Genetic and molecular mechanisms of early embryonic patterning in Danio rerio, Oryzias latipes and Kryptolebias marmoratus
Almatwari, Hussein Abed Saud
Date: 24 July 2017
University of Exeter
PhD in Biological Sciences
The aim of this project is to investigate genetic mechanisms of early development of vertebrate embryos using model fish species. Zebrafish (Danio rerio) and medaka (Orizias latipes) have been used extensively for molecular genetics and developmental biology studies because these fish produce many eggs, which can be manipulated from ...
The aim of this project is to investigate genetic mechanisms of early development of vertebrate embryos using model fish species. Zebrafish (Danio rerio) and medaka (Orizias latipes) have been used extensively for molecular genetics and developmental biology studies because these fish produce many eggs, which can be manipulated from the 1 cell stage and are ideally suited for analysing gene expression, function, and embryonic phenotypes. These species have already been extensively used to generate many mutants which show clear phenotypes during early embryonic development. The development of other model species for mutant screening and analyses is likely to provide scope to analyse gene function from uncharacterised/under-characterised genes. Therefore we have developed and tested a small number of early developmental mutants from the mangrove killifish (Kryptolebias marmoratus). To achieve my aim, embryos from zebrafish, medaka and the mangrove killifish have been used as models to study gene function and understand the molecular mechanisms for early patterning genes. We focused in particular on development of neural ectoderm and non-neural ectoderm (epidermis) and anterior-posterior patterning (head, trunk and tail development). As different model animals have different advantages, we used these model animals for different purposes. Zebrafish and medaka were used with chemical treatment (specific inhibitors of target genes) and morpholino analyses because they give many synchronized eggs every morning allowing highly replicated analyses. On the other hand, the mangrove killifish were used for developing and testing novel mutants and associated loss (or gain) of gene function. Firstly, zebrafish was used to study maternal fibroblast growth factor (FGF) signaling at pre-maternal zygotic transition (Pre-MZT) and consequent neural induction at the gastrula stage (Chapter 3). This study found the important role of acquiring maternal FGF signaling in stem cells to achieve neural induction during the zygotic gene expression stage. An FGF signaling inhibitor SU5402 was tested using RNA–seq, ATAC-seq, in.situ hybridization and immunohistochemistry methods. Through these techniques, we found that the maternal FGF signaling provides competence to the ectodermal stem cells for neural induction possibly via epigenetic modification of histone trimethylation. To examine the role of a specific FGF molecule (FGF2), gene knockdown was conducted to study fgf2 gene function during early development in zebrafish (Chapter 4). In situ hybridization and immunostaining with tissue-specific markers at the gastrula stage were used to discover a novel role for fgf2 in development of the epidermis. The final stage of my project involved characterization of mutations underlying two mutant phenotypes (short tail/stl and ball tail/stl), that exhibit defects in tail development using the self-fertilizing mangrove killifish (Chapter 5). Using a small scale RNA-seq, the mutated genes responsible for the stl and btl mutations were instantly identified as noto and msgn1 respectively. The mutant phenotype was phenocopied by morpholino injections in medaka. This study revealed crucial roles of the two genes in tail bud development. Defects of these genes affected the motility of progenitor cells in the tail bud by suppressing cell translocation to the axial mesoderm in the noto mutation and to the paraxial mesoderm in the msgn1 mutation. The study demonstrated similarity of gene function and redundancy in the mangrove killifish and medaka that is different from the function of these genes in zebrafish, revealing the importance of research on different model animals to fully characterise the gene function. From these data, it can be considered that mangrove killifish is very powerful model for mutation screening, suggesting that this animal model can be applied in various genetic studies alongside or in addition to other vertebrate models.
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