EM demonstrates dystrophic axons and axonal spheroids in PBS- and rhGas6-treated mice.

<p>A. Axonal spheroids (>10 µm) are shown in PBS-treated mice (black arrow). Myelin debris (arrowhead) and lipid droplets (white arrow), are also noted. B – 400 ng/ml, C – 4 µg/ml of rhGas6 demonstrate dystrophic axons ranging in size from 3.0–9.5 µm in diameter (black arrow). Note microglial cell process (white arrow, B), and astrocytes in rhGas6-treated mice (gray arrow B and C), ×5000.</p

MBP immunostaining shows that rhGas6 treatment enhances remyelination following cuprizone toxicity.

<p>Relative to untreated mice (A) there is demyelination of the corpus callosum of mice fed cuprizone for 4 weeks (B and C). A comparison of remyelination assessed by MBP staining of the PBS treated mice (D) versus rhGas6 treatment for 14 days following cuprizone withdrawal (E – 400 ng/ml; F - 4 µg/ml; G – 40 µg/ml of rhGas6) shows that the extent of remyelination is greater in rhGas6-treated mice, ×400.</p

EM shows more myelinated axons in rhGas6-treated mice.

<p>Relative to PBS (A), the number of myelinated axons was significantly increased in mice treated with 400 ng/ml (B) and 4 µg/ml (C) rhGas6. Black arrows denote myelinated axons; white arrows show demyelinated, naked axons, ×5000.</p

Treatment with rhGas6 improves axonal integrity after cuprizone challenge.

<p>SMI32 and APP immunostaining of corpus callosum sections from mice treated for 14 days with PBS (A and E), 400 ng/ml (B and F), 4 µg/ml (C and G), 40 µg/ml of rhGas6 (D and H). Magnification ×50 (A–D), and ×400 (E–K). Increased number of SMI32- positive axonal swellings is observed in the PBS-treated mice (A–E) versus three doses of rhGas6-treated mice (B–H). Arrows show several axonal swellings indicating breakdown in axonal structure. I–K demonstrate APP immunostaining in mice treated with PBS (I), 400 ng/ml (J) and 4 µg/ml of rhGas6 (K). Arrows show APP positive deposits in the corpus callosum, ×400.</p

Similar numbers of Iba1-positive microglia are present in PBS- and rhGas6-treated mice.

<p>A. Following cuprizone ingestion for 4 weeks an extensive Iba1-positive microglial activation within the corpus callosum is observed. There was significant reduction of Iba1-postive microglia in both PBS- (B), and rhGas6-treated mice (C and D) at 14 days post treatment relative to 4 week cuprizone treated mice (p<0.02). Arrows show Iba1-positive microglia.</p

Treatment with rhGas6 reduces Oil-Red-O positive deposits after cuprizone intoxication.

<p>A–F, corpus callosum of mouse brain after PBS treatment. Extensive deposition of Oil-Red- O positive droplets can be seen (arrows). Brain sections from mice treated with 400 ng/ml (G and J), 4 µg/ml (H and K), and 40 µg/ml (I and L) of rhGas6 demonstrate a beneficial effect for all tested rhGas6 doses. Arrows indicate the clearance of debris from the corpus callosum. Low and high magnification, ×50 and ×400.</p

Increased number of myelinated axons is observed in mice treated with rhGas6.

a<p>- data was collected from four photomicrographs, ×5000; 3 mice/group.</p>b<p>- 100 randomly selected axons were measured per mouse.</p><p>*- g-ratio of cuprizone untreated mice was 0.802±0.01 (mean ±s SD).</p><p>**- P<0.05.</p

Number of Olig1-positive mature oligodendrocytes is increased in rhGas6-treated mice.

<p>Olig1-positive nuclear and cytoplasmic localization in cells (A–C). Increased numbers of cells with Olig1-positive cytoplasmic localization (arrows) in mice treated with 400 ng/ml (B), and 4 µg/ml rhGas6 (C) versus PBS-treated mice (A), are shown, ×400.</p

Conditional <i>Klf6</i> inactivation in vivo causes selective loss of differentiating oligodendrocytes.

<p><b>(A–F)</b> Confocal and morphometric analysis of oligodendrocyte numbers in developing <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and control <i>Klf6</i>^<i>fl/fl</i> spinal cords. Panel <b>(A)</b> shows samples from E12.5, immunostained for Olig2 and either Mnr (upper panels) or NeuN (lower panels). At E12.5, Olig2⁺ numbers in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and control samples are identical, and no differences are seen in neuronal markers. In controls, Olig2⁺ cell numbers then increase from E16.5 through P14, but no increase is seen in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> samples <b>(B–D)</b>. Areas outlined in panel <b>(B)</b> are shown at higher magnification, inset. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s006" target="_blank">S4B Fig</a>. At P1, myelin proteins are expressed in controls, but not in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> spinal cords <b>(C)</b>. <b>(E,F)</b> Analysis of stage-specific markers. Early events in differentiation, such as Nkx2.2 co-expression at E14.5, occur normally in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mice, but differentiating cells are selectively lost at subsequent timepoints <b>(E)</b>. In contrast, numbers of Olig2⁺Sox9⁺ OLP remain identical to controls <b>(F)</b>. There are no changes in Mnr⁺ motor neurons, which share the same origin as ventral OLP in the pMN domain (<b>A</b> upper panels, <b>B, G</b>). <b>(H,I)</b> Selective loss of differentiating cells is associated with increased apoptosis <b>(H)</b>, but OLP proliferation is unaffected <b>(I)</b>. <b>(J–L)</b> Postnatal stage-specific analysis confirms absence of differentiating (Apc⁺) oligodendrocytes from <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mice <b>(J,L)</b>, whereas OLP (Olig2⁺Ng2⁺) numbers are comparable to controls <b>(K,L)</b>. Representative cells are arrowed. <b>(M)</b> Analysis of <i>NG2creER</i>^<i>–</i>:<i>Klf6</i>^<i>fl/fl</i> mice, in which <i>Klf6</i> inactivation is inducibly targeted to OLP. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s006" target="_blank">S4C and S4D Fig</a>. Similar to <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> embryos, these mice also display selective loss of differentiating oligodendrocytes. Data are mean ± SEM. Statistics, <b>(D–I,L)</b> ANOVA plus Bonferroni post test, <b>(M)</b> Student’s <i>t</i> test, <i>***p <</i> 0.001. Scale: <b>(A,B)</b> 100 μm, inset 20 μm, <b>(C)</b> 250 μm, <b>(J,K)</b> 10 μm. Data shown are from lumbar sections of two to six mice per genotype per timepoint. Thoracic sections showed compatible findings. Individual values are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s002" target="_blank">S1 Data</a>.</p

Klf6 is induced by gp130-Stat3 signals and is required for CNS myelination.

<p><b>(A)</b> Stage-specific markers in differentiation. Following specification to the Olig2⁺ oligodendrocyte lineage, oligodendrocyte progenitors (OLP) express Ng2, Sox9, and Pdgfrα. Pro-myelinating signals induce differentiation to immature oligodendrocytes (iOL), marked by co-expression of Nkx2.2 and acquisition of Apc and O4. These undergo terminal differentiation to mature OL, which express myelin proteins. <b>(B)</b> Klf/Sp family responses to the pro-myelinating factors Cntf (100ng/ml) and T3 (40ng/ml), as determined by quantitative PCR (qPCR). <b>(C)</b> Immunoblotting for Klf6 in Oli-neu cultures pretreated with 0–1,000 nM Jak Inhibitor I 2 h, then exposed to 100 ng/ml Cntf 1 h. <b>(D)</b> Confocal imaging for Klf6 and the gp130 effector Stat3 in mouse OLP exposed to 100 ng/ml Cntf or vehicle 1 h. Cntf upregulation of Klf6 is associated with translocation to the nucleus, where it colocalizes with Stat3. <b>(E,F)</b> Klf6 expression visualized via confocal imaging in vivo. <b>(E)</b> In the postnatal CNS (spinal cord shown), immunoreactivity is heterogeneous in Olig2⁺ cells and more homogeneous in astrocytes (Gfap⁺) and neurons (NeuN⁺). Boxes and arrows highlight representative cells. <b>(F)</b> Klf6 is highly expressed (arrowheads) in OLP (left panel). In contrast, expression is lower (arrowhead) or undetectable (asterisks) in more mature Apc⁺ iOL (center panel), and Klf6 is not seen in mature Mag⁺ cells (right panel). <b>(G)</b> Still from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s015" target="_blank">S1 Video</a> comparing a P11 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mutant (cko, right) with sex-matched <i>Klf6</i>^<i>fl/fl</i> littermate (ctrl, left). The mutant is ataxic. <b>(H)</b> CNS white matter tracts in P14 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mice display hypomyelination (arrowed), whereas <i>mGfapCre</i>:<i>Klf6</i>^<i>fl/fl</i> mice and <i>Klf6</i>^<i>fl/fl</i> littermate controls show no gross abnormalities. Brains are shown at the same magnification (scale bar, upper right). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s003" target="_blank">S1 Fig</a>. <b>(I,J)</b> Confocal analysis of lumbar spinal cords of P14 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and <i>mGfapCre</i>:<i>Klf6</i>^<i>fl/fl</i> mice and <i>Klf6</i>^<i>fl/fl</i> controls. Myelin proteins are almost absent from <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> spinal cord, whereas the peripheral nervous system (PNS) appears normal (arrowed). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s004" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s005" target="_blank">S3 Figs</a>. <b>(K,L)</b> Electron micrographs of P14 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and <i>Klf6</i>^<i>fl/fl</i> spinal cords and optic nerves. Almost no myelin sheaths are present in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> CNS samples. <b>(M)</b> Gene ontology of BeadArray profiling of P1 CNS from <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> pups and sex-matched <i>Klf6</i>^<i>fl/fl</i> littermates. The five most significant results are shown for physiologic and disease relevance. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s006" target="_blank">S4 Fig</a>. Data are mean ± SEM. Statistics: <b>(B,J)</b> ANOVA plus Bonferroni test, *<i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001. Data are representative of two to four mice per genotype (for confocal imaging data) or three mice per genotype (for electron microscopy data). Scale, <b>(A)</b> 5 μm, <b>(E)</b> 20 μm, <b>(F)</b> 10 μm, <b>(H)</b> 3 mm <b>(I)</b> 150 μm, inset 15 μm. Magnifications, <b>(K)</b> x5,000, inset x20,000, <b>(L)</b> x3,000. Individual values are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s002" target="_blank">S1 Data</a>.</p