Establishment Of Myelinogenesis By Sipl/Smad Signaling Pathway In The Central Nervous System

Objective:To evaluate the effect of Smad interacting protein-1 (Sip1/Zfhxlb) on oligodendrocyte differentiation and maturation, and to identify the direct downstream target genes of Sipl. To explore the mechanisms how Sip1 promotes oligodendrocyte differentiation and initiates Myelinogenesis.Methods:1. The effects of Sipl/Zfhxlb on oligodendrocyte differentiation and maturation:(1)Immunostaining was used to detect Sip1 expression in oligodendrocyte-enriched regions of CNS. (2) in situ hybridization (ISH) was used to detect Sipl expression in Spinal cord.(3)oligodendrocyte-lineage specific Sip1 knockout mice model was used to detect phenotype and survival affects of ko mice. (4) Immunostaining and ISH were used to evaluate oliodendrocyte markers in Sip1cKO mice. (5)electron microscopy was used to evaluate myelinogenesis in SiplcKO mice. (6) Immunostaining was used to detect oligodendrocyte differentiation and maturation by deleting Sipl in vitro. (7) ISH was used to evaluate OPC markers in Sip1cKO mice.2. The mechanisms of Sipl/Zfhxlb regulates OLs differentiation and maturation and initiates myelinogenesis2.1 The mechanisms of Sipl/Zfhxlb regulates OLs differentiation and maturation by affecting related signaling pathways(1) Luciferase reporter assay was used to detect OLs development-related genes and myelin genes. (2) Co-IP and western blotting Assays were used to detect expression of smad signaling pathway (3) Immunostaining was used to detect Smadl phosporylation in Sip1cKO mice. (4) Immunostaining was used to detect OPC differentiation and maturation after Sip1 overexpression. (5) Real-time qPCR was used to detect OLs-regulated genes after Sip1 overexpression. (6) ISH, immunostaining and RT-qPCR were used to evaluate the expression of myelin related genes and proteins. (7) CHIP was used to identify whether Sip1 binds to the promoters of myelin-regulated factors.2.2 identifing the direct downstream genes of Sip1/Zfhxlb in OLs differentiation and maturation(1) Gene-chip microarray was used to identify the direct downstream genes of Sip1. (2) ISH was used to evaluate the expression of Smad7 in spinal cord development. (3) immunostaining was used to evaluate the protein expression of Smad7 in OLs lineage cells of spinal cord. (4) ISH was used to evaluate the expression of myelin-related genes by Smad7 electroporation in chick neural tube. (5) immunostaining and RT-qPCR were used to evaluate the expression of Smad7 and Sip1 both in OPC and OLs.(6) CHIP was used to evaluate whether Sip1 binds to Smad7 promoters. (7) RT-qPCR was used to evaluate the expression of Smad7 after Sip1 overexpression. (8) Immunostaining was used to evaluate whether Smad7 overexpression rescues Sip1-deficient phenotype in vitro. (9) Western blotting assay was used to detect expression of BMP pathway related proteins. (10) ISH was used to evaluate the expression of myelin related genes in Smad7cKO mice.Results:1.1. Sip1 is required for oligodendrocyte maturation and myelination (1) Sip1 transcripts were highly enriched in the white matter tracts of the corpus callosum, spinal cord and cortex. High levels of Sip1 were obviously detected in CC1+ differentiated oligodendrocytes, in contrast to its relatively weak expression in PDGFRα+ OPC. (2) E14.5～16.5, Sip1 was not expressed in white matter tracts of spinal cord. It was observed in ventral region of spinal cord at P0 and increased as mice growed up. (3) all mutant Sip1 flox/flox;OliglCre+/- mice (referred to as SiplcKO), but not their control littermates, developed generalized tremors, hindlimb paralysis and seizures from postnatal week one and died around postnatal week 2. (4) Expression of myelin genes such as myelin basic protein(Mbp) and proteolipid protein(Plpl) and myelin protein such as CC1 is essentially undetectable in the forebrain, cerebellum and spinal cord of mutant mice at P14. (5) In contrast to a large number of myelinated axons that are observed in control mice at P14, they were completely absent in the optic nerve and spinal cord of SiplcKO mutants (6) In lenti-GFP transduced cells, we observed an increase of mature MBP+ oligodendrocytes bearing a complex morphology over time during differentiation In contrast, no MBP+ oligodendrocytes were detected in lenti-CreGFP infected cells at any time points.. (7) The number of OPC and their proliferation rate (the percentage of Ki67+ proliferating OPC) were comparable to the control2. Sipl antagonizes the inhibitory effect of receptor-activated Smad signaling on the oligodendrocyte differentiation program2.1 Sipl/Zfhxlb interacts with Smadl to regulate oligodendrocyte differention program:(1) overexpression of Sipl antagonized the inhibitory effects mediated by BMPRCA/Smadl/p300 expression on the Mbp promoter activity while repressing the promoter activity of Id2, Id4 and Hesl activated by BMP/Smad signaling. (2) In the absence of BMPRCA Sipl interacted weakly with p-Smadl as long as Smad4 was present. This interaction increased dramatically when BMPRCA was introduced; in OPC culture system, Sip1 preotein was obviously increased after 5 days incubation with OLs differentiation medium. Smad1, as a bridge, connected Sip1 with p300 to regulation of oligodendrocyte differentiation inhibitors. (3) pSmadlwas comparable to the control in white matter tracts of ventral spinal cord. (4) Sip1 overexpression led to a drastic increase of RIP+ differentiated oligodendrocytes that harbored complex processes and also yielded a reduced number of PDGFRα+ OPC. (5) a significant upregulation of myelin genes such as Cnp, Cgt and Mbp and of differentiation activators such as Sox10, MRF and Olig2 in Sipl-transfected cells compared to the control; Conversely, we observed significant downregulation of negative regulators of differentiation, including of Id2, Id4, Hesl, Hes5, and BMPR1α. (6) a downregulation of myelin genes such as Cgt and of differentiation activators such as Sox10, MRF in Sip1cKO mice compared to the control. (7) Sipl binds to the promoters of differentiated activators MRF and Sox10,and differentiated inhibitors Id2, Id4 and Hesl.resulting in OLs differentiation and maturation.2.2 Sipl/Zfhxlb activates Smad7 expression to regulate oligodendrocyte differentiation program:(1)mRNA microarray profiling analysis from the spinal cord of control and Sip1cKO mice at P14 found the Smad7 gene significantly downregulated in Sip1cKO mice. (2) Smad7 mRNA expression appears in the ventral spinal cord at PO and increases strongly in the white matter of the cord at perinatal stages, and persisted into adulthood (3) Intense Smad7 antigen staining was detected in differentiated CC1+ oligodendrocytes but not in PDGFRa+ OPC in the developing spinal cord。(4) Overexpression of Smad7 was able to promote ectopic expression and dispersal of the OPC marker PDGFRa and the differentiated oligodendrocyte marker Sox10 in the electroporated side compared to the non-electroporated side (5) In cell culture, Smad7 staining was very low in OPC but increased significantly in mature MBP+ oligodendrocytes, similar to Sipl, which was also demonstrated by qRT-PCR analysis in oligodendrocytes and OPC. (6) Sipl was recruited to the Smad7 promoter region carrying Sip1 consensus binding sites in oligodendrocytes but hardly detectable in OPC. (7) overexpression of Sipl in HCN significantly promoted Smad7 expression assayed by qPCR。(8) Introducing Smad7 should rescue the defect caused by Sipl deletion. (9) When cotransfected with Smurfl, Smad7 substantially downregulated BMPR1a, phospho-Smadlandβ-catenin steady state protein levels relative to Gapdh. (10) the expression of the myelin genes Mbp and Plpl was diminished in the white matter of the brain and spinal cord in contrast to robust expression in control mice; The OPC marker PDGFRa was detected throughout the spinal cord and their number was comparable to that of control littermatesConclusion: (1) Abalation of Sipl significantly inhibits oligodendrocyte differention and maturation(2) Deletion of Sip1 in vivo fails to induce myelinogenesis in mice spinal cord and optic nerve(3) Sipl directly interacts with Smadl to reverse BMP effect on OLS development and binds to the promoters of myelin-differentiated regulators, which further promote oligodendrocyte differentiation and maturation(4) Sipl indirectly regulates OLs differentiation and maturation via activating Smad7 expression to antagonize Smad1 phosphorylationSip1 is essential for oligodendrocytes differentiation and maturation. Sip1 affects oligodendrocyte differentiation related factors by inhibiting receptor-activated Smad signaling pathway. In the mean time, Smad7, a direct target gene of Sip1, inhibits activation of BMP signaling pathway. Taken together, Sip1 promotes myelinogenesis in CNS via antagonist of BMP signaling pathway both in positive and negative manners.