Investigating the roles of Gfi1/1b transcription factors, Lsd1 histone demethylase and long non coding RNA 1/2 genes in haematopoiesis during zebrafish embryogenesis
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...
| Main Author: | |
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
| Published: |
2024
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| Subjects: | |
| Online Access: | https://eprints.nottingham.ac.uk/77312/ |
| _version_ | 1848800985758040064 |
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| author | Khalaf, Samer Naji |
| author_facet | Khalaf, Samer Naji |
| author_sort | Khalaf, Samer Naji |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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
xix
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. |
| first_indexed | 2025-11-14T21:00:16Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-77312 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T21:00:16Z |
| publishDate | 2024 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-773122025-02-28T15:19:57Z https://eprints.nottingham.ac.uk/77312/ Investigating the roles of Gfi1/1b transcription factors, Lsd1 histone demethylase and long non coding RNA 1/2 genes in haematopoiesis during zebrafish embryogenesis Khalaf, Samer Naji 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 xix 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. 2024-07-16 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/77312/1/Khalaf%2C%20Samer%2C%2020220581%2C%20Corrections.pdf 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. Cellular differentiation; zebrafish embryogenesis; Transcriptional repressors; Haematopoiesis |
| spellingShingle | Cellular differentiation; zebrafish embryogenesis; Transcriptional repressors; Haematopoiesis Khalaf, Samer Naji Investigating the roles of Gfi1/1b transcription factors, Lsd1 histone demethylase and long non coding RNA 1/2 genes in haematopoiesis during zebrafish embryogenesis |
| title | Investigating the roles of Gfi1/1b transcription
factors, Lsd1 histone demethylase and long non
coding RNA 1/2 genes in haematopoiesis during
zebrafish embryogenesis |
| title_full | Investigating the roles of Gfi1/1b transcription
factors, Lsd1 histone demethylase and long non
coding RNA 1/2 genes in haematopoiesis during
zebrafish embryogenesis |
| title_fullStr | Investigating the roles of Gfi1/1b transcription
factors, Lsd1 histone demethylase and long non
coding RNA 1/2 genes in haematopoiesis during
zebrafish embryogenesis |
| title_full_unstemmed | Investigating the roles of Gfi1/1b transcription
factors, Lsd1 histone demethylase and long non
coding RNA 1/2 genes in haematopoiesis during
zebrafish embryogenesis |
| title_short | Investigating the roles of Gfi1/1b transcription
factors, Lsd1 histone demethylase and long non
coding RNA 1/2 genes in haematopoiesis during
zebrafish embryogenesis |
| title_sort | investigating the roles of gfi1/1b transcription
factors, lsd1 histone demethylase and long non
coding rna 1/2 genes in haematopoiesis during
zebrafish embryogenesis |
| topic | Cellular differentiation; zebrafish embryogenesis; Transcriptional repressors; Haematopoiesis |
| url | https://eprints.nottingham.ac.uk/77312/ |