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In vitro and in situ study of the role of Sema5A in the developing mouse telencephalon

The Semaphorina5A (Sema5A) is a transmembrane protein that has been shown to have a role as bifunctional axon guidance molecule during axon elongation and cell migration in the development of diencephalon and its connections. It was also shown that the down- regulation of SEMA5A expression is present in patients with autism spectrum disorder and haploinsufficiency for SEMA5A in Cri-du-chat mental retardation. According to the recent data (Allan Brain Institute), the transcripts of Sema5A gene are present in proliferative zones of the telencephalic vesicle in rodents at age E13.5 E15.5 and P14. Aiming to disclose the role of Sema5A in the development of telencephalon and its neocortical connections, we firstly, conducted functional in vitro study. The neocortical explants from mouse embryo (E15) were cultivated 72h on Sema5a uniform or alternately presenting carpet (Sema5A transfected Human Embryonic Kidney- 293 cells membranes). The indirect immunohistochemical method for Sema5A protein was performed on postmortem brain samples from early postnatal mouse brains to disclose the spatial and temporal expression of Sema5A protein throughout the developing telencephalon. The in vitro assays showed that Sema5a influence neocortical axons by promoting their outgrowth and elongation when it is uniformly presented on the carpet. The study also shows Sema5a doesn’t steer axons or modulate collateralization on Sema5A- alternating carpets. The expression of Sema5A protein revealed by immunohistochemical staining was more prominent at P15 than at P5, in the entire cortex with clear regional differences in the expression. The layer II/III, V and VIa of the neocortex show transmembrane expression of the Sema5A protein at the soma and the initial dendrite segment at P15. At P5 Sema 5A is expressed in layer V and layer VIb. The insular and the piriform region of the cortex show prominent expression of Sema5A protein on the neuronal soma membrane at P15. At the same time, the Sema5A protein was not expressed in the cingulate cortex. Further postmortem-in situ, in vitro and in vivo studies are needed to get a better understanding of Sema5a roles in the developmental processes of the telencephalon in health and disease (Fund: KK.01.1.1.01.0007, CoRE – Neuro).

cortex ; axon guidance ; thrombospondin repeats ; autism spectrum disorder

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