Genetic Pathways Controlling CNS Development the Role of Notch Signaling in Regulating Daughter Cell Proliferation in Drosophila

Linköping University Medical Dissertations No. 1542

Genetic pathways controlling CNS development The role of Notch signaling in regulating daughter cell proliferation in Drosophila

Caroline Bivik Stadler

Department of Clinical and Experimental Medicine Faculty of Medicine and Health Sciences Linköping University, SE-581 85 Linköping, Sweden

Linköping 2016

Front cover: Confocal image of a Drosophila CNS, immunostained with the neural daughter cell marker Prospero (green), the NB5-6 marker lbe(k)-lacZ (ß-gal; blue) and a PH3 marker for mitosis (red).

Published articles I-IV have been reprinted with permission of the copyright holders.

Printed in Sweden by LiU-Tryck, Linköping, Sweden, 2016

ISBN: 978-91-7685-665-9 ISSN: 0345-0082

“Be stubborn about your goals, and flexible about your methods” SUPERVISOR Stefan Thor Department of Clinical and Experimental Medicine Faculty of Medicine and Health Sciences Linköping University, Sweden

CO-SUPERVISOR Jan-Ingvar Jönsson Department of Clinical and Experimental Medicine Faculty of Medicine and Health Sciences Linköping University, Sweden

FACULTY OPPONENT Allison Bardin Department of Developmental Biology and Cancer Institut Curie Paris, France ABSTRACT

The human central nervous system (CNS) displays the greatest cellular diversity of any organ system, consisting of billions of neurons, of numerous cell sub-types, interconnected in a vast network. Given this enormous complexity, decoding the genetic programs controlling the multistep process of CNS development remains a major challenge. While great progress has been made with respect to understanding sub-type specification, considerably less is known regarding how the generation of the precise number of each sub-type is controlled. The aim of this thesis was to gain deeper knowledge into the regulatory programs controlling cell specification and proliferation. To address these questions I have studied the Drosophila embryonic CNS as a model system, to thereby be able to investigate the genetic mechanisms at high resolution. Despite the different size and morphology between the Drosophila and the mammalian CNS, the lineages of their progenitors share similarity. Importantly for this thesis, both species progenitors show elaborate variations in their proliferation modes, either giving rise to daughters that can directly differentiateinto neurons or glia (type 0), divide once (type I), or multiple times (type II). The studies launched off with a comprehensive chemical forward genetic screen, for the very last born cell in the well-studied lineage of progenitor NB5-6T: the Ap4 neuron, which expresses the neuropeptide FMRFa. NB5-6T is a powerful model to use, because it undergoes a programmed type I>0 daughter cell proliferation switch.An FMRF-eGFP transgenic reporter was utilized as readout for successful terminal differentiation of Ap4/FMRFa and thereby proper lineage progression of the∼20 cells generated. The strongest mutants were mapped to genes withboth known and novel essential functions e.g., spatial and temporal patterning, cell cycle control, cell specification andchromatin modification. Subsequently, we focused on some of the genes that showed a loss of function phenotype with an excess of lineage cells. We found that Notch is critical for the type I>0 daughter cell proliferation switch in the NB5-6T lineage and globally as well. When addressing the broader relevance of these findings, and to further decipher the Notch pathway, we discovered that selective groups of E(spl) genes is controlling the switch in a close interplay with four key cell cycle factors: Cyclin E, String, E2F and Dacapo, in most if not all embryonic progenitors. The Notch mediation of the switch is likely to be by direct transcriptional regulation. Furthermore, another gene identified in the screen, sequoia, was investigated. The analysis revealed that sequoia is also controlling the daughter cell switch in the CNS, and this partly through context dependent interactions with the Notch pathway. Taken together, the findings presented in this thesisdemonstrate that daughter cell proliferation switches in Drosophila neural lineages are genetically programmed, and that Notch contributes to the triggering of these events. Given that early embryonic processes is frequently shown to be evolutionary conserved, ou y can speculate that changeable daughter proliferation programs could be applied to mammals, and contribute to a broader understanding of proliferation processes in humans as well.

POPULÄRVETENSKAPLIG SAMMANFATTNING

DET CENTRALA NERVSYSTEMETS CELLANTAL KONTROLLERAS AV GENETISKA REGLERPROGRAM Vårt centrala nervsystem (CNS) utgörs av hjärnan och ryggmärgen. Det är det mest komplexa organet i vår kropp, bestående av miljarder av celler , var och en med en specifik uppgift, interagerande i enorma nätverk. Tidigt under utvecklingen finns omogna celler, stamceller, som är stamfäder till alla de celltyper som bygger uppvåra organ. Dessa stamceller har den unika egenskapen att kunna dela sig på så sätt att de dels bildar förnybara kopior av sig själva och dels nya celltyper med mer specifika funktioner, såsom de nervceller som styr våra muskler eller de som tar emot känselsignaler. Det finns fortfarande många outforskade genetiska program i vårt DNA som bestämmer när, var, till vad och hur många gånger dessa stamceller ska dela sig. Vidare dikterar dessa reglerprogram för de specifika celltyperna, vilken uppgift och vilken funktion de ska få. Syftet med avhandlingsarbetet var att fördjupa kunskapen kring de genetiska reglerprogram i CNS som kontrollerar att rätt celltyperproduceras i rätt antal, vilket resulterar i att hjärnan varken blir för liten, växer sig för stor eller bildar cancertumörer av celler. Då den mänskliga hjärnan består av hundra miljarder celler valdes den mindre och mer lättstuderade bananflugan Drosophilas embryonala CNS med cirka 15 000 celler som modellsystem. I sin basala uppbyggnad harDrosophilas CNS många likheter med däggdjurens, i synnerhet under den tidiga utvecklingen. Vidare har majoriteten av de gener som studerats i denna avhandling en tydlig motsvarighet hos däggdjuren. Avhandlingsarbetet bygger på forskningsresultat presenterade i fyra separata publikationer. I det första projektet genomfördes en genetisk screen där Drosophilas DNA punktförstördes (muterades) slumpmässigt med kemikalien EMS. I denna screen fann vi en mängd nya reglergener som är essentiella för utvecklingen av en enda stamcell i CNS och den grupp av specifika nervceller som denna ger upphov till. Vi valde att fortsätta studera en av de identifierade generna, benämnd Notch, mer i detalj. Denna gen står för en viktig, hittills okänd, kontrollmekanism i CNS; Notch styr antalet producerade nervceller som en stamcell ger upphov till genom att påverka celldelningen. Vi visade även att flera reglerprogram kan verka sida vid sida i stamcellen. Fascinerande nog räcker det att endast eliminera två kontrollmekanismer samtidigt för att stamcellen helt ska förlora kontrollen över sin cellproduktion och istället för normal vävnad generera ett stort tumörliknande kluster av celler. Notch utgör denna kontrollerande funktion i hela . CNS Grundliga analyser visade på att genen arbetar genom att hämma stamcellens livscykelgenom repression av cellcykelfaktorer via selektivt aktiverande av en genfamilj kallad E(spl)-C. Resultaten presenterade i avhandlingen har bidragit till ökad förståelse för hur reglergener arbetar med att kontrollera cellantal på molekylär nivå i Denna CNS. information kan hjälpa till i sökandet efter potentiella angreppspunkter för utvecklingen av läkemedel mot nerv-, utvecklings- och cancersjukdomar.

TABLE OF CONTENTS

List of papers ...... 11 Abbreviations ...... 13 Introduction ...... 15 Developmental neurobiology ...... 15 All cells arise from one single stem cell ...... 15 The same DNA but different gene expression ...... 16 Cancer is uncontrolled cell proliferation ...... 16 The vertebrate central nervous system (CNS) ...... 18 Neurons and glia ...... 18 Development of the CNS ...... 19 Control mechanisms in development ...... 20 Drosophila melanogaster as a model system ...... 22 Genetic similarities between human and Drosophila ...... 22 Development of the Drosophila VNC ...... 22 Specification of the neuroectoderm ...... 23 Lateral inhibition ...... 24 Proliferation through asymmetric division ...... 25 Three types of neuroblast proliferation modes ...... 27 Binary lineage decisions ...... 29 Temporal progression ...... 30 The cell cycle ...... 31 Cell cycle exit ...... 32 Notch signaling pathway ...... 34 Multiple functions of Notch signaling ...... 34 Notch dosage sensitivity and disease ...... 34 The canonical Notch signaling pathway ...... 35 The Notch receptor ...... 36 Nuclear events in Notch signaling ...... 39 Regulation of the Notch signaling pathway ...... 42 Cis and trans signaling ...... 42 Degradation of ligand and receptor ...... 42 Glycosylation of the Notch receptor ...... 43 Modifications of the chromatin landscape ...... 43 Aims of the Thesis ...... 45 Methods ...... 47 Immunohistochemistry (IHC) ...... 47 Microscopy ...... 48 Fluorescence ...... 48 Confocal laser scanning microscopy ...... 48 Image analysis ...... 49 Detection and analysis of DNA-protein interactions...... 49 Chromatin immunoprecipitation (ChIP) ...... 49 DNA adenine methyltransferase identification (DamID) ...... 50 Characterizing EMS-induced mutations by WGS ...... 50 Next generation sequencing (NGS) ...... 51 Manipulating the genome ...... 51 The Gal4-UAS system ...... 51 EdU labeling ...... 52 Statistical analysis...... 52 Results and Discussion ...... 53 Paper I ...... 53 Paper II ...... 56 Paper III ...... 58 Paper IV ...... 61 Conclusions ...... 63 Future perspectives ...... 65 Acknowledgements ...... 67 References ...... 69 LIST OF PAPERS

LIST OF PAPERS

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals (I-IV):

PAPER I Caroline Bivik, Shahrzad Bahrampour, Carina Ulvklo, Patrik Nilsson, AnnaAngel, Fredrik Fransson, Erika Lundin, Jakob Renhorn and Stefan Thor. Novel genes involved in controlling specification ofDrosophila FMRFA neuropeptide cells. Genetics, 2015 Aug; 200(4):1229-44.

PAPER II Carina Ulvklo, Ryan MacDonald, Caroline Bivik, Magnus Baumgardt, Daniel Karlsson and Stefan Thor. Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression. Development, 2012 Feb; 139(4): 678-89.

PAPER III Caroline Bivik, Ryan MacDonald, Erika Gunnar and Stefan Thor. Control of neural daughter cell proliferation by multi-level Notch/Su(H)/E(spl)-HLH signaling. PLoS Genet, 2016 Apr; 12(4).

PAPER IV Erika Gunnar, Caroline Bivik, Annika Starkenberg and Stefan Thor. sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system. Development, 2016 Oct; 143(20):3774-3784.

11 12 ABBREVIATIONS

ABBREVIATIONS

Ap Apterous A-P Anteroposterior bHLH Basic helix-loop-helix CDK Cyclin dependent kinase ChIP Chromatin immunoprecipitation CNS Central nervous system CSL CBF1/RBP-J (mammals), Suppressor of hairless (Drosophila) and Lag1 (C.elegans) CycE Cyclin E DamID DNA adenine methyltransferase identification Dap Dacapo (mammalian p21cip1/ p27kip1, p57kip2) Dl Delta DSL Delta-Serrate-Lag2 D-V Dorsoventral eGFP Enhanced green fluorescent protein E(spl)-C Enhancer of split complex FMRFa FMRFamide GMC Ganglion mother cell HES Hairy and enhancer of split Mam Mastermind NB Neuroblast NECD Notch extracellular domain NICD Notch intracellular domain NGS Next generation sequencing Pros Prospero RGC Radial glial cell Seq Sequoia Ser Serrate Stg String (mammalian Cdc25) Su(H) Suppressor of hairless TF Transcription factor VNC Ventral nerve cord

13 14 INTRODUCTION

INTRODUCTION

DEVELOPMENTAL NEUROBIOLOGY Developmental biology attempts to answer the question ofhow a complex functional mature organism can emerge from one single fertilized egg. This field deals with many fundamental concepts, such as growth, differentiation, metamorphosis, regeneration after injuries, and stem cell biology. In addition to understanding basic concepts of biology, developmental biology may help identify new avenues for curing disease (Slack, 2013). A major goal in the subfield of developmental neurobiology is to understand how the vast set of neurons and glia in the central nervous system (CNS), that displays such a remarkable diversity, is generated from a relatively small set of precursor cells i.e., how is neuronal diversity established? As an example of this complexity we can take the human brain, the most cognitively able of all brains known, which contains a large cerebral cortex essential for coordinating our body in response to the environment. Moreover, it makes highly sophisticated features as memory, learning and language as well as social behavior and self-awareness possible. An astonishing number of 86 billion cells and thousands of different cell types are interconnected in a huge network of circuitries that together governs these functions (Azevedo et al., 2009; Muotri and Gage, 2006). Another major challenge in the field is the attempt to understand cell proliferation control. Different parts of the CNS have different size i.e., the forebrain is much larger that the hindbrain. In addition, within each region different neuron and glia subtypes are generated in different numbers. These regional and subtype differences point convoluted to programs controlling proliferation, but these are not well characterized.

ALL CELLS ARISE FROM ONE SINGLE STEM CELL Stem cells can act as founder cells of all the cells in the body. They have the capacity to both reproduce themselves, and give rise to daughter cells that construct our tissues and organs. The stem cell classification is made according to versatility and plasticity to produce specific cell types. The development of a multicellular organism starts with the first initial stem cell, the fertilized egg. This diploid cell is totipotent i.e., it could give rise to an entire human being. As development proceeds, het stem cells get more and more specialized. This transformation and specialization is crucial for various tissues with diverse functions to be created. After about four days of development, after the fifth round of division, the stem cell competence is reduced to pluripotency i.e., committed to generating all the tissue types in the body such as brain, heart, liver and skin, but not a new individual. A multipotent stem cell is even more restricted, and can solely generate all the cells of a specific tissue e.g., a neural stem can only generate cells in the CNS. A neural progenitor is a further specialized version, functional to produce specific subtypes in the CNS (Condic, 2014).

15 INTRODUCTION

THE SAME DNA BUT DIFFERENT GENE EXPRESSION Each organism carries a species-specific genome, coding for a developmental plan that gives a unique set of characteristics, which is inherited from generation to generation. Despite the great differences in morphology and function between the multiple cell types in the body, all cells contain the same basic genetic composition. However, an organism never expresses all genes in a cell at the same time.The selective use of the genome depends upon several levels of gene regulation, with one of the more extensive ones being at the level of transcriptional regulation. A gene is transcribed when RNA polymerase II (Pol II) is synthesizing asingle stranded mRNA copy of the gene. Most genes are not expressed in all cells and their level of specificity varies dramatically, from myosin that is expressed by all muscle cells to the neuropeptide RFRP that only is expressed in a small subset of mouse hypothalamic neurons. This elaborate transcriptional gene regulation depends upon several entities, such as transcription factors (TF), cofactors, histone modifications, and DNA-folding. These entities combine to generate cell and tissue specific gene expression, and thereby cell and tissue specialization. Gene expression is also highly dynamic through development and a gene may even play different roles at different time points within the same cell (Hartl and Ruvolo, 2012). The study of when, where and why different genes are expressed is a critical part of understanding the biological functions of an organism. A typical gene most often have several enhancers of ∼200bp-1kb length, which can be located both 5´and 3´, and can work over long distances. The enhancers contain multiple binding sites for TFsand co-factors that could act as activators or repressors for Pol II. There are additionally several general TFs such asthe common TFIID: TATA-box binding protein (TBP) and its associated factors (TAFs). TFIID directs Pol II to dock into the transcription start site on the core promotor. TF activated enhancers loop into this promotor region. Furthermore, other cis-regulatory complexes as silencers and insulators are involved in the transcription activation (Bulger and Groudine, 1999; Levine and Tjian, 2003; Spitz and Furlong, 2012).

CANCER IS UNCONTROLLED CELL PROLIFERATION A general aim in the studies of developmental neurobiology is the search for targets for future treatments to human CNS related diseases, such as Alzheimer’s, Parkinson’s, psychiatric disorders and brain cancer. The definition of cell proliferation is an increase in total cell number achieved through cell division and cell growth. Many factors affect proliferation: The size of the progenitor pool, the number of progenitor divisions, the time period of cell division and the proliferation potentialthat the daughter cells possess (Homem and Knoblich, 2012).

Genetic programs are tightly regulating the generation of the proper amount of cells in the correct regions of the brain at the correct time points. This requires an extensive spatial and temporal control, keeping the correct balance between proliferation and differentiation. Even small failures in the production process could result in malformation,

16 INTRODUCTION overproduction or even tumor formation. This delicate balance differs between species, but also between CNS regions, due to alterations through evolution.

Cancer is not one but a collection of diseases that all have in common that cells in a specific area of the bodybecomes abnormal and start to divide without control.Nearly all metazoans can get cancer except from theblind mole- and naked mole-rats that have interesting anti-tumorigenic capabilities (Gorbunova et al., 2012). Cancer has a genetic origin, and is caused by inheritance or acquisition of one or often several errors (mutations) over time. Proto-oncogenes, such as MYC and RAS that normally act to stimulate growth, inhibit differentiation or halt apoptosis, and tumor suppressor genes such as p53 and PTEN that normally arrest growth and activate DNA repair genes, are important players. A common theory proposes that there are a small subpopulation of cancer stem cell like cells in a tumor that sustain the cancer through self-renewal, which could explain metastasis and relapse of the disease after treatment (Visvader, 2011).

17 INTRODUCTION

THE VERTEBRATE CENTRAL NERVOUS SYSTEM (CNS) NEURONS AND GLIA Animals are required to interact with the environment to survive and thereby need to have the capacity to sense stimuli with their senses, such as touch, hearing, eyesight, smell and taste. Furthermore, this information needs to be quickly processed in the CNS and generate a suitable output response. The CNS is the control center of the body and is grossly composed of the brain and the spinal cord. The spinal cord is a structure that connects the brain with the peripheral nervous system (PNS) and is also the center for automatic reflex responses. In human and vertebrates the spinal cord is located on the dorsal side (back), but in arthropods it is ventrally located (abdomen). In the CNS there are two main groups of cells interconnected in an elaborate network: neurons and glia. It isestimated that the human brain contains neurons and glia an equal number of 86 x 109 (Azevedo et al., 2009; Lodisch et al., 2000). The father of modern neuroscience, the Spanish Nobel laureate Santiago Ramón y Cayal, is entitled with the discovery of the neurons as single units. Neuronal sub-types are distinguished on the basis of their unique location, morphology, connectivity, function and molecular properties (Lopez-Munoz et al., 2006). However, they all have in common that they have a cell body to which information is transmitted from surrounding areas via dendrites, and information is passed out via a long axon. There are many types of neurons, all specialized for different tasks. Motor neurons innervate and control muscles and glands, sensory neurons receive signals from the sensory organs in the periphery, and interneurons carry information between the two.Moreover, there are different sub-groups of neurons classified upon other characteristics, such as morphology, function, location, branching and expression of neurotransmitters.

Figure 1. There are two broad classes of cells in the CNS: neurons and glia.

18 INTRODUCTION

The glia cells play instrumental roles as supporting cells, but have not been as extensively studied as neurons. Observations show that glia constitute a heterogeneous group of cells, and carry out a number of specific tasks, including axon guidance, homeostasis, nutritional support, and neurogenesis. There are three general types glial of cells: upporting S astrocytes, myelin producing oligodendrocytes and immune active microglia (Fig1) (Dimou and Gotz, 2014; Lodisch et al., 2000).

DEVELOPMENT OF THE CNS During vertebrate neural development the neural plate is formed by neuroepithelialcells from a part of the ectoderm denoted the neuroectoderm. The neural plate folds into a single layer of pseudostratified cells, the neural tube (neurulation). Along the dorsoventral (D-V) axis, the neural tube is subdivided into the dorsal roof plate, the alar and basal plate, and the ventral floor plate. Patterning cues acting at these early stages include bone morphogenic proteins (BMPs) and WNT ligands, secreted from the dorsal roof plate, and Sonic hedgehog (Shh) secreted from the ventral floor plate and the notochord. Together, the BMP, WNT and Shh ligands establish gradients along the D-V axis, which guide and specify the progenitor cells within the neural tube. Additional patterning cues are chiefly retinoic acid and FGF. Along the anteroposterior (A-P) axis, the neural tube islater developed into forebrain, midbrain, hindbrain and spinal cord (Rowitch and Kriegstein, 2010).

The wall of the neural tube is lined with elongated neuroepithelial cells , which after neural tube closure convert into more fate -restricted differentiated radial glial cells (RGC). RGCs show hallmarks both of astrocytes and neuroepithelial cells.Furthermore, the RGCs are polarized in an apical-basal-orientation, and show a distinctive interkinetic nuclear migration through the cell cycle. Most of the RGC cell bodies are locatedtowards the apical inside during mitosis, which becomes the ventricular zone, to migrate basally for the cell cycle S-phase. The basal side later becomes the outer surface of the CNS and the apical side the ventricular zone. As an effect of patterning along the D-V and A-P axis, differently located RGCs generate different types of neurons and glia over time (Anthony et al., 2004).

The neural progenitors are multipotent and have the capacity to self-renew. The cell divisions could either be proliferative i.e., symmetric, to generate two cells with the same fate and potential, or differentiative and asymmetric, to generate one progenitor and one daughter cell witha more restricted potential (Paridaen and Huttner, 2014). The neuroepithelial cells divide symmetrically, during the proliferative phase, to expand the progenitor pool. During the neurogenic phase, neuroepithelial cells switch to RGC fate, and typically divide asymmetrically to produce more differentiated cell populations. At the peak of neurogenesis the RGCs almost exclusively undergo asymmetric divisions (Paridaen and Huttner, 2014; Rakic, 1995). Symmetric versus asymmetric division requires an equal versus unequal distribution of cellular constituents to the daughter cells. During asymmetric divisions, the daughter cells can either directly differentiate, divide

19 INTRODUCTION once to make two neurons/glia, or act as an intermediate progenitor cell (IPC), that will divide multiple times to generate several neurons (Gotz and Huttner, 2005).

There is a clear temporal order in neuronal and glia cell generation and specification. Sequential generation of neural subtypes is crucial for CNS development. RGCs become progressively more restricted and cannot reverse and give rise to neurons in a deeper layer. This is believed to be controlled by an intrinsic mechanism, whichalso is shown to be applicable on ESCs in culture (Desai and McConnell, 2000; Gaspard et al., 2008; Kohwi and Doe, 2013). In mouse, at the end of neurogenesis, most RGCs undergo a terminal symmetric division and a fraction remain to continue to produce glial cells i.e., astrocytes and oligodendrocytes. This switch is regulated by multiple factors e.g., Notch signaling, JAK/STAT signaling, Ngn1 downregulation, NF1A and Sox9 upregulation( Morrison et al., 2000; Rowitch and Kriegstein, 2010).

CONTROL MECHANISMS IN DEVELOPMENT The average cell cycle length is shown to increaseduring the development of the mammalian brain. This progression was first shown by S-phase labeling studies in mouse VZ (Takahashi et al., 1995). According to the “cell cycle length hypothesis” the cell cycle phases must be long enough for cell fate determinants to induce differentiation. Neurogenic divisions is longer when compared to proliferative divisions, and this time difference is sufficient to trigger the transition between the two division modes. In particular, the G1 phase and the S phase of the cell cycle are shown to be lengthened in the beginning of neurogenesis (Calegari et al., 2005; Calegari and Huttner, 2003). There is also a prolongation of the cell cycle between the RGCs and the more restricted IPCs (Arai et al., 2011). Cell cycle lengthening ofthe G1 phase, by the use of pharmaceuticals, such as Cdk2/CycE or Cdk4/Cyclin D inhibitors, triggers premature neurogenesis in rodents (Calegari and Huttner, 2003; Lange et al., 2009). In the less extensively studied primate VZ, a cell cycle lengthening of the neural progenitors is also observed in the beginning of neurogenesis. However, in mid neurogenesis, during neurogenesis of the cortical layers the cell cycle accelerate (Kornack and Rakic, 1998).

It is well-established that the apical-basal orientation, and thereby the perpendicular cleavage orientation, of the mitotic spindle in RGCs is deterministic for the type of division mode. In general, if the cleavage lanep is vertical (apical domainbisecting), then both daughter cells inherit both the apical and basal processes, which triggers a proliferative division. Conversely, if the cleavage plane is horizontal, the daughter cell that only inherits the basal processes delaminates from the VZ and differentiates into a neuron or IPC i.e., the result is a neurogenic division( Florio and Huttner, 2014; Konno et al., 2008). Even subtle changes in the mitotic spindle orientation during development can disturb the ratio between symmetric vs. asymmetric divisions (Kosodo et al., 2004). It has lately been discussed how predictive the spindle orientation is for division mode outcome, for later stages of development (Homem et al., 2015; Mora-Bermudez and Huttner, 2015). Strong correlations between centrosome inheritance and daughter cell fate have been observed

20 INTRODUCTION

(Wang et al., 2009). Notch signaling is furthermore known in rodents to maintain a self- renewing state and to inhibit neurogenesis(Pierfelice et al., 2011). Notch oscillatory expression patterns of HES1, proneural factor neurogenin 2 (NGN2) and delta-like1 is also involved in the embryonic brain development (Shimojo et al., 2008). The activation of Notch leads to HES-mediated repression of proneural genes, such as Neurogenin2 (Ngn2) and Ascl1, that keeps the cell proliferating. High Notch signaling keeps the cell in a self- renewing state and low signaling initiates neural differentiation. In differentiating cells the oscillations cease, Notch expression goes down and proneural gene expressi on goes up and induces change (Dong et al., 2012; Shimojo et al., 2008).

21 INTRODUCTION

DROSOPHILA MELANOGASTER AS A MODEL SYSTEM For the past century the fruit fly Drosophila melanogaster (Drosophila) has been used widely as a scientific research tool, and has contributed invaluably to our understanding of neurodevelopment. The pioneering American geneticist Thomas Hunt Morgan began using Drosophila at Columbia University in 1908, studying chromosomal heredity, research for which he later was awarded the Nobel price (Morgan, 1910). Ten years later the geneticist H.J. Muller demonstrated the mutational effect of radiation on Drosophila, and started to produce genetic modified flies with different deficiencies randomly created by the use of X-ray; an important milestone for the field of genetics (Muller, 1927; Muller, 1928).

GENETIC SIMILARITIES BETWEEN HUMAN AND DROSOPHILA The embryonic Drosophila CNS was chosen as a model system formy thesis studies, mainly due to the powerful genetics.Despite ∼700 million yearsof evolutionary divergence between human andDrosophila (Hedges et al., 2015), he t extent of conservation is high and the two species share a core set of genes; almost 60% of the Drosophila genes have homologs in the more extensive human genome (Chien et al., 2001 ; Rubin et al., 2000). Furthermore, nearly 75% of human disease genes haveequivalent orthologues in Drosophila (Reiter et al., 2001). The human ∼3.08 GB long haploid DNA sequence encoding ∼20,000 genes, organized in 23 pairs of chromosomes, tightly packed together in nucleosomes by histone octamers. The Berkeley Drosophila Genome Project and Celera Genomics sequenced the entireDrosophila genome in the year 2000. The genome was reported to have a compact genome size of 180Mb, encoding for approximately 13,600 genes, distributed on four pairs of chromosomes (Adams et al., 2000; Ezkurdia et al., 2014; International Human Genome Sequencing, 2004). The more complex an organism is the more complex is the gene regulation. However, the number of genes or the length of DNA is not the most important measurement for developmental progress. Only 1% of the human genome encodes proteins and less than 10% of the genome is thought to be functional (Ezkurdia et al., 2014; Rands et al., 2014).

DEVELOPMENT OF THE DROSOPHILA VNC One of the key limiting factors in neuroscience is the difficulties in being able to study an isolated group of neurons, follow their progression and trace changes in detail. Compared to the human CNS, with approximately 86 billion neurons, the 100, 000 neurons of the Drosophila CNS are relatively few( Azevedo et al., 2009). Neuroblasts (NBs) are Drosophila neural stem cell-like multipotent neural progenitors in the CNS, and their lineages have during the past 20 years grown into an extensively used key model system for neural stem cell biology( Saini and Reichert, 2012). A relatively small number of ∼1,200 NBs are generated in the embryo to give rise to the all the diverse neurons and glia produced in the larval CNS (Birkholz et al., 2013 ; Bossing et al., 1996 ; Schmid et al., 1999 ; Schmidt et al., 1997; Urbach et al., 2016; Urbach et al., 2003; Wheeler et al., 2009).

22 INTRODUCTION

The central brain, the optic lobe and the ventral nerve cord (VNC), forms the embryonic and larval CNS in Drosophila. In this thesis the well-mapped embryonic VNC (Fig 2a) with its estimated 10,000 cells, which is functionally equivalent to the mammalian spinal cord, was chosen as a model system to study molecular genetic networks controlling proliferation and specification during development. The VNC is segmented and could be subdivided into 13 subunits along the A-P axis: 3 thoracic T1-T3 segments and 10 abdominal A1-A10 segments (Fig 2b) (Birkholz et al., 2013).

Figure 2. The Drosophila embryonic CNS. a) A side view of a Drosophila embryo. Anterior (A), posterior (P), ventral (V) and dorsal (D). b) A ventral view of the Drosophila VNC with 13 segments visualized; thoracic T1- T3 and abdominal A1-A10. Three specific lineages are highlighted: NB3-3 (green), NB5-6 (red), and NB7-3 (purple) (modified from Baumgardt et.al., 2016).

SPECIFICATION OF THE NEUROECTODERM The Drosophila embryonic period last for less than 24h, which is a great advantage compared to other slower model systems such as rodents. The early development of the CNS includes different phases: Patterning of the ectoderm, formation and delamination of neural progenitor cells, as well as migration and formation of neurons through asymmetric division. It is becoming increasingly clear that many of the regulatory processes in the neurogenesis are controlled by geneswith well conserved orthologues in mammals (Hartenstein and Wodarz, 2013). The neurogenesis of the VNC and the brain is divided into two periods: a short embryonic and an extensive larvae period when 90% of the adult neurons are produced (Urbach and Technau, 2004).

In Drosophila the unfertilized egg is polarized by gradients of maternally loaded mRNAs, creating an A-P axis, with bicoid deposited anteriorly, and nanos and caudal posteriorly. Upon fertilization an early D-V axis is shaped by the influence of Spätzle, dl and the EGF receptor. During early embryogenesis the maternal segmentation genes , belonging to three families, continue to pattern the embryo in a hierarchical order: First expressed are Gap genes (Nusslein-Volhard and Wieschaus, 1980), which specifies the A-P axis into broad

23 INTRODUCTION regions i.e., hunchback, krűppel, huckebein, knirps, giant and tailess. Second member of the family is the Pair-rule genes, activated by the Gap genes, which further contributes to the patterning of the A-P axis, each expressed in seven evenly stripes (e.g. even-skipped, hairy, paired).

During gastrulation three germ layers are formed by two major invaginations: ectoderm, mesoderm and endoderm. The VNC originates from the two neurogenic regions of the ventrolaterally located ectoderm, the neuroectoderm. The gap and pair rule genes together pre-pattern the embryo, upon which the third family member of the segmentation genes act: The “segment polarity genes” that are all expressed in all 14 embryonic segments (i.e. hedgehog, engrailed, frizzled, wingless, gooseberry, patched), which subdivides the neuroectoderm into distinct rows (Bhat, 1999; Skeath, 1999). These segment polarity genes are expressed in the beginning of neurogenesis, and NBs born in each A-P strip inherits the expression of these genes (Skeath, 1999).

Moreover, perpendicular to the A-P axis, the neuroectoderm is divided into three longitudinal regions, with one columnar homeodomain-containing gene expressed in each i.e., ventral nervous system defective (vnd) (Chu et al., 1998; McDonald et al., 1998), intermediate neuroblast defective (ind) (Weiss et al., 1998) and muscle segment homeodomain (msh) (Isshiki et al., 1997), respectively. NBs born in each column acquire different fates. Removal or ectopic expression of one of these identity columnar regulators is shown to alter the identity of the neurons born within that region( Skeath and Thor, 2003).

The columnar and segment polarity genes are expressed in repeatingpattern in each segment. The gap and pair-rule genes interact to regulate a fourth class of genes: The homeotic genes (Hox), whose transcription determine the developmental fate of each body segment, and contribute to diversity. The bithorax complex (Bx-C) is expressed posteriorly in the VNC and specifies the ten abdominal segments; A1-A10. The Antennapedia (Antp) complex is expressed more anteriorly in the VNC, and is responsible for the formation of more anterior segments (Gehring and Hiromi, 1986; Pearson et al., 2005).

LATERAL INHIBITION The A-P segment polarity, and D-V columnar gene complexes together establish a Cartesian molecular coordinate system that subdivides the neuroectoderm into neuronal equivalence groups of cells withsimilar developmental potential. Shortly after gastrulation, one cell from each equivalence group will be singled out to adapt NB fate, while the others will become epidermal cells or simply stay undifferentiated (Skeath and Thor, 2003). This Notch-dependent process is denoted “lateral inhibition” (Fig 3). Initially, all the cells in the homogenous equivalence groups have equal neural potential. A dynamic competition in regulatory loops is important for the decision of which cell should be the Notch signaling sending or receiving cell. The NB-becoming cell will turn out to be Notch off, and will during the process inhibit the immediate neighbors from adapting the same fate (Doe and Goodman, 1985; Guruharsha et al., 2012).

24 INTRODUCTION

Figure 3. Schematic illustration of the process oflateral inhibition. Neuroblasts (NBs) are selected from equivalence groups in the neuroectoderm by Notch signaling. The NB-becoming cell will at the end be Notch off. The Notch signaling pathway is activated by Delta ligand binding, whichresults in transcription of E(spl)-complex (E(spl)-C) genes. Subsequently, E(spl)-C genes repress the expression of the basic-helix-loop-helix (bHLH) proneural genes: achaete (ac), scute (sc) and lethal of scute (l´sc). The proneural genes activate Delta expressionin a genetic positive feedback loop. A diminished proneural expression consequently leads to a diminution in Delta expression (Garcia-Bellido, 1979; Heitzler et al., 1996). The cell that in the end achieves the highest proneural expression and thereby the highest level of Delta ligand is Notch-off and will develop into a NB (Heitzler et al., 1996; Skeath and Carroll, 1992; Skeath and Doe, 1996).

In five sequential waves∼ 30 NBs per hemisegment are specified. The NBs enlarge, delaminate and migrate from the external surfaceof the neuroectoderm into the inner region of the embryo. The detachment from the apical surface triggers mitosis. Because of the early patterning events, each NB has a specific identity,expresses a unique combination of regulatory genes and is born at a stereotyped time. The NBs are named based on position according to the nomenclature i.e., subdivided into seven rows and six columns e.g., NB5-6 is born in row 5 and column 6 (Doe, 1992).

PROLIFERATION THROUGH ASYMMETRIC DIVISION After segregation the NBs start to divide asymmetrically to bud off smaller daughter cells meanwhile renewing themselves. Extensive studies in Drosophila have resulted in the identification of a core asymmetric machinery based on unequal inheritance of polarity proteins and cell fate determinants, which dictates the fate of the daughter . cells

25 INTRODUCTION

Asymmetric division is fundamental to all lineage alterations and the basic mechanisms are conserved in vertebrates as well, thus not extensively at a molecular level (Knoblich, 2008).

During asymmetric division of the NB (Fig 4), protein complexes gather on opposing poles of the cell to coordinate the mitotic spindle in an apical-basal direction, directing the segregation of cell fate determinants( Li, 2013). The homeodomain transcription factor Prospero (Pros) (Chu-Lagraff et al., 1991; Matsuzaki et al., 1992), the adaptor protein Miranda (Mira) (Shen et al., 1997), the Notch antagonist Numb (Uemura et al., 1989), the adaptor protein Partner-of-Numb (Pon) (Lu et al., 1998), and the growth regulator Brain tumor (Brat) (Sonoda and Wharton, 2001), are localized at the basal cortex. These proteins will be translocated exclusively to the NB daughter cell, denoted ganglion mother cell (GMC), after cytokinesis. The basally localized factors promote differentiation and limit the mitotic potential to only one round of GMC division. Pros is synthesized at the onset of mitosis, but is prevented to act in the NB by the adaptor protein Mira that tethers Pros to the basal side of the membrane. Mira is phosphorylated and degraded after completion of cell division, and Pros is released to segregate in to the nucleus of the GMC (Ikeshima- Kataoka et al., 1997; Knoblich et al., 1995; Spana and Doe, 1995; Vaessin et al., 1991). In the nucleus Pros represses the cell cycle factors: Cyclin E (Cyc E), Cyclin A, E2F, String (stg; cdc25) and the retinoblastoma-family protein (Rbf), meanwhile activating Dacapo (Dap; mammalian p21cip1/ p27kip1, p57kip2) (Choksi et al., 2006; Li and Vaessin, 2000). Brat, a translation repressor, is another Mira cargo that segregates to the GMC, and might be a posttranscriptional inhibitor of dMyc and regulate ribosomal protein biosynthesis. In the absence of Brat Pros is cytoplasmic and Brat is further suggested to either stabilize Pros-Mira interactions or promote Pros transcription by indirect effects (Betschinger et al., 2006; Lee et al., 2006).

The protein complexes distributed to the apical cortex have the primary function to promote the maintenance of the self-renewal potential of the NB. Two protein complexes, connected by the scaffolding protein Inscuteable (Insc) (Kraut et al., 1996), are exclusively localized on the apical side: the Par/aPKC-complex and the Pins/Gαi complex. The Par- complex, which consists of Bazooka (Baz/Par3), DmPar6 and Drosophila atypical protein kinase C DaPKC ( ), is crucial for setting up and maintaining the apical-basal axis of polarity. The Par/aPKC-complex is also responsible for the basal location of cell fate determinants through phosphorylation events (Betschinger et al., 2003; Knoblich, 2008). Insc binds to Baz and recruits the second apicalcomplex by binding toPartner of Inscuteable (Pins). Pins binds to Gαi that subsequently recruits Mud, which exert a pulling force on the mitotic spindle important for its orientation (Izumi et al., 2006; Kang and Reichert, 2015). Defects in the asymmetric machinery could result in loss of differentiated cells and uncontrolled overgrowth of NBs.

26 INTRODUCTION

When transplanting Drosophila larval NBs lacking: Pros, Numb, Mira or Pins, into wild type hosts, the clones reveal a hyperproliferative phenotype. Furthermore, the clones are shown to grow into 100 times their initial size and become immortalized. This effect is presumably due to a switch from asymmetric to symmetric divisions (Caussinus and Gonzalez, 2005; Gomez-Lopez et al., 2014).

Figure 4. Asymmetric division in Drosophila. The main components localized to the basal side of the neuroblast (NB) is important for daughter cell specification and the main components localized to the apical side is crucial for maintenance of progenitor fate.

THREE TYPES OF NEUROBLAST PROLIFERATION MODES After segregation the NBs starts to proliferate for a short period during embryogenesis to generate all the neurons and glia that will make up the early larval CNS. Each NB gives rise to a fixed cell lineage, both in size and identity, producing neurons and glia in a strict stereotypic order. These lineages vary in both in production time, number of cells (2-40 cells) and functions. In each of the ten abdominal VNC hemisegments, the ∼30 NBs generate ∼320 neurons and 25-30 glia, during the embryonic neurogenesis. Slightly more cells, around 500 neurons and glia, are generatedin each of the three thoracic VNC hemisegments (Bossing et al., 1996; Schmid et al., 1999; Schmidt et al., 1997). There are three main classes of cells in each VNC hemisegment: interneurons (80%), motor neurons (10%) and glia cells (10%) (Schmid et al., 1999; Technau et al., 2006).

There are three types of NB division modes: type 0, type I and type II(Fig 5). The most common division mode during Drosophila neurogenesis, in fact the only mode known for many years, is denoted type I (Boone and Doe, 2008). During type I division the NB gives rise to one self -renewing NB and one smaller daughter (GMC), which has a more restricted proliferation potential. The GMC divides once (hence the name type I) to generate two post mitotic daughter cells, which terminally differentiates into neurons and/or glia cells (Skeath and Thor, 2003).

27 INTRODUCTION

The type II NBs were discovered in 2008(Bello et al., 2008; Boone and Doe, 2008; Bowman et al., 2008); eight in each brain lobe. Type II NBs divide asymmetrically to renew themselves and to generate transit amplifying secondary progenitors, denoted intermediate neural progenitors (INP). Thenewly born immature iINPs develops into mature mINPs after a 4-6h time period. The mINPs continue to divide asymmetrically for 3-5 rounds, renewing themselves and generating GMCs. Each GMC terminally divides once more to generate two neurons and/or glia. Theype t II NB differ fromtype I molecularly by the lack of expression of the proneural transcription factor Asense and the cell fate determinant Pros. In contrast to ypet I division this proliferation amplification mode allows these lineages to grow very large, containing up to 500 cells(Bello et al., 2008).

Figure 5. There are three types of asymmetric division modes in Drosophila: type 0, type I and type II. The NB division modes differ by generating daughter cells that directly differentiate, divide once or multiple times.

28 INTRODUCTION

Recently a third proliferation mode of lineage progression in the VNC has been identified, type 0. Studies in our lab and the work in this thesis have contributed to this observation. Here, the NB divides to renew itself to generate daughters that directly differentiate into neurons, without any intermediates (Baumgardt et al., 2009; Karcavich and Doe, 2005; Ulvklo et al., 2012 ). It have also been discovered that many developing NB lineages switch in division mode over time from type I to type 0 (type I>0). This have been shown in the NB5-6T lineage, NB3-3A, NB7-3A, and as a global phenomenon in the entire VNC as well (Baumgardt et al., 2014; Baumgardt et al., 2009; Ulvklo et al., 2012)

BINARY LINEAGE DECISIONS Asymmetric cell division is important for the generation of cell diversity.The GMC daughter cells divides asymmetrically in most if not all cases (Doe, 2008; Knoblich, 2008). This GMC asymmetric division is in part controlled in a similar manner as the NB division and gives rise to progeny with different cell fates. Notch signaling pathway is involved to generate diversity between the two daughter cells . Both of the daughter sibling cells receive a Notch activation signal from Delta ligands presented on the surrounding cells. However, Numb segregates exclusively to one of the daughter cells, a localization process requiring functional Insc (Kraut et al., 1996; Spana and Doe, 1996). Numb is a well characterized and evolutionary conserved Notch inhibitor. During interphase Numb is distributed around the membrane to form a crescent. In metaphase and telophase, Numb segregates into one of the two daughter cells. Exactly how Numb inhibits Notch is not entirely clear. Genetic and molecular studies in PNS SOP cells indicates that Numb functions through polarizing the distribution of α-adaptin to the Numb-side of the cell. α-adaptin is involved in receptor - mediated endocytosis as a subunit of the trafficking adaptor-protein 2 (Ap2) complex that internalizes cargo into clathrin coated vesicles. By somewhat unclear mechanisms, it is hypothesized that Numb recruits Notch to these vesicles, where Notch subsequently gets degraded and cell fate B (Notch off) is promoted. The absence of Numb in the other daughter cell leads toan active Notch pathway and fate “A” (Notch on). This binary difference in Notch activity thereby produces two different cell fates (Berdnik et al., 2002; Couturier et al., 2012 ; Santolini et al., 2000 ). Sanpodo (Spdo) (O'Connor-Giles and Skeath, 2003) also contribute to the specification of the Notch-dependent A fate. spdo encodes a four-pass transmembrane protein localized in the cell membrane, which is known to directly interact with and positively regulate the Notch receptor. Drosophila Numb acts to reduce membrane-accumulation of Spdo through direct interaction by regulating endocytosis (Babaoglan et al., 2009 ; Cotton et al., 2013 ; O'Connor-Giles and Skeath, 2003 ; Skeath and Doe, 1998).

Notch A and B fate plays a role in selecting between pre -existing developmental programs, such as axonal targeting, dendrite innervation or cell survival. DuringDrosophila PNS development, asymmetric division plays an important role in the development of the sensory bristles from the sensory organ precursor (SOP) lineages, by generation offive distinct cell fates in these lineages (Bardin et al., 2004). The first step is a process of lateral

29 INTRODUCTION inhibition, in which SOP precursors are specified from groups of ectodermal cells. When the SOP cell is dividing,an interplay between Numb and Spdo guides the endocytic trafficking of the Notch receptor to exclusively one of the daughter cells, which leads to adaption of different fates. Numb promotes pIIb cell fate (B-fate) and suppress pIIa cell fate (“Notch on”; A-fate). The pIIa and pIIb use Notch for three more rounds of division to generate daughters with different fates i.e. socket cell (A), hair cell (B), sheath cell (A), neuron (B) and a glial cell (B) that later undergoes apoptosis (Gho et al., 1999; Guo et al., 1996; Hutterer and Knoblich, 2005; Rhyu et al., 1994).

TEMPORAL PROGRESSION Studies of NB lineage development have revealed that the GMCs/neurons/glia are born in a fixed birth order. This fascinating temporal progression has been found to beregulated by a stereotyped intrinsic temporal series of transcription factors that alter the progenitor competence over time. Strikingly, NBs grown in culture continue to undergo this temporal program (Brody and Odenwald, 2000). The temporal factors are importantboth in Drosophila and vertebrates for the control of proper daughter cell fate specification and generation of unique sub-types of neurons and glia over time (Okano and Temple, 2009; Pearson and Doe, 2004). Each GMC, and thereby neurons and glia, continues for a short time to express the temporal genes that are expressed by the NB when it is born (Isshiki et al., 2001). During Drosophila embryonic development ive f genes are sequentially expressed in a very precise cascade inmost if not all NBs; hunchback (Hb) krüppel (Kr) pdm (nubbin and pdm2; collectively referred to as pdm) castor (cas)  grainy head (grh) (Fig 6) (Baumgardt et al., 2009 ; Brody and Odenwald, 2000 ; Isshiki et al., 2001 ; Kambadur et al., 1998; Novotny et al., 2002). In addition to this, sub-temporal genes are expressed, acting downstream the temporal genes, to fine-tune the cascade to generate cellular diversity (Baumgardt et al., 2009; Benito-Sipos et al., 2011; Stratmann et al., 2016). In all NBs analyzed, this cascade starts with the expression of Hb, independent on time of delamination (Isshiki et al., 2001 ). These series of transcription factors can however vary some between lineages in different tissues.

Figure 6. Temporal progression of Drosophila VNC neuroblasts (NBs). The NBs sequentially express the transcription factors Hunchback (Hb), Krüppel (Kr), Pdm, Castor (Cas) and Grainy head (Grh). The daughter cells born in each temporal window will retain the expression pattern present at the time of birth.

30 INTRODUCTION

Both feed-forward and feedback regulation is involved in the temporal transition of expression, to thereby ensure that the genes do not start to express the next temporal gene too early or too late. Each temporal gene promotes the expression of the next gene in the cascade, represses the former gene and the next plus one gene (Isshiki et al., 2001; Kohwi and Doe, 2013). The connection between the temporal genes and distinct cell fate has been studied in several specific lineages. Importance of temporal gene expression cascades have also been reported in the vertebrate CNS, cerebral cortex, hindbrain, retina and spinal cord (Okano and Temple, 2009).

THE CELL CYCLE The process when a cell divides, denoted the cell cycle, is strictly controlled (Fig 7). A number of cell cycle factors govern the proper transfer between each of the four cell cycle phases: Gap 1 phase (G1), Synthesis phase (S1), Gap 2 phase (G2) and mitosis phase (M). Cyclin dependent kinases (Cdks) are expressed throughout the cell cycle, but acquire binding to Cdk-specific regulatory cyclins to get activated. Cyclins are produced andare being degraded in an oscillatory cycling manner. The activeyclin C-Cdk enzymatic complexes catalyzes the phosphorylation of cell cycle regulating proteins on serines and threonines (Budirahardja and Gonczy, 2009).

The cell cycle in Drosophila is less complex when compared to mammals and presents fewer protein families involved. The first part of the cell cycle is called interphase and starts with G1, which is a growth phase. In each G1 phase, an essential decision for the cell to continue dividing and entry the S -phase or exit the ce ll cycle is taken ; the G1 checkpoint . The levels of Cyclin D-Cdk4/6 (Datar et al., 2000) play a role for cell growth and later Cyclin E-Cdk2 (Knoblich et al., 1994) is required for the initiation ofthe next S-phase (Bertoli et al., 2013). Cyclin E (CycE) is thereafter degraded by the Skp1-Cullin-Fbox (SCF)/ Archipelago complexes, which trigger ubiquitin-mediated degradation (Vodermaier, 2004). The E2F/Dp transcription factor complex promotes the expression of CycE and genes important for DNA replication and mitosis. The retinoblastoma protein homolog (Rbf1) prevents entry to the S-phase by negatively regulation of E2F, which in complex with Rbf1 acts as a repressor. When cells are stimulated to entry S-phase Rbf1 gets phosphorylated by cyclin-Cdks and is detached from E2F (Du et al., 1996; Duronio et al., 1996).

Figure 7. A schematic model of the cell cycle regulation in Drosophila (modified from Baumgardt et.al., 2014).

31 INTRODUCTION

S-phase is a short phase of DNA replication and is followed by G2, in which further protein synthesis and growth preparing for the mitotic M phase takes place. The second important checkpoint is the G2/M transition controlled by Cdc2/Cdk1 associated withCyclin A, Cyclin B and Cyclin B3. However, single mutants have revealed that only Cyclin A is required for mitosis (Jacobs et al., 1998; Knoblich and Lehner, 1993 ; Lehner and O'Farrell, 1989). The phosphatase String (Stg; mammalian Cdc25) activates Cdc2/Cdk1 by removing inhibitory phosphate groups (Edgar and O'Farrell, 1989 ). These G2/M cyclins are degraded in anaphase by anaphase promoting complex (APC).

CELL CYCLE EXIT Programmed cell death (PCD), or apoptosis, is central during development, for sculpting, deleting structures and for eliminating damaged cells. The PCD machinery is evolutionary conserved throughout the metazoan kingdom. In the mammalian CNS, an overproduction of cells is observed during development, and the same is reported in Drosophila, albeit not to the same extent (Rogulja-Ortmann et al., 2007 ). When a cell goes through PCD the D NA is condensed, fragmented and the cell shrinks. The PCD could be induced in various ways from both intrinsic and extrinsic factors (Yamaguchi and Miura, 2015).

In Drosophila the NBs demonstrate a cell cycle length of 40 -50 min and the GMC s a longer one of 100 min (Baumgardt et al., 2014; Hartenstein et al., 1987). During this short time period the cell grows, replicates its DNA and goes through cytokinesis. At the end of embryogenesis, when the NBs have finished the neurogenic divisions and created a complete cell lineage, they need to exit the cell cycle to terminate proliferation. The precise time at which each NB stop dividing is crucial for the final number of cells and is therefore tightly regulated. This stop -time and thereby the intrinsic regulatory molecular mechanism, differs between NBs, and is dependent to some extent upon the region (Reichert, 2011). There are three lineage stop mechanisms observed: To stop by directly undergoing PCD; to exit the cell cycle and later undergo PCD; to exit the cell cycle and enter quiescence, in order to later resume cell divisions at the larvae stage (Baumgardt et al., 2014; Bivik et al., 2016). In the brain and the thoracic region of the VNC, it is most common, with a few exceptions, with arrest of the cell cycle in G1 to G0, to remain in a quiescent state until larvae stage. In the abdominal region however, cell cycle exit followed by PCD is the more frequent event (Homem and Knoblich, 2012; Sousa-Nunes and Somers, 2013). Both the temporal cascade and the Hox homeotic system are involved in the cell cycle exit decision (Baumgardt et al., 2014; Baumgardt et al., 2009; Karlsson et al., 2010; Tsuji et al., 2008). In the larvae, hormonal and nutritional signals mediated by glial cells and the fat body triggers the NBs to reenter the cell cycle to continue to proliferate (Chell and Brand, 2010; Sousa-Nunes et al., 2011).

Recently, 21 cell cycle factors were analyzed for their relevance in theembryonic VNC development, which revealed crucial roles for CycE, E2F and Dap determining the proper timing for cell cycle exit (Baumgardt et al., 2014). dap expression needs to be gradually upregulated in G2 before the final division, which leads to a cell arrest in G1. dap is off or

32 INTRODUCTION low in the early embryo and show a robust expression late(Baumgardt et al., 2014; de Nooij et al., 1996 ; Lane et al., 1996 ). Dap is functionally a Cdk inhibitor (CKI) that inhibits CycE-Cdk2 activity by directly binding to the complex. Knoblich et.al. showed that CycE needs to be downregulated for proper cell cycle exit. However, overexpression of CycE only leads to another round of cell division and the same effect has loss of function of Dap (de Nooij et al., 1996; Knoblich et al., 1994).

33 INTRODUCTION

NOTCH SIGNALING PATHWAY The Notch gene was first described in 1917 by Thomas Hunt Morgan, as a sex -linked gene that when heterozygously mutated caused a notched wing margin phenotype in Drosophila (Morgan, 1917). In the 1930’s Poulson published a mutation inNotch that affected the development and resulted in an overproduction of NBs in the embryonic CNS,which he denoted a neurogenic phenotype (Poulson, 1937). By now, the Notch pathway has hence been extensively studied for nearly a century, which has revealed an exceptionally complex genetic circuitry, still challenging to dissect at the molecular level. Furthermore, Notch has been studied in various model organisms, ranging from invertebrates, such asC. elegans and Drosophila, to vertebrates such as zebrafish,Xenopus , mouse and human. Both the Notch receptor and many essential pathway components are highly conserved throughout metazoan evolution, and insight obtained from analysis of Drosophila can in many cases be applicable to all species, humans included. In this section I will focus on discussing Notch signaling in Drosophila and mammals.

Notch, together with a small set of other conserved signaling pathways, regulates fundamental cell fate processes, by the tran smission of information from the exterior to the interior of cells, during embryogenesis, development and adulthood. While many other signaling pathways operate via diffusible ligands and acting at long-range, the Notch pathway belongs to a smaller groupf opathways that operate via cell-cell contact. Perturbations of the Notch pathway, leading to gain or loss of function, have severe consequences for the organism and contribute to genetic disorders, syndromes and diseases (Andersson et al., 2011; Chitnis and Bally-Cuif, 2016).

MULTIPLE FUNCTIONS OF NOTCH SIGNALING Notch is demonstrated to regulate multiple processes, such as cell fate decisions, differentiation, proliferation, maintenance, self-renewal and apoptosis. There are a myriad of roles reported and the pathway is highly pleiotropic throughout development(Bray, 2006; Guruharsha et al., 2012). In its essence, Notch governs binary decisions by selecting between preexisting developmental programs, to thereby dictate different fates of two neighboring cells. A major aim in the field is to understand why and how Notch triggers different outcomes in different contexts and how this could change over time. Early in mammalian development Notch acts to prevent differentiation of progenitor cells to promote self-renewal and at later time points Notch is involved in inducing differentiation of these cells (Kopan and Ilagan, 2009; Schwanbeck, 2015).

NOTCH DOSAGE SENSITIVITY AND DISEASE Notch signaling is very sensitive to changes in gene dosage, and both increased and decreased Notch signaling often results in phenotypic effects. This is consistent with the notion that the gene shows both haploinsufficiency (heterozygosity phenotypes) and triplomutant (extra copy-number) effects (Louvi and Artavanis-Tsakonas, 2012). Alongside the JAK/STAT and Sonic hedgehog (Shh) pathways, Notch belongs to a smaller

34 INTRODUCTION sub-group of signaling pathways that do not operate by extensive amplification of the signaling cascade. Rather, the signaling mechanism relies on activation of a transcription factor(s) complex, rather than enzymatic second-messenger amplification: Each Notch receptor is activated irreversibly once and one Notch molecule simply generates one signal in the nucleus. However, transcriptional feedback mechanisms could regulate receptor and ligand expression and thereby amplify the effect (Guruharsha et al., 2012).

Because Notch signaling is crucial for the organism, and plicated im in the balance of diverse processes, it is not surprising that dysregulated Notch could lead to disease (Louvi and Artavanis-Tsakonas, 2012). In 1991, Ellisen et.al, published the first evidence of Notch involvement in cancer; in - Tcell acute lymphoblastic leukemia (T-All) (Ellisen et al., 1991). In a subsequent study of patients with T-ALL, NOTCH1 activating mutations were detected in nearly 56% of all cases (Weng et al., 2004). Since then, Notch activity has been observed directly or indirectly in nearly all mayor solid tumors (Ranganathan et al., 2011). The common belief is that Notch promotes tumorigenesis as an oncogene, but there is growing evidence that Notch under other circumstances should be considered as a potent tumor suppressor e.g., in skin cells, pancreatic epithelium, hepatocytes and bladder (Hanlon et al., 2010; Lobry et al., 2011; Nicolas et al., 2003; Rampias et al., 2014; Viatour et al., 2011). Notch has also been reported to have both oncogenic and tumor suppressor functions in the same tissue (Klinakis et al., 2011; Lobry et al., 2014). Mutations of the components of the Notch signaling pathway could be of a - lossoff-function character (frameshift or nonsense mutations) or lead to aberrant activation e.g., affect the stability or promote ligand independent processing(Wang et al., 2011). Notch signaling haploinsufficiency i.e., only one functional copy of the ligand geneJAG1 , is strongly correlated with Alagille syndrome (Oda et al., 1997), as well as of NOTCH1 with aortic valve disease (McKellar et al., 2007). Notch amplification as well as overexpression is widely known to act oncogenically.NOTCH3 copy number is linked to ovarian serous carcinoma (Park et al., 2006) and both gain of function and amplification of NOTCH2 is associated with diffuse large-B-cell lymphomas (Lee et al., 2009).

THE CANONICAL NOTCH SIGNALING PATHWAY To be able to unravel the molecularly link between the pathophysiology and the symptoms in these multiple diseases, the normal function of Notch in the organismare being rigorously studied. Extensive genome-wide screens have been performed in both Drosophila and in mammalian systems , aimed at identifying and expanding the list of core - factors as well as co-factors in the pathway. Hundreds of genes have been found and it is suggested that many still remains unknown. The canonical Notch signaling pathway (Fig 8) describes the basic principles of the most common core signal transduction (Guruharsha et al., 2012).

Notch signaling is a short -range signal that links the cell fate of two cells. The transmission of the signal depends upon a juxtacrine interaction, where the membrane attached Notch receptor physically binds to a Delta-Serrate-LAG2 ligand (DSL-ligand) on a neighboring

35 INTRODUCTION cell. The receptor gets activated through this step of ligand binding together with three steps of proteolysis, which results in release of Notch intracellular domain (NICD). NICD gets translocated to the nucleus to associate with a DNA binding transcription complex containing CBF1/RBP-jκ/Su(H)/Lag1 (CSL) that recruits Mastermind (Mam) (Weigel et al., 1987) and cofactors, which in turn actives transcription of downstream target genes (Bray, 2006; Kopan and Ilagan, 2009). In addition to the canonical Notch pathway there are recent reports describing a non-canonical Notch pathway, which I will not address further here. In short, it suggests that the pathway could act in various ways e.g., in a ligand- or CSL-independent, or in a Deltex (Dx) dependent manner( Sanalkumar et al., 2010).

Figure 8. A schematic cartoon of the Notch signaling cascade. The DSL ligand binds to Notch receptor (N). As a result of two proteolytic cleavages (S2 and S3), NICD is released from the membrane, translocated to the nucleus and bound to the CSL together with coactivators. NICD-CSL act to activate target genes.

THE NOTCH RECEPTOR The number of each Notch pathway component differs between species. Evolution has resulted in more components and paralogues in the mammalian Notch signaling compared to Drosophila, and thereby a more complex signaling pathway. While Drosophila only has one Notch receptor gene, four paralogues are described in vertebrates Notch1-4( ) (Bray, 2006). However, recent studies have revealed that at least the function of Notch1 and Notch2 is functionally equivalent (Liu et al., 2015). The Drosophila Notch receptor (Fig 9) was cloned and sequenced for the first time in 1980’s, which provided greater insight into the structure and function of the pathway, and represented a major breakthrough for the field. A large transmembrane protein withan extracellular surface receptor and an

36 INTRODUCTION intracellular domain was revealed. This opened up for a discussion of cell-cell interaction and intracellular response cascades (Artavanis-Tsakonas et al., 1983; Kidd et al., 1986; Kidd et al., 1983 ; Wharton et al., 1985 ). The Notch receptor is a single -pass transmembrane protein that after S1 cleavage consists of two parts: Notch intracellular (NICD) and extracellular domains (NECD). NECD consists of 29-36 tandem epidermal growth factor (EGF) repeats and a negative regulatory region (NRR) composed of three cysteine rich LIN12 repeats and a heterodimerization domain(Kopan and Ilagan, 2009). The extracellular domain could bind in trans to ligands presented on an adjacent cell. The EGF- like repeat 11-12 is shown to be both essential and sufficient for this type of DSL interaction (Chillakuri et al., 2012; Rebay et al., 1991). The receptor could also bind in cis to ligands located in the same cell membrane, and for this interaction repeats -2429 are involved. NICD consists of a RAM-domain (RBPjk association module), seven ankyrin repeats (ANK), a transcriptional activation domain (TAD) and a C -terminally located Pro- Glu-Ser-Thr (PEST) domain. The NICD further contains two nuclear localization signals (NLS), which guides the NICD to the nucleus when cleaved from the membrane (Kopan and Ilagan, 2009).

Notch receptors are synthesized as one long 300-350kDa polypeptides and require three proteolytic cleavages to be functionally activated, as well as maturation process to change both structure and trafficking behavior. The first cleavage, at site 1 (S 1) between TMD and NRR, is performed by Furin convertases during exocytosis of the Notch precursor protein from the trans-Golgi apparatus. The receptor gets separated into two parts: NECD and NICD (Blaumueller CM, 1997; Logeat F, 1998) that bind to each other at the cell surface as a heterodimer associated by noncovalent bonds(Kopan and Ilagan, 2009). The importance and the exact function of the S1 cleavage is unclear (Gordon et al., 2009; Lake et al., 2009). In addition to the S1 cleavage, the Notch precursor protein goes through posttranslational modifications and becomes glycosylated, an action important for the ligand binding and response, as well as context-dependent activity.

The Notch receptor is activated upon ligand-binding to a neighboring cell. The EGF-like repeats of NECD nteracts i with the N-terminal part of the Delta-Serrate-LAG2 ligand (DSL) (Fig 9). The Notch DSL ligands are transmembrane proteins as well. They consist of three structural motifs: several numbers of EGF repeats, specialized EGF repeats (DOS) and an N-terminal motif. While the Drosophila genome solely encodes two ligands: Delta (Vassin et al., 1987) and Serrate (Fleming et al., 1990), the vertebrates have two families of ligand paralogues: Delta-like (DLL1, -3, -4) (Chitnis et al., 1995; Henrique et al., 1995) and Jagged (JAG1, -2) (Lindsell et al., 1995). The main difference lies in the presence (Jagged/Serrate) or absence (Delta) of a cysteine-rich domain. The paralogue protein sequences show homology, but display differences both in expression profiles and functions (Bray, 2006; D'Souza et al., 2010).

37 INTRODUCTION

Figure 9. The structure of the DSL ligand and the Notch receptor. Three proteolytic cleavages is shown to be important for the activation of the Notch receptor: S1 during synthesis and S2-S3 upon ligand binding.

The DSL ligand binding of the Notch receptor is followed by ligand and receptor trans- endocytosis and recycling in the endosome of the signal-sending cell. This internalization will generate an essential mechanical pulling force on the Notch receptor bound to the ligand, which dissociates NECD from the Notch transmembrane protein (TMD)(Parks AL, 2000; Weinmaster and Fischer, 2011). This process, which for a long time only was a hypothesis, was finally proven last year by Gordon et.al, using a single-molecule high- throughput magnetic tweezer assay( Gordon et al., 2015). The pulling force induces unfolding of the protective NRR region, which allows the second proteolytic cleavage ta site 2 (S2) to happen, performed by the disintegrin and metalloprotease Kuzbanian (Kuz) (Adam10/Adam17 in mammals) (Bozkulak and Weinmaster, 2009; Pan and Rubin, 1997; Rooke et al., 1996).

The discovery of the enzyme involved in the third and last proteolytic cleavage, the γ-secretase complex, was made in 2002 during studies of the pathogenesis of Alzheimer´s disease, based upon its function in cleaving the transmembrane amyloid precursor protein. γ-secretase is an enzyme complex containing Presenilin (the catalytic subunit), Nicastrin, PEN-2 (presenilin enhancer 2) and APH1 (anterior pharynx-defective). The γ-secretase cleavage is facilitated by the former S2 cleavage, and is performed at site 3 (S3) in the transmembrane domain of the Notch receptor. The γ-secretase cleavage leads to membrane release of the transcriptionally active 120kDa NICD (De Strooper et al., 1999; Fortini, 2002). In Drosophila and mammals presenilin or nicastrin loss of function, due to null mutation, prevents the NICD from being released from the plasma membrane upon Notch activation and inhibits the pathway (Struhl and Greenwald, 1999).

38 INTRODUCTION

NUCLEAR EVENTS IN NOTCH SIGNALING NICD becomes directly translocated to the nucleus, guided by the NLS (Fig 10). The RAM domain is shown to mediate association to a hydrophobic pocket in the DNA binding transcription factor CSL (CBF1/RBP-J [mammals], suppressor of hairless (Su(H)) [Drosophila] and Lag1 C.elegans [ ]) (Christensen et al., 1996; Fortini and Artavanis- Tsakonas, 1994). The conservation between human and Drosophila CSL crystal structure is high (84%) (Kovall and Hendrickson, 20 04; Tamura et al., 1995 ). NICD-CSL interaction facilitates for the binding of the transcriptional-activator co Mastermind (Mam; Mastermind-like in mammals) to the complex, which thus becomes trimeric (Bray, 2006; Petcherski and Kimble, ).2000 Furthermore, the complex recruits histone acetyltransferases (HATs) such as p300/CBP and PCAF, as well as other chromatin remodeling complexes (e.g. BRM, TRA1/TRAPP, Dom), which act to “loosen up” the chromatin, promoting transcription of effector genes (Bray, 2006; Wallberg et al., 2002). In the absence of NICD and Notch activity, CSL can recruit a co-repressor complex and histone deacetylases (HDAC), which keep the chromatin condensed and suppresses target gene transcription (Kao et al., 1998). In Drosophila, Hairless, Groucho (Gro), C-terminal- binding-protein (CtBP) and histone deacetylases (HDACs) are known members of this complex (SHARP, SMRT/NCor, CIR, KyoT2 etc. in mammals), and probably there are many additional factors yet to be identified (Borggrefe and Oswald, 2009; Borggrefe and Oswald, 2016). In the original static binding model it is suggested that CSL is bound to enhancers and upon the binding of NICD there is a displacement of co-repressors and an exchange to transcriptional activators, which activates transcription of downstream genes. This model has recently been challenged and a new hypothesis is that the entire CSL complex is dynamically exchanged upon Notch activation i.e., the CSL-CoR is binding with a low stability and CSL-CoA with a high stability. This is partially based upon the result that there is a significant increase of Su(H) occupancy on the regulatory elements of the E(spl)-C target genes after Notch activation, and furthermore that RBPJ is binding weaker to enhancers in absence of NICD (Bray, 2016; Castel et al., 2013; Krejci and Bray, 2007).

The active CSL complex activates the transcription of Notch target genes in a tissue- and cell-specific manner, to thereby execute the functions of Notch. The cell response is also greatly dependent on temporal features i.e., developmental stage. One of the broadest expressed and best characterized groups oftarget genes are the basic helix-loop-helix (bHLH) transcription factors encoded by Hairy and the Enhancer of split complex of genes (E(spl)-C) (Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995). The mammalian counterparts are the evolutionary conservedHairy and E(spl) genes (HES): mainly HES1, HES5 and HES7, as well as the closely related HERP/Hey family: Hey1, Hey2 and HeyL (Borggrefe and Oswald, 2009; Kageyama et al., 2007). E(spl)-C was originally found to act during neurogenesis, and encodes 12 Notch responsive genes of different classes, seven of which are closely related(m8, m7, m5, m3, mβ, mγ and mδ) bHLH transcriptional repressors (Knust et al., 1992; Wurmbach et al., 1999).

39 INTRODUCTION

Figure 10. NICD is translocated to the nucleus to bind the CSL-complex and further recruit Mam, histone acetyltransferases (p300/CBP and PCAF), as well as other chromatin remodeling complexes (eBRM, TRA1/TRAPP, Dom). In absence of NICD CSL is bound by co-repressors. The E(spl)-C-HLH proteins contain a C-terminal WRPW motif, an Orange-domain and a bHLH domain, and binds to DNA as ahetero - or homo-dimer (Fig 11). The repressing mechanisms are still under investigation, but one of the most well established is repression by recruitment of the co-repressor Groucho (TLE1-4 in mammals) to the WRPW motif, which in turn recruits HDACs that in turn alter the chromatin structure (Chen et al., 1999; Fisher et al., 1996). However, the WRPW domain is not always required, and it has been shown that HES1, acting through the orange domain, can repress its own promotor and Cdkn1a (Dap; p21WAF1) through recruitment of co-repressors (Iso et al., 2003). The HERP/HEY-family is closely related to the HES/E(spl) familybut have a YRPW motif instead of a WRPW. E(spl)-c-HLH and Hey may work independently in some tissues and together as a heterodimer in some (Iso et al., 2003).

In Drosophila, the seven E(spl)-C-HLH genes are very similar I structure and function and show a high level of genetic redundancy. The individual roles of the E(spl)-C-HLH genes have been difficult to separate; i.e., mutating one gene has a small or no impact on the phenotype and fitness of the organism. This is because E(spl) other -C-HLH genes compensate for the function of the mutated gene, partially or completely (Nagel et al., 2004; Wurmbach and Preiss, 2014). Gene redundancy could evolve via gene duplication, which is a rather frequent event during evolution. The reason why redundancies have been kept through evolution has been discussed and the answer is most likely because they have a beneficial function for the moment, and are not due to potential benefits creating a robust system for future mutations as a backup. However, even if redundancy is not selected for during evolution, it has great impact on health and development of an organism(Zhang, 2012).

40 INTRODUCTION

Figure 11. a) The E(spl)-C is located on the 3rd chromosome and covers an area of ∼50kb. b) The structure of the E(spl)-C-bHLH protein.

Notch has more target genes and many of them probably only expressed in a context specific manner. The cell cycle factors Cyclin D, p27KIP1 and p21Waf1/CIP1, and the growth regulator c-Myc have been linked to Notch signaling during cancerogenesis (Koch and Radtke, 2007), and CD25, GATA2-3, and Runx1 as downstream targets in the hematopoietic system (Pajcini et al., 2011). Furthermore, the Notch- pathway components Nrarp (Pirot et al., 2004) and Deltex1 have been reported as downstream targets (Cheng et al., 2015) in zebrafish. However, these genes, together with HES and HERP/HEY, cannot explain all varied consequences of Notch activation observed, suggesting that there are yet undetected effectors to be found. Frequently, it is troublesome to determine if a factor is a direct target of Notch or a secondary target, which makes the search more challenging(Borggrefe and Oswald, 2009).

41 INTRODUCTION

REGULATION OF THE NOTCH SIGNALING PATHWAY The phenotypic outcomes of Notch signaling are diverse, and an important issue for the field is to understand why and how different target genes are activated in different tissues and at different developmental time points. Some Notch targets are only expressed in some tissues, such as neuro-ectodermal factor Pax6, which is regulated by Notch in ESC but not in mesodermal cells (Meier-Stiegen et al., 2010 ). Furthermore, during muscle development is Twist recognized as a required co-activator of Notch (Bernard et al., 2010). Dynamic fluctuations in Notch activity also hasan instructive value, with one of the clearest examples of this being the regulation of the sequential formations of the somites in the mesoderm, in which Notch and HES is oscillatingand dependent on transient signaling (Dequeant et al., 2006; Hirata et al., 2002; Jouve et al., 2000). Oscillations by HES1 have also been demonstrated with real time imaging to regulate Ngn2 and Dll1 in the maintenance of neural progenitors (Shimojo et al., 2008).

Mechanisms must exist that direct NICD to the appropriate enhancer. Moreover, Notch signaling must be correctly regulated and fine-tuned in multiple ways, through: cis-trans signaling, proteolytic processing, degradation and recycling, miRNA control, glycosylation, chromatin modifications, absence/presence of other TFs etc. Notch could also activate expression of transcriptional repressors that inhibit their own expression and/or that of positive Notch targets( Bray, 2016; Chitnis and Bally-Cuif, 2016). Cross- talk between Notch and other pathways has also been described, where WNT, hypoxia and p53 are the best established cases (Collu et al., 2014; Dotto, 2009; Gustafsson et al., 2005). These facts together presumably underlie the scenario why Notch can induce lineage specific transcription factors, which in turn would be the key reason why Notch has different functions in different tissues.

CIS AND TRANS SIGNALING When a ligand presented on one cell interacts with a Notch receptor on the opposing cell it is called trans-interaction, and the receptor gets activated as previously described in the canonical Notch pathway. However, if both the ligand and the receptor are presented on the same cell, the interaction is of a cis-character, which leads to inhibition of the receptor due to it being blocked from binding other ligands in trans. The differences between cis- and trans-regulation is important for the switch between the state of being a signal sending cell, with expression of a high amount of ligands, to the state of being a signal receiving cell, with a high number of receptors on the surface. In this way, as in the process of lateral inhibition, an initial small ifference d in the amount of Notch activation between neighboring cells can gradually be amplified( Guruharsha et al., 2012; Sprinzak et al., 2010).

DEGRADATION OF LIGAND AND RECEPTOR It is necessary that Notch is switched on at the right spatiotemporal moment. Moreover, the durability and the termination of the signal is also of great importance. NEDC, NICD

42 INTRODUCTION and the unbound Notch-receptors constantly go through recycling/degradation programs. Binding of the NICD to the CSL complex results in rapid NICD turnover(Bray, 2006; Kopan and Ilagan, 2009). The degradation of NICD, and a short half-time of the signal, is crucial for the effective short pulse action by Notch. In mammals, MAM is recruiting Cyclin-dependent-kinase 8 (CDK8) to multi-phosphorylate NICD on degrons within its PEST domain. This phosphorylation leads to that the E3 ubiquitin ligases Fbw7/Ago/SEL10 ubiquitinate Notch, and targets it for proteasomal degradation. However, this degradation pathway in Drosophila is still poorly understood (Fryer et al., 2004; Oberg et al., 2001).

The Notch DSL ligands often require endocytosis to be functional. The RING E3 ubiquitin ligase Neuralized (Neur) (Lai et al., 2001) and Mindbomb (MIB) (Itoh et al., 2003) cooperatively ubiquitinate the cytoplasmic intracellular tail of the ligands, to thereby enhance Epsin-mediated endocytosis in Drosophila. Even if these two proteins have almost no structural similarities they can partly rescue the mutant defect of one another (Le Borgne et al., 2005; Parks AL, 2000; Wang and Struhl, 2005). Neur does not play an essential role in Notch signaling in mammals, because the ligand epitope has evolved and is no longer evident. In mammals, Mindbomb 1 (Mib1) is the only known E3 ligase that activates DSL ligands through ubiquitination, while Mib2 and Neur are dispensable (Guo et al., 2016; Koo et al., 2007). Finally Notch signaling could be regulated and controlled by E3 ligase activity, such as by the Bearded-related family, which negatively regulates Neur and thereby reduces Notch activity (Bardin and Schweisguth, 2006).

GLYCOSYLATION OF THE NOTCH RECEPTOR After translation, the Notch receptor becomes post-translationally modified by two forms of O-linked glycosylations i.e., attachments of sugars to oxygen residues in the EGF-like- repeats, which also play a key role in regulating Notch signaling. This is important for maturation of this large glycoprotein, and ensures correct activity, productive ligand binding, as well as proper structure and function. In the endoplasmic reticulum (ER), the glycosyltransferase O-fut adds fucose to the Notch receptor right after synthesis, while Rumi adds O-glucose (Acar et al., 2008; Okajima et al., 2003). In the Golgi apparatus, Notch becomes further modified by Fringe, which catalyzes glycosylation by adding -N acetylglucosamine on the added O-fucose (Rana and Haltiwanger, 2011). Studies have shown that some glycosyl-modifications could alter the ligand-binding ability to Notch. O-fut overexpression increases the binding of Serrate and decreases the binding of Delta. Fringe overexpression gives the opposite effect with increased Delta binding as a result (Chillakuri et al., 2012; Okajima et al., 2003; Rebay et al., 1991).

MODIFICATIONS OF THE CHROMATIN LANDSCAPE The transcription of Notch target genes is regulated by the chromatin landscape i.e., by DNA-methylation, histone modifications and chromatin folding, which all can act to affect the DNA accessibility (Perino and Veenstra, 2016). DNA-methylation located in a promotor region usually acts to repress transcription, by reducing binding affinity of TFs

43 INTRODUCTION or recruiting transcription repressing complexes to the DNA. Cytosine can be methylated in eukaryotes, and a methylation on the 5 position of cytosine (5-methylcytosine) leads to silencing of the gene.However, Drosophila does almost not show any cytosine methylation at all (Capuano et al., 2014). Histones are chiefly modified upon their N- terminal-tail, where arginines and lysines are typically acetylated or methylated, one, two or three times. The result of each methylation/acetylation depends on which amino acid in which histone that is modulated and how many times. Histone modifications such as trimethylations can both promote transcriptional activation e.g., H3K4me3, or repress it e.g., H3K27me3 and H3K9me3. H3K4me3 has been shown to be necessary for activation of genes and thereby resolve the poised state downstream Notch in both Drosophila, ESCs and mice. H3K4me3 and H3K27me3 are also hypothesized to direct the NICD/MamL/CSL complex to the Notch target genes. Histone acetylation occurs to the N-terminal lysine residues and is catalyzed by histone acetyl transferases (HAT) (Schwanbeck, 2015). The histone acetyl transferases p300/CBP and PCAT act with NICD, MAML1 and RBP-J to acetylate the residues of the histone tail to transcriptionally activate the chromatin (Wallberg et al., 2002).

44 AIMS OF THE THESIS

AIMS OF THE THESIS

The general aim of the work presented in thesis was to investigate the proliferation mechanisms ensuring that the exact number of each neural cell subtype is generated in the CNS. More specifically, the projects were aimed to:

PAPER I To perform a compressive forward genetic screen for genes affecting the generation and specification of a defined subset of embryonic Drosophila VNC neurons originating from NB5-6T; the Ap cluster neurons and the Ap4/FMRFa neuron in particular. This screen was designed in order to identify genes involved in the different phases of embryonic progenitor lineage development.

PAPER II To investigate the function of Notch signaling pathway in the NB 5-6T lineage, to hence answer the question of what the origin of the excess of Ap neurons and the frequently double FMRFa cells in the Notch mutants found in the forward genetic screen was.

PAPER III To shed light upon the molecular and genetic mechanisms by which Notch is mediating a type I>0 switch in daughter cell proliferation mode in the NB5 -6T lineage and the possible interplay between Notch andkey cell cycle factors. Furthermore, to elucidate if the involvement of Notch in the daughter cell proliferation switch could be extended to the entire VNC.

PAPER IV To address and characterize the function of the gene sequoia in the VNC and to study the possible interaction between sequoia and Notch signaling pathway in governing the type I>0 daughter cell proliferation switch.

46 METHODS

METHODS

A number of methods were used throughout the work of this thesis.The main methods used are explained in brief in the following section. For more detailed information about the experimental procedures please see Paper I-IV.

IMMUNOHISTOCHEMISTRY (IHC) Most cells are colorless and cannot be seen by a regular light microscope. Immunohistochemistry (IHC) has since its discovery in 1941 (Coons et al., 1941), become a widely used technique to label and visualize the distribution, quantity and localization of different proteins in a tissue by the use ofspecific monoclonal or polyclonal antibodies (Chen et al., 2010).

In Paper I-IV Drosophila embryos were staged according to standard criteria (Campos- Ortega and Hartenstein, 1997), dissected into fillets by adhering the embryo to poly-lysine coated glass slides, and stained by IHC. Embryos were fixed with paraformaldehyde (PFA), for preserving the tissue architecture and cell morphology, by crosslinking of macromolecules. This was followed by incubation with detergent (Triton-X) and blocking agent (horse serum), which permeabilizes the cell membranes and blocks the background, respectively. Primary antibodies, designed to recognize and bind to particular epitopes on proteins of interest, were added to the fixed tissue. Subsequently, secondary antibodies conjugated with fluorescent markers (fluorophores) were added to bind the species specific FC domain of the primary antibodies (Fig 12). A fluorophore is capable of emitting light upon irradiation, which makes it possible to visualize it in a microscope.

Figure 12. The basic concepts of immunohistochemistry (IHC).

47 METHODS

For achieving a maximum 4-color separation in our analysis,secondary antibodies conjugated to the following fluorophores were used: AMCA or Dylight 405 (emission max ∼420-450nm), Alexa Fluor 488 or FITCemission ( max ∼520nm), Rhodamine Red-X (emission max ∼590nm) and Alexa Fluor 647 or Cy5emission ( max ∼670nm). Alternatively the enzymatic substrate (X-gal) was added to detect ß-galactosidase, which can catalyze the hydrolysis of X-gal into a blue precipitate that can be visualized under microscope. The slides were finally mounted in Vectashield® to inhibit photo bleaching of the dye.

MICROSCOPY Microscopy is used to highly magnify a tiny object of interest to be able to study it more in detail. This technique is based on the feasibility to converge light rays using one or more lenses. There are a widerange of different microscopes for different purposes: light-, fluorescence-, confocal laser scanning-, electron microscopes- etc.

FLUORESCENCE Fluorescence is emission of radiation by a substance when molecules are returning from electronically excited states. When a fluorophore is illuminated, in our case conjugated to a secondary antibody, it is hit by photons that excite its electrons via absorption to a higher energy level. When the electrons return to ground state the fluorophore loose energy emitted as light. The emitted light has less energy, longer wavelengths, compared to the excitation light and the difference between the two is called Stokes shift (Lichtman and Conchello, 2005).

CONFOCAL LASER SCANNING MICROSCOPY In a wide-field fluorescence microscope the entire specimen gets illuminated at the same time, which yields a good lateral resolution. However, this microscope suits thin specimens the best, due to problems with background noise.Confocal laser scanning microscopy (CLSM) was developed in the late 1980’s to improve the images from the wide-field fluorescence microscope (White et al., 1987). It performs a point illumination on a discrete part of the specimen with a laserlight spot and a point detection of the emission. For all fluorescent images in this thesis, tissues were stained with IHC and analyzed by the use of CLSM to achieve a high optical resolution. The data were captured with: Zeiss LSM700, Zeiss LSM700 inverted, Zeiss META510 or Zeiss LSM5 confocal microscopes, using a 40x oil immersion objective. The basic principle for all four confocals is that high energy laser beams (405nm, 488nm, 555nm or 633nm) are projected into a specimen (Fig 13). The emitted light is collected into a small pinhole, excluding out-of-focus light, resulting in that only light from the right focus point hits the recording detector. Further, the beam of light scans over the sample, back and forth in a horizontal plane square, producing a 1µm optical sectioning effect. By merging different Z-stacks (sets of images collected at different focal depth) a 3D-projection can be produced. The optical resolution of a imagining system implicates the ability of the microscope to resolve details in an object

48 METHODS i.e., defined as the shortest distance between two points that can still be distinguished as separate entities (Amos and White, 2003).

IMAGE ANALYSIS Zeiss LSM image browser software, Carl Zeiss Zen software and Adobe Photoshop (CS4- 6) were used for studying single optical sections, for merging confocal Z-stacks into a projection, to quantify cell numbers and to measure emission intensity. Adobe Illustrator (CS4-6) was used for compiling informative images.

Figure 13. The emission specra from IHC using Dylight 405, Alexa fluor 488, Rhodamine Red-X and Alexa Fluor 647.

DETECTION AND ANALYSIS OF DNA-PROTEIN INTERACTIONS CHROMATIN IMMUNOPRECIPITATION (CHIP) The ChIP technique was used in Paper III to analyze the interactions between tran scription factors and specific DNA regions i.e., enhancers/promotors, to identify binding sites (Gilmour and Lis, 1984 ; Gilmour et al., 1991 ; Solomon and Varshavsky, 1985 ). Chromatin preparation was carried out according to a protocol frommod- ENCODE (Nègre et al., 2007). Immunoprecipitation was conducted according to MERCK Millipore protocol (Magna ChIP protein A/G beads) using αFLAG (m) (BPS Bioscience cat: 25003). In principle, DNA and DNA-binding proteins were crosslinked with formaldehyde. Following the crosslinking, the cells were ly sed and the DNA-complexes were fragmented into smaller pieces by sonication. Protein specific antibodies were added to label the DNA - complexes, in our case α-FLAG to capture Flag-tagged E(spl)-C proteins. The fragments were immunoprecipitated via magnetic separation through addition of magneticbeads coupled to IgG binding recombinant protein A/G. Finally the DNA fragments were purified and sequenced on the Illumina Hiseq2500 platform (see sequencing).

49 METHODS

DNA ADENINE METHYLTRANSFERASE IDENTIFICATION (DAMID) The DamID method was used in Paper III to map DNA-protein interactions, utilized as a validation and complementary method to the ChIP experiments (van Steensel et al., 2001; van Steensel and Heinkoff, 2000). The DamID was performed according to a slightly modified protocol based on Vogel et.al. (Vogel et al., 2007) and a collaboration between Brand and Meadows lab (Brand and Meadows, 2012). In brief, DNA adenine methyltransferase (Dam), originally from E.coli, was fused to a protein of interest.Dam methylates the adenines (A) in “GATC” in the vicinity of this specific proteins’ binding site on the DNA. Embryos were collected and DNA was extracted (Qiagen Blood and Tissue DNA Extraction Kit). An a ddition of the restriction enzyme Dpn1 cleaved the DNA sequences at the Dam methylated GATC-sites. Subsequently, a double stranded adaptor oligonucleotide was ligated to these blunt ends to mark them for amplification. Addition of the restriction enzyme DpnII cutun- methylated GATC sites and thereby eliminated them from amplification. The sequences were amplified in a PCR protocol with primers identical to the adaptor oligonucleotides. The PCR product, the PCR-amplicons, were sequenced on the Illumina Hiseq2500 platform (see sequencing).

CHARACTERIZING EMS-INDUCED MUTATIONS BY WGS In Paper I, a subset of 23 mutants with interesting phenotypes identified in the forward genetic screen were mapped by whole genome sequencing ( WGS). DNA was purified from 20-30 wandering third instar larvae (L3) according to Blumenstiel et.al. 2009 (Blumenstiel et al., 2009; Gerhold et al., 2011). Since the mutants identified in our screen were homozygous lethal we needed to sequence the individualsas heterozygous. To avoid sequencing non-mutant chromosomes and to increase the efficiency from each sequencing run we crossed two mutant alleles and sequenced the -heterozygo trans us L3 larvae progeny. Because of the amplification of euchromatinic DNA during the larval stages, this furthermore increased the sequencing coverage of gene-rich regions (Blumenstiel et al., 2009). Furthermore, all the EMS mutants were induced on the same genetic background, and could thereby act as internal sequencing controls to each other. The sequencing files were analyzed in DNASTAR aligned to a reference GenBankDrosophila genome, searching for mutations in protein- coding sequences only represented on one allele i.e., >29% of the reads in a specific region. We searched for - nonsynonymous mutations (missense and nonsense) in particular, which could possible cause the severe lethal phenotypes. Mutations commonly presented on both alleles were believed to be polymorphisms, naturally occurring in the parental line.A 45mM dose EMS causes an average of 11 mutations affecting coding sequences per chromosome( Blumenstiel et al., 2009). An average of seven candidate genes was identified per mutant pair, based on known functions and upon previous created gene expression profiles. The candidates were confirmed or rejected by complementation tests to smallgenomic deficiencies and available alleles.

50 METHODS

NEXT GENERATION SEQUENCING (NGS) In Paper I and III, high-throughput next generation sequencing (NGS) was used for WGS, DamID- and for ChIP-sequencing. Sequencing was carried out on the Illumina HiSeq2500 platform by GENEWIZ. Briefly the input DNA sample is randomly fragmented into short 100-150 bp sequences, followed by 5´and 3´adapter ligation and PCR amplification. The prepared DNA fragment library is loaded on a flow cell to hybridize to its surface-bound adapters. Single fluorescent labeled nucleotides are incorporated into the DNA strands in cycles and the emission from the sequences are recorded (Illumina, 2016). A quantity of ∼20-30 million reads of a length of 50-100 bp are recommended for statistical purposes, sequencing a medium genome such as Drosophila. We used 50 bp (ChIP, WGS) or 100 bp (DamID, WGS) single read configurations. The bioinformatics software DNASTAR Seqman NGN (DNASTAR, Inc. version 12.2) was used forassembly and alignment of sequence reads to the Drosophila genome. Normalization was done with RPM, while Qseq was used for peak detection and the wig-files were aligned to genome assembly dm6 on the UCSC genome browser for visualization (Karolchik et al., 2014).

MANIPULATING THE GENOME One of the greatest advantages with the Drosophila model system is the extensive genetic toolbox available. Many of the techniques are based on parts of already existing biological processes, which have been transferred from their ordinary environment to be conformed for a new directed function. Furthermore, these techniques often include homologous recombination and/or introduction of transgenes into the Drosophila genome by injection.

THE GAL4-UAS SYSTEM The Gal4–UAS system (Brand and Perrimon, 1993; Fischer et al., 1988) has been used throughout the work of this thesis to selectively express genes ectopically in a spatially or temporally specific manner. This system is widely utilized in the Drosophila model, and is becoming increasingly used also in the zebrafish (Halpern et al., 2008; Scheer and Campos-Ortega, 1999) and successfully trialed in mouse (Lewandoski, 2001; Ornitz et al., 1991). Many ectopic expression profiles are not compatible with vitality. However, by the use of Gal4-UAS you can turn on a protein at a specific given time point in a specific tissue. In short, the two parts of the system, the Gal4 and the UAS is initially separated into two transgenic lines. The Gal4 gene, encoding a yeast transcription factor, is fused to any promotor/enhancer e.g., to a tissue specificcontrol- element. The UAS u (pstream activating sequence) consists of several GAL4 binding sites. UAS is fused to a geneof interest e.g., a reporter gene such as GFP. DNAis introduced into the genome, by the means of P element or phiC31 (Bischof et al., 2007) mediated transposon transgenesis. When the two transgenic lines are crossed, the progeny that inherits both transgenic elements activates the UAS transgene. The GAL4 protein is translated and binds to the UAS target enhancer, consequently activating the transcription of the gene that is under control of the UAS promoter, whichthen will be ectopically expressed. The Gal4-UAS

51 METHODS system can be used for overexpression of genes, both for a loss of function, a dominant- negative effect, as well as for expression of marker genes (e.g. GFP/RFP) (Duffy, 2002).

EDU LABELING 5-ethynyl-2’deoxyuridine (EdU) is a synthetic analogue to the nucleotide thymidine. EdU can be incorporated into the DNA in- phase S during replication and is used to label proliferating cells (Chehrehasa et al., 2009). The EdU detection is made by covalently binding a fluorescent labeled azide (Alexa Fluor) to the terminal acetylene of theEdU molecule via “Click-It™” chemistry reaction (Li et al., 2010). In Paper II, EdU was used to determine sibling relationships in the NB5-6T lineage. Stage 11-13 embryos were posteriorly injected with EdU and incubated at 26°C to continue to develop. At stage 18AEL the CNSs were dissected, attached to poly-L-lysine-coated slides, fixed with 4 % paraformaldehyde and treated with “Click-It™ reaction”. After subsequent IHC adding other markers, the EdU-Alexa Fluor was detected by confocal microscopy. Only neurons born sequentially can be separated by EdU staining, thus two cells born in the same divisions cannot. By studying the EdU pattern we got supporting information about if a cell is born in type 0 or type I division mode.

STATISTICAL ANALYSIS For statistical calculations in Paper II-IV GraphPadPrism software was used, and graphs were compiled in Adobe Illustrator (CS4-CS6). Student’s t-test was used to test statistical significance between two normally distributed groups. When comparing more than two groups, ANOVA with Bonferroni’s post hoc test were used. Nonparametric - Mann Whitney U test, Kruskal Wallis with Dunn´s posthoc or Wilcoxon signed rank test was applied in the case of non-Gaussian distribution of variables. For analysis of proliferation frequencies in Paper IV in the NB3-3A, NB5-6T and NB7-3A, Chi-square was used. Evaluation of cell cycle gene expression levels in Paper III-IV was conducted by dissecting CNSs from mutants/overexpression and control embryos on the same slide in at least two different experiments. The intensity of staining inmitotic NBs (pH3+) was measured in Adobe Photoshop (CS4-6) or Image J (1.49d). To compensate for size when comparing results from separate experiments, the means were normalized and multiplied by volume or area in Adobe Photoshop or ImageJ. Student’s-test t was performed to quantify differences on the whole dataset. P-values < 0.05 (*), < 0.01 (**) and < 0.001 (***) were considered significant.

52 RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

PAPER I “NOVEL GENES INVOLVED IN CONTROLLING SPECIFICATION OF DROSOPHILA FMRFA NEUROPEPTIDE CELLS” The thoracic neuroblast 5-6 (NB5-6T) lineage, consisting of 23 neurons and glia, has been well mapped. The lineage can be identified by the expression of GFP/lacZ reporters under control of an enhancer fragment from the ladybird early gene, lbe(K) (Baumgardt et al., 2007; De Graeve et al., 2004). The NB5-6T progression starts with nine rounds of type I proliferation. At stage 12-13 there is a switch in division mode, and the lineage continues with five rounds of type 0 division. The NB5-6T generates a unique group of interneurons at the end of the lineage, hence born in the type 0 mode directly from the NB. After production of its designated cells the NB5-6T stops proliferating, exits the cell cycle and undergoes apoptosis (Baumgardt et al., 2014; Baumgardt et al., 2009). The group of last born interneurons have in common that they are all expressing the LIM-homeodomain transcription factor Apterous (Ap) and are thus denoted Ap neurons (Fig 14). Five Ap neurons are produced in the T1 segment and four in T2-T3 (Lundgren et al., 1995). The Ap neurons can be identified by the expression of the transcription factor Eyes absent (Eya) (Miguel-Aliaga et al., 2004), and be sub-divided based on their specific neuropeptide expression. The first Ap-neuron (Ap1; also denoted Tv1) is expressing the Neuropeptide- like precursor 1 (Nplp1) and last born cell in lineage (Ap4; also denoted Tv4) is expressing FMRFamide (FMRFa) (Baumgardt et al., 2009; Schneider et al., 1993). Furthermore, the Ap4 neuron has a unique axonal trajectory, innervating a secretory gland outside the CNS, the dorsal neurohemal organ (DNH). Upon axonal targeting the DNH is sending a TGFβ/BMP retrograde signal critical for FMRFa expression (Allan et al., 2003; Marques et al., 2003). Solely six neurons out of 10,000 in the embryonic Drosophila VNC express FMRFa, 28 cells Nplp1, and 90 cells Ap (Baumgardt et al., 2007).

A forward genetic screen is a powerful tool for identifying genes involved in a specific biological process. When designing such a screen it is of a great value to utilize a refined model system with a clear readout, to thereby be able to retrieve as much information as possible from the outcome. The mapping of the NB5 -6T lineage provided us with extensive lineage information, creating a framework that made it possible to set up a screen with a high single cell resolution. We took advantage of the fact thatFMRFa is exclusively expressed in Ap1/FMRFa; and hence that successful terminal differentiation of the Ap4 neuron makes it a selective readout for addressing many aspects of neural development. We were aiming at finding additional cues for how proliferation control and subtype specification is achieved in the NB5-6T lineage, and in particular and how the fine precision to specify the four late born Ap clusteris regulated. cells An ethylmethanesulfonate (EMS) forward genetic screen, utilizing FMRFa-eGFP transgenic reporter, was conducted in the attempt to resolve these questions.

53 RESULTS AND DISCUSSION

The FMRFa-eGFP construct was injected into w1118 flies, and subsequently mobilized by the use of transposase. One insert on the 2nd and one on the 3rd chromosome, showing robust eGFP expression in stage 18 AEL embryos, were identified. For mutagenesis, EMS treatment was used to trigger point mutations by alkylation in the gametes of isogenic FMRFa-eGFP male flies. A total of 9,781 balanced stocks were established and initially screened in larvae for aberrant phenotypes, which resulted in 611 mutated strains with altered eGFP expression. After further screening, 277 mutants, showing a robust phenotype in both larvae and embryo , were selected to be studied at higher resolution. The mutants were categorized into five different phenotypic es class based upon eGFP expression: None, weak, variable, ectopic, or altered expression. The mutants were subsequently sub-grouped into different phenotypic classes based on immunostaining phenotype i.e., proFMRFa, Eya, Dimmed, Nplp1 and phosphorylated Mad expression. Complementation tests against genes already known to play important roles in theNB5 - 6T lineage specification resulted in re-identification of a number of genes, which validated the screen set-up. Most mutants were mapped against the Bloomington Stock Center Deficiency Kit, based upon the assumption that the gene ofinterest was homozygous lethal. In the latter phase of the project a group of alleles were mappedby whole genome sequencing. The region mapping was followed by subsequent sub-mapping with smaller deletions and finally testing against putative candidate genes. The screenresulted in the identification of 43 genes (79 alleles), strikingly all of those had conserved human orthologs.

This genetic screen was extensive and the mapping of mutants by deletion mappingwas labor-intensive to perform. However, NGS was a considerably more rapid way to identify mutations. Several other groups have successfully used this approach before, both in Drosophila and C.elegans (Blumenstiel et al., 2009; Haelterman et al., 2014; Smith et al., 2008). With the progress and price drop in NGS it is likely to become the method of choice for mapping mutations in genetic screens.

The exact % coverage in a genetic screen is traditionally calculated by determining how many loci were identified, and how many loci had one versus two or more alleles. However, more recently, the exact calculation of saturation has been debated, and we have not attempted to calculate saturation for our screen. Based upon the fact that we only identified multiple hits for a few genes, it is most likely that the screen had a low saturation (Pollock and Larkin, 2004). You could hypothesize about a number of factors that are difficult to dissolve, which decreased the outcome of our screen.First , only the large 2nd and 3rd chromosomes were chosen to be screened, whilst the X (20% of genome) and the 4th (1%) chromosome were excluded. Second, we only choose to map genes presenting a strong, easy scored and interesting phenotype. Third, genes whose function are very broad and essential during early development were discarded early on, due to the fact that they probably were early lethal or severely malformed long before 18AEL. Finally, genes whose RNA or protein is maternally loaded, and thus protected during embryogenesis, may have failed to come up in the screen because they showed no phenotypic alteration.

54 RESULTS AND DISCUSSION

The genes identified in the screen belonged to diverse groups important for different parts of the NB5-6T progression (Fig 14): The spatial Hox gene Antp, columnar gene ind and NB5-6T-specific lbe. The temporal genes cas, grh and seven up. Chromatin modification factors such as brahma, Ctr9 and polycomb group. The axonal retrograde transport/BMP signaling factors Gbb and Mad. The Ap4/FMRFa cell fate determinants collier and eya. The Notch signaling genes uz k and neur. The cell cycle genes dap (p21Cip1/p27Kip1/p57Kip2), CycE, E2F and Cyclin T. The RNA splicing factors Peanuts (Pea), Processing factor (Prp8), Brr2 and jhl-1. As well as several genes not categorized into a group. We discovered new functions of 16 genes that have not previously been observed to play roles during CNS development e.g., seq, Ctr9, 18w, Notum, mop, mute, Prp8 and pea. Some of the novel genes were chosen to be investigated further, which hopefully will help us to achieve a better understanding of cell fate specification and neural development. To summarize, the screen conducted in Paper I was fruitful andidentified genes that covered many aspects of NB5-6T development.

Figure 14. Summary of the identified genes in the genetic screen performed in . Paper I 79 alleles of 43 genes belonging to subgroups important for the development and progression of the NB5-6T lineage were identified. The screen was based on the Ap neuron (red) and Ap4/FMRFa (green/red) phenotypes (adapted from Bivik et.al., 2015).

55 RESULTS AND DISCUSSION

PAPER II “CONTROL OF NEURONAL CELL FATE AND NUMBER BY INTEGRATION OF DISTINCT DAUGHTER CELL PROLIFERATION MODES WITH TEMPORAL PROGRESSION” In the forward genetic screen (Paper I), two genes in the Notchsignaling pathway were identified in the group of mutants displaying additional Ap4/FMRFa neurons and a n excess number of Ap neurons. Seven alleles of kuz; implicated in the S2 cleavage of NECD (Pan and Rubin, 1997; Rooke et al., 1996) and one allele of neur; encoding an E3 ubiquitin ligase important for the internalization and endocytosis of the Delta ligand, were mapped (Yeh et al., 2001). Based upon the identification of the interesting joint phenotype of these Notch signaling pathway mutants, we wanted to elucidate the mechanisms by which absence of Notch could alter the lin eage to produce more Ap neurons and frequently double Ap4 neurons at the end of the lineage. This was all the more interesting as our laboratory had recently identified a switch in daughter proliferation mode in this lineage, from type I to type 0 (Baumgardt et al., 2014).

We began by analyzing a number of core components of the Notch pathway with regard to Ap cluster alterations, to be able to confirm the importance of the canonical pathway. Notch pathway mutants are often difficult to work with, since the signaling pathway is pleiotropic and crucial formany aspects ofdevelopment , hence resulting in severe embryogenesis defects. In this study we took advantage of Notch pathway genes that are maternally loaded i.e., their RNAs and proteins are deposited in the egg, and thereby provide gene function during early development even in a zygotic mutant background. Through this developmental protection mechanism we could analyze genes affecting the late linage without suffering from early lethal complications. Intriguingly, in all Notch mutants, RNAi and dominant negative components analyzed, we only observedNotch proliferation problems and no effect on neuron specification in the NB5-6T lineage. Furthermore, we never saw more than two Ap4/FMRFa cells perAp cluster. Our first hypothesis was that this increase in Ap cell number could be due to increased number of progenitor cells and thereby NB lineages, on the basis of Notch’s important role during lateral inhibition. Further analysis with the NB5-6T transgenic lineage marker lbe(K)- lacZ/GFP revealed that this was indeed true for strong Notch perturbations, such as neur and Dl. However, in mutants of the maternally loaded gene kuz, we did not find any extra neuroblasts. Analysis of mitotic events in kuz mutants allowed us to conclude that Notch only is important in the latter part of the lineage. We postulated three additional hypotheses for the extra Ap cluster cells in Notch pathway mutants: 1) premature Ap neuron production, 2) failure for the NB to exit th e cell cycle, or 3) failur e to switch division mode from type I>0. We could exclude the two former alternatives by analyzing kuz mutants for the expression of the genes in the temporal cascade, which revealed a normal progression, and by staining for cleaved-caspase 3, a marker for apoptosis, which revealed that the NB was normally undergoing apoptosis at St16. We began to elucidate the third hypothesis,

56 RESULTS AND DISCUSSION and through mitotic analysis of the GMC we found 37% dividing daughter cells late in the lineage in kuz mutants, something never observed in wild type. A final experiment with EdU staining labeling S-phase of different Notch perturbations showed that in the cases when double FMRFa or Nplp1 was observed , these cells were always generated as siblings. Combined, this verified our last theory and demonstrated that there is a failure to switch from type I to type 0 daughter cell proliferation mode in Notch pathway mutants. To our knowledge this is the first publication showing the importance of Notch, or anygene, in this context. We continued to demonstrate thatthe Notch target gene E(spl)m8 is increasingly activated in NBs over time and likely activate this switch on a threshold.

Prospero, a well-known component in the asymmetric machinery, was known to restrict proliferation in the GMC through acting on cell cycle genes. We found that in the NB5-6T development, Pros is acting early in the lineage, restricting the mitotic potential of daughters in the type I mode. However, in the late lineage, in the type 0 mode, although Prospero is expressed, it has no obvious function. Notch and Pros was shown to act simultaneously to control GMC proliferation, but atan individual level without cross- regulation. However, mutating both pathways at the same time resulted in a massive overproduction of neurons.

Since there were no indications f ocross-regulation between Notch and the temporal cascade (hypothesis I) it allowed us to set up a topology-temporal interplay experiment. We wanted to manipulate the daughter cell proliferation andcell specification simultaneously but independently. This was achieved by studying the effects of a double mutant of kuz and the sub-temporal gene nab (that affects the transition from Ap1 to Ap2 cell fate). In this manner we managed to generate a NB5-6T lineage with four Ap1/Nplp1 neurons as a combined consequence of a failure to switch and an inhibition in temporal progression i.e., alteration in both cell fate and cell numbers.

To summarize, in Paper II we demonstrated that the NB5-6T lineage uses two mechanisms to control daughter cell proliferation and the generation of distinct neural subtypes over time. Pros controls daughter proliferation mechanisms acting early in the NB5 -6T lineage, in the type I mode, and Notch activity is successively superimposed on Pros to further limit the mitotic capacity and hence trigger the NB to switch from producing GMCs (type I) to directly generate neurons and glia (type 0) (Fig 15). In Notch mutants the switch does not occur and the lineage progresses in type I mode until the NB exits the cell cycle, hence resulting in the double amount of Ap cluster cells. We further demonstrated that the switch in NB5-6T is the only observed process affected in the Notch mutants analyzed, while NB selection, cell specification, temporal transitions as well as NB cell cycle exit seem to progress normally.

57 RESULTS AND DISCUSSION

Figure 15. Summary of findings in Paper II. a) In the NB5-6T lineage Notch signaling pathway is mediating a switch in daughter cell proliferation modes from type >I 0. In absence of Notch the switch is prevented from happening. b) The NB5-6T lineage. c) In Notch mutants e.g., kuz, the switch is prohibited and the lineage continues in type I mode. d) In pros mutants the early lineage daughter cell proliferation control is lost. e) In kuz, pros double mutations both early and late daughter cell proliferation control are lost, resulting in a massive amount of neurons and glia (adapted from Ulvklo et.al., 2012 and Baumgardt et.al., 2016).

Paper III “CONTROL OF NEURAL DAUGHTER CELL PROLIFERATION BY MULTI-LEVEL NOTCH/SU(H)/E(SPL)-HLH SIGNALING” In Paper III we were interested in i nvestigating if Notch is only controlling the proliferation in one progenitor linage i.e., NB5 -6T or if the mechanism is globally utilized. Furthermore we wanted to elucidateif Notch signaling mediates the type I>0 switch in other NB lineages in the embryonic VNC. In the canonical Notch pathway, NICD binds to the CSL complex in the nucleus, recruits Mam and co-activators, which drives the expression of several target genes. Among the most well-studied target genes inDrosophila are the Enhancer-of-split-complex (E(spl)-C) gene family (HES in mammals), consisting of seven members of bHLH-O transcriptional repressors, located in a ~50 kbp genomic region on the 3rd chromosome. Notch signaling frequently acts through these HES genes, but it is unclear which one(s) if any would be involved in the type I>0 proliferation switch, and if there may be differential usage of HES genes in different NBs.

We begin by addressing how applicable Notch mediation of the type I>0 switch is in the whole VNC. Through detailed lineage analysis we demonstrated that Notch indeed can control another known type I>0 switch;at St11 in the NB3-3A lineage. By further analyzing mitotic GMC events in both the thoracic and abdominal parts of the VNC we could conclude that in kuz mutants there are approximately 50% more GMC divisions at St14 and St15, when compared to wild type. However, we did not observe any significant difference in NB divisions. These global findings were in line with our findings on NB5-

58 RESULTS AND DISCUSSION

6T and NB3-3A i.e., that Notch signaling is critical for daughter proliferation globally but not for NB cell cycle exit.

Proneural genes are centralplayers in the early event of lateral inhibition. However, studying the expression of the three main proneural genes;achaete , scute and lethal of scute, in the early VNC at the stage of the switch (St12) did not reveal any evidence for proneurals to play a role in the switch. The proneural genes were sparsely expressed, and not obviously in the NBs at all. These resultsindicate d that only certain aspects of canonical Notch signaling are involved in the switch.

Aiming to gain deeper understanding into how Notch controls the switch, we studied the E(spl)-C-HLH (E(spl)-C) downstream genes. We conducted anE(spl) -C functional analysis by using a number of available deficiencies in the E(spl)-C region. Corresponding to earlier studies of the Notch pathway inDrosophila , this revealed that several of the E(spl)-C genes are involved in NB selection, but additionally that E(spl)-C seemed to affect the type I>0 switch. A major problem with the E(spl)-C genes are that they show a high degree of genetic redundancy (Bray, 2006), which has made it difficult to discriminate between the genes and their possible separate function, and before Paper III no point mutations in the E(spl)-C genes had been identified. With the purpose of identifying if all or only some of the E(spl)-C genes were involved in the switch, we ordered TILLING analysis performed on the E(spl)-C genes, from the Seattle TILLING Consortium (Cooper et al., 2008). This fortunately resulted in the identification of point mutations in all seven individual genes of the E(spl)-C. In addition, in collaboration with Francois Schweisguth, Institut Pateur, we generated recombineering and Crispr/Cas9 mutants in some of the E(spl)-C genes. By analyzing these novel mutants in a sensitized background over a deficiency for the whole region, we found that E(spl)m5, m7, m8 and mdelta are involved in the type I>0 switch in the both the NB5-6T and NB3-3A lineage. Strikingly, different E(spl)-C genes were shown to be involved in different lineages: E(spl)m7 showed altering of Ap neuron numbers in the NB5-6T lineage, but gave no effect on the NB3-3A lineage. Meanwhile E(spl)m8 gave us the opposite result. Analysis revealed that extra Ap neurons resulted both from additional NB5-6T neuroblasts, as well as from defects in the type I>0 switch. This indicates that in spite of the reported high degree of redundancy between E(spl)-C genes we observed an interesting context-dependent usage of these regulators and effects in all single gene mutants.

Since mutants of the Notch pathway could prevent the switch from happening weere w curious to find out if over-expression of one E(spl) gene, E(spl)m8, could force the switch to happen earlier. To this end, we constructed - FLAGtagged UAS-m8 transgenes. However, when trying to visualize these constructs with apros- Gal4 driver we observed little if any FLAG-tag signal. This led us to suspect rapid degradation of the E(spl) protein, a likely assumption since rapid degradation, due to miRNA control, has been reported (Bray, 2006). Based upon previous publications, we mutated critical Casein kinase (CK) phosho-degron sequences in E(spl)m8, and constructed new degradation-stabilized FLAG-

59 RESULTS AND DISCUSSION tagged transgenes. In two of these transgenes we observed improved FLAG-tag detection. Analyzing proliferation patterns with these transgenes – UAS-E(spl)m8-FLAG-CK2 and UAS-E(spl)m5-FLAG-CK2 – we found a significantly lower number of both NB and GMC divisions globally, indicating on an earlier switch to Type 0 direct neuron generation, but also a Notch effect on NB proliferation.

Proliferation control in the developing Drosophila VNC requires precise regulation of the expression of four key cell cycle factors shown to be instrumental for the switch: The activators CycE, E2f, String (cdc25) and the inhibitor Dap. Mutation or misexpression of these three genes results failure in of the type I>0daughter proliferation control (Baumgardt et al., 2014). To elucidate the possible connection between Notch signaling and the cell cycle, we analyzed expression of these cell cycle proteins in various Notch mutant backgrounds, which revealed that expression all four was affected. To further study the relationship between the cell cycle genes and Notch signaling, rescue experiments were performed. As anticipated, the Notch phenotype in kuz mutants was successfully rescued by UAS-dap. Moreover, we observed genetic interactions betweenkuz and dap mutants, and E(spl)m5, m7 and CycE.

To address if this regulatory interplay results from direct transcriptional regulation, we conducted genome-wide DNA-binding analysis for two of the E(spl)-C proteins, m5 and m8, as well as for the common pathway factor Su(H). This was conducted using two different approaches; DamID-seq and ChIP-seq. These analysis revealed direct transcriptional control through binding of m5, m8 and Su(H) to the E(spl)-C as well as the cell cycle genes: CycE, stg, E2F and dap, which verified our previous results but also indicated a multi-level Notch signaling cascade.

These results, combined with previous studies, adds a third aspect of Notch signaling during CNS development. Notch plays a crucial role in the early lateral inhibition process in the neuroectoderm involving the Notch-E(spl)-proneural cassette. Furthermore Notch acts in the binary decision between daughter cell fates involving a cell fate A versus B decision. Paper II and III addsthat later re-activation of the Notch pathway in NBs, involving Notch-E(spl)-cell cycle genes, triggers a programmed daughter proliferation switch, involving different E(spl)-C genes in different lineages (Fig 16b). We suggest a multi-level Notch signaling cassette where Notch activates E(spl)-C and cell cycle genes i.e., primarily dap, but also CycE, stg and E2f on a primary level, and subsequently a second level Notch signaling where E(spl)-C acts to repress E(spl)-C genes and the four cell cycle genes, primarily CycE, stg and E2F (Fig 16a). This is revealing a complicated process where balancing repressor and activator activitymodulate daughter cell proliferation decision avoiding cell cycle exit. However, additional studies need to be done to investigate the exact process further, especiallyit will be interesting to analyze ifthe factors involved could be oscillating.Moreove r, the type I>0 switch is controlled by several other cues. Previous data revealed that the Hox gene Antp and the temporal pathway genes cas and grh is controlling the switch through activation of Dap, thus there

60 RESULTS AND DISCUSSION are no evidence for cross-regulation with the Notch pathway. These pathways appear to act independently and simultaneously, to control the type I>0 switch (Baumgardt et al., 2014).

Figure 16. Summary of the findings in Paper III. Notch signaling controls the global type I>0 switch via cell cycle control. a) Analysis of the NB5-6T and NB3-3A lineages revealed different functions of the E(spl) genes in the type I>0 switch. b) A multi-level model of the Notch downstream mechanisms in the switch (adapted from Bivik et.al., 2016).

PAPER IV “SEQUOIA CONTROLS THE TYPE I>0 DAUGHTER PROLIFERATION SWITCH IN THE DEVELOPING DROSOPHILA NERVOUS SYSTEM” In Paper IV we wanted to assemble more cues for how the onset of Notch signaling in a NB lineage was controlled. Notch pathway is off as NBs form, a prerequisite for NB determination, but is activated during lineage progression. Our studies in Papers II and III demonstrated that activation of Notch signaling triggers the type I>0 daughter proliferation switch in many NB lineages. In the forward genetic screen for genes affecting the specification of the Ap neurons (Paper I), in the group of mutants showing additionalAp cells and but no FMRFa expression, one strain was mapped to the sequoia (seq) gene. seq was identified in a genetic screen for bristle formation in the larval PNS named after the appearance of the extensive dendritic branching in a loss of function mutant( Brenman et al., 2001; Gao et al., 1999). seq encodes an 873 amino acid long protein that contains multiple OPA repeats and two zinc fingers. The Seq zinc finger domain shows strong homology to the zinc finger domain of the Drosophila Tramtrack protein (Ttk); a member of the BTB-ZF family (Brenman et al., 2001), and also some similarities to the mouse Prdm10 and Vezf1 zinc finger proteins (human ZNF161/DB1), involved in tumor suppression (Hohenauer and Moore, 2012; Zannino and Sagerstrom, 2015). Pan-neuronal seq mRNA expression was observed in the nervous system in most stages of embryonic

61 RESULTS AND DISCUSSION development (Brenman et al., 2001). A number of studies show links between seq, ttk and the PRDM family, and Notch signaling(Hohenauer and Moore, 2012). Specifically, seq acts on a common target (Pax2) with Notch (Andrews et al., 2009) and ttk is identified as a likely downstream target of Notch(Guo et al., 1996) during PNS development. Furthermore, the PRDM family, which includes mammalian Prdm10, which Seq show homologues similarities to,were demonstrated to be involved in Notch signaling (Hohenauer and Moore, 2012). This prompted the idea that seq may help shed light on the gating of the Notch signaling in NB lineages, and how this relates the Notch-mediated type I>0 daughter proliferation switch.

By analyzing the phenotype ofseq mutants we found that the number of Eya cells is increased. By analyzing the NB5-6T and NB3-3A lineages we observed an increase of cells in the entire lineages, and that aberrant proliferation extended into late embryogenesis (Fig 17). Mitotic analysis revealed that this was a global VNC phenotype. Analyzing the mechanism behind this proliferation, we observed that theseq mutants showed an increased NB proliferation together with a failure to undergo theype t I>0 switch. Additionally, we investigated cell cycle expression in seq mutant and detected an increase of E2f and CycE expression.

Because the results show similar patterns to the Notch pathway perturbations, and because several other studies have indicated genetic interactions between the Notch pathway and seq, we addressed this possible connection with regards to the type I>0 switch. Genetic interaction analysis revealed connections betweenseq and Notch pathway transheterozygotes, such as kuz and E(spl)-C. We previously found that Notch is gradually activated in NBs (Paper II), and in this study we observed that Seq levels gradually decline in NB. Intriguingly, in seq mutants, Notch pathway activation was found to be premature.

In summary, the findings in Paper IV point to a model where gradually declining levels of Seq plays a role in controlling the onset of Notch signaling to thereby ensure a timely type I>0 switch, and where seq also acts by repressing the important balance of several Notch downstream genes such as E(spl), as well as CycE and E2F. Seq may be a stronger repressor of the cell cycle genes than E(spl) and work together with E(Spl) repressing the same factors.

Figure 17. Summary of Paper IV. The gene Sequoia is mediating the type I>0 switch, in the NB5-6T, 3-3A as well as globally, through interaction with the Notch signaling pathway (adapted from Gunnar et.al., 2016).

62 CONCLUSIONS

CONCLUSIONS

The series of four papers presented in this thesis has focused on cell specification and daughter cell proliferation control in the development of the embryonic Drosophila CNS. The principal findings are as follows:

PAPER I Through a forward genetic chemical mutagenesis (EMS) screen in Drosophila, scoring 9,781 mutated lines, 611 lines were identified with alteration of the Ap4/FMRFaneuron in the NB5-6T lineage. Genetic mapping of the strongest mutants revealed 43 genes (79 alleles) important for neural progenitor specification and proliferation. The gene functions covered diverse areas of development ranging from cell cycle and temporal factors , to Hox genes and chromatin landscape modifiers.

PAPER II We present a novel mechanism of the Notch signaling pathway in regulating the type I>0 switch in daughter cell proliferation mode in the - NB56T lineage. Wefurthermore demonstrated that the asymmetric componentP rospero controls daughter proliferation early in the lineage during the type I division mode, and that Notch is superimposed upon Prospero to trigger the type I>0 proliferation switch.

PAPER III We found that Notch mediates the type I>0 daughter proliferation switch in the entire Drosophila nerve cord. The NICD-CSL complex activates transcription of Dacapo (Dap; mammalian p21cip1/ p27kip1, p57kip2) and the E(spl)-C genes, which in turn represses the cell cycle genes: Cyclin E, String (Cdc25) and E2F. This events combine to trigger the type I>0 switch. Intriguingly, the switch is mediated through activation of different E(Spl)-C genes in different neuroblasts.

PAPER IV We found that the transcription factor Sequoia interacts with the Notch signaling pathway to fine-tune the timing of the t ype I>0 switch. Sequoia furthermore controls neuroblast exit from the cell cycle.

63 64 FUTURE PERSPECTIVES

FUTURE PERSPECTIVES

The generation of the specific number of each differentneural sub-type in the CNSis regulated by multiple mechanisms throughout development. The size of each progenitor pool is limited by the number of rogenitor p divisions, the different temporal window length, the time for cell NB cycle exit and the PCD reduction of daughters. The same subtype could furthermore be produced by several different progenitors.It is becoming increasingly suggested that vertebrates and invertebrates must share multiple similar basic regulatory processes controlling the early development of the CNS and the sequential generation of neurons and glia (Jacob et al., 2008). In mammals, the asymmetrical dividing NB could be compared to the radial glial ( cellFranco and Muller, 2013). In both Drosophila and vertebrates it is demonstrated that the progenitor daughter cell could have three different behaviors i.e., mitotic potentials, resulting in different lineage topologies: Differentiate directly to a neuron/glia (type 0), divide once to generate two neurons and/or glia (type I), or divide multiple times before generating neurons and/or glia (type II) (Brand and Livesey, 2011; Gotz and Huttner, 2005; Kriegstein et al., 2006). Additionally our group has identified that NB lineages can switch in daughter cell division modes from type I>0 mode in most, if not all, lineages in Drosophila (Baumgardt et al., 2014; Baumgardt et al., 2009; Ulvklo et al., 2012). According to the intermediate progenitor hypothesis, the cortical development in gyrencephalic animals could be partlydue to division of intermediate progenitors that leads to a fast amplification of neurons without a need of an increased size of the SVZ (Kriegstein et al., 2006). Given this information, the work in this thesis elucidating the Notch mechanisms regulating the type I>0 switch, and thereby limiting the lineage topology , could be of great importance. You could hypothesize that switches in proliferation modes, and thereby also the regulatory mechanisms, could be conserved to mammals, thus within more elaborated lineage trees. The possibilities utilizing the Drosophila model system are wide. In spite of the many genetic tools available, unwiring the molecular mechanisms is still a challenge, but even a greater challenge in the vertebrate systems.

In paper II we demonstrated that mutations in two tumor suppressor genes , Notch and pros, that plays different roles in daughter cell proliferation, can cooperatively generate extensive over proliferation. Thisdespite the fact thatthey only generate minor proliferative problems on their own. Notch is shown to mainly work as a proto-oncogene in vertebrates, but recent publications have pointed to both a tumor suppressor function and dual functions in the same lineage. Predicting that even more pathologies will be associated with Notch, making Notch a highly relevant therapeutic target. It is of a growing commercial and clinical relevance to study mechanistic details ofN otch activation and nuclear activity, due to the great pleiotropy Notch shows and the complex control of the pathway. New upcoming techniques as CRISPR, generation of disease specific IPCs, big data approaches and mathematical models, means new possibilities in the search of

65 FUTURE PERSPECTIVES mechanistic cues. The challenges of Notch biology can thereby be addressed through many approaches, resulting in deeper understanding of Notch signaling and the nature of context - dependent signaling, and point to new therapeutic targets.

66 ACKNOWLEDGEMENTS

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to everyone who have given me support, contributed or in other ways helped me making this thesis possible.

First and foremost I would like to thank my supervisor Stefan Thor for generous guidance, support, and positive encouragement. Thank you for sharing your exceptional knowledge and contagious enthusiasm for science. Your remarkable ability to make everything seem interesting is motivating and truly an inspiration.

My co-supervisor Jan-Ingvar Jönsson for scientific support, fruitful discussions and for always being updated and sharing your view on interesting current topics.

My co-authors for good teamwork, ideas and motivation.

To the members of the Thorlab group for all the good times in- and outside the lab, making it a cheerful place to be. Erika for your company on conference trips all over the world and for being the best office-mate for the past years. Annika, Carolin, Helen and Anna for excellent technical support, for always keeping good track on all lab-equipment and for taking such a good care of Thorlab. Magnus, Daniel and Carina for introducing me to confocal microscopy and fly pushing.Ryan for your positive attitude and for not giving up in the search for E(spl)-mutants. Johannes for your help with ChIP experiments and bioinformatics, Shaz for always bringing a happy smile and Behzad for being a living dictionary. Josefin, Ignacio, Jonathan and Jesús for keeping me company late hours at the lab. To past members and students. Time really flies when you are having fun!

To past and present colleagues at “Floor 13”, for sharing working environment and making it a stimulating atmosphere: Group Sigvardsson, Larsson, Jönsson, Svensson, Hinkula and Alenius. Thank you for all laughs and nice chats in the coffee room, discussing ups, downs and p-values. You are all together my extended lab group; always someone there to answer your questions and encourage you.

The “MedBi-crew” Jenny, Lina, Angelika, Anna E and Anna F. It is invaluable to have friends who share your scientific interests and help you stay motivated. Thank you for all interesting discussions, barbecues, travels and mushroom hunts. To the ”appendix”: Martin, Christian and Ulf, for being good friends and for adding funny non-scientific subjects to our discussions.

My dear non-scientific friends for being curious about my work and for filling my life with joy. What should I do without you? Special thanks to: Filip, Mélica, Magnus and Fanny for sharing many years in the amazing student corridor “Loungen”. The past “Flamman- workers”, for all good times and memories. My childhood friends in Växjö, Emelie and Sofie, for warm hugs and laughter. Malin, Henrik, Pontus, Joanna, Emma and John for nice dinners and occasional get-togethers.

67 ACKNOWLEDGEMENTS

To Lena, Svante and Sofia, since you have so warmly taken me into your family. Thanks for all kinds of support, delicious dinners, boat trips and l ong evenings playing trivia games at Hällebäck. Thanks also to my bonus-relatives for all celebrations and parties. Especially to Karin and Owe for your warm hearts and care.

Sist men inte minst.

Till Mamma och Pappa för all kärlek, allt stöd och all support. Tack för att ni uppfostrat mig till att bli nyfiken och vetgirig. Tack för att ni även bryr er omhur mina labbflugor mår och om jag muterat fram något kul på sistone. Till min fina systeryster Cecilia för att du alltid finns ett telefonsamtal bort. Tack för att du peppar mig och ger mig de bästa råden, ofta utan att du ens märker det. Dan, Isabelle och Ida för att ert hem alltid står öppet och välkomnande.

Till underbara Niklas för ditt tålamod, för att du tror på mig och för att du är du. Du ger mig perspektiv, gourmetmatlådor och får mig att skratta när jag behöver det som mest. Tack för alla äventyr vi hunnit med och alla de som komma skall. Agnes, du är så liten och så oerhört älskad. Ditt skratt och dina leenden ger mig en sådan energi. Jag älskar er! ♥

Thank you all! Caroline Bivik Stadler, November 2016

68 REFERENCES

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