Invertebrates show enhanced immunity and even specific primed immunity in response to repeat infections, analogous to vertebrate adaptive immunity. Little is known of the mechanism for this phenomenon, or which molecules are involved. A candidate gene for the underlying mechanism for a pathogen-specific response in invertebrate immunity is Down syndrome cell adhesion molecule (Dscam). Dscam can produce thousands of different protein isoforms through the mutually exclusive splicing of many exon variants contained within variable regions of the gene. It is an important receptor of the invertebrate nervous system but has been implicated in having a role in immunity. Dscam has been shown to be involved in phagocytosis across members of the Pancrustacea, and it has been reported to respond in a pathogen-specific manner in mosquitoes and crayfish. In this thesis, I have investigated the splicing of Dscam in response to diverse pathogens in different host species. In the Anopheles mosquito, I cloned and sequenced a fragment of Dscam spanning across two of its variable exon regions to enable me to detect mutually exclusively splice variants and their associations in different treatments (Chapter 2). I discovered that the expression diversity of the hypervariable Dscam is higher in parasite-exposed mosquitoes. In Chapter 3, I extended the study to the more experimentally amenable Drosophila fruit fly. A new Illumina-based sequencing assay was developed and implemented to examine more closely Dscam expression in response to diverse pathogens. The new method successfully quantified non-random expression of Dscam variable exons 4 and 6. I also describe a small but detectable effect of pathogen-exposure on the expression of Dscam exon 4 variants. In Chapter 4, I expanded the work of Chapter 3 to study tissue-specific Dscam expression in response to well-characterised immune elicitors of Drosophila. I describe how exon 4 variants were expressed in a tissue-specific manner, but not exon 6 variants. I also found a small exon 4-by-tissue-by-pathogen effect, which although detectable, did not dominate over the tissue effects. Finally, in Chapter 5, I turned to the crustacean, Daphnia, to study Dscam expression in a natural host-parasite interaction and in a clonal organism. I describe the non-random expression of exons 4 and 6, and another small effect of pathogen-exposure on the expression of Dscam exon 4. My work aimed to further investigate the putative pathogen-specific alternative splicing of the hypervariable Dscam receptor. The data presented quantified the constitutive expression of Dscam exons 4 and 6 in different pancrustacean species. The data also suggest that infection-responsive splicing of Dscam may occur but that effects are small, and may be diluted within the background of the highly important Dscam expression of the nervous system if they exist at all. The study supports the high-throughput sequencing method for future studies of alternative splicing and Dscam expression.

Hemocytes are an important part of a crayfish’s immune system in helping tackling both virus and bacterial infections. Dscam is a protein that can be found in hemocytes, as well as many other tissues like the brain. In the brain, Dscam is thought to be important in the establishment of neuronal connections. Previous studies have found that the neurons in the crayfish brain do not replenish themselves, but instead are replenished by hemocytes that enter through a vascular cavity that pass through the neurogenic niche. There might be a specific type of hemocyte that is drawn to the niche and because of the link between Dscam and establishment of neuronal connections, Dscam have been chosen as a potential factor for this attraction. Dscam could be upregulated at many places along the way from the HPT to the brain. In this study, antibodies have been used to view BrdU and Dscam presence in hemocytes from crayfish P. leniusculus to find out where Dscam is upregulated and in what cells they are located. It was found that Dscam is not present on newly synthesized cells but rather on more differentiated cells, suggesting that Dscam is upregulated in older HPT cells or in circulation. It was found that LPS injections are an efficient way to upregulate Dscam in hemocytes and that expression of extracellular Dscam is peaking 24 hours post LPS injection.

The over-expression of Down syndrome cell adhesion molecules (DSCAMs) is partially responsible for the mental retardation associated with Down syndrome. Previous work in our lab showed that a DSCAM homolog in C. elegans, Y32G9A.8, is expressed at all developmental stages and appears to be crucial for survival. In an effort to map the expression pattern, I used the Genome Sciences Centre’s primer design program (http://elegans.bcgsc.bc.ca/gfp_primers/) to design a GFP promoter fusion product that was used to monitor gene expression. The results indicate that Y32G9A.8 is expressed in the animal’s gut, suggesting that it may function in the worm’s innate immune response. I also designed a primer set to amplify the Y32G9A.8 transcript. RT-PCR of the entire Y32G9A.8 coding region resulted in a single product; there appears to be no alternative splicing. Although this gene shows homology to other N-CAMS, results indicate that this gene may function in the innate immune system of C. elegans.

S'il est de plus en plus évident que le sommeil joue un rôle crucial dans la consolidation de la mémoire, nous ne connaissons toujours pas le rôle exact joué par le sommeil lors de ce processus. L'objectif de ma thèse a été de mieux comprendre les interactions sommeil/mémoire. Pour cela, nous avons choisi d'étudier chez la drosophile l'effet de courtes privations de sommeil sur la consolidation de la mémoire aversive olfactive. Nous avons utilisé un protocole de conditionnement pavlovien qui permet aux drosophiles d'associer une odeur à des chocs électriques. En fonction du conditionnement qui est réalisé, deux types de mémoires consolidées peuvent être formées: la Mémoire Résistante à l'Anesthésie (MRA) et la Mémoire à Long Terme (MLT). Contrairement à la MRA, la MLT dont la formation est dépendante d'une néo synthèse protéique est couteuse en énergie. Les résultats obtenus démontrent que MRA et MLT sont toutes deux affectées par de courtes privation de sommeil, mais qu'elles deviennent insensibles à de telles privations si le test mnésique est réalisé en fin de journée. Ces résultats mettent à jour : i) une interaction fonctionnelle entre les étapes de consolidation et de rappel mnésiques, et ii) l'influence de facteurs circadiens sur la sensibilité de la mémoire aux privations de sommeil. De plus, mes résultats montrent qu'une privation de sommeil ayant lieu juste après le conditionnement exerce un effet bénéfique sur la consolidation de la mémoire. Ce travail met donc en évidence la corrélation complexe qui existe entre le rythme circadien, le sommeil, et les étapes de consolidation et de rappel des mémoires consolidées

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Neurons exhibit highly polarized structures, including two morphologically and functionally distinct domains, axons and dendrites. Dendrites and axons receive versus send information, and proper execution of each requires different sets of molecules. Differential distribution of membrane proteins in distinct neuronal compartments plays essential roles in neuronal functions. The major goal of my doctoral thesis was to study the molecular mechanisms that govern the differential distribution of membrane proteins in neurons, using the Drosophilalarval mushroom body (MB) as a model system. My work was initiated by an observation of differential distribution of distinct Dscam isoforms in neurons. Dscam stands for Down Syndrome Cell Adhesion Molecule, which is a Drosophila homolog of human DSCAM. According to genomic analysis, DrosophilaDscam gene can generate more than 38,000 isoforms through alternative splicing in its exons 4, 6, 9 and 17. All Dscam isoforms share similar domain structures, with 10 immunoglobulin domains and 6 fibronectin type III repeats in the ectodomain, a single transmembrane domain and a cytoplasmic endodomain. There are two alternative exons in exon 17 (17.1 and 17.2), which encodes Dscam’s transmembrane domain. Interestingly, in ectopic expression, Dscam isoforms carrying exon 17.1 (Dscam[TM1]) can be preferentially localized to dendrites and cell bodies, while Dscam isoforms carrying exon 17.2 (Dscam[TM2]) are distributed throughout the entire neuron including axons and dendrites. To unravel the mechanisms involved in the differential distribution of Dscam[TM1] versus Dscam[TM2], I conducted a mosaic genetic screening to identify the possible factors affecting dendritic distribution of Dscam[TM1], established an in vivoTARGET system to better distinguish the differential distribution of Dscam, identified the axonal and dendritic targeting motifs of Dscam molecules and further showed that Dscam’s differential roles in dendrites versus axons are correlated with its localization. Several mutants affecting dendritic distribution of Dscam[TM1] have been identified using a MARCM genetic screen. Three of these mutants (Dlis1, Dmn and p24) are components of the dynein/dynactin complex. Silencing of other dynein/dynactin subunits and blocking dynein function with a dominant-negative Glued mutant also resulted in mislocalization of Dscam[TM1] from dendrites to axons. However, microtubule polarity in the mutant axons was maintained. Taken together, this was the first demonstration that the dynein/dynactin complex is involved in the polarized distribution of membrane proteins in neurons. To further examine how dynein/dynactin is involved in the dendritic distribution of Dscam[TM1], I compromised dynenin/dynactin function with dominant-negative Glued and transiently induced Dscam[TM1] expression. The results suggested that dynein/dynactin may not be directly involved in the targeting of newly synthesized Dscam[TM1] to dendrites. Instead, it plays a role in maintaining dendritic restriction of Dscam[TM1]. Notably, dynein/dynactin dysfunction did not alter distribution of another dendritic transmembrane protein Rdl (Resistant to Dieldrin), supporting involvement of diverse mechanisms in distributing distinct molecules to the dendritic membrane. To identify the targeting motifs of Dscam, I incorporated the TARGET (Temporal and regional gene expression targeting) system into mushroom body (MB) neurons, and this allowed the demonstration of the differential distribution of Dscam[TM1] and Dscam[TM2] with more clarity than conventional overexpression techniques. Using the TARGET system, I identified an axonal targeting motif located in the cytoplasmic juxtamemebrane domain of Dscam[TM2]. This axonal targeting motif is dominant over the dendritic targeting motif located in Dscam’s ectodomain. Scanning alanine mutagenesis demonstrated that two amino acids in the axonal targeting motif were essential for Dscam’s axonal distribution. Interestingly, swapping the cytoplasmic juxtamembrane portions between TM1 and TM2 not only reversed TM1’s and TM2’s differential distribution patterns but also their functional properties in dendrites versus axons. My thesis research also involved studying endodomain diversity of Dscam isoforms. Besides the diversity originally found in the ectodomain and transmembrane domain of Dscam, my colleagues and I further demonstrated the existence of four additional endodomain variants. These four variants are generated by skipping or retaining exon 19 or exon 23 through independent alternative splicing. Interestingly, different Dscam endodomain isoforms are expressed at different developmental stages and in different areas of the nervous system. Through isoform-specific RNA interference, we showed the differential involvement of distinct Dscam endodomains in specific neuronal morphogenetic processes. Analysis of the primary sequence of the Dscam endodomain indicated that endodomain variants may confer activation of different signaling pathways and functional roles in neuronal morphogenesis. In Summary, my thesis work identified and characterized several previously unknown mechanisms related to the differential distribution of membrane proteins in neurons. I showed that there may be a dynein/dynactin-independent mechanism for selective transport of dendritic membrane proteins to dendrites. Second, dynein/dynactin plays a maintenance role in dendritic restriction of Dscam[TM1]. Third, different membrane proteins may require distinct combinations of mechanisms to be properly targeted and maintained in certain neuronal compartments. Further analysis of the mutants indentified from my genetic screen will definitely help to resolve the missing pieces of the puzzle. These findings provide novel mechanistic insight into the differential distribution of membrane proteins in polarized neurons.