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Wnt Signalling and Peroxisome Dynamics in the Zebrafish (Danio Rerio)

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Wnt Signalling and Peroxisome Dynamics in the Zebrafish (Danio Rerio) Lauren Porter 620007286 Wnt signalling and peroxisome dynamics in the zebrafish (Danio rerio) Submitted by Lauren Porter to the University of Exeter As a thesis for the degree of Masters by Research in Biosciences On 20th September 2019 This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. I certify that all the material in this thesis which is not my own work has been identified and that no material has been previously submitted and approved for the award of a degree by this or any other University. Signature …………………………………………………. 1 Lauren Porter 620007286 Abstract Cell-cell or paracrine signalling is a form of cellular communication in which a cell produces a signal that influences the behaviour of neighbouring cells, which is important because it allows for the local coordination of the activities of groups of cells. This coordination is indispensable during development; for example, paracrine Wnt signalling is fundamental to body patterning in all metazoans where it helps to determine cell fate in a developing embryo. Wnt can regulate the transcription of target genes including cyclin and peroxisome-proliferator activated receptor-ẟ. The importance of Wnt signalling is not temporally limited, and Wnt has roles throughout the life of an organism such as the management of stem cells and the cellular abundance of mitochondria, the ‘sister organelle’ of the peroxisome. The peroxisome is a single membrane-bound organelle with diverse roles in healthy development and life, inclusive of the breakdown of very long chain fatty acids (VLCFAs) and the production of plasmalogens for efficient nervous conduction. The relationship between the Wnt signalling pathway and peroxisomes is unknown. Here I investigate the influence of Wnt signalling on peroxisome dynamics in zebrafish. To do so, canonical wnt8a was knocked out and knocked down using the genomic engineering tool CRISPR and Morpholino oligomers. The number and morphology of peroxisomes was observed in Wnt8a- deficient zebrafish embryos and appeared to be aggregated and less numerate than in wild type zebrafish. Consistently, in zebrafish embryos overexpressing wnt8a, peroxisomes were visualised as highly numerate singular puncta. I hypothesise that - in addition to a set of functions in development and tissue homeostasis - Wnt signalling has a novel role in regulating peroxisome proliferation in zebrafish. 2 Lauren Porter 620007286 Abstract 2 1.0 Introduction 6 1.1 The history of Wnt discovery 9 1.2 The canonical Wnt pathway 11 Figure 1: A schema of the ꞵ-catenin pathways triggered by the reception of Wnt at the cell membrane, and the genes targeted (Strutt, 2003). 14 1.3 Wnt signalling and its roles in model organisms 16 1.4 Transport of Wnt proteins 18 1.5 The wnt8a locus 20 Figure 2: A representation of the wnt8a locus, adapted from Wylie and colleagues (2014). 21 1.6 Role of wnt8a in zebrafish 21 1.7 Zebrafish as an experimental model 23 1.8 Peroxisomes in health and disease 24 1.9 Peroxisome dynamics 26 Figure 3: The mechanism of growth and division from pre-existing peroxisomes. 28 1.10 Following PEX and Wnt through developmental trajectories 29 1.11.0 CRISPR/CRISPRi 31 1.11.1 CRISPR in comparison to other knock-out strategies 33 Table 1: A comparison of Morpholino oligomers, TALENs, BACs, CRISPR and CRISPRi for manipulating gene transcription. 36 1.11.2 CRISPRi - a flexible method of knocking down genes 36 1.12 Summary of aims and objectives 40 2.0 Materials and Methods 40 2.1 Cas9 and dCas9 retransformation and transcription 40 2.2 Zebrafish maintenance, husbandry and spawning 42 2.3 Sequencing the wnt8a locus 43 2.4 gRNA design 44 Table 2: gRNA target sites in wnt8a. PAM sequence is underlined and bolded. 45 Figure 4: Representation of wnt8a1 and wnt8a2. 46 2.5 Phenotypic Analyses 46 Table 3: Morpholino target sites on open reading frames 1 and 2 of wnt8a. 47 2.6 In situ hybridisation 48 2.7 RT-qPCR 48 Table 4: Primer sequences against target genes indicated in the left column. 50 3 Lauren Porter 620007286 2.8 Cell culture and peroxisome visualisation 51 2.9 Visualising peroxisomes in live embryos 52 Table 5: Composition of injection mixes injected into zebrafish embryos. 55 3.0 Results 55 3.1 Testing dCas9 and Cas9 RNA with tyrosinase gRNA 55 Figure 5: Zebrafish embryos with CRISPR and CRISPRi targeting tyrosinase. 56 Figure 6: Quantification of pigmented area between the MHB and 1st somite of wild type control embryos. 57 3.2 In vitro digestion using wnt8a gRNAs 57 Figure 7: In vitro digestion of zebrafish wnt8a1 DNA after CRISPR. 58 3.3 Phenotypic analysis of injected embryos 58 Figure 8: Representation of phenotypes generated by injection. 61 Figure 9: Quantification of embryos grouped into 3 categories; WT, mild and severe. 62 Figure 10: Phenotypes of zebrafish embryos injected with Morpholino oligomer targeted to wnt8a. 63 3.4 Visualising the expression patterns of wnt8a1 and wnt8a2 at 70-80% epiboly 63 Figure 11: In situ hybridisation of zebrafish embryos at 70-80% epiboly using a probe against wnt8a1. 65 Figure 12: In situ hybridisation of zebrafish embryos at 70-80% epiboly using a probe against wnt8a2. 66 3.5 Quantifying wnt8a1 and wnt8a2 expression at 70-90% epiboly 66 Figure 13: Expression of wnt8a after targeting with CRISPR or CRISPRi. 68 Figure 14: Primer detection of wnt8a overexpression. 68 Figure 15: Expression of downstream Wnt target genes after targeting wnt8a with CRISPR or CRISPRi. 69 Figure 16: Impact of injection of endonuclease RNA without gRNA. 70 3.6 Peroxisomes visualised in human AGS cells 70 Figure 17 A: Human AGS cell (top) and zebrafish PAC2 cell (bottom) in culture transfected with SKL-GFP (green) and membrane-associated mCherry (red). 72 Figure 17 B: Human AGS cells transfected with SKL-GFP (green) and membrane- associated mCherry (red) and treated with BIO (2.5 µm, top) and IWR1 (25 µm, bottom) 72 Figure 18: Quantification of the average number of peroxisomes per cell in human AGS cell culture. 73 3.7 Peroxisomes visualised in vivo in zebrafish embryos 73 Figure 19: Peroxisome-like structures (green) in the live zebrafish embryo. 74 3.8 Peroxisomes in embryos injected with CRISPR/CRISPRi constructs 74 Figure 20: Peroxisomes (green) in zebrafish embryos with varying levels of wnt8a expression. 79 4 Lauren Porter 620007286 Figure 21: The proportion of peroxisome-like structures per cell in control, Morpholino, wnt8a RNA overexpression and Cas9 + wnt8a gRNA embryos. 80 Figure 22 A: The distribution of sizes of peroxisome-like structures within live uninjected zebrafish embryos (control sample). 81 Figure 22 B: The distribution of sizes of peroxisome-like structures within live zebrafish embryos injected with Morpholino against wnt8a ORF1 and ORF2. 81 Figure 22 C: The distribution of sizes of peroxisome-like structures within live zebrafish embryos injected with wnt8a RNA. 82 Figure 22 D: The distribution of sizes of peroxisome-like structures within live zebrafish embryos injected with CRISPR Cas9 and gRNA targeted to wnt8a ORF1 and ORF2. 82 4.0 Discussion 83 4.1 Overview of the Wnt-peroxisome relationship 83 4.1.1 Wnt signalling and organelles 84 4.2 Peroxisomes and peroxisomal diseases 87 4.3 The function of peroxisome proliferator activated receptors - PPARs 89 4.4 Wnt8a and its role in zebrafish 91 4.5.0 Phenotypic changes in zebrafish embryos injected with CRISPR/CRISPRi constructs 94 4.5.1 Targeting tyrosinase with CRISPR and CRISPRi 95 4.5.2 Designing gRNA to target wnt8a with CRISPR and CRISPRi 95 4.5.3 Measures taken for controls 96 4.5.4 Implementation of CRISPR and CRISPRi to target wnt8a and phenotypic observations 97 4.5.5 Comparison between targeting the first and second open reading frames of wnt8a 99 4.5.6 Comparison between targeting wnt8a with CRISPR or CRISPRi 101 4.6 Changes in expression pattern of wnt8a at epiboly 102 4.7 Quantifying changes in expression levels of wnt8a downstream genes 106 4.8 Peroxisomes in human cell culture 111 4.9 Peroxisomes in vivo in zebrafish embryos with varying levels of wnt8a expression 115 4.10 Future Studies 119 5.0 Conclusion 125 Acknowledgements 127 References 128 5 Lauren Porter 620007286 Abbreviations acox1 – acyl-CoA oxidase 1 AGS – gastric adenocarcinoma AP – antero-posterior APC - adenomatous polyposis coli protein apoa1 – apolipoprotein A1 BACs – bacterial artificial chromosomes Bcl9 – B-cell CLL/lymphoma protein BIO - 6-bromoindirubin-3’-oxime BMP – bone morphogenetic protein βTrCP – β-transducin repeats-containing proteins CBP – CREB-binding protein Cdc42 – cell division control protein 42 homolog CKIa – casein kinase Ia CLEM – correlative light-electron microscopy CRISPR – clustered regularly spaced palindromic repeats CRISPRi - CRISPR interference crRNA – CRISPR ribonucleic acid dCas9 – deactivated Cas9 enzyme Dkk-1 – dickkopf 1 DLP1 – dynamin-like protein 1 DMSO – dimethyl sulfoxide DSB – double-strand break Dvl – dishevelled EGF-CFC – epidermal growth factor-Cripto-FRL1-Cryptic EGFR – epidermal growth factor receptor ER – endoplasmic reticulum ERK1/2 – extracellular signal-regulated kinase 1/2 fgf8 – fibroblast growth factor 8 6 Lauren Porter 620007286 fis1 – mitochondrial fission 1 gbx1 – gastrulation brain homeobox 1 gDNA – genomic DNA gRNA – guide ribonucleic acid GSK-3 – glycogen synthase kinase 3 GFP – green fluorescent protein GTP – guanosine triphosphate hpf – hours post fertilisation hPSC – human pluripotent stem cell IRD – Infantile
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