Neural function depends on precise wiring of axon during development. Previous studies have demonstrated that the erythropoietin producing hepatocellular carcinoma (Eph) family of tyrosine receptor kinases is crucial for the proper development of a number of neural circuits in the mammalian central nervous system (CNS). Mice lacking Eph receptors have been shown to exhibit deficits in pathways which include the thalamocortical, callosal, retinal and corticospinal tract. Due to the large number of Eph family members, the relative contribution of each receptor to axon pathfinding and neural function remains elusive. In this thesis, I have addressed the function of EphA4, EphB2 and EphB3 in the regulating the formation of interhemispheric projections within the forebrain and motor axon connections within the spinal cord using EphA4, EphB2 and EphB3and combinatorial null mice. To perform a detailed examination of the process of axon guidance regulated by these receptors within the forebrain, high resolution magnetic resonance imaging (MRI), immunofluorescence and in vivo stereotactic fluorescent labeling were performed. This work resulted in the development and validation of MRI-based analytic tools performed using EphB2 mutants which we have previously shown to exhibit specific morphologic defects in the anterior commissure (AC). Analysis of EphA4 null mice using high resolution MRI revealed for the first time that in addition to errors of midline crossing, loss of EphA4 activity results in a positional reorganization of the rostral AC. Results demonstrate that while EphB2 and A4 each regulate distinct aspects of guidance within ACpp, these receptors also operate cooperatively to control the guidance of axons in the pars anterior of the AC, a pathway not been previously implicated in Eph-mediated guidance. With respect to the spinal cord, mice deficient in EphB2 and EphA4 display prominent axon guidance errors in the medial subsets of the lateral motor column (LMCm); neurons which normally innervate ventral limb musculature. Finally, I have addressed the functional effect which Eph mutants exhibit with respect to motor behavior by examining a detailed set of motor coordination parameters

Axon guidance is the developmental process where developing neurons navigate their processes based on attractive and repulsive cues. C. elegans has been an instrumental model in the study of neurobiology with one of the key benefits being a relatively simple nervous system which comprises of only 302 neurons. The Eph Receptors are a canonical class of axon guidance molecules and in C. elegans there is only Eph receptor VAB-1. To understand axon guidance it is useful to study the mechanosensory neurons, and in particular a pair of neurons called the PLM (Posterior Lateral Microtubule). In this thesis I undertook a series of projects involving new techniques, and identified gene products that may interact with VAB-1 in the PLMs. I demonstrate that the use of the PLM Length and PLM/Body length ratio at the L1 stage offers an improved way of detecting axon guidance phenotypes. I show proof of concept that use of a light induced cell ablation technique can help study the developing nervous system. Further, I show that with the use of a tissue specific RNAi technique the role of lethal genes in axon guidance can be analyzed. Finally I conducted a screen that identified new effectors of the VAB-1 signal transduction pathway. / Thesis (Master, Biology) -- Queen's University, 2012-10-29 11:31:54.764

During neural development, each neuron sends an axon out from its cell body. Extending axons are guided by interactions between environmental factors and axonal receptors for these factors. It has been suggested that certain proteins of the immunoglobulin-like superfamily are among the molecules involved in axon guidance. In particular, TAG-1, Ll and NrCAM have previously been implicated in the guidance of dorsal spinal commissural axons at the ventral midline region known as the floor plate. To establish whether these molecules have such roles in mice, the dorsal spinal axons of TAG-1, L1 or NrCAM mutant mouse embryos were traced. There were no significant differences between the results from mutant embryos and their wild type counterparts. This indicated that these three proteins are individually not essential for the normal development of mouse dorsal spinal projections. However, results from TAG-MLI double mutant embryos suggested that TAG- I and LI might affect the ability of commissural axons to extend out of the floor plate. Analysis of ephrin B3 mutant embryos indicated that ephrin B3 might also be important for floor plate exit. As the TAG-1 null mutation includes a lacZ construct, this reporter gene was used to further investigate the roles of TAG-1. Its expression was used to determine distribution of TAG-1 gene activity in the developing mouse nervous system. As the pattern of reporter expression was found to be comparable with that of TAG-1 protein, the TAG-1 null allele was used as a marker for TAG-1-expressing cells in mutant embryos. Most of the structures that normally express TAG-1 seemed to be unaffected by an absence of the protein. However, the hypoglossal nerve was significantly less likely to extend towards the tongue in TAG-1 null homozygous embryos than in heterozygotes. This suggested that TAG-1 might be important for the guidance of hypoglossal axons.

Neural function depends on precise wiring of axon during development. Previous studies have demonstrated that the erythropoietin producing hepatocellular carcinoma (Eph) family of tyrosine receptor kinases is crucial for the proper development of a number of neural circuits in the mammalian central nervous system (CNS). Mice lacking Eph receptors have been shown to exhibit deficits in pathways which include the thalamocortical, callosal, retinal and corticospinal tract. Due to the large number of Eph family members, the relative contribution of each receptor to axon pathfinding and neural function remains elusive. In this thesis, I have addressed the function of EphA4, EphB2 and EphB3 in the regulating the formation of interhemispheric projections within the forebrain and motor axon connections within the spinal cord using EphA4, EphB2 and EphB3and combinatorial null mice. To perform a detailed examination of the process of axon guidance regulated by these receptors within the forebrain, high resolution magnetic resonance imaging (MRI), immunofluorescence and in vivo stereotactic fluorescent labeling were performed. This work resulted in the development and validation of MRI-based analytic tools performed using EphB2 mutants which we have previously shown to exhibit specific morphologic defects in the anterior commissure (AC). Analysis of EphA4 null mice using high resolution MRI revealed for the first time that in addition to errors of midline crossing, loss of EphA4 activity results in a positional reorganization of the rostral AC. Results demonstrate that while EphB2 and A4 each regulate distinct aspects of guidance within ACpp, these receptors also operate cooperatively to control the guidance of axons in the pars anterior of the AC, a pathway not been previously implicated in Eph-mediated guidance. With respect to the spinal cord, mice deficient in EphB2 and EphA4 display prominent axon guidance errors in the medial subsets of the lateral motor column (LMCm); neurons which normally innervate ventral limb musculature. Finally, I have addressed the functional effect which Eph mutants exhibit with respect to motor behavior by examining a detailed set of motor coordination parameters

Hohenheim, Univ., Diss., 2008.

Tübingen, Univ., Diss., 2004.

Universiẗat, Diss., 2004--München.

Berlin, Freie Universiẗat, Diss., 2004. / Dateiformat: zip, Dateien im PDF-Format.

Axons are up-to-a-meter-long cable-like cellular processes of neurons. The proper function of nervous systems requires that axons grow and wire up correctly during development or regeneration. The uniquely challenging architecture of axons has to be sustained for an organism's lifetime, and renders them key lesion sites during healthy ageing, in injury and neurodegenerative diseases. Notably, axon degeneration is considered as the cause rather than consequence for neuron decay in the context of various neurodegenerative diseases. The structural backbones of axons are formed by parallel bundles of microtubules (MTs) which also provide the highways for life-sustaining long-distance transport between cell bodies and the growth cones or synaptic endings. To better understand axon development, regeneration, maintenance and degeneration during ageing, my PhD project has focused on mechanisms underpinning the regulation of MT bundles in axons. For this, I have capitalised on fast and genetically and experimentally amenable research possible in Drosophila neurons, both in primary culture and in vivo. I have used systematic combinatorial genetics and pharmacological approaches to unravel mechanisms and roles of actin as well as the cortical collapse factor Efa6 in MT regulation during axon formation and maintenance. I was able to gain a number of novel mechanisms contributing to the de novo alignment and maintenance of ordered MT bundles. First, it has been proposed that Spectraplakins (large actin-microtubule linkers) guide the extension of polymerising MTs along cortical F-actin, thus directly laying axonal MTs out into parallel bundles. Here, I have used manipulations of actin networks as well as hybrid constructs of Shot where the actin binding domain was replaced by actin associating domains of other molecules. My data strongly suggest that Shot's ABD domain has unique properties that can sense specific properties of F-actin networks, and this is important for its ability to appropriately regulate MT behaviours. Second, using combinations of actin and Shot manipulations, I found that Shot displays not only these actin-dependent guidance functions, but it displays novel actin-independent function in MT bundle maintenance for which I present a working hypothesis. Third, I found a novel and Shot-independent role of axonal actin in maintaining MTs and promoting axon growth, and my results suggest that these functions involve promotion of MT polymerisation. MT maintenance is therefore mediated through two complementary mechanisms involving Shot on the one hand and actin on the other, and simultaneous removal of Shot and actin leads to entire loss of axons. Finally, I have unravelled novel axonal functions of the cortical collapse factor Efa6 which serves as a check point in MT bundle maintenance by eliminating "off track" MTs that have escaped the axonal bundle organisation. In the absence of this factor, a gradual increase of disorganised, criss-crossed MTs occurs as a matter of days. These new mechanisms strongly suggest that different MT-regulatory mechanisms act in parallel in axons and complement each other in one common mechanism of MT bundle formation and maintenance. I propose here a local homeostasis model of axonal MT bundle maintenance which provides new ways to think about problems of ageing as well as a range of different neurodegenerative diseases.

Cyclin Dependent Kinase (Cdk) family members play a role in CNS development. Cyclin Dependent Kinase 5 (Cdk5) is well known for its fundamental role in neuronal development and axogenesis, as well as, cell death. Other Cdks include Pctaire and Pftaire. Inhibition of Pctaire results in increased axon outgrowth, however, the role and function of Pftaire is unknown. Pftaire1 is a novel member of the Cdk family that was initially detected in a screen for cdc2-like kinases. Unpublished data from our lab reveals that Pftaire1 (Eip63E) deficiency in Drosophila melanogaster results in defects in the axon and neuronal structure of the ventral nerve cord (VNC). In mammals, Pftaire1 is highly, expressed in the CNS. Here, we proposed that Pftaire1 might have a role in axon outgrowth. To investigate the role of Pftaire1 in mammals, the first germline Pftaire1 knockout mice were generated. Considering the severe effects of Eip63E deficiency in Drosophila and the homology between mammalian and fly Pftaire1, CNS defects in the mouse were anticipated. However, to date, no gross abnormalities have been detected in the overall morphology, fertility, life span, or anatomical brain structures of the Pftaire1 deficient mice. This may be due to the presence of other post-mitotic Cdk proteins that are highly similar to Pftaire1. For instance, mammals possess Pftaire (1, and 2), as well as, Pctaire (1, 2, and 3), while Drosophila only possess the Pftaire1 orthologue where the Pftaire2 and Pctaire (1, 2, and 3) are absent. Furthermore, the mice were of mixed background. In spite of this, we demonstrated that Pftaire1 deficient neurons showed increased axon length, in the initial phases of culture. This was confirmed by expression of dominant negative (DN) D228N-Pftaire1 in wild type neurons. Also classification of axons into different ranges, reveals a higher percentage of hyperextended neurites in D228N and Pftaire1 knockout mice. The mechanism by which Pftaire1 controls axon outgrowth is unknown. In this study we show that, Pftaire1 interacts physically with the small GTPase proteins Rac1, Cdc42, and RhoA. Importantly, we showed that Pftaire1 phosphorylates GDP-RhoA on a serine residue. We propose that this regulates RhoA activity, which in turn controls axon outgrowth.