Fig 1.
Klf6 is induced by gp130-Stat3 signals and is required for CNS myelination.
(A) 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) Klf/Sp family responses to the pro-myelinating factors Cntf (100ng/ml) and T3 (40ng/ml), as determined by quantitative PCR (qPCR). (C) 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. (D) 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. (E,F) Klf6 expression visualized via confocal imaging in vivo. (E) 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. (F) 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). (G) Still from S1 Video comparing a P11 Olig1Cre:Klf6fl/fl mutant (cko, right) with sex-matched Klf6fl/fl littermate (ctrl, left). The mutant is ataxic. (H) CNS white matter tracts in P14 Olig1Cre:Klf6fl/fl mice display hypomyelination (arrowed), whereas mGfapCre:Klf6fl/fl mice and Klf6fl/fl littermate controls show no gross abnormalities. Brains are shown at the same magnification (scale bar, upper right). See S1 Fig. (I,J) Confocal analysis of lumbar spinal cords of P14 Olig1Cre:Klf6fl/fl and mGfapCre:Klf6fl/fl mice and Klf6fl/fl controls. Myelin proteins are almost absent from Olig1Cre:Klf6fl/fl spinal cord, whereas the peripheral nervous system (PNS) appears normal (arrowed). See S2 and S3 Figs. (K,L) Electron micrographs of P14 Olig1Cre:Klf6fl/fl and Klf6fl/fl spinal cords and optic nerves. Almost no myelin sheaths are present in Olig1Cre:Klf6fl/fl CNS samples. (M) Gene ontology of BeadArray profiling of P1 CNS from Olig1Cre:Klf6fl/fl pups and sex-matched Klf6fl/fl littermates. The five most significant results are shown for physiologic and disease relevance. See also S4 Fig. Data are mean ± SEM. Statistics: (B,J) ANOVA plus Bonferroni test, *p < 0.05, ** p < 0.01, *** p < 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, (A) 5 μm, (E) 20 μm, (F) 10 μm, (H) 3 mm (I) 150 μm, inset 15 μm. Magnifications, (K) x5,000, inset x20,000, (L) x3,000. Individual values are in S1 Data.
Fig 2.
Conditional Klf6 inactivation in vivo causes selective loss of differentiating oligodendrocytes.
(A–F) Confocal and morphometric analysis of oligodendrocyte numbers in developing Olig1Cre:Klf6fl/fl and control Klf6fl/fl spinal cords. Panel (A) shows samples from E12.5, immunostained for Olig2 and either Mnr (upper panels) or NeuN (lower panels). At E12.5, Olig2+ numbers in Olig1Cre:Klf6fl/fl 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 Olig1Cre:Klf6fl/fl samples (B–D). Areas outlined in panel (B) are shown at higher magnification, inset. See also S4B Fig. At P1, myelin proteins are expressed in controls, but not in Olig1Cre:Klf6fl/fl spinal cords (C). (E,F) Analysis of stage-specific markers. Early events in differentiation, such as Nkx2.2 co-expression at E14.5, occur normally in Olig1Cre:Klf6fl/fl mice, but differentiating cells are selectively lost at subsequent timepoints (E). In contrast, numbers of Olig2+Sox9+ OLP remain identical to controls (F). There are no changes in Mnr+ motor neurons, which share the same origin as ventral OLP in the pMN domain (A upper panels, B, G). (H,I) Selective loss of differentiating cells is associated with increased apoptosis (H), but OLP proliferation is unaffected (I). (J–L) Postnatal stage-specific analysis confirms absence of differentiating (Apc+) oligodendrocytes from Olig1Cre:Klf6fl/fl mice (J,L), whereas OLP (Olig2+Ng2+) numbers are comparable to controls (K,L). Representative cells are arrowed. (M) Analysis of NG2creER–:Klf6fl/fl mice, in which Klf6 inactivation is inducibly targeted to OLP. See also S4C and S4D Fig. Similar to Olig1Cre:Klf6fl/fl embryos, these mice also display selective loss of differentiating oligodendrocytes. Data are mean ± SEM. Statistics, (D–I,L) ANOVA plus Bonferroni post test, (M) Student’s t test, ***p < 0.001. Scale: (A,B) 100 μm, inset 20 μm, (C) 250 μm, (J,K) 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 S1 Data.
Fig 3.
Klf6 is required for differentiation regardless of the initiating stimulus.
(A–H) Results of confocal analysis of primary mouse OLP subjected to Klf6 silencing as described in Materials and Methods, and treatments indicated. Klf6 silencing has no effect on active proliferation, shown by BrdU immunoreactivity in proliferating cultures (A), nor on cell cycle exit index (Ki67-BrdU+/BrdU+) in differentiating OLP cultures (B). (C–F) Early events in differentiation, such as Nkx2.2 co-expression, occur normally in Klf6-silenced OLP differentiated with either 40 ng/ml T3 or 100 ng/ml Cntf (C). However, expression of markers of subsequent stages of differentiation, such as O4 and Apc, is delayed in silenced cultures compared with non-targeting (NT) controls, and these cultures fail to mature to Mbp+ mOL (D–F). See also S4G Fig. These changes are associated with increased apoptosis (G,H). (I–L) Confocal imaging analysis of primary mouse OLP nucleofected with GFP-labeled Klf6-overexpression construct or GFP vector control, and either left untreated or exposed to 40 ng/ml T3 for 72 h. Klf6 overexpression accelerates differentiation triggered by a pro-myelinating signal (T3), as measured by induction of O4 and Mbp (I–K). However, Klf6 overexpression alone does not initiate differentiation (L). (M-O) Confocal imaging analysis of Klf6-silenced and NT control mouse OLP pretreated with 2 μM caspase inhibitor Q-VD-OPh or vehicle for 2 h, then differentiated with T3 for 48 h. Apoptosis is almost absent from cultures exposed to the inhibitor, and Olig2+ cell numbers in Klf6-silenced and control conditions are almost identical (M,N). However, rescue of viability does not restore differentiation, as measured by the percentage of O4+ cells (M,O). Statistics, (A,E,G,J,K,L) Student’s t test, (B,C,D,H,N,O) ANOVA plus Bonferroni post-test, *p < 0.05, **p < 0.01, ***p < 0.001. Data are representative of at least three independent studies in separate cultures. Individual values are in S1 Data.
Fig 4.
RNA sequencing identifies Klf6-dependence of gp130-driven transcriptional patterns.
(A) Overview of approach used to define key Klf6 effectors. Initial RNA-seq analysis of primary mouse cultures identifies 212 unique Klf6-regulated transcripts in OLP and 91 in iOL, of which 40 are shared. See S1–S3 Tables and S5A and S5B Fig. (B,C) Results of functional inference (B) and GO analysis (C) of RNA-seq data, from primary OLP (red), iOL (blue), or both (purple). In (B), numbers of Klf6-regulated genes are indicated for each function. Implicated signaling pathways in OLP are presented as a smaller Venn diagram, inset. (D) Examples of qPCR validation of RNA-seq data for select OLP, iOL, and shared genes. A larger cohort of validation data is presented in S5C Fig. (E) Gp130 sensitivity of select validated differentially expressed transcripts. Results are shown from Oli-neu cells treated with Cntf (100 ng/ml) for up to 24 h. Colored areas indicate the time period before peak response of Klf6 to Cntf. See also S5D Fig. (F) Klf6-dependence of Cntf-induced responses. qPCR analysis of Klf6-silenced and control Oli-neu cells treated with Cntf for up to 24 h. Cntf sensitivity of differentially expressed targets is blunted in Klf6-silenced samples. Note also that some genes are Cntf-independent but Klf6-dependent during differentiation. Data are mean ± SEM. Statistics, (D) Student’s t test, (F) Two-way ANOVA plus Bonferroni post-test, *p < 0.05, **p < 0.01, ***p < 0.001. Data are representative of two to three independent studies. RNA-seq data are presented in full in S1–S3 Tables and are available on the GEO website (http://www.ncbi.nlm.nih.gov/geo/) (Accession number GSE79245). Individual values for all other quantifications are in S1 Data.
Fig 5.
Stat3 inactivation in vivo produces failure of myelination, similar to Klf6 inactivation.
(A) Graphical overview of genome-scale chromatin occupancy (ChIP-seq) data for Klf6 in primary mouse OLP and iOL. ChIP-seq analysis identifies 577 peaks of Klf6 chromatin binding within 20 kb of transcription start sites in OLP, 416 in iOL, and 337 that are shared. See also S4 Table. (B) Cross-referencing this ChIP-seq dataset to RNA-seq data in S1 and S2 Tables identifies 20 genes as prospective directly-regulated Klf6 effectors. This cohort is presented in S5 Table. (C,D) ChIP-seq identifies Klf6 binding to the promoter of the gp130 gene (C), which is also amongst multiple gp130-Stat3 signaling pathway components identified by RNA-seq as Klf6-regulated (D, and see S1–S3 Tables). These findings suggest that Klf6 exerts feedback upon the gp130-Stat3 pathway that drives its expression (E). (F) Confocal images of spinal cords of E16.5 Olig1Cre:Klf6fl/fl and Klf6fl/fl control embryos labeled for Stat3 and Olig2. Areas illustrated are from grey matter (GM). Levels of Stat3 are reduced in the oligodendrocyte lineage in Olig1Cre:Klf6fl/fl samples. Arrowheads mark representative cells, which are shown at higher magnification below. See also S6A Fig. (G–M) Confocal analysis of spinal cords from mice with Stat3 inactivation targeted to OLP (Olig1Cre:Stat3fl/fl and PdgfracreERTM:Stat3fl/fl). The oligodendrocyte phenotype of these mice resembles that produced by conditional Klf6 inactivation (see Fig 2). At E12.5–14.5, OLP numbers in conditional Stat3 mutants are comparable to Stat3fl/f controls, and early events in differentiation occur normally (G,H). However, selective loss of differentiating cells is seen from E16.5 onward (H,I), and myelination is profoundly disrupted (J,K, see also S6B and S6C Fig). Stat3 inactivation produced by Olig1Cre also results in loss of motor neurons, which share the same origin in the pMN domain (K, and see S6D Fig). (L) Spinal cords of E16.5 Olig1Cre:Stat3fl/fl and control Stat3fl/fl embryos, labeled for Klf6 and Olig2. The border between grey (GM) and white matter (WM) is marked, and representative cells are arrowed. Conditional Stat3 inactivation produces defective Klf6 expression. (M) Conditional Stat3 inactivation in OLP using PdgfracreERTM results in selective failure of OLP differentiation and myelination. Motor neurons are unaffected. Data are mean ± SEM. Statistics, (G–J) ANOVA plus Bonferroni test, (M) Student’s t test, **p < 0.01, ***p < 0.001. Scalebars, (F,L) 5 μm, (K) 250 μm. Data are representative of two to six mice per genotype per time point. Results in (F–M) are from lumbar sections. ChIP-seq data are presented in full in S4 Table, RNA-seq and ChIP-seq intersect data are presented in full in S5 Table, and both datasets are available on the GEO website (http://www.ncbi.nlm.nih.gov/geo/) (Accession number GSE79245). Individual values for all other quantifications are in S1 Data.
Fig 6.
Klf6 promotes differentiation via transactivation of importin-α5.
(A–C) ChIP-seq (A), qPCR (B), and confocal imaging data (C) identifying Klf6 regulation of the downstream importin effector Impα5. Data are also compatible with RNA-seq results in S1–S3 Tables. (A) ChIP-seq in primary mouse OLP and iOL demonstrates direct Klf6 binding to the promoter region of Impα5, which RNA-seq also identifies as Klf6-regulated (see S3 Table). (B) QPCR for importin family members in Klf6-silenced versus NT control primary mouse OLP, and O4+Apc+Mbp- iOL differentiated with T3 for 18 h. Note that Klf6 control of Impα5 expression is selective. (C) Confocal imaging of lumbar spinal cords of E16.5 Olig1Cre:Klf6fl/fl and Klf6fl/fl control embryos (upper panels), and Olig1Cre:Stat3fl/fl and Stat3fl/fl control embryos (lower panels), showing reduced Impα5 expression in oligodendrocyte lineage cells in mice with conditional Klf6 or Stat3 inactivation. Arrowheads in white matter areas (outlined) mark representative cells. Collectively, findings in (A–C) are compatible with the hypothesis that gp130-driven Klf6 uses selective control of Impα5 to regulate oligodendrocyte development (D). (E–G) qPCR, confocal imaging, and immunoblotting data from primary mouse OLP silenced for Impα5, then differentiated with T3 for 24–72 h. In (E), data for the myelin marker Cnp are from 72 h; other data are from 24 h. Note that differentiation markers are strongly reduced in Impα5-silenced cultures at both the RNA (E) and protein levels (F,G). See also S6F Fig. (H) OLP cultures from Klf6fl/flRosa26fl/fl mice were exposed to Ad5CMVCre or Ad5CMV control, then nucleofected with either an Impα5 expression construct or empty vector. Cultures were treated with 40 ng/ml T3 and harvested at 72 h. Notably, recombinant Impα5 significantly increased the proportion of cells expressing the differentiation marker O4. Recombinant Impα5 also partially rescued differentiation in Klf6-deficient cultures. Numbers in panels (G,H) refer to proportion of cells positive for the maturation marker per field at 20x magnification, for at least four fields per condition. Data are mean ± SEM. Statistics, (B,E,G) Student’s t test, (H) ANOVA plus Bonferroni post-test, *p < 0.05, ** p <0.01, *** p <0.001. Scalebars, (C,G,H) 20 μm. Data are representative of at least three independent studies. ChIP-seq data are available on the GEO website (http://www.ncbi.nlm.nih.gov/geo/) (Accession number GSE79245) and in S4 Table. Individual values for all other quantifications are in S1 Data.