Semaphorin 5A (Sema5A) functions in neocortical development In vivo

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Interdepartmental Graduate Program of Neuroscience, 2008.Proper nervous system function critically depends upon the proper assembly of an intricate network of neuronal connections. A limited number of axon guidance cues have been identified that regulates growth cone guidance and directly participate in neural network development and patterning. A key question in developmental neurobiology is: How can a limited number of axon guidance cues create such an enormous complexity of neuronal connections? Emerging new concepts include “contextdependent” function of guidance cues, hierarchies among guidance cues, and bi-functionality (i.e. attracting or repelling growth cones by the same guidance cue depending on the presence of different co-receptors). Understanding how specific axon guidance cues exert their diverse function at the cellular and molecular level is of considerable interest both biologically and clinically. The focus of my thesis work was on Semaphorin 5A (Sema5A), a type-1 transmembrane protein with a large ectodomain composed of a Sema-domain followed by 7 type-1 thrombospondin repeats. I will show that Sema5A is a bi-functional axon guidance cue that is essential for the proper formation of the fasciculus retroflexus (FR) in vitro. Sema5A bi-functional activity is regulated in a proteoglycan-dependent manner. In the presence of heparan sulfate proteoglycans (HSPGs), Sema5A exerts an attractive response towards habenular neurons. Conversely, in the presence of chondroitin sulfate proteoglycans (CSPGs), Sema5A exerts an inhibitory response towards the same population of neurons. Structural and functional analyses indicate that HSPGs and CSPGs interact with Sema5A directly. Proteoglycans mediate Sema5A bi-functional responses via interaction with its first four type-1 thrombospondin repeats. Further work has also identified the neuronal heparan sulfate proteoglycan, syndecan-3, as a direct binding partner for Sema5A. In order to study the role of Sema5A in vivo, a Sema5A conditional mutant mouse was generated using cre-loxP technology. Western blot analysis and immunohistochemistry of Sema5A homozygous mutant mice revealed that Sema5A-/- are null for Sema5A protein. Detailed expression analyses indicate that during embryogenesis, Sema5A is prominently expressed in the ventricular zone of the dorsal telencephalon. Sema5A shows a lateral-high to medial-low gradient of expression across the ventricular/subventricular zone (VZ/SVZ) between E11-E16 in the mouse. Furthermore, robust expression of Sema5A is seen in the dentate gyrus (DG) postnatally and continues throughout adulthood. Both the VZ/SVZ and the DG are areas known for their neurogenic potential. During normal cortical development, neurons are born in the VZ/SVZ, migrate up into the cortex along radial glia scaffolds, and subsequently colonize their appropriate laminar layer within the cortex. BrdU birthdating studies reveal that mice lacking Sema5A exhibit abnormal corticogenesis owing to impaired radial migration. Sema5A null mice demonstrate delayed migration of newborn cortical neurons towards the cortical plate, resulting in a larger number of BrdU positive neurons residing within the intermediate zone, compared to their wild-type counterparts. Conversely, over-expression of Sema5A under the transcriptional control of a nestin promoter results in an increased number of BrdU positive cells in the marginal zone of the neocortex. Together these studies uncover a novel role for Sema5A in radial neuronal migration in vivo. Moreover, examination of axonal trajectories of major fiber pathways, including the FR of Sema5A deficient mice revealed no gross abnormalities. Further expression studies of class V semaphorins revealed an overlap of expression within the embryonic brain for Sema5A and Sema5B, suggesting a role for functional redundancy between the two mammalian members during development. Preliminary results suggest that the FR is defective in Sema5A/Sema5B double mutant mice and that the migration defects observed in Sema5A mutant mice in the cortex are enhanced in Sema5A/Sema5B null mice. These studies show for the first time that class 5 semaphorins function in cortical development and axon guidance in vivo