Severe hypomyelination of the murine CNS in the absence of myelin-associated glycoprotein and fyn tyrosine kinase

Abstract

The analysis of mice deficient in the myelin-associated glycoprotein (MAG) or Fyn, a nonreceptor-type tyrosine kinase proposed to act as a signaling molecule downstream of MAG, has revealed that both molecules are involved in the initiation of myelination. To obtain more insights into the role of the MAG-Fyn signaling pathway during initiation of myelination and formation of morphologically intact myelin sheaths, we have analyzed optic nerves of MAG-, Fyn- and MAG/Fyn-deficient mice. We observed a slight hypomyelination in optic nerves of MAG mutants that was significantly increased in Fyn mutants and massive in MAG/Fyn double mutants. The severe morphological phenotype of MAG/Fyn mutants, accompanied by behavioral deficits, substantiates the importance of both molecules for the initiation of myelination. The different severity of the phenotype of different genotypes indicates that the MAG-Fyn signaling pathway is complex and suggests the presence of compensatory mechanisms in the single mutants. However, data are also compatible with the possibility that MAG and Fyn act independently to initiate myelination. Hypomyelination of optic nerves was not related to a loss of oligodendrocytes, indicating that the phenotype results from impaired interactions between oligodendrocyte processes and axons and/or impaired morphological maturation of oligodendrocytes. Finally, we demonstrate that Fyn, unlike MAG, is not involved in the formation of ultrastructurally intact myelin sheaths.

Fig. 1.

Visualization of unmyelinated axons in optic nerves of different genotypes. Unmyelinated retinal ganglion cell axons were visualized in optic nerves of 7-month-old wild-type (a) and age-matched Fyn- (b) and MAG/Fyn- (c) deficient mice by immunohistochemistry using polyclonal antibodies to the neural adhesion molecule L1. Only a few L1-positive unmyelinated axons are detectable in optic nerves of wild-type mice (a), whereas they are numerous in nerves of Fyn-deficient animals (b). Note that the density of L1-positive axons is significantly increased in mice lacking both MAG and Fyn (c) when compared with Fyn single mutants (compare c with b). Scale bar (inc): a–c, 100 μm.

Fig. 2.

Ultrastructure of optic nerves of Fyn- and MAG/Fyn-deficient mice. Optic nerves of 2-month-old Fyn-deficient mice (a) contain a high number of small-sized unmyelinated axons (some labeled with asterisks). Compared with Fyn mutants, a significantly increased number of unmyelinated axons (some labeled with asterisks) is present in optic nerves of age-matched MAG/Fyn double knock-out mice (b). Some myelin sheaths in MAG/Fyn double mutants form redundant myelin (white dots inb) or contain noncompacted regions of myelin (curved arrows in b). Ax, Axon. Scale bar (in b): a,b, 1 μm.

Fig. 3.

The percentage of unmyelinated axons in the optic nerve of adult mice of different genotypes. The percentage of unmyelinated axons from the total axon population was determined in optic nerves of 2- (a), 4- (b), and 9- (c) month-old wild-type (open bars), MAG- (hatched bars), Fyn- (stippled bars), and MAG/Fyn- (filled bars) deficient mice. Bars for 4-month-old wild-type and MAG-deficient mice represent the mean value (±SD) of five and four animals, respectively. Bars for all other genotypes and developmental ages represent the mean value (±SD) of six animals. Note that the number of unmyelinated axons is significantly increased in optic nerves of MAG/Fyn double mutants when compared with Fyn single mutants (p < 0.0001 according to the unpaired t test).

Fig. 4.

Visualization of PLP/DM-20-positive cells in optic nerves of different genotypes. PLP/DM-20-positive cells were visualized in optic nerves of 7-month-old wild-type (a), MAG- (b), Fyn- (c), and MAG/Fyn- (d) deficient mice by in situ hybridization. Note the similar density and labeling intensity of positive cells in optic nerves of different genotypes. Scale bar (in d):a–d, 200 μm.

Fig. 5.

Ultrastructure of myelin sheaths in optic nerves of Fyn- and MAG/Fyn-deficient mice. Most myelin sheaths in optic nerves of 2-month-old MAG/Fyn mutants (a) lack a well developed periaxonal cytoplasmic collar, and some sheaths form redundant myelin coursing away from the axon (whitedots in a) or show degenerative alterations (asterisk in a). Myelin sheaths in optic nerves of age-matched Fyn mutants (b), in contrast, appear morphologically intact. Note the well developed periaxonal cytoplasmic collar (arrowheads in b) of most sheaths.Ax, Axon; M, myelin. Scale bar (inb): a, 0.5 μm; b, 0.25 μm.

Fig. 6.

Frequency of morphologically affected myelin sheaths in different genotypes. A, The percentage of myelin sheaths with an oligodendrocyte periaxonal cytoplasmic collar spanning less than half of the axonal circumference was determined in optic nerves of 2- (a) and 9-month-old (b) wild-type (open bars) and MAG- (hatched bars), Fyn- (stippled bars), and MAG/Fyn-deficient (filled bars) mice. Each bar represents the mean value (±SD) of four animals. Note that the formation of well developed periaxonal cytoplasmic collars is dependent on the presence of MAG but not on the presence of Fyn.B, The frequency of multiply myelinated axons, myelin sheaths with regions of noncompacted myelin, myelin sheaths forming redundant myelin, and myelin sheaths with signs of intramyelinic or periaxonal degeneration was determined in optic nerves of 2- (a) and 9-month-old (b) wild-type (open bars), MAG- (hatched bars), Fyn- (stippled bars), and MAG/Fyn-deficient (filled bars) mice. Each bar represents the mean value (±SD) of four animals. Note that Fyn is not involved in the formation of ultrastructurally intact myelin sheaths.