Data supporting the role of Fyn in initiating myelination in the peripheral nervous system

Transgenic mice, which express active Fyn tyrosine kinase under the control of a glial fibrillary acidic protein promoter, have been produced. This promoter induces protein expression in the initiation stage of myelination in the peripheral nervous system (PNS) “Phosphorylation of cytohesin-1 by Fyn is required for initiation of myelination and the extent of myelination during development (Yamauchi et al., 2015 [1])”. Herein we provide the data regarding myelination-related protein markers and myelin ultrastructure in transgenic mice.


a b s t r a c t
Transgenic mice, which express active Fyn tyrosine kinase under the control of a glial fibrillary acidic protein promoter, have been produced. This promoter induces protein expression in the initiation stage of myelination in the peripheral nervous system (PNS) "Phosphorylation of cytohesin-1 by Fyn is required for initiation of myelination and the extent of myelination during development (Yamauchi et al., 2015 [1])". Herein we provide the data regarding myelination-related protein markers and myelin ultrastructure in transgenic mice. &

Value of the data
This data set is of value to the scientific community to need the information for molecules controlling myelination.
The data can provide the method of studying the initiation of myelination in vivo.
The data may promote further research on signaling molecules controlling myelination in vivo.

Data
The data shared in this article is the biochemical analysis for myelination-related proteins in active Fyn transgenic mice. The data also provides myelin ultrastructure in transgenic mice.

Experimental design, materials and methods
We generated transgenic mice expressing active Fyn at the relevant developmental stage. The sciatic nerves of these mice were then analyzed through electron microscopy at 3 days postnatal and through immunoblotting of proteins such as myelin markers.

Data from Fyn transgenic mice
In immunoblotting, neonatal transgenic mice expressing active Fyn exhibited increased expression levels of myelin marker proteins such as myelin protein zero (MPZ, also called P0) and 2 0 ,3 0 -cyclicnucleotide 3 0 -phosphodiesterase (CNPase) ( Fig. 1A and B). In electron microscopic analysis, transgenic mice exhibited smaller g-ratios, indicating increased myelin thickness, in the sciatic nerves (0.73 70.045 in the transgenic mice compared to 0.78 70.060 in the control mice). Since the g-ratio is the numerical ratio of an axon's diameter to the diameter of the axon's outermost myelinated fibers [1][2][3], a smaller g-ratio indicates a thicker myelin sheath ( Fig. 2A-D). In immunoblotting with an antibody specific for phosphorylated Akt kinase (active Akt), increased phosphorylation was observed in samples from transgenic mouse nerves ( Fig. 3A and B). Akt is one of the central signal transducers controlling myelination [2][3][4][5]. The myelination-associated transcription factor Krox20 [4,5] was also increased in transgenic mouse nerves (Fig. 4, A and B). On the other hand, levels of Sox10 (Fig. 5A and B) and Oct6 (Fig. 6A and B), transcription factors expressed in Schwann cell lineage cells [4,5], were comparable in transgenic mice and controls.

Generation of active Fyn transgenic mice
A DNA fragment ( 4.5 kb) containing the SV40 enhancer, a mouse glial fibrillary acidic protein (GFAP) promoter specific for the neonatal stage of Schwann cells in the PNS [1,6,7], V5-epitopetagged active Fyn (isolated Src homology domain 1 [1]), an artificial intron, and human chorionic gonadotropin polyA units [1,7] was digested from the vector backbone ( 3.5 kb) with NcoI, purified, and injected into fertilized BDF1 oocytes. Transgenic founder mice and established transgenic mice were routinely identified using the KAPA genomic PCR kit (KAPA Biosystems, Wilmington, MA, USA) with the specific primer pair 5 0 -CCGGAATTCGAATATTAGCTAGGAGTTTCAGAAAGGGGGCCTG-3 0 and 5 0 -CCGGAATTCACTAGTGGGACTATGGTTGCTGACTAATTGAGATGC-3 0 . PCR was performed in 35 cycles, each consisting of denaturation at 94°C for 0.5 min, annealing at 65°C for 0.5 min, and extension at 0.5°C for 1 min. The transgenic allele yielded PCR bands for 322 bases. One transgenic founder was obtained from every 240 fertilized oocyte injections. Transgenic founders were mated to wild type C57BL/6JJms mice. The transgene was stably maintained for at least 3 generations. Male mice were used for experiments when their gender was distinguishable.

Electron microscopic analysis
Mouse sciatic nerves were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1% cacodylate buffer [1,7]. The tissues were postfixed with buffered 2% osmium tetroxide, dehydrated with an ethanol gradient, treated with acetone, and embedded in epoxy resin. Ultrathin sections of cross sections were stained with uranyl acetate and lead citrate, then observed and photographed with the Hitachi H-7600 or JEOL JEM-2010 electron microscope system. Myelinated nerves in the cross sections were randomly selected, and the g-ratio was calculated for each axon and as an average.

Statistical analysis
Data are presented as means 7SD from independent experiments. Intergroup comparisons were performed using unpaired Student's t test. Differences were considered significant when p value was less than 0.05.