Developmental Cell, Volume 40

Supplemental Information

miR-219 Cooperates with miR-338 in Myelination

and Promotes Myelin Repair in the CNS

HaiboWang, Ana Lis Moyano, ZhangyanMa, Yaqi Deng, Yifeng Lin, Chuntao Zhao, LiguoZhang, Minqing Jiang, Xuelian He, Zhixing Ma, Fanghui Lu, Mei Xin, Wenhao Zhou, SungOk Yoon, Ernesto R. Bongarzone, and Q. Richard Lu

SUPPLEMENTARY FIGURES

Figure S1. Normal neurogenesis in the spinal cord of miR-219-1/2-/- embryos. Related to

Figure 1.

A) Immunostaining for Islet1 (red) and Olig2 (green) in the spinal cords from heterozygous control

(Ctrl) and miR-219-1/2-/- mice at E10.5. At least three pairs of littermate embryos were analyzed.

Scale bar, 50 µm.

B) Immunostaining for Tuj1 (red) and Olig2 (green) in the spinal cords from control and miR-219-

1/2-/- mice at E10.5. Scale bar, 50 µm.

C) Immunostaining for Islet1 (red) and Olig2 (green) in the spinal cords from control and miR-

219-1/2-/- mice at E12.5. Scale bar, 50 µm.

D) Immunostaining for Pax6 (green) in the spinal cords from control and miR-219-1/2-/- mice at

E14.5. Scale bar, 50 µm.

Figure S2. Myelination deficits in miR-219 mutant mice. Related to Figure 2.

A) qRT-PCR analysis of the level of miR-219 relative to the Plp1 in the cortex (Ctx), corpus

callosum (CC), spinal cord (SC) and optic nerve (ON) of P12 wild type mice. Data are means ±

SEM, n = 5 mice per regions (one-way ANOVA with Tukey's multiple-comparison test).

B) Immunostaining for astrocyte marker GFAP (green) in coronal sections of P14 Ctrl and miR-

219-dCKO forebrains. Scale bar, 100 µm.

C) Immunostaining for neuron marker NeuN (red) in coronal sections of P4 Ctrl and miR-219-

dCKO mouse forebrains. Scale bar, 100 µm.

D) Quantification of NeuN+ neurons at cortices of Ctrl and miR-219-dCKO mice at P4. Data are

means ± SEM, n = 3 mice per genotype (Student’s t test).

E) Immunostaining for MBP (red), CC1 (green), and Olig2 (blue) in the cortices of Ctrl, miR-219-

1 cKO, miR-219-2 cKO, and miR-219-dCKO at P30. Scale bar, 250 µm.

F) RNA in situ hybridization for Mbp and Plp1 in the cortices of representative control and miR-

219-dCKO mice at P215. Scale bar, 100 µm.

Figure S3. miR-338 expression during remyelination and effects of vehicle treatment on

myelination in the spinal cord of adult miR-219-iKO. Related to Figure 4.

A) qRT-PCR analysis of miR-338 levels in spinal cord lesion tissues over vehicle injected

control spinal cords at dpl 0, 8, and 15. Data are means ± SEM, n = 4 animals/stage (*p < 0.05,

one-way ANOVA with Tukey's multiple-comparison test).

B) EM images of the ventral spinal white matter from vehicle-treated 8-week-old Ctrl (Plp-

CreERt;miR-219-1/2 fl/+) and miR-219-iKO (Plp-CreERt;miR219-1/2 fl/fl) mice at 21 days after

tamoxifen injection. Scale bar, 2 µm.

C) The percentage of myelinated axons in the ventral spinal white matter of adult Ctrl and iKO

mice at 21 days after tamoxifen injection. Data are means ± SEM, n = 4 animals/genotype

(Student’s t test).

Figure S4. Oligodendrocytes and OPCs in the vehicle-injected white matter and transgene

expression in OPCs in the LPC lesion in miR-219-Tg spinal cord. Related to Figure 5.

A,B) Quantification of CC1+ OL (A) and PDGFRα+ OPC (B) cell numbers in the vehicle-

injected spinal white matter of WT and miR-219-Tg mice at P49. Data are means ± SEM, n = 3

animals/genotype (Student’s t test).

C) Immunostaining for PDGFRα (red) and the transgene reporter GFP (green) in the LPC lesion

of miR-219-Tg spinal cord at dpl 14. Scale bar, 50 µm.

Figure S5. miR-219 target differentiation inhibitors for OL differentiation. Related to Figure

6.

A) Schematic diagram depicting the miR-219 binding sites (green) in Nfia, Nfib, and Etv5 mRNAs.

Coding regions (CDS) are in yellow, 3’ UTRs are in white box. Regions of transcripts used for

reporter assays are indicated as blue lines.

B) Sequences of the putative miR-219 binding sites in Nfia, Nfib, Etv5, and Lingo1 mRNAs. CDS

regions are indicated in yellow, 3’ UTRs are indicated as red straight lines, and miR-219 binding

sites are indicated as red arches.

C) Relative luciferase activity of reporter constructs carrying the Nfia, Nfib, and Etv5 segments

containing the miR-219 binding sites or the mutated binding sites. Reporter genes were transfected

into 293T cells that expressed control vector (pCIG) or pCIG-miR-219. Data are means ± SEM, n

= 3 transfections (**p < 0.01, ***p < 0.001, Student’s t test).

D) Immunostaining for MBP (red) in rat OPCs transfected with pCIG or pCIG-Etv5 vectors after

culture under differentiation conditions for 3 days, GFP (green) indicates transfected cells. Scale

bar, 50 µm.

E) The percentage of MBP+ OLs in an OPC culture transfected with pCIG or pCIG-Etv5 vectors.

Data are means ± SEM, n = 3 experiments (***p < 0.001; Student’s t test).

F) qRT-PCR analyses of indicated OL differentiation associated genes in OPCs transfected with

pCIG or pCIG-Etv5 vectors. Data are means ± SEM, n = 3 experiments (**p < 0.01, ***p < 0.001,

Student’s t test).

G) Immunostaining for MBP (red) and Olig2 (green) in rat OPCs transfected with scrambled

control siRNA (siCtrl) or siEtv5 after culture under differentiation conditions for 3 days. Scale bar,

50 µm.

H) The percentage of MBP+ OLs among Olig2+ cells in an OPC culture transfected with scrambled

siCtrl or siEtv5. Data are means ± SEM, n = 3 experiments (*p < 0.05; Student’s t test).

I) Schematic diagram for miRNA transfection in OPCs and biotin-tagged affinity capture of miR-

219-mRNA complex.

Figure S6. miR-219 enhances remyelination in the LPC-lesioned CNS Related to Figure 7.

A-C) The quantification of CC1+ (A), PDGFRα+ (B) and cleaved Caspase3+ (C) cells in the spinal

cords of vehicle or miR-219 mimic-treated wildtype adult mice at 21 days post injection. Data are

means ± SEM, n = 3 animals per group (Student’s t test).

D, E) Orthogonal reconstruction of confocal images in the corpus callosum (CC) of the mice

infused intranasally with (D) Cy3-labeled control miRNA (Cy3-miR) or (E) control vehicle (i-

Fect). Scale bar, 10 µm. Nuclei were counterstained with DAPI.

F) Immunostaining for MBP in the LPC lesions at dpl 8 in the CC of mice treated with vehicle (i-

Fect), control miRNA (Ctrl mimic), miR-219 mimic, and unlesioned CC of mice treated with Ctrl

mimic. Scale bar, 50 µm.

Figure S7. Histology and immune responses after miR-219 mimic treatment in EAE mice.

Related to Figure 8.

A) Immunostaining for MBP (green) at the stereological serial sections in the DIP29 lumbar spinal

cords from EAE mice treated with control or miR-219 mimics. Scale bar, 500 µm.

B-D) Quantification of MHCII+ (B), CD19+ (C) and Ly6G+ cell density in the lesion areas at PID

29 in the lumbar spinal cord of the mice treated with Ctrl or miR-219 mimic. Data are means ±

SEM, n = 5 mice treated with Ctrl mimic and 6 mice treated with miR-219 mimic (Student’s t

test).

E) qRT-PCR analysis of expression of the genes iNOS, TNF-α, Ifng, IL-12, IL-23, IL-17 and IL-5

in the lumbar spinal cord of the mice treated with Ctrl or miR-219 mimic at PID29. Data are means

± SEM, n = 5 mice per group (Student’s t test).

Figure S8. Normal myelination in oligodendrocyte lineage-specific Par3-deficient brains.

Related to Figure 6.

RNA in situ hybridization for Mbp, Plp, and PDGFRα in coronal brain sections of controls Par3fl/+

or Par3fl/+:Olig1-Cre+/- mice, and Par3 mutant (Par3fl/fl :Olig1-Cre+/-) mice at P14. Scale bar, 100

µm.

Supplemental Table S1. Expression of the genes implicated in oligodendrocyte development and myelination altered in miR-219 dCKO optic nerves.

Related to Figure 6

Gene ID Fold change (Ctrl/dCKO) p valueMal 4.02 0.0039Omg 2.27 0.0030Olig1 2.20 0.0002Fa2h 2.20 0.0020Plp1 2.16 0.0006Gsn 2.11 0.0035Ugt8a 2.11 0.0021Tspan2 2.03 0.0023Gpr17 2.01 0.0057Nkx2-2os 1.98 0.0039Nkx2-2 1.94 0.0031Nkx6-2 1.92 0.0062Gal3st1 1.89 0.0004Mag 1.81 0.0010Mobp 1.79 0.0005Enpp2 1.77 0.0030Sox8 1.77 0.0036Cnp 1.75 0.0003Trf 1.68 0.0020Myrf 1.68 0.0022Aspa 1.66 0.0064Pou3f1 1.66 0.0157Mbp 1.65 0.0004Nfasc 1.64 0.0010Sox10 1.62 0.0024Scn8a 1.59 0.0000Pllp 1.57 0.0009Gjc3 1.54 0.0230Jam3 1.54 0.0018Fyn 1.54 0.0009Zeb2 1.52 0.0113Myoc 1.52 0.0002Sirt2 1.50 0.0027Slc8a3 1.49 0.0163Bace1 1.44 0.0053Atrn 1.43 0.0017Serinc5 1.42 0.0006Zfp488 1.41 0.0093Arhgef10 1.38 0.0030Qk 1.35 0.0207Hdac11 1.31 0.0225Mpp5 1.30 0.0188Rtn4 1.29 0.0046

Supplemental Table S1. Expression of the genes implicated in oligodendrocyte development and myelination altered in miR-219 dCKO optic nerves （continued） Related to Figure 6

Gene ID Fold change (Ctrl/dCKO) p valuePtprj 1.27 0.0238Dicer1 1.26 0.0037Bmp4 1.24 0.0032Sox6 1.24 0.0115Dusp10 1.24 0.0319Olig2 1.22 0.0278Scd2 1.19 0.0168Gnpat 1.18 0.0121Cd9 1.18 0.0399Tsc1 1.18 0.0295Tnfrsf21 1.16 0.0078Sod1 0.86 0.0521Ski 0.85 0.0049Rheb 0.84 0.0027Apoe 0.84 0.0156Acsbg1 0.84 0.0563Trpc4 0.83 0.0561Dag1 0.83 0.0037Clu 0.83 0.0349Megf10 0.82 0.0056Zbtb18/ZFP238 0.81 0.0114Pou3f2 0.81 0.0382Cntnap1 0.80 0.0291Ank2 0.79 0.0010Vtn 0.78 0.0178Erbb2 0.77 0.0035Nfia 0.76 0.0044Ntrk2 0.75 0.0072Fgfr3 0.75 0.0019Marveld1 0.75 0.0062Pax6 0.74 0.0246Aldh1a2 0.74 0.0299Gpr126 0.72 0.0075Mal2 0.72 0.0724Id4 0.69 0.0226Sox9 0.68 0.0097Gli3 0.66 0.0177Lingo1 0.65 0.0307Nkx6-1 0.63 0.0046Elovl7 0.62 0.0061

Supplemental Table 2. Top overlapping genes between the genes upregulated in miR-219 dCKO optic nerves at P12 and those down-regulated in OPCs transfected

with miR-219 mimic. Related to Figure 6.

Optic nerve OPC with miRNA mimic Gene_ID dCKO/Ctrl p value Ctrl/miR-219 p value

1 Cxadr 1.336 0.014 3.469 0.025 2 Apc 1.302 0.030 2.229 0.011 3 Ntrk3 1.303 0.009 2.148 0.015 4 Kif5a 1.428 0.018 2.078 0.006 5 Tmod2 1.395 0.032 2.058 0.018 6 Padi2 1.340 0.010 1.815 0.051 7 Slc6a6 1.459 0.016 1.790 0.007 8 Nfib 1.255 0.004 1.755 0.054 9 Ncoa1 1.213 0.010 1.717 0.036

10 Alcam 1.370 0.037 1.587 0.054 11 Nfia 1.313 0.004 1.584 0.017 12 Slc38a1 1.453 0.005 1.559 0.039 13 Ptk7 1.270 0.001 1.550 0.013 14 Rapgef3 1.246 0.043 1.476 0.040 15 Farp1 1.247 0.003 1.462 0.010 16 Slc23a2 1.319 0.033 1.460 0.008 17 Nfic 1.213 0.014 1.447 0.051 18 Unc119 1.214 0.001 1.402 0.005 19 Twsg1 1.263 0.017 1.624 0.015 20 Sirpa 1.243 0.034 1.532 0.012 21 Fbxo44 1.330 0.028 1.480 0.034 22 Lingo1 1.548 0.031 1.469 0.004 23 Ntrk2 1.335 0.007 1.451 0.044 24 Tmem255a 1.303 0.013 1.433 0.024

Supplemental Table 3. Top overlapping genes between the genes upregulated in miR-219 dCKO optic nerves at P12 and those down-regulated in iOL transfected

with miR-219 mimic. Related to Figure 6.

Optic Nerve iOL with miRNA mimic Gene_ID dCKO/Ctrl p value Ctrl/miR-219 p value

1 Sh3pxd2a 1.350 0.004 6.617 0.001 2 Myo10 1.278 0.005 4.703 0.001 3 F3 1.688 0.031 4.615 0.020 4 Tubb6 1.362 0.041 4.084 0.013 5 Adora1 1.271 0.034 3.698 0.027 6 Smoc1 1.352 0.018 3.408 0.039 7 Gatm 1.330 0.007 2.948 0.053 8 Etv5 1.220 0.009 2.911 0.007 9 Ednrb 1.566 0.015 2.779 0.000

10 Zfp36l1 1.291 0.032 2.645 0.023 11 Cpe 1.259 0.015 2.471 0.008 12 Scg3 1.361 0.008 2.435 0.002 13 Cox6b2 1.416 0.026 2.432 0.032 14 Cdc42ep4 1.354 0.008 2.220 0.004 15 Rapgef3 1.246 0.043 2.175 0.008 16 Cpt1a 1.241 0.009 2.048 0.053 17 Slc6a1 1.279 0.022 1.982 0.008 18 Fgfr1 1.338 0.001 1.835 0.006 19 Sfxn5 1.493 0.003 1.833 0.018 20 Epdr1 1.347 0.024 1.805 0.005 21 Idh2 1.347 0.010 1.764 0.007 22 Sfxn1 1.321 0.023 1.732 0.038 23 Ldhb 1.247 0.007 1.722 0.010 24 Nfib 1.255 0.004 1.712 0.003 25 Slc38a1 1.453 0.005 1.703 0.017 26 Tob1 1.299 0.027 1.690 0.011 27 Tusc3 1.246 0.025 1.626 0.041 28 Ltbr 1.319 0.001 1.603 0.041 29 Aldh2 1.315 0.005 1.593 0.008 30 Oat 1.219 0.007 1.591 0.021 31 Uap1l1 1.216 0.042 1.582 0.028 32 Ppap2b 1.297 0.020 1.576 0.002 33 Ckb 1.310 0.002 1.571 0.047 34 Ptk7 1.270 0.001 1.557 0.012 35 Nfic 1.213 0.014 1.512 0.032 36 Tnfrsf1a 1.221 0.011 1.492 0.047 37 Atl2 1.278 0.044 1.448 0.026 38 Slc1a4 1.350 0.017 1.437 0.050 39 Racgap1 1.214 0.048 14.881 0.041 40 Cdkn1a 1.396 0.039 5.661 0.052 41 Cst3 1.242 0.004 5.015 0.018 42 Igfbp2 1.485 0.015 4.669 0.024 43 Lhfp 1.220 0.010 4.067 0.003 44 Cspg5 1.301 0.046 3.943 0.012 45 Gadd45g 1.507 0.029 3.046 0.011 46 Sord 1.282 0.001 2.584 0.006 47 B9d1 1.260 0.028 2.462 0.002 48 Jund 1.321 0.012 2.459 0.031 49 Anxa6 1.227 0.029 2.422 0.027 50 Nck2 1.362 0.008 2.402 0.020 51 Glrx 1.222 0.039 2.388 0.005 52 Jun 1.329 0.019 2.325 0.004 53 Tmem255a 1.303 0.013 2.323 0.011 54 Scpep1 1.224 0.002 2.117 0.017 55 Slc25a18 1.899 0.006 2.095 0.009 56 Ctnnd2 1.246 0.009 2.010 0.014 57 Pxmp2 1.337 0.004 2.009 0.020 58 Twsg1 1.263 0.017 1.960 0.033 59 Nnat 1.490 0.007 1.865 0.025 60 Herpud1 1.349 0.011 1.793 0.003 61 Mt3 1.401 0.003 1.765 0.038 62 Dnajc4 1.211 0.040 1.659 0.019 63 Nr2f1 1.429 0.001 1.567 0.011 64 Acadl 1.398 0.039 1.556 0.013 65 Pygb 1.316 0.027 1.554 0.008 66 Zfp385a 1.377 0.023 1.537 0.041 67 Pfkm 1.253 0.005 1.520 0.051