Schwann Cell Secreted Sema3b Modulates Peripheral Axonal Radial Sorting

BackgroundIn the peripheral nervous system（PNS）,Schwann cells are glial cells that are in intimate connection with axons throughout developmental stage.Axo-Schwann cell interdependence has a significant role in peripheral morphological structures and performance.Schwann cells,neural crest-derived cells in the PNS,include myelinating Schwann cells（m SCs）and non-myelinating Schwann cells（NMSCs）that perform different functions.Recognition and segregation of a large（larger than 1μm）caliber axon by a m SC are characterized by reaching a 1:1 relationship with axons.The myelinated axons are mainly associated with the fast conduction velocity of action potentials.As Schwann cell proliferation and axonal segregation processes,multiple small（smaller than 1μm）caliber axons are embraced by a NMSC,which subsequently differentiated into a Remak bundle.Therefore,NMSCs are known as Remak cells.The axons that termed as unmyelinated axons are concerned with the slow conduction velocity of action potentials.Thus,axonal radial sorting is a crucial morphogenetic process which include axonal myelination and Remak bundle formation.Radial sorting,one of the ways that the interaction of Schwann cells and axons,requires molecular modulation of Schwann cells and axons.Neuregulin1（Nrg1）-Erb B2/3,the canonical signaling pathway,has been indicated to a significant factor of radial sorting.The choice to myelinate or not is based on the amount of NRG1 exposed on the membrane of axons with which Schwann cells are in touch.Neuronal reduced expression of NRG1 induces hypomyelination,and disproportionately unmyelinated,aberrantly ensheathed axons of Remak bundles.Such a series of clinic symptoms as demyelination or neuropathic pain will be led to considering abnormal molecular expression in developmental stage of radial sorting.Therefore,uninterrupted exploration of molecular mechanisms of radial sorting is particularly imperative based on the fact that the molecular mechanisms have not been clearly understood.The roles of Semaphorin family are deemed to the suppression of tumors by inducing apoptosis and inhibition of axonal extension.The gene Sema3b,which belongs to one of the members of semaphoring family and as a tumor suppressor,acts on the oppression of breast cancer,lung cancer and renal cancer et al.The importance of Sema3b in axonal radial sorting remains elusive.Objective and significanceThe research focus on the molecular mechanisms of axonal radial sorting by means of specific techniques such as immunostaining,electron microscopy and behavioral test et al,aiming at providing new insights into the development of radial sorting and neuropathic research.Methods1. Systematic observations of developmental process of axonal radial sorting（1）The process of myelination was investigated by labeling the myelinated axons,unmyelinated axons and myelin with NF200,peripherin and MBP antibody,respectively;（2）Electron microscopy was used to research the formation of Remak bundles.2. Screen the underlying candidate Sema3b which expresses on Schwann cells by mRNA sequencing（1）To ablation of Schwann cells,inject proper dosage of diphtheria toxin in Dhh ^Cre;R26^{i DTR}mice at P4 asΔSC group,and Dhh ^Creor R26^{i DTR}mice who were handled with the same dosage of diphtheria toxin as Control group;（2）RNA sequencing of sciatic nerves from Control andΔSC mice at P14 and screen the underlying gene Sema3b who expresses specifically on Schwann cells;（3）Verify the specificity of Sema3b via FISH.3. Construct the animal models of Sema3b knockout（Sema3b CKO）mice,and explore morphological and behavioral phenotypes in the process of axonal radial sorting（1）Construct the Sema3b CKO mouse model,and inspect the morphological phenotypes using electron microscopy;（2）Discover behavioral phenotypes by behavioral tests;4.Check out the morphological and behavioral phenotypes in Sema3b inducible knockout（Sema3b i CKO）mice.5.Construct Nrp1 CKO mouse model,and explore the morphological phenotype.6.Verify the performance of Sema3b-Nrp1/2 signaling pathway in developmental process of axonal radial sorting in vitroResults1.The process of radial soring is finished at postnatal day 28（P28）（1）Almost all myelinated axons which were labeled with NF200 antibody were wrapped by myelin which were labeled with MBP antibody in sciatic nerves of postnatal day 28（P28）mice;（2）The amount of Schwann cell cytoplasm is present among unmyelinated axons in Remak bundles in sciatic nerves of postnatal day 28（P28）mice.2.Sema3b was screened as an underlying candidate by RNA sequencing（1）The amount of Sema3b was significantly decreasing after ablation of Schwann cells compared with the control mice;（2）Sema3b was expressed specifically on Schwann cells by the way of FISH;（3）Sema3b was expressed specifically on NMSC by the way of immunostaining.3.The morphological phenotype of late radial sorting arrest/delay was observed in Sema3b CKO mice（1）Conditional deletion of Sema3b in Schwann cells has a negative effect on the number of proliferating and apoptotic Schwann cells;（2）Conditional deletion of Sema3b in Schwann cells resulted in a morphological phenotype that large-caliber（larger than 1μm）axons were observed in Remak bundles besides multiple small ones;（3）Amount of NF200⁺myelinated axons were not wrapped by myelin which was labeled with MBP antibody in sciatic nerve of Sema3b CKO mice;（4）Measurement of mechanic allodynia using Von Frey Filaments showed a lower threshold in Sema3b CKO mice compared with the Control mice.And rotarod test exhibited equivalent motor function in the Control and Sema3b CKO mice;（5）Assessment of thermal stimuli using hot plate and tail flick suggested a lower threshold in Sema3b CKO mice compared with the Control ones.4. The morphological phenotype of late radial sorting arrest/delay was observed in Sema3b conditional knockout mice（Sema3b i CKO）at P21.While Tamoxifen-induced ablation of Sema3b in adult mice altered neither the morphological structure nor statistical analysis,suggesting a crucial role for Sema3b in initiating the developmental process rather than maintaining it.5. Amount of axons whose calibers were larger than 1 um were not wrapped by myelin in sciatic nerves of Nrp1 CKO mice,which was similar to the phenotype of Sema3b CKO mice.6. Schwann cell modulates radial sorting during developmental stage via Sema3b-Nrp1/2 signaling pathway in vitroConclusionThis research firstly exposed the result that axonal radial sorting was finshed at P28,and the gene Sema3b was expressed on NMSC and its receptors Nrp1/2 were secreted by sensory axons.Then the morphological phenotype that late radial sorting delay/arrest was observed in Sema3b CKO mice.And the outcome that Schwann cell modulates axona radial sorting through Sema3b-Nrp1/2 signaling pathway was evaluated in vitro.Altogether,this studies revealed the molecular mechanism of radial sorting and provided new insights into the development of peripheral axonal radial sorting and the treatments of neuropathy.