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Title: Zebrafish Gfi1 paralogs play redundant roles in primitive and definitive haematopoietic development
Author: Ucanok, Deniz
ISNI:       0000 0004 6349 1695
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2016
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Haematopoietic stem cells (HSCs) are multipotent progenitors that can self-renew and give rise to cells of all blood lineages. In vertebrates, HSCs are specified during embryogenesis from haemogenic endothelial cells (HECs) in the ventral wall of the dorsal aorta (vDA). Previously, a transposon-based gene trap approach was used in zebrafish to identify a transgenic line called qmc551 that expresses a GFP reporter gene in primitive red blood cell (prRBC) progenitors and in HECs of the vDA. In this line, GFP expression is retained as the cells undergo endothelial to haematopoietic transition and seed sites of larval and adult haematopoiesis. In the adult kidney marrow, GFP expression is found in progenitors, lymphocytes and myelomonocytes. The transposon that encodes qmc551:GFP is located in intron 1 of gfilaa, one of three zebrafish gfil paralogs along with gfilab and gfilb. In this study, it was shown that qmc551 :GFP is expressed from a spliced transcript that carries the GFP reading frame downstream of exon 1 of gfilaa, demonstrating that GFP expression is under the control of the gfilaa promoter. Consequently, GFP recapitulates gfilaa expression in development. The presence of the transposon in intron 1 interferes with normal splicing of the gfilaa transcript leading to a loss of gfilaa mRNA in primitive and definitive haematopoietic cells as shown in WISH and qRT-PCR experiments. Gfilaa depletion is compatible with the normal development of prRBC which retain the expression of the paralogue, Gfilb. Morpholino-mediated depletion of Gfilb in homozygous qmc551 embryos blocked prRBC maturation, leading to a severe reduction in haemoglobinization. This phenotype is rescued by Gfilaa in a dose dependent manner as morphant embryos carrying one or two wt copies of gfilaa have increasingly normal levels of haemoglobin. This shows that Gfilaa and Gfilb play essential redundant roles in prRBC maturation. During the onset of definitive haematopoiesis, gfilaa expression is induced downstream of Vegf and Notch signalling and is parallel to Runxl in HECs of the vDA. Its depletion in qmc551 homozygous embryos does not interfere with HSC specification or maintenance which is likely due to the Runxl dependent upregulation of its paralog, gfilab, in the vDA. Interestingly, the loss of Gfilb in qmc551 homozygous embryos leads to the hyperproliferation of haematopoietic stem and progenitor cells (HSPCs) in the caudal haematopoietic tissue (CHT), suggesting that these genes, like their mammalian counterparts, are involved in the maintenance of HSPC quiescence. Together, results presented here show that, in contrast to previously published morphant data, zebrafish Gfil factors and their mammalian homologs play functionally conserved roles during primitive and definitive haematopoiesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available