The nuanced evolutionary consequences of duplicated genes

Gene duplication is postulated to facilitate evolutionary change by providing new genetic material which can gain or lose functions over evolutionary time. There are many instances throughout metazoan evolution which implicate gene duplications as forerunners of innovation. Though often these exist in the literature as implications only. Remarkably, there are still many facets of paralogous gene divergence which are poorly understood. These facets form the basis of questions to be addressed in this thesis. How do new genes acquire new functions? What is the reason for the expansion of certain gene families in certain taxa? Why do some paralogous genes remain redundant for millions of years of evolution? Are paralogues destined to become less essential than the genes from which they were derived? Caenorhabditis elegans and its close relatives represent powerful and tractable models for answering these questions. With their unrivalled suite of genomic and molecular genetic resources, the Caenorhabditis genus will be the stage on which this quest to understand the evolutionary consequences of duplicated genes will play out. But Caenorhabditis nematodes are not the main characters in this story. Our characters, as it were, will be four very different gene families. Here, the fates of duplicated genes are explored in light of the various complexities of paralogue diversification. While gene family size will provide an overall focus for this study, insights garnered from paralogue dynamics within families are expected to illuminate the mechanisms by which gene duplications can be a vehicle for evolutionary change. Factors such as the evolution of domain architecture following tandem duplication, as well as asymmetric gene diversification, overlapping functionality between paralogues, and the role of rapid paralogue divergence in major evolutionary events, such as the formation of new species, will all be explored. In Chapter 3, the taxon-specific dynamism of the T-box family of transcription factors in the Caenorhabditis genus is investigated, with particular focus on the functional evolution of the tbx-35 and tbx-36 gene pair only found in C. elegans. Despite the accumulation of severe deleterious mutations in gene pair among various wild populations, it is shown that the tbx-35 and tbx-36 have each acquired very different roles in the early embryo. Chapter 4 characterises the pervasiveness of redundancy in a slightly smaller, more conserved, gene family. The redundancy relationships in the Warthog family in C. elegans are reconciled with the evolutionary fates members therein have adopted, following which it is seen that particular clades of the family specialise in particular aspects of postembryonic development. Chapter 5 focusses on the consequences (and underpinnings) of asymmetric paralogue divergence for gene family dynamics in the Drd family of oxidoreductases, finding that asymmetric paralogue divergence can be underpinned by the loss of biological function, as well as the gain. Lastly, in Chapter 6, changes to domain architecture following gene duplication — and the associated developmental consequences thereof — are explored through the Myrf family of TFs. It is found that subfunctionalisation, while shown elsewhere in this thesis to occur via regulatory change, can also occur via the complementary degeneration of domain architecture. Taken together, these results characterise hitherto poorly understood aspects of duplicated gene evolution. But this is not to say that this thesis is a collection of just-so stories to try and persuade you of the importance of gene duplication to complex, multifaceted evolutionary processes. It is more than that. Rather, it will be argued that only with the kind of careful genetic approach that is employed in this thesis can we truly understand not only the fates that duplicated genes adopt, but meaningfully comprehend the roles they play in evolution. Together, these findings will provide a new framework for furthering our understanding of the very many evolutionary consequences of duplicated genes

Time-Resolved Quantification of Centrosomes by Automated Image Analysis Suggests Limiting Component to Set Centrosome Size in C. Elegans Embryos

The centrosome is a dynamic organelle found in all animal cells that serves as a microtubule organizing center during cell division. Most of the centrosome components have been identified by genetic screens over the last decade, but little is known about how these components interact with each other to form a functional centrosome. Towards a better understanding of the molecular organization of the centrosome, we investigated the mechanism that regulates the size of the centrosome in the early C. elegans embryo. For this, we monitored fluorescently labeled centrosomes in living embryos and developed a suite of image analysis algorithms to quantify the centrosomes in the resulting 3D time-lapse images. In particular, we developed a novel algorithm involving a two-stage linking process for tracking entrosomes, which is a multi-object tracking task. This fully automated analysis pipeline enabled us to acquire time-resolved data of centrosome growth in a large number of embryos and could detect subtle phenotypes that were missed by previous assays based on manual image analysis. In a first set of experiments, we quantified centrosome size over development in wild-type embryos and made three essential observations. First, centrosome volume scales proportionately with cell volume. Second, beginning at the 4-cell stage, when cells are small, centrosome size plateaus during the cell cycle. Third, the total centrosome volume the embryo gives rise to in any one cell stage is approximately constant. Based on our observations, we propose a ‘limiting component’ model in which centrosome size is limited by the amounts of maternally derived centrosome components. In a second set of experiments, we tested our hypothesis by varying cell size, centrosome number and microtubule-mediated pulling forces. We then manipulated the amounts of several centrosomal proteins and found that the conserved centriolar and pericentriolar material protein SPD-2 is one such component that determines centrosome size

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

An evolutionary perspective on germ cell specification genes in insects

This dissertation investigates the embryonic specification of a specific group of cells: the germ cells. Germ cells, which give rise to sperm and egg, are the only cells in sexually-reproducing animals that directly contribute hereditary information to the next generation. Germ cells are therefore a universal cell type across animals, and represent a profound novelty that likely arose near the base of the animal phylogeny. Yet despite their conserved, essential function in all animals, there is surprising diversity in the mechanisms that specify these cells during embryonic development. In this dissertation, I address the diversity of germ cell specification mechanisms in insects. I focus on two species, the milkweed bug Oncopeltus fasciatus (Hemiptera) and the cricket Gryllus bimaculatus (Orthoptera), which both branch basally to the Holometabola (those insects which undergo metamorphosis, including the well-studied fruit fly Drosophila melanogaster), and thus provide important phylogenetic breadth to our understanding of germ cell specification across insects. Using functional genetic approaches, I show that germ cell specification in both Oncopeltus and Gryllus differs fundamentally from germ cell specification in Drosophila. Specifically, I provide evidence that germ cells arise via inductive cell signaling during mid-embryogenesis, rather than via maternally-supplied cytoplasmic determinants localized in the oocyte, as is the case for Drosophila. These data suggest that Drosophila employs an evolutionarily derived mode of germ cell specification. In further support of this hypothesis, I show that several of the genes required for Drosophila germ cell specification perform other functions in both Oncopeltus and Gryllus. I demonstrate that one of these genes, oskar, which is the only gene both necessary and sufficient for germ cell specification in Drosophila, instead functions in nervous system of the cricket, both during embryonic development and in the adult brain. I suggest that the evolution of the derived mode of germ cell specification seen in Drosophila may have involved co-opting oskar into the germ cell specification pathway from an ancestral role in the nervous system

Feeling the Heat: Investigating the dual assault of Zymoseptoria tritici and Heat Stress on Wheat (Triticum aestivum)

As a result of climate change, field conditions are increasingly challenging for crops. Research has shown how elevated temperatures affect crop performance, yet the impact of temperature on host-pathogen relationships remains unknown. Understanding the effects of combined abiotic and biotic stresses on crop plants and the plant-microbial interaction is crucial in developing strategies to improve crop stress tolerance and manage diseases effectively. Lipids sense, signal, and mitigate temperature elevation effects, and lipid remodelling plays a key role in the plant and fungal response to heat stress. Our study uses a systems approach to examine the Z. tritici wheat model system, combining transcriptomics, lipidomics, and phenotyping to decipher the impact of high-temperature stress on the plant-pathogen interaction. Microscopy in vivo and RNA-Seq analyses confirmed that Z. tritici responds to high-temperature treatments with morphological and transcriptomic changes. Temperature-related configuration of the transcriptome was associated with the accessory chromosomes and expression of ‘accessory’ pan-genome-derived genes. Metabolism-related gene expression predominated, indicated by GO enrichment and analysis of KOG classes, and large-scale lipid remodelling was likely given the proportion of lipid transport and metabolism-related expression changes in response to temperature. Changes in lipid content and composition were then validated by LC-MS analysis. Heat-responsive fungal genes and pathways, including scramblase family genes, are being tested by reverse genetics to ascertain their importance for fungal adaption to elevated temperatures. Elevated temperature schemes were applied to wheat to study the impact of combined stress on the plant-pathogen interaction, based on long-term climate data from Rothamsted Research, using transcriptomic, lipidomic and phenotypic analyses. Comparing non-infected and infected wheat plants under typical and elevated temperatures. Our initial analysis of the transcriptomic data indicates a delay in the development of Z. tritici, followed by its adaptation to the warmer environment. Once the infection was established, the fungus exhibited resilience to the impact of higher external temperatures. Our results indicate that temperature elevations associated with climate change directly impact plant-pathogen interactions. Furthermore, the study demonstrates a need for further detailed understanding to sustain crop resilience

Xenopus

This book focuses on the amphibian, Xenopus, one of the most commonly used model animals in the biological sciences. Over the past 50 years, the use of Xenopus has made possible many fundamental contributions to our knowledge in cell biology, developmental biology, molecular biology, and neurobiology. In recent years, with the completion of the genome sequence of the main two species and the application of genome editing techniques, Xenopus has emerged as a powerful system to study fundamental disease mechanisms and test treatment possibilities. Xenopus has proven an essential vertebrate model system for understanding fundamental cell and developmental biological mechanisms, for applying fundamental knowledge to pathological processes, for deciphering the function of human disease genes, and for understanding genome evolution. Key Features Provides historical context of the contributions of the model system Includes contributions from an international team of leading scholars Presents topics spanning cell biology, developmental biology, genomics, and disease model Describes recent experimental advances Incorporates richly illustrated diagrams and color images Related Titles Green, S. L. The Laboratory Xenopus sp. (ISBN 978-1-4200-9109-0) Faber, J. & P. D. Nieuwkoop. Normal Table of Xenopus laevis (Daudin): A Systematical & Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis (ISBN 978-0-8153-1896-5) Jarret, R. L. & K. McCluskey. The Biological Resources of Model Organisms (ISBN 978-1-0320-9095-5

Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation

Poster number: P-T099 Theme: Neurodegenerative disorders & ageing Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation. References Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7. Cunningham C (2013). Glia 61: 71-90. Heneka MT et al. (2015). Lancet Neurol 14: 388-40

Xenopus

This book focuses on the amphibian, Xenopus, one of the most commonly used model animals in the biological sciences. Over the past 50 years, the use of Xenopus has made possible many fundamental contributions to our knowledge in cell biology, developmental biology, molecular biology, and neurobiology. In recent years, with the completion of the genome sequence of the main two species and the application of genome editing techniques, Xenopus has emerged as a powerful system to study fundamental disease mechanisms and test treatment possibilities. Xenopus has proven an essential vertebrate model system for understanding fundamental cell and developmental biological mechanisms, for applying fundamental knowledge to pathological processes, for deciphering the function of human disease genes, and for understanding genome evolution. Key Features Provides historical context of the contributions of the model system Includes contributions from an international team of leading scholars Presents topics spanning cell biology, developmental biology, genomics, and disease model Describes recent experimental advances Incorporates richly illustrated diagrams and color images Related Titles Green, S. L. The Laboratory Xenopus sp. (ISBN 978-1-4200-9109-0) Faber, J. & P. D. Nieuwkoop. Normal Table of Xenopus laevis (Daudin): A Systematical & Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis (ISBN 978-0-8153-1896-5) Jarret, R. L. & K. McCluskey. The Biological Resources of Model Organisms (ISBN 978-1-0320-9095-5