, which is nucleated in zebrafish but not in mammals. Zebrafish that ubiquitously express the Brainbow construct and contain a blood specific CreERT2 were treated with tamoxifen at various embryonic stages. Treated fish were grown to adulthood, and peripheral blood and marrow samples were subjected to confocal and FACS analysis to observe color barcodes. Using automated cluster analysis on normalized intensities of the Brainbow fluorescent proteins, we detected multiple and unique barcoded cells (average of 4-8) in all lineages of the blood as far as 8 months post treatment. The efficiency of recombination in this system allows for labeling of 20-80% of the mature blood pool. Transplants of Brainbow-labeled whole kidney marrow near limiting dilution into irradiated recipients showed strong monoclonal engraftment, suggesting that HSCs are being labeled. Using this system, we will investigate mutations that are necessary or sufficient for generation of clonal dominance. Our studies will provide insight on early clonal events that regulate normal and premalignant hematopoiesis.
Somatic mutations in hematopoietic stem and progenitor cells can provide proliferative advantages, leading to clonal expansion and a potential pre-leukemic state. The goal of this project is to identify factors that promote clonal dominance in zebrafish hematopoiesis through clonal barcoding and genetic manipulation.
| Henninger, Jonathan; Santoso, Buyung; Hans, Stefan et al. (2017) Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development. Nat Cell Biol 19:17-27 |
