Investigating the roles of long-non-coding RNA genes in zebrafish haematopoiesisTools Alzaqqah, Fatemah (2020) Investigating the roles of long-non-coding RNA genes in zebrafish haematopoiesis. PhD thesis, University of Nottingham.
AbstractIn vertebrates, haematopoiesis, the process of blood formation, is governed by a complex network of gene regulators that include numerous transcription factors, members of signalling pathways as well as regulatory RNA species. In a previous study, a transposon-mediated gene trap screen performed in zebrafish identified the gene trap line qmc551 that expresses green fluorescent protein (GFP) in primitive red blood cells (prRBCs) and in definitive haematopoietic progenitors. This line carries the transposon in intron 1 of the gene that encodes the transcriptional repressor Gfi1aa. The gene trap interferes with its haematopoietic expression and expresses GFP in its place. A transcriptome analysis performed on GFP-positive and GFP-negative cells isolated from 30 hour-old qmc551 heterozygous embryos revealed that two long-noncoding RNAs (si:ch211-214p16.1 (lncRNA1) and si:ch211-214p16.2 (lncRNA2)) are specifically expressed in GFP-positive cells. Whole-mount RNA in situ hybridisation on wt and cloche mutant embryos confirmed the blood-specific expression patterns of lncRNA1 and 2, and analysis of mindbomb mutant embryos showed that both prRBCs, as well as immature definitive blood progenitors expressed the two lncRNAs. In the prRBCs, lncRNA1 and 2 expressions are subject to Gfi1aa-mediated repression, as embryos homozygous for the qmc551 mutant allele of gfi1aa displayed elevated lncRNA1 and 2 levels. While all early embryonic blood cells express higher levels of lncRNA2, lncRNA1 alone displays high-level expression in T-cell progenitors in the thymus. Altogether, their expression patterns suggested a specific role for the lncRNAs in early blood development. To study this role, the CRISPR/Cas9 technology was used to delete their genes individually and in combination from zebrafish chromosome 17, where they occupy adjacent positions at a common lncRNA1/2 locus. Here, the generation and preliminary characterisation of the lncRNA1/2 locus mutant alleles qmc221, qm222, qmc223 and qmc224 are described. These experiments form the foundation for future investigations into the roles that these lncRNAs play within the gene regulatory network that controls haematopoiesis.
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