Saudi Cultural Missions Theses & Dissertations
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Item Restricted Unravelling the role of P4HB in skeletal development using zebrafish as a disease model(University of Sheffield, 2024-08-02) Zafar, Myar; Balasubramanian, MeenaOsteogenesis imperfecta (OI), commonly known as brittle bone disease, is a genetic disorder characterized by fragile bones and connective tissue abnormalities, primarily caused by mutations in collagen-related genes such as COL1A1 and COL1A2. Recent research has implicated the P4HB gene in OI, particularly in the Cole-Carpenter syndrome variant. This study aims to explain the role of P4HB in skeletal development using zebrafish as a model organism due to their genetic similarity to humans, transparency during embryonic development, and rapid reproduction. The research specifically investigates the differential expression of the genes sox9b and bmp2b in wild-type (WT) zebrafish compared to those with mutations in the P4HB gene, hypothesizing a reduction in expression levels in the mutated models. To test this hypothesis, a knock-in model of zebrafish with the P4HB mutation was developed, allowing for the assessment of gene function and expression. In-situ hybridization techniques were used to analyze the expression patterns of sox9b and bmp2b. These genes are integral to skeletal development and are thought to be affected by disruptions in P4HB function. The results showed no significant reduction in the expression of the sox9b genes in P4HB mutant zebrafish compared to WT, in comparison to the bmp2b mutant zebrafish that showed significant reduction in expression compared to the WT. These results suggest that the P4HB mutation, which disrupts type I collagen formation, may not have a major impact on the expression of sox9b and bmp2b which are involved in cartilage and bone development. This study enhances the understanding of the genetic mechanisms underlying OI and highlights the benefit of zebrafish as a model for studying skeletal diseases. The findings also emphasize the importance of P4HB in maintaining normal skeletal development, providing potential insights for therapeutic targets in treating conditions like OI.24 0Item Restricted Investigations into the role of Gfi1 proteins in the exocrine pancreas and the intestine in zebrafish embryos(University of Nottingham, 2024-02-08) Alshanqiti, Aisha; Gering, MartinGfi1 is a transcriptional repressor that plays important biological roles in the development and maintenance of different cell types. Gfi1 knock-out mice suffer from a defect in the exocrine pancreas and intestine. Yet, the exact physiological role of Gfi1 in these organs remains unclear. This study chose to explore the role of Gfi1/b in exocrine pancreatic and intestinal cell differentiation in zebrafish during embryonic stage. Zebrafish were chosen as model here because research on mice has shown that Gfi1 knockout mice suffer from severe neutropenia and die a few months after birth. Our gfi1aaqmc551/qmc551 mutant line showed that adult fish were viable and well fertile with no phenotypic abnormalities. However, full-length bulk RNA-sequencing showed that the qmc551 allele is not a null allele in the exocrine pancreas of zebrafish. RNA in situ hybridisation experiments showed that loss of gfi1aa disrupted the normal expression of the amy2al1 gene but not the expression of prss1 or amy2a in acinar cells of zebrafish embryos. Data from single cell RNA sequencing showed that acinar and centroacinar cells express gfi1aa in the exocrine pancreas of zebrafish embryos. Single cell RNA-sequencing and RNA in situ hybridisation experiments showed that the gfi1aa gene is the only member of the Gfi1/b transcription factor family expressed in exocrine pancreatic tissue in zebrafish embryos. β-cells in the endocrine component of the pancreas develop normally in Gfi1 null zebrafish embryos. Intestinal goblet cells were found in null gfi1aasa11633/sa11633 homozygous zebrafish embryos. These data suggested that Gfi1 is not required for early differentiation of acinar and goblet cells in zebrafish at the embryonic stage.19 0Item Restricted Roles of Notch Receptors in Epithelia Cell Fate Selection in Intestinal Development During Embryogenesis of Zebrafish(Saudi Digital Library, 2023-11-27) Allayati, Samah; Wallace, KennethDuring zebrafish embryogenesis, differentiation and specialization of intestinal epithelial cells begin at the larva stage (72 hpf). Notch signaling is used multiple times during intestinal development to specify cell fate decisions and commitment. There are four Notch receptors and five ligands in zebrafish. Notch signals transmit between adjacent cells by lateral inhibition. Activation of Notch receptors by secretory cells initiates differentiation of enterocytes, while loss of Notch signaling in zebrafish intestine results in many additional secretory cells. At Dr. Wallace's lab, they identified a group of intestinal secretory cells at the interfold base that receive Notch signaling (Notch receiving secretory cells NRSCs). The interruption of Notch signaling during embryogenesis results in the elevation of cell proliferation in the interfold region due to the interruption of NRSC production. In this work, we hypothesized that two Notch receptors work redundantly to guide epithelial cell differentiation and production towards secretory cell fate, and simultaneously, two Notch receptors work redundantly to signal NRSC differentiation. To test the hypotheses, simultaneous null mutant embryos were created using previously identified heterozygous mutant adult fish in the four zebrafish Notch genes. Screening of combined null mutant intestines and determining the average number of secretory cells in each null mutant combination allows us to decide which gene combination causes the dramatic increase of secretory cell number in intestinal development during embryogenesis. The Notch receptors control NRSCs during their development, and differentiation was identified using the same null mutants of notch genes. We created double 4 mutant embryos to determine which combination of receptors causes the most dramatic reduction in NRSC numbers during embryogenesis when unavailable. To visualize NRSCs, we used a Ncre zebrafish transgenic line with a Notch responsive Cre driver and responder transgene, which turns on nuclear mCherry to label and fate map Notch receiving cells.9 0Item Restricted Application of fish models for studying mechanisms of human fetal alcohol spectrum disorders (FASDs)(Saudi Digital Library, 2022-12-23) Alsakran, Amena Ali; Kudoh, TetsuIn this project, embryos from two small fish species, zebrafish (freshwater) and Arabian killifish (AKF) (seawater and brackish water), have been used as suitable models to study the mechanism of human fetal alcohol spectrum disorders (FASDs). A. dispar was used as a novel model species for ethanol (EtOH) toxicity research. FASDs are caused by an elevated alcohol level in the pregnant mother’s body. The symptoms of FASDs include microcephaly, holoprosencephaly, craniofacial abnormalities, and cardiac defects with a birth defect in severe cases and milder cases, leading to developmental and learning disabilities. The action of alcohol on embryo development is highly complicated, having many target tissues and cellular events, including gene expression, cell migration, metabolism, cell cycle regulation, DNA replication, cell differentiation and others. Therefore, the actual mechanism of alcohol effect (direct and indirect) on each tissue development is not fully understood. Here, we hypothesised that early cell movement and gene expression change at the gastrula stage might cause direct and indirect consequences to induce later-stage embryonic abnormalities, including microcephaly, eye deformity, short body axis and many other defects. To test this hypothesis, we aim to investigate the mechanism of the impact of alcohol on cell movement and gene expression during the gastrulation stage. Firstly, we overview the studies on FASDs (general introduction in Chapter 1 and In Chapters 3 and 5, we discuss with a specific focus the mechanisms by which alcohol alters cell migration during early embryogenesis, including blastula, gastrula and organogenesis stages, which later cause morphological defects in the brain and other tissues. In Chapter 4, we describe the AKF embryonic staging in detail that was needed to use the species as a novel model for embryonic toxicological studies. Developmental stages of embryos were named with the hour post fertilisation at 28˚C. We found that A. dispar can also tolerate a wide range of temperatures (Chapter 4). Toxicities of EtOH were investigated using zebrafish (Chapter 3) and AKF (Chapter 5). The early embryogenesis data with EtOH exposure were collected from WT and TG strains: WT zebrafish (Danio rerio), TG (H2A:GFP), TG (Gsc:GFP) and Zebrabow. WT Arabian killifish A. dispar, TG (EF1α:Kaede) and TG (beta-actin:Dsred). In the zebrafish, the effects of alcohol were observed in many places during embryo development, from cell lineage-specific gene expression at the blastula/gastrula stage, gastrulation cell movement, morphogenesis of the central nervous system and neuronal development. ACQUIFER multiwall live Imaging Machine microscope was used to image the EtOH treated embryos. With a large data set, quantitative and qualitative analyses were accomplished. The data revealed that EtOH suppresses convergence-extension and epiboly cell movement at the gastrula stage and causes the failure of normal neural plate formation, brain development, eye development and body extension. In addition to the malformation of the body, reduction of the cell pigment (melanophore) was observed with a higher dose of EtOH treated embryos (Chapter 3). The transparent zebrafish embryo offers an ideal model system to investigate the genetic, cellular and organismal response to alcohol. However, there is a limitation in using zebrafish as a model because the short time for independent feeding and irregular shape of the blastoderm cause a compromise of the precise measurement of the epiboly cell movement in the ACQUIFER multiwell imaging with random orientation. Therefore, in this thesis, besides zebrafish, AKF embryos were also used for examining the effect of EtOH (Chapter 5). Finally, a new transgenic line of A. dispar fish was developed using the EF1α:Kaede transgene. We intend to employ it for single-cell tracing by UV photo conversion in embryos with control and EtOH treated. Our data of Kaede fish with Zeiss LSM suggest we can trace and analyse single-cell migration at the gastrula stage. It would further clarify our understanding of the effect of EtOH on different cell movements, including involution, convergence-extension and epiboly cell movement. Overall, these results present that gastrulation cell movement will become a very sensitive, quantitative, and specific biomarker for chemical toxicities. In addition, it has been proved that our novel TG (EF1α:Kaede) AKF would contribute to the study of alcohol toxicity’s effect on cell migration during embryo development. It demonstrates the potential advantages of using this species in future studies of chemical toxicity research.8 0