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Khalaf, Samer Naji (2024) Investigating the roles of Gfi1/1b transcription factors, Lsd1 histone demethylase and long non coding RNA 1/2 genes in haematopoiesis during zebrafish embryogenesis. PhD thesis, University of Nottingham.

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Abstract

Cellular differentiation entails the activation of mature cell markers and the

repression of genes that are characteristic for immature cell states and alternative

differentiation paths. Their repression involves transcriptional repressors and

numerous non-coding RNAs that act to suppress gene expression at a

transcriptional and post-transcriptional level. During zebrafish embryogenesis, two

long ncRNAs, si:ch211-114p16.1 and si:ch211-114p16.2, as well as members of

the Gfi1/1b repressor family and their interaction partner and co-repressor

Lsd1/Kdm1a are expressed during blood cell differentiation from the lateral

mesoderm. Here, I show that while the long ncRNAs are useful lineage markers

they are neither required for haematopoiesis nor needed for the survival of the

zebrafish. The zebrafish genome encodes two orthologues of mouse Gfi1, Gfi1aa

and Gfi1ab, in addition to a single orthologue of the mouse Gfi1b protein. Just like

mouse Gfi1/1b double mutant embryos, zebrafish gfi1aa/1ab/1b triple mutant

embryos are anaemic at the time when primitive red blood cells (prRBCs) populate

the circulation of a wildtype embryo. Here, to find out more about the fate of the

mesodermal prRBC progenitors I have performed a single cell RNA-sequencing

experiment. To increase the number of embryos available for this experiment, I used

the latest CRISPR/Cas9 technology to establish generation zero (G0) triple mutant

embryos. These faithfully phenocopied the prRBC defect of the stable triple mutants.

For the scRNA-seq experiment, the blood progenitors of interest were sorted from

embryos that carried two reporter transgenes, qmc551:GFP and gata1:dsRed. The

transcriptomic data on the GFP and dsRed single and double positive cells

demonstrates a complete loss of mature prRBCs, as well as a severe reduction in

the number of haematopoietic progenitors among the sorted G0 triple mutant cells.

It also shows a concomitant increase in neutrophil granulocytes. These data

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highlight the important roles that Gfi1/1b proteins play in haematopoietic progenitor

maintenance and mesodermal progenitor cell commitment to the prRBC lineage.

Interestingly, loss of the Gfi1/1b interaction partner Lsd1/Kdm1a does not lead to

severe anaemia in either stable or G0 mutant embryos. The latter were generated

as part of this project. They will serve as a valuable source for Lsd1/Kdm1a mutant

cells for future single cell RNA-sequencing experiments in which to explore the

differential need for Gfi1aa/1ab/1b and Lsd1/Kdm1a in mesodermal prRBC

progenitors.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Gering, Martin Wilkinson, Rob
Keywords:	Cellular differentiation; zebrafish embryogenesis; Transcriptional repressors; Haematopoiesis
Subjects:	Q Science > QL Zoology > QL951 Embryology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	77312
Depositing User:	Khalaf, Samer
Date Deposited:	16 Jul 2024 04:40
Last Modified:	16 Jul 2024 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/77312

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