FUNCTIONAL GENETIC APPROACHES TO PROVIDE EVIDENCE FOR THE ROLE OF TOOLKIT GENES IN THE EVOLUTION OF COMPLEX COLOR PATTERNS IN DROSOPHILA GUTTIFERA

Toolkit genes are set of genes that orchestrate the development of basic body plan of animals, and they are highly conserved in all animals. The co-option of the toolkit genes into the pigmentation pathway has led to the evolution of novel species. This study focuses on understanding how the complex color patterns in animals develop by using the Drosophila species in the quinaria group as models. We developed an mRNA in situ hybridization (ISH) protocol, which allowed us to study gene expression patterns in the abdomen of developing pupae of non-model Drosophila species (Chapter 2). Through ISH, we found that the pigmentation gene y foreshadowed the adult D. guttifera abdominal pattern. Following the discovery of the wingless gene’s (a toolkit gene) expression on the wings of D. guttifera by my advisor, Dr. Thomas Werner, we used ISH to screen 110 putative toolkit genes in the abdomen of D. guttifera to identify putative upstream activators of the pigmentation gene y. We identified five toolkit genes, wingless (wg), hedgehog (hh), abdominal-A (abd-A), decapentaplegic (dpp), and zenknullt (zen) that may collectively orchestrate the patterning in the abdomen of D. guttifera. Using the transgenic technique for modifying non-model Drosophila species (Chapter 1), Dr. Raja Komal (a former Ph.D. student in our lab) deployed the reporter assay technique to investigate the cis-regulatory elements (CREs) that control y gene expression. Dr. Raja showed that only one CRE (gut y spot CRE) controls all six rows of spots, and that this CRE contains a stripe-inducing CRE at its core. In an attempt to provide direct genetic evidence for the roles of toolkit genes in complex pattern formation, we ectopically expressed the cDNAs of the toolkit genes, using the gut y stripe CRE as a driver for gene overexpression. Our intention was to manipulate the adult color pattern on the D. guttifera abdomen to change it into a stripe pattern. Unfortunately, the gut y stripe CRE could not drive the toolkit genes’ expression at a time when it matters for the induction of pigmentation. Unfortunately, no earlier acting abdominal CRE is available for this species to ectopically express our toolkit genes. Furthermore, we performed RNAi knockdown experiments of the toolkit genes, which resulted in gross developmental abnormalities in the fruit flies, causing them to die before the pigmentation patterns could be observed. We suggest that this outcome may be due to the vital roles that these developmental genes play at every stage of Drosophila development. To understand how novel animal color patterns evolved, it is essential to query how the formation of color patterns varies among closely related species. Therefore, we compared the expression patterns of terminal pigmentation genes (Dopa decarboxylase (Ddc), tan (t), and yellow (y)) in three fruit fly species in the quinaria species group – D. guttifera, D. palustris, and D. subpalustris. Our results show that the genes y, t, and Ddc are co-expressed in modular and identical patterns in the pupal abdomens in each species, which correlate with the adult abdominal pigmentation pattern

Investigation into the cohesin loading complex of Caenorhabditis elegans

Sister chromatid cohesion is important to ensure faithful chromosome segregation occurs during mitosis and is mediated by the multisubunit cohesin complex. In Saccharomyces cereviside the cohesin complex is loaded onto chromosomes by a protein complex composed of Scc2 and Scc4. Caenorhabdifis elegans PQN-85 is readily identified as the orthologue of Scc2 and the sequence similarity between PQN-85 and its orthologues from all organisms suggests that their function in cohesin loading is conserved throughout evolution. Data presented in this thesis shows that in accordance with its suspected role in cohesin loading RNA interference against pqn-85 results in chromosome segregation defects in C elegans embryos similar to those observed when the cohesin subunit scc-3 is depleted. Although it has been problematic to identify homologues of Scc4 in metazoans, it has recently been proven that C elegans MAU-2 is distantly related to Scc4. Co-depleting scc-3 with either pqn-85 or mau-2 result in exacerbation of the chromosome segregation defects with very similar phenotypes suggesting that mau-2 as well as pqn-85 functions in chromosome segregation. Data is also presented demonstrating that MAU-2 physically interacts with the N-terminus of the PQN-85 protein in a directed yeast two-hybrid assay consistent with the idea that PQN-85 and MAU-2 form a complex. Microscopic examination of mau-2:: GFP embryos in this thesis demonstratesth at MAU-2:: GFP localises to the nucleus in accordancew ith a role in cohesin loading, whilst depletion of pqn-85 by RNAi in mau-2:: GFP embryos resulted in the reduction of MAU-2 :: GFP within the nucleus and concurrent accumulation within the cytoplasm. Together these results suggest that the cohesin loading complex is conserved in C elegans whilst the extensive homology between PQN-85, MAU-2 and their metazoan orthologues suggests that it is also likely that the cohesin complex is conserved amongst all metazoans.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

GENE EDITING OF DND BY TALENS IN MEDAKA EMBRYOS AND ITS ROLE IN PGC FORMATION

Ph.DDOCTOR OF PHILOSOPH

A transgenic mouse with PMP-22 directed GFP expression - a model for schwann cell behaviour

In the peripheral nervous system the myelin sheath is produced by the spiral wrappings of the Schwann cell (SC) membrane around the axon. This provides insulation and increases the velocity of impulse propagation. The structure of myelin is maintained by a group of myelin proteins. Peripheral myelin protein-22 (PMP-22) is a 22 KDa glycoprotein, originally identified following nerve crush injury, that is found within SCs and is identical to the growth arrest specific protein GAS-3. The PMP-22 gene is regulated by two alternative promoters immediately upstream of two alternative non-coding exons. In order to study temporal and spatial expression of the PMP-22 gene and regulation of SC ensheathment and myelination, a transgenic mouse expressing the green fluorescent protein (GFP) driven by the myelin specific PMP-22 promoter was produced. To achieve this the PI promoter isolated from genomic DNA was initially incorporated into a plasmid containing the EGFP gene. In vitro transfection studies demonstrated appropriate expression of EGFP fluorescence. Microinjection of the transgene into pre- implantation fertilised embryos gave rise to three transgenic lines as confirmed by Southern blot and PGR. One founder expressed the transgene in a tissue specific manner. Mosaicism of expression both within an individual and between individuals was noted. In vitro manipulations showed that the expression patterns observed were independent of axonal contact and myelination but could be influenced by the extracellular matrix. These GFP expressing transgenic mice potentially provide a means to determine the dynamics of SC-axon interactions during myelination and the behaviour of transplanted SCs into myelin deficient regions and the SCs response to injury. Preliminary reports of this work are found in abstract form: British Neurosci. Assoc. Abstr., Vol 15, pi04, 1999

Efficiency and safety of gene editing in pigs: Evaluated in an experimental and in a disease relevant model

In this thesis, the feasibility, efficiency, and safety of gene editing technology were analysed in the pig. Gene editing has the potential to complement traditional breeding techniques and help improve animal health and welfare. However, concerns regarding the safety of this new technology prevent its application outside of research. Because pigs are physiologically very similar to humans and play an important role in agriculture, they are a suitable model to analyse the potential and risks of gene editing for applications in agriculture and potential therapeutic applications in patients. In this study the FUT1 gene which mediates resistance to Oedema disease was edited in vitro in somatic cells and in vivo in porcine embryos. The desired base specific G>A nucleotide exchange at position 307 was performed precisely and efficiently using CRISPR/Cas9 expression vectors in combination with ssDNA repair templates. The generation of OD resistant animals by GE was not planned within the scope of this project. The second part of this project focused on the investigation of off-target mutations caused by CRISPR/Cas9 technology, the most widely used GE tool. One blastomere of porcine two-cell-stage embryos was microinjected with CRISPR/Cas9 expression vectors to generate mosaic foetuses. The edited and non-edited cell populations from two mosaic foetuses were separated and analysed by whole genome sequencing. This approach facilitates the differentiation between natural mutations that occur during embryogenesis and off-target mutations caused by gene editing technology. The frequency of SNVs in embryos edited by CRISPR/Cas9 was higher than expected but within the spontaneous mutation rate. Because none of the mutations aligned with the predicted off-target sites they are likely artefacts caused by PCR amplification during library generation. A more precise evaluation of off-target mutations could be obtained by avoiding the cell culture step and by utilising PCR-free Next Generation Sequencing technology. Cells from the same embryo are the best possible control group but a final distinction between off-target effects and natural mutations remains difficult because even these have certain genomic differences.In dieser Arbeit wurde die Machbarkeit, Effizienz und Sicherheit der Genom-Editierung beim Schwein analysiert. Genom Editierung könnte in Zukunft traditionelle Züchtungsmethoden ergänzen und somit zur Verbesserung der Tiergesundheit und des Tierwohls beitragen. Allerdings verhindern Zweifel bezüglich der Sicherheit dieser neuen Technologie ihre Anwendung außerhalb der Forschung. Schweine sind Menschen physiologisch sehr ähnlich und spielen eine wichtige Rolle in der Landwirtschaft. Deshalb sind sie ein geeignetes Model, um die potenziellen Risiken der Genom-Editierung vor einer möglichen Anwendung in der Landwirtschaft oder Humanmedizin zu erforschen. Hier wurde das FUT1 Gen, welches die Resistenz gegenüber der Ödemkrankheit vermittelt in vitro in somatischen Zellen und in vivo in Schweine Embryos editiert. Der erwünschte G>A Basenaustausch an Position 307 konnte präzise und effizient durchgeführt werden. Hierzu wurden CRISPR/Cas9 Expressionsvektoren in Verbindung mit einzelsträngigen DNA-Reparaturvorlagen verwendet. Die Generierung gegen Ödemkrankheit resistenter Tiere mittels Genom-Editierung war im Rahmen dieses Projektes nicht vorgesehen. Der zweite Teil dieses Projektes beschreibt die Erforschung von Off-Target Mutationen durch CRISPR/Cas9, das am häufigsten Verwendete Werkzeug für die Genom-Editierung. Eine Blastomere eines Zwei-Zell Embryos wurde mit CRISPR/Cas9 Expressionsvektoren mikroinjiziert, um mosaike Föten zu erzeugen. Die editierten und nicht editierten Zellpopulationen wurden separiert und durch Sequenzierung des gesamten Genoms analysiert. Diese Vorgehensweise erlaubt eine Abgrenzung zwischen natürlichen Mutationen, welche während der Embryogenese entstehen und jenen, welche durch die Genom-Editierung verursacht werden. Die Rate an Polymorphismen in den editierten Embryos war höher als erwartet, lag aber innerhalb der spontanen Mutationsrate. Keine der Mutationen stimmte mit den vorhergesagten off-target Sequenzen überein. Vermutlich handelte es sich hierbei um Artefakte, welche durch die PCR Amplifikation zur Erstellung der Sequenzierungsbibliothek verursacht wurden. Eine präzisere Evaluierung von Off-Target Mutationen könnte in Zukunft durch Vermeidung des Zellkultur Schrittes und durch Verwendung PCR-freier Sequenzierungstechnologie erreicht werden. Zellen vom selben Embryo stellen die bestmögliche Kontrollgruppe dar, doch selbst diese weisen gewisse genomische Unterschiede auf. Deshalb bleibt eine klare Abgrenzung zwischen natürlichen und off-target Mutationen schwierig

Cloning and characterisation of SSV genes from Saccharomyces Cerevisiae

In order to investigate the pleiotropic effects exhibited by mutations of S. cerevisiae defective in genes involved in vacuole biogenesis, a number of mutants were chosen for further study. Three mutants which had been isolated for salt sensitivity (ssv mutants) also exhibited defects in a number of unrelated functions including protein targeting and vacuole inheritance. Attempts were made to clone the corresponding wild type genes defined by these mutations. Plasmid pMLY38 was thought to confer an advantageous growth effect on the ssv7-l mutant strain, complementing the salt sensitivity and enhancing its growth on YPDA media supplemented with 1.5M salt. Further analysis of growth curves indicated that pMLY38 did not enhance the growth of ssv7-l. Subsequent sequencing indicated that pMLY38 contained the SSV7 gene. However, an open reading frame (ORFl) was detected downstream of SSV7 and the sequence was analysed. A gene disruption of ORFl indicated that it was not an essential gene. Computer analysis of the DNA sequence showed that it encoded a 310 amino acid protein which has a highly acidic C-terminal end. Searches of the data bases showed limited homology to SCYKL202W1 and Nucleolar Transcription Factor 1, the significance of which is unknown. Characterisation of ssv 16 strains indicated cells with class B vacuoles. Immunoprecipitation results showed the mutants also secrete large quantities of CPY. They also display a semi-dominant phenotype for CPY mis-sorting. In the mutant/wild type diploid CPY continues to be secreted indicating that the mutant phenotype is not completely suppressed by the wild type gene. Cloning of SSV16 isolated the previously sequenced gene, SISl. A study of the growth curves, secretion of CPY and chromosome mapping of SSV16 failed to ascertain whether SISl is identical to SSV16.Immunofluorescence of the ssv 17-101 strains indicated that it belongs to a small group of mutants defective in vacuole segregation. Four transformation methods were employed in order to clone SSVl 7 via complementation from a gene bank. Difficulties encountered with these methods resulted in a chromosome mapping technique being employed to locate the gene. This placed the SSV17 gene on chromosome XIV. Attempts to narrow down the position on the chromosome by meiotic mapping were unsuccessful. Information that a VACl homologue was present on chromosome XIV led to its cloning by PGR in order to determine if it complemented the ssvl7-101 mutation