Calcineurin/NFAT signaling is required for neuregulin-regulated Schwann cell differentiation

Abstract

Schwann cells develop from multipotent neural crest cells and form myelin sheaths around axons that allow rapid transmission of action potentials. Neuregulin signaling through the ErbB receptor regulates Schwann cell development; however, the downstream pathways are not fully defined. We find that mice lacking calcineurin B1 in the neural crest have defects in Schwann cell differentiation and myelination. Neuregulin addition to Schwann cell precursors initiates an increase in cytoplasmic Ca2+, which activates calcineurin and the downstream transcription factors NFATc3 and c4. Purification of NFAT protein complexes shows that Sox10 is an NFAT nuclear partner and synergizes with NFATc4 to activate Krox20, which regulates genes necessary for myelination. Our studies demonstrate that calcineurin and NFAT are essential for neuregulin and ErbB signaling, neural crest diversification, and differentiation of Schwann cells.

Fig. 1

CnB1 is deleted in the PNS by Wnt1^cre.(A) X-gal staining of E9.0 neural tube from a NFAT-reporter mouse line harboring LacZ under control of the DSCR1 enhancer region counterstained with nuclear fast red. Arrows, migrating neural crest cells; arrowheads, interneuron progenitors. (B) Whole-mountX-gal staining of E10.5 Rosa26; Wnt1^cre mouse embryo. Arrows, sensory ganglia. (C) Immunostaining of newborn DRG and peripheral nerves at thoracic limb region by using CnB1-specific antibody. Arrows, CnB1-positive motor axons of peripheral nerves; arrowheads, ventral roots. (D and E) Immunoblots of CnB1 and NFATc3 and c4 from E13.5 DRG cocultures at day 0 (D) and day 12 (E). Heat shock protein 90 (Hsp90) is a loading control. Arrows, NFATc4 proteins. *Cross-reactive protein.

Fig. 2

Schwann cell differentiation is defective in CnB1 mutant mice. (A) Schwann cells in newborn mutant sciatic nerves fail to establish one-to-one relations with axons. Low-power electron microscopic images showing overall structure of sciatic nerves. Asterisks, sorted axons; arrows, unsorted axon bundles. (B and F) Quantification of axonal sorting (number of axons in bundles) of sciatic nerves and ventral motor roots of spinal cord (n = 4 per group; error bars, +SEM; P < 0.001; two-tailed t test). (C) Immunoblots of newborn sciatic nerves showing expression levels of myelin-specific proteins. Results from two different litters of animals are shown. Arrows, NFATc3 and c4 proteins. *Cross-reactive protein. Hsp90 is a loading control. (D) Immunostaining of cryosections of newborn sciatic nerves using MBP-specific antibody (green). (E) Electron microscopic images of ventral motor roots. Asterisks, sorted axons; arrows, unsorted axon bundles. All experiments were performed with at least four littermate pairs of mice.

Fig. 3

Calcineurin/NFAT is required for NRG1 and ErbB signaling. (A) Ca²⁺ influx stimulated by NRG1. DRG cocultures were preloaded with the Ca²⁺ sensor Fura-2AM in the presence or absence of PLC-γ inhibitor U73122, 30 min before stimulation. Ca²⁺ influx was detected, and average Ca²⁺ levels were measured. (B) Dephosphorylation of NFATc3 and c4 is not induced in mutant cocultures after NRG1 stimulation, as examined by immunoblotting (ib). Arrows, dephosphorylated (active) NFATc3 and c4. (C) Tyrosine phosphorylation levels of ErbB3 following the activation by NRG1. ErbB3 were isolated by immunoprecipitation (ip). Tyrosine phosphorylation was detected by immunoblotting using phosphotyrosine-specific antibody. The blot was reprobed with ErbB3-specific antibody to verify protein expression levels. (D) Calcineurin/NFAT-dependent transcription is stimulated by NRG1. E13.5 DRG cocultures were transfected with luciferase reporter constructs containing the DSCR1 enhancer, NFATc4 promoter, Krox20 promoter, or Krox20 MSE. Luciferase activities were measured after 20 hours of NRG1 stimulation. Fold activation is relative to unstimulated cells (n = 6; error bars, +SEM; **P < 0.01; ***P <0.001; t test).

Fig. 4

Synergistic effects of NFATc4 and Sox10 on myelin gene expression. (A) NFATc4 and Sox10 synergistically activate the Krox20 MSE and P₀ promoter. DRG cocultures were transfected with luciferase reporter constructs containing the Krox20 MSE, P₀ promoter or Krox20 promoter. Expression constructs encoding constitutively active calcineurin A (CnA*), NFATc4, and Sox family members were co-transfected. Luciferase activities were measured. Fold activation is relative to reporter baseline activities (n = 6, error bars, +SEM, ***P < 0.001; t test). (B) Model of myelination process regulated by calcineurin/NFAT signaling pathway. Solid arrows, calcineurin/NFAT signaling pathway; dotted arrows, pathways defined by previous literature.

Fig. 4

Synergistic effects of NFATc4 and Sox10 on myelin gene expression. (A) NFATc4 and Sox10 synergistically activate the Krox20 MSE and P₀ promoter. DRG cocultures were transfected with luciferase reporter constructs containing the Krox20 MSE, P₀ promoter or Krox20 promoter. Expression constructs encoding constitutively active calcineurin A (CnA*), NFATc4, and Sox family members were co-transfected. Luciferase activities were measured. Fold activation is relative to reporter baseline activities (n = 6, error bars, +SEM, ***P < 0.001; t test). (B) Model of myelination process regulated by calcineurin/NFAT signaling pathway. Solid arrows, calcineurin/NFAT signaling pathway; dotted arrows, pathways defined by previous literature.