The nervous system integrates multiple sources of sensory information that ultimately controls motor neurons to generate complex movements. Motor neurons form topographically organised ‘myotopic maps’ between the nerve cord and muscles in the periphery to ensure that correct pre-motor inputs into motor dendrites are relayed through corresponding axons to the appropriate muscle groups. Therefore understanding the development and assembly of motor neuronss is crucial for understanding how animals execute various motor outputs. In adult Drosophila, ~50 motor neurons are topographically organized between each leg and the nerve cord in a highly stereotyped manner (Baek and Mann, 2009). In this thesis, I describe a novel group of transcription factors that act in a combinatorial manner to specify the projections of distinct Drosophila leg motor neurons. Our studies suggest that morphological transcription factors regulate various downstream cell-surface genes that are involved in the assembly of motor circuitry. Using in vivo live imaging I describe the developmental steps involved in Drosophila leg motor neuron axon targeting during metamorphosis and the spatial expression patterns of a novel hetero-binding Ig domain transmembrane protein family – the DIPs and Dprs (Ozkan et al., 2013) in leg neuro-musculature. I further describe a function between interacting partners DIP-alpha and Dpr10, expressed in subsets of leg motor neurons and muscles respectively, in establishing the final stereotyped terminal axon branching of corresponding motor neurons. The combinations of such interactions throughout development between leg motor neurons, not only with muscles in the periphery, but also among themselves, with leg sensory neurons and other components in the central nervous system may ultimately lead to synaptic specificity and stereotyped morphologies of Drosophila leg motor neurons.

Neuronal migration is an essential process in the development of the nervous system. Neurons are born in one location and migrate sizable distances to their final location. In many other developmental processes, cells migrate as collectives, where the migration of one cell influences the migration of another cell; this process has yet to be shown in the developing central nervous system. Using the conserved tangential migration of facial branchiomotor neurons (FBMNs), I aim to determine the nature of the collective migration in the developing nervous system. Here, two models of FBMN collective migration are tested: the “Pioneer” model, where following FBMNs migrate intimately on the axon of the first neuron to migrate and the “Contact inhibition of locomotion (CIL)” model, where transient cell-cell contacts are the driving influence of the proper caudal migration of FBMNs. Using fixed tissue imaging, it was found that early born FBMNs do not contact the axon. In contrast, they are more likely to make soma-soma contact and display morphology typical of CIL. FBMNs that do contact the axon do not display an elongated morphology that is predicted of a cell using the leader axon as a substrate for migration. Further, wild-type FBMNs are able to rescue PCP-deficient FBMNs. Therefore, blastula-stage transplantation of PCP-deficient neurons into wild-type hosts allows us to live image the method of collective migration. CIL events were observed between PCP-deficient neurons and wild-type neurons, indicating that PCP is not required for CIL. In addition, PCP-deficient neurons making sustained contact with wildtype axons were not rescued, arguing against the Pioneer model. Taken together, these observations are more consistent with the “CIL” model of FBMN collective migration in which transient soma-soma interactions are required for the coordinated movement of neurons as they migrate in the developing nervous system.

Les exosomes sont des vésicules d’origine endosomale sécrétées par les cellules dans leur environnement après fusion à la membrane plasmique des endosomes multivésiculés. Les exosomes représentent un nouveau mode de communication entre les cellules en permettant un transfert direct de protéines, de lipides et d’ARN. L’objectif de ma thèse était d’étudier le rôle des exosomes dans la communication entre les neurones. Précédemment, le laboratoire a montré que les neurones sécrètent des exosomes de manière régulée par l’activité synaptique. Nous avons observé que les exosomes neuronaux ne sont endocytés que par les neurones. Après avoir montré qu’ils ne contiennent que des ARN courts, nous avons réalisé un séquençage complet de leurs microARN et observé que ces microARN étaient sélectivement exportés dans les exosomes. Nos observations suggèrent que les microARN contenus dans les exosomes peuvent modifier la physiologie des neurones receveurs. Nos résultats renforcent l’hypothèse du rôle des exosomes dans la communication entre les neurones via le transfert de microARN. / Exosomes are vesicles of endocytic origin released by cells into their environment following fusion of multivesicular endosomes with the plasma membrane. Exosomes represent a novel mechanism of cell communication allowing direct transfer of proteins, lipids and RNA. The goal of my PhD thesis was to study that exosomes represent a novel way of interneuronal communication. Our team has previously reported that neurons release exosomes in a way tightly regulated by synaptic activity. We observed that exosomes released by neurons are only endocytosed by neurons. We found that exosomes contain only small RNA and did a deep sequencing of all their microRNA. MicroRNA are selectively exported into exosomes. It seems that exosomal microRNA can modify the physiology of receiving neurons. Our results strengthen the hypothesis of the role of exosomes in the interneuronal communication by the way of microARN transfert.

No description available.

A neurone model (the FORMON) is proposed which provides a mathematical explanation for a range of psychological phenomena and has potential in Artificial Intelligence applications. A general definition of organisation in terms of entropy and information is formulated. The concept of microcodes is introduced to describe the physical nature of organisation. Spatio-temporal pattern acquisition and processing functions attributable to individual neurones are reviewed. The criterion for self-organisation in a neurone is determined as the maximisation of mutual organisation. A feedback control system is proposed to satisfy this criterion and provide an integrated long-term memory of spatio-temporal pattern. This pattern acquisition system is shown to be applicable to dendritic pattern recognition and axonal pattern generation. Provision is also made for adaptation, short-term memory and operant learning. An electro-chemical model of transmission and processing of neural signals is outlined to provide the pattern acquisition functions of the Formon model. A transverse magnetic mode of electrotonic propagation is postulated in addition to the transverse electromagnetic mode. Configurations of the Formon are categorised in terms of possible pattern processing functions. Connective architectures are proposed as self-organising models of acquisitive semantic and syntactic networks.

Inhibitory neurons exert control the expression of many aspects of behaviour by regulating the effectiveness of excitatory neural function. By comparison with excitatory neural systems, relatively little is known about the development of inhibitory neurons and the influence which these neurons exert on the development of other neural systems. Two issues which relate to the development of inhibitory neurons are of particular interest. First, a paradox arises when inhibitory neurons are considered in terms of modern models of synaptic development which involve activity-dependent mechanisms of synaptic plasticity. Second, there is some evidence that inhibitory neurotransmitters may act in a special trophic manner during the early development of nervous systems. Investigations of these issues would be greatly facilitated in a neural system in which it was possible to experimentally control aspects of the development of individual pre- and postsynaptic cells. The aim of the results presented in this thesis was to characterise the normal development of one such system: the GABAergic inhibitory system of the Australian freshwater crayfish, Cherax destructor. The ontogeny of the inhibitory neurotransmitter GABA across the embryonic period of 30% to 100% development was investigated using immunohistochemical techniques. GABA-like immunoreactive cells and fibres were first detected in the embryonic brain region. The expression of GABA-like immunoreactivity progressed along a rostro-caudal gradient, with GABA-like immunoreactive cells being detected in the most anterior thoracic ganglia at 45% development and in all ganglia by 65% development. GABA-like immunoreactive fibres were evident in peripheral nerves as early as 55% development and ramified extensively throughout the neuropil of the nervous system by 65% development. By contrast, immunoreactivity to the primary excitatory neurotransmitter, glutamate, was not detected until 60-65% development. Glutamate-like immunoreactivity at 60-65% development was evident only in the form of punctate staining in the midline of the ventral nerve cord. Cell body staining was observed only at 90% development and was restricted to only a few cells on the periphery of the ventral nerve cord. Radio-labelled ligand binding methods and autoradiography were used to study the expression of putative GABA receptors in the Cherax embryos from 30% to 100% development. Specific binding was evident in the earliest embryos studies at 30% development. There was an initial increase in binding from 30% to 40% development, followed by a dramatic drop to almost zero binding at 50-55% development. This was followed by a gradual increase in binding levels with age, reaching a plateau at 85% development. Preliminary pharmacological evaluation of binding indicated that at least three GABA receptor types were expressed during embryonic development. Methods for culturing, dissociated neural tissues explanted form Cherax embryos at 85% development were established. The success of cultures was demonstrated by neurite extension, and neuronal networks in which neurons appeared to form connections with other neurons and with explanted muscle cells after two days in culture. Immunohistochemical studies demonstrated that some explanted neurons expressed GABA-like immunoreactivity within two days of explanting. These studies have provided a comprehensive description of the development of GABAergic neurons and their receptors in Cherax destructor embryos. The very early expression of GABA-like immunoreactivity, coupled with the early onset of specific GABA binding, strongly indicates that the GABAergic neurons are functional and able to exert an effect on other cells during much of the period of nervous system development in crayfish embryos. These results support the hypothesis that inhibitory neurons may play an important role as regulators of the overall process of assembly and maturation of the nervous system and provide a substantial basis for future experimental studies in which the specific action of inhibitory neurons on the development of discrete components of the crayfish nervous system may be investigated.

The survival of motor neurons is controlled by multiple factors, which regulate different aspects of their physiology. The identification of these factors is important because of their relevance for motor neuron disease. This thesis began with a search for novel growth factors that naturally keep these neurons alive. Members of the TGF-β superfamily, including Mullerian inhibiting substance (MIS) and bone morphogenetic protein 6 (BMP6), were identified as putative survival factors following a cDNA microarray analysis of a mouse model of motor neuron disease. MIS is a gonad-derived hormone with a male bias. It induces the degeneration of the female reproductive tract during development and it was thought to have no physiological function outside of the reproductive system. In this thesis, multiple techniques were used to show that adult motor neurons produce MIS and its receptors. The copy number of MIS mRNA in motor neurons was comparable with that of the testis, whereas the mRNA of the MIS type II receptor (MISRII) in motor neurons appeared to be the most abundant receptor of the TGF-β superfamily. These results were confirmed using Western blot and immunohistochemistry. Thus, MIS may exert its function through an autocrine or a paracrine mechanism between neighbouring motor neurons. The function of MIS was examined using a culture system and a mouse null mutation of MISRII. The in vitro assays showed strong neurotrophic effects of MIS on embryonic motor neurons with the maximum extent of survival being similar to that achieved by the classical motor neuron survival factor, GDNF. MIS has a male bias in utero raising the issue of whether motor neurons are sexually dimorphic. Consistent with this, the number of motor neurons in the lumbar lateral motor column of neonatal male MISRII+/+ mice was 13 % greater than in female mice (P = 0.01). The nuclei of male motor neurons were approximately 20 % larger than their female counterparts (P = 0.000). MISRII-/- male mice had 18 % fewer motor neurons than wild-type males (P = 0.01) and the mean size of their motor neurons was 20 % smaller (P = 0.000). The number and size of motor neurons in the MISRII-/- males was not different to those of MISRII+/+ females. These results implicate MIS as being responsible for neuronal survival as well as producing sexual dimorphism of the limb innervating motor neurons. Since MIS does not appear to be expressed in the embryonic neuromuscular system, it is postulated that MIS is a gonad-derived neurotrophic factor for developing motor neurons. The BMP type II receptor (BMPRII) was the second most abundant receptor of the TGF-β superfamily expressed by motor neurons. One of its ligands, BMP6, was found to have a neurotrophic effect on motor neurons in culture but was slightly less potent than MIS. BMP6 mRNA was detected in nerve, skeletal muscle and spinal cord, but not in motor neurons. BMP6 immunoreactivity was mainly associated with the myelinated Schwann cells and satellite glia that surround motor neurons. In skeletal muscles, immunoreactivity was not detected in muscle fibers, nor the postsynaptic region of the neuromuscular junction (NMJ). BMP6 was, however, associated with the interstitial cells of skeletal muscles. Double nerve ligations were used to examine whether Schwann cell-derived BMP6 interacts with motor neurons. Consistent with this, BMP6 was retrogradely transported in motor axons. These observations collectively suggest that BMP6 is a glia-derived regulator of motor neurons. MIS and minority of BMP6 were anterogradely transported towards the NMJ. Their receptors, MISRII and BMPRII, were detected in the postsynaptic portions of the adult NMJ. These observations raised the possibility that MIS and BMP6 may be regulators of the adult NMJ. Since functional redundancy amongst the members of the TGF-β superfamily has been suggested, the function of MIS/BMP6 signaling at the NMJ was therefore examined in mice with muscle-specific deletion of Smad4, a central mediator of TGF-β superfamily pathways. More than 75% of animals lacking Smad4 in muscles died before embryonic day (E) 14 and none survived postnally. This was due to the loss of functional Smad4 in developing cardiac myocytes, which resulted in severe heart defects and early death of embryos. Thus, the function of MIS/BMP6 signaling at the adult NMJ could not be studied. Finally, this thesis briefly examined the phenotypes of mice carrying double null mutations of MISRII and TGF-β2. The animals died at an early stage and showed a more severe phenotype than either of the single null mutants. This suggests that functional redundancy among members of the TGF-β superfamily exists in many organs. In summary, motor neurons require multiple sources of growth factors for their survival. MIS and BMP6 were discovered as novel survival factors for motor neurons in this study. MIS was implicated as a regulator of sexual dimorphism in developing motor neurons, whereas both MIS and BMP6 appear to regulate mature motor neurons, and possibly the NMJ.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Identification de déterminants génétiques impliqués dans la composante vasculaire de la MA, par analyses transcriptomiques, génétiques et moléculaires<br />La maladie d'Alzheimer (MA) est une maladie dégénérative du cerveau qui provoque la démence, avec une perte graduelle de mémoire, du jugement, et des fonctions cognitives. Cette maladie apparaît généralement chez les personnes âgées de plus de 65 ans, mais certaines formes moins fréquentes peuvent apparaître plus précocement. Les bases génétiques et moléculaires de la MA sont encore mal connues. L'hérédité des formes à début précoce est liée à des mutations dans trois gènes différents: le gène du précurseur de la protéine amyloïde (APP) sur le chromosome 21, le gène de la préséniline 1 (PS1) sur le chromosome 14 et le gène de la préséniline 2 (PS2) sur le chromosome 1. Cependant, ces mutations expliquent moins de 1% des cas de MA. Dans la grande majorité des cas, la génétique apparaît beaucoup plus complexe car résultant de l'interaction entre des facteurs environnementaux et divers gènes de susceptibilité. Malgré le consensus sur l'importance de la composante génétique de la MA, seul l'allèle ε4 du gène de l'apolipoprotéine E (APOE) a été retrouvé comme un facteur constant de vulnérabilité. Toutefois, plus de 200 gènes ont déjà été proposés comme déterminants génétiques de la MA, mais aucun consensus n'a pu être obtenu pour l'un d'entre eux en raison du manque de robustesse des associations observées au sein de populations indépendantes. Tout d'abord, nous avons cherché à reproduire l'association entre la MA et des polymorphismes localisés dans 3 gènes candidats (VEGF, PON1 et GAB2). Deuxièmement, afin la sélection de nouveaux gènes candidats, nous avons combiné les informations issues de carte génétique avec le profil d'expression de gènes. Cette stratégie résulte de deux grandes observations: (i) l'expression de nombreux gènes est modifiée au cours de l'étiologie de la MA, (ii) les polymorphismes dans les promoteurs de l'APOE, PS1, PS2 et APP gènes ont été associés à l'apparition de la MA. Par conséquent, nous avons supposé que les gènes situés dans les régions chromosomiques définies par des études de liaisons génétiques et présentant une expression différentielle entre des patients et des témoins, pourraient constituer des gènes candidats implique dans la MA. Nous avons effectué l'analyse transcriptomique de 2741 gènes situés dans les régions chromosomiques d'intérêt définies dans le cadre de la MA. Les niveaux de l'expression génique ont été évalués à partir d'ARN totaux provenant de tissus cérébraux post-mortem de malades et de témoins. Cent six gènes ont été retrouvés différentiellement exprimés. Ensuite, nous avons évalué, au sein de ces gènes, l'impact de polymorphismes sur le risque de développer la MA. Le polymorphisme le plus intéressant, situé sur le gène IL33, a été associé à la MA dans 3 populations cas-témoins indépendantes. En outre, nous avons pu montrer une implication de ce gène dans un processus physiopathologie touchant le réseau vasculaire cérébral au cours de la MA. De façon intéressante, IL33 est préférentiellement exprimé dans les cellules vasculaires. En effet, un nombre croissant de données suggèrent un rôle central des facteurs de risque cardiovasculaire, de la modification des parois artérielles, amenant à une hypoperfusion chronique du cerveau et au développement de la physiopathologie de la maladie. Ces données sont basées sur des études épidémiologiques, physiopathologiques, de neuro-imagerie, de neuropathologiques et d'études pharmacologiques. Toutes ces observations indiquent qu' une altération du réseau vasculaire pourrait être un facteur important dans le processus conduisant à la neurodégénérescence chronique dans la MA.

The remodeling of the central nervous system (CNS) during metamorphosis in Drosophila melanogaster is a prime model system in which to study the genetic control of the sexual dimorphisms in the abdominal ganglion of the CNS. I have been using a P[tau-lacZ] enhancer trap line, 4.078, to label a segmentally repeated subset of abdominal motorneurons in order to assess the function of the sex determination hierarchy in controlling sex-specific development of the adult nervous system. In both the male and female larva there are 8 sets of these labeled abdominal motorneurons but only six sets in males and five sets in females survive in the adult. When this P[tau-lacZ] reporter construct is placed into a doublesex (dsx) mutant background, all 8 sets of these labeled abdominal motorneurons survive in both male and female adults. These results strongly suggest that dsx plays a role in the sex-specific survival of larval neurons that have functions in the adult. During the construction of mutant strains containing the sex determining genes transformer (tra) and transformer-2 (tra2), a genetic interactor was discovered in the P[tau-lacZ] 4.078 line. Female flies heterozygous for either tra or tra-2 alleles and the P[tau-lacZ] 4.078 developed with masculinized external and internal sex-specific structures. The external sex-specific structures, such as the genitalia, and ventral muscles are dependent on dsx gene function and a dorsal sex-specific muscle is dependent on fruitless (fru) gene function. From standard genetic crosses, I have characterized and demonstrated that the genetic interaction is linked to the P-element insertion site, which maps to the 85-87 region on the right arm of the third chromosome. By genetic analysis, this new genetic interactor appears to interfere with the tra and tra2 regulated female specific functions of both dsx and fru, potentially by reducing the female-specific splicing of the primary transcripts of the genes dsx and fru. To test the possibility that this newly described genetic interactor was allelic to a known gene, B52, that maps to the same region of the chromosome and alters dsx splicing, complementation tests were conducted which showed that the P[tau-lacZ] is not allelic B52. Additional phenotypes were observed in the crosses that first detected the interaction, suggesting that this newly described locus may affect other gene functions as well. Among the phenotypes observed were XX intersexes, male-female gynandromorphs (XX//XO mosaics), and non-disjunction events evident as XO males and XXY females. This new locus may represent a new member of the family of genes that influence regulated splicing events. / Graduation date: 1999