No description available.

No description available.

DNA methylation-dependent gene silencing, catalyzed by DNA methyltransferases (DNMTs) and mediated by methyl binding domain proteins (MBDs) and histone deacetylases (HDACs), is essential for mammalian development, with the nervous system demonstrating particular sensitivity to perturbations. Little is known, however, about the role of DNA methylation in the stage-specific differentiation of neurons. In the olfactory epithelium (OE), where neurogenesis is continuous and the cells demonstrate a laminar organization with a developmental hierarchy, we identified sequential, transitional stages of differentiation likely mediated by different DNMT, MBD and HDAC family members. Biochemically, HDAC1 and HDAC2 associate with repressor complexes recruited by both MBD2 and MeCP2. HDAC1 and HDAC2, however, are divergently expressed in the OE, a pattern that is recapitulated in the brain. Rather than simultaneous inclusion in a complex, therefore, the individual association of HDAC1 or HDAC2 may provide specificity to a repressor complex in different cell types. Furthermore, distinct transitional stages of differentiation are perturbed in the absence of MBD2 or MeCP2. MeCP2 is expressed in the most apical immature olfactory receptor neurons (ORNs), and is up-regulated with neuronal maturation. In the MeCP2 null OE there is a transient delay in ORN maturation and an increase in neurons of an intermediate developmental stage. Two protein variants of MBD2 are expressed in the OE, with MBD2b expressed in cycling progenitor cells and MBD2a in the maturing ORNs. MBD2 null ORNs undergo increased apoptotic cell death. There is also a significant increase in proliferating progenitors in the MBD2 null OE, likely due, at least in part, to feedback from the dying ORNs, acting to up-regulate neurogenesis. Increased cell cycling in the MBD2 null is also observed post-lesion, however, in the absence of feedback back from the ORNs, a phenotype that is recapitulated by an acute inhibition of HDACs with valproic acid. Therefore, disruptions at both transitional stages of ORN differentiation are likely in the MBD2 null mouse. Together, these results provide the first evidence for a sequential recruitment of different MBD proteins and repressor complexes at distinct transitional stages of neuronal differentiation.

DNA methylation-dependent gene silencing, catalyzed by DNA methyltransferases (DNMTs) and mediated by methyl binding domain proteins (MBDs) and histone deacetylases (HDACs), is essential for mammalian development, with the nervous system demonstrating particular sensitivity to perturbations. Little is known, however, about the role of DNA methylation in the stage-specific differentiation of neurons. In the olfactory epithelium (OE), where neurogenesis is continuous and the cells demonstrate a laminar organization with a developmental hierarchy, we identified sequential, transitional stages of differentiation likely mediated by different DNMT, MBD and HDAC family members. Biochemically, HDAC1 and HDAC2 associate with repressor complexes recruited by both MBD2 and MeCP2. HDAC1 and HDAC2, however, are divergently expressed in the OE, a pattern that is recapitulated in the brain. Rather than simultaneous inclusion in a complex, therefore, the individual association of HDAC1 or HDAC2 may provide specificity to a repressor complex in different cell types. Furthermore, distinct transitional stages of differentiation are perturbed in the absence of MBD2 or MeCP2. MeCP2 is expressed in the most apical immature olfactory receptor neurons (ORNs), and is up-regulated with neuronal maturation. In the MeCP2 null OE there is a transient delay in ORN maturation and an increase in neurons of an intermediate developmental stage. Two protein variants of MBD2 are expressed in the OE, with MBD2b expressed in cycling progenitor cells and MBD2a in the maturing ORNs. MBD2 null ORNs undergo increased apoptotic cell death. There is also a significant increase in proliferating progenitors in the MBD2 null OE, likely due, at least in part, to feedback from the dying ORNs, acting to up-regulate neurogenesis. Increased cell cycling in the MBD2 null is also observed post-lesion, however, in the absence of feedback back from the ORNs, a phenotype that is recapitulated by an acute inhibition of HDACs with valproic acid. Therefore, disruptions at both transitional stages of ORN differentiation are likely in the MBD2 null mouse. Together, these results provide the first evidence for a sequential recruitment of different MBD proteins and repressor complexes at distinct transitional stages of neuronal differentiation.

DNA methylation-dependent gene silencing, catalyzed by DNA methyltransferases (DNMTs) and mediated by methyl binding domain proteins (MBDs) and histone deacetylases (HDACs), is essential for mammalian development, with the nervous system demonstrating particular sensitivity to perturbations. Little is known, however, about the role of DNA methylation in the stage-specific differentiation of neurons. In the olfactory epithelium (OE), where neurogenesis is continuous and the cells demonstrate a laminar organization with a developmental hierarchy, we identified sequential, transitional stages of differentiation likely mediated by different DNMT, MBD and HDAC family members. Biochemically, HDAC1 and HDAC2 associate with repressor complexes recruited by both MBD2 and MeCP2. HDAC1 and HDAC2, however, are divergently expressed in the OE, a pattern that is recapitulated in the brain. Rather than simultaneous inclusion in a complex, therefore, the individual association of HDAC1 or HDAC2 may provide specificity to a repressor complex in different cell types. Furthermore, distinct transitional stages of differentiation are perturbed in the absence of MBD2 or MeCP2. MeCP2 is expressed in the most apical immature olfactory receptor neurons (ORNs), and is up-regulated with neuronal maturation. In the MeCP2 null OE there is a transient delay in ORN maturation and an increase in neurons of an intermediate developmental stage. Two protein variants of MBD2 are expressed in the OE, with MBD2b expressed in cycling progenitor cells and MBD2a in the maturing ORNs. MBD2 null ORNs undergo increased apoptotic cell death. There is also a significant increase in proliferating progenitors in the MBD2 null OE, likely due, at least in part, to feedback from the dying ORNs, acting to up-regulate neurogenesis. Increased cell cycling in the MBD2 null is also observed post-lesion, however, in the absence of feedback back from the ORNs, a phenotype that is recapitulated by an acute inhibition of HDACs with valproic acid. Therefore, disruptions at both transitional stages of ORN differentiation are likely in the MBD2 null mouse. Together, these results provide the first evidence for a sequential recruitment of different MBD proteins and repressor complexes at distinct transitional stages of neuronal differentiation. / Medicine, Faculty of / Graduate

The nematode <i>Caenorhabditis elegans</i> is a small invertebrate whose nervous system, general anatomy, and normal development are all (comparatively) extremely simple and reproducible, and have all been well characterised. This dissertation describes work based on two different approaches to the study of the control of neural development in <i>C. elgans</i>. In the first part the course of neural outgrowth in the region of the ventral nerve cord was followed from electron microscope reconstructions of a series of fixed embryos. Following this, neurons whose processes grew out early were removed by laser ablation of their parent cells and the effect on subsequent nerve outgrowth was assayed by electron microscope reconstruction. The first process to grow along the ventral cord is that of AVG, and its presence is required for the normal, highly asymmetrical structure of the cord. Two more examples of dependency on particular nerve processes for correct guidance can be deduced from experiments in which cells at the back of the animal were removed. The observations of normal development and the ablation experiments can in some cases be related to defects seen in <i>uncoordinated</i> mutants with defective nerve process organisation. The second approach was to establish and analyse a computer data base containing all the synaptic connectivity data obtained by White et al. (1986), who reconstructed at an electron microscope level the entire central nervous system regions of two <i>C. elegans</i> specimens. Since the circuitry is highly reproducible, comparisons of connections between equivalent pairs of cells can be used to infer properties of synapse formation. Overall, the <i>C. elegans</i> circuitry is anatomically highly directional, and what little chemical synaptic feedback that is seen is mostly part of reciprocal synaptic connections. There is also evidence for physical organisation of the nerve processes in subbundles of the main process tract in the central nervous system.

No description available.

No description available.

No description available.

The formation of neural connections in the mammalian nervous system is a complex process. During development, axons are initially overproduced and compete for limited quantities of target-derived growth factors. Axons which participate in functional circuits and secure appropriate amounts of growth factors are stabilized, while those axons that are either inappropriately connected or do not obtain sufficient concentrations of growth factors are eliminated in a process termed ‘axon pruning’. In this thesis, I examined the mechanisms that regulate pruning of peripheral, NGF-dependent sympathetic neurons that project to the eye. I determined that pruning of these projections in vivo requires the p75 neurotrophin receptor (p75NTR) and synthesis of brain-derived neurotrophic factor (BDNF) from the activity-dependent exon IV promoter. Furthermore, analysis of an in vitro model of axon competition, which is regulated by the interplay between nerve growth factor (NGF) and neuronal activity, revealed that p75NTR and BDNF are also essential for axon competition in culture. In this model, in the presence of NGF, neural activity confers a competitive growth advantage to stimulated, active axons by enhancing downstream TrkA (NGF receptor) signaling locally in axons. More interestingly, the unstimulated, inactive axons deriving from the same and neighboring neurons acquire a "growth disadvantage" due to secreted BDNF acting through p75NTR, which induces axon degeneration by suppressing TrkA signaling that is essential for axonal integrity. These data support a model where, during developmental axon competition, successful axons secrete BDNF in an activity-dependent fashion which activates p75NTR on unsuccessful neighboring axons, suppressing TrkA signaling, and ultimately promoting pruning by a degenerative mechanism.