Peña, Oscar A; (2019) Modelling haematopoietic malignancies using zebrafish. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Primitive and definitive haematopoiesis were studied during embryonic and larval development in three zebrafish models of human haematological diseases: TEL-AML1 expressing acute lymphoblastic leukaemia (ALL), GATA2 haploinsufficient disorders, and MDS with isolated del(5q) (MDS5q). The t(12;21)(p13;q22) chromosomal translocation resulting in the TEL-AML1 fusion gene is the most common translocation in childhood ALL. The translocation arises in utero and can be found in approximately 1% of new-born infants. However, less than 1% of these children develop leukaemia and this translocation is not observed in adult ALL. This suggests that the cell of origin resulting in the eventual transformation to BCP-ALL is present and disrupted early during development. Early developmental haematopoiesis was studied in transgenic zebrafish expressing the human TEL-AML1 fusion protein. The numbers of granulocytes are transiently increased by the expression of TEL-AML1 fusion protein in these animals. The GATA2 gene encodes a transcription factor that plays a major role in haematopoiesis. Recently, germline GATA2 mutations have been identified as the cause of a range of haemato-vascular disorders including Emberger syndrome, Monomac and DCML deficiency all with predisposition to MDS. We took advantage of the presence of two GATA2 paralogs (gata2a and gata2b) in zebrafish to dissect the role of GATA2 in early haematopoietic and vascular development. Gata2a and Gata2b were shown to have different functions in haematopoiesis. Gata2a regulates primitive myeloid cell development and vascular morphogenesis. The first wave of lymphocytes and haematopoietic progenitors derived from the dorsal aorta are also affected. A novel role for the Gata2b was identified, in the development of myeloid cells in the caudal haematopoietic tissue. Double heterozygous fish carrying mutations in both gata2a and gata2b were used to model germinal mutations in GATA2 found in patients. Ribosomal protein gene 14 (RPS14), located on 5q, has been shown to be the likely genetic determinant of anaemia in patients with MDS5q. Our group has used TALENs to generate an rps14 mutant zebrafish. Fish carrying a heterozygous mutation in rps14 gene show defects in erythropoiesis and granulopoiesis, and the severity of these defects is enhanced by haemolytic or cold stress. The rescuing effects of a TLR7/8 agonist, imiquimod, identified previously in a small molecule screen for modifiers of the anaemia phenotype associated with Rps14 loss, were studied in rps14 mutant fish. Pharmacological evidence suggests a role for TLR7 in erythroid development, as exposure to a TLR7 agonist partially rescues the phenotype of heterozygous rps14 mutant fish, while treatment with a TLR8 agonist has no effect.

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