Abstract
In colon cancer, mutation of the Wnt repressor APC (encoding adenomatous polyposis coli) leads to a state of aberrant and unrestricted high-activity signaling. However, the relevance of high Wnt tone in non-genetic human disease is unknown. Here we demonstrate that distinct functional states of Wnt activity determine oligodendrocyte precursor cell (OPC) differentiation and myelination. Mouse OPCs with genetic Wnt dysregulation (high tone) express multiple genes in common with colon cancer, including Lef1, Sp5, Ets2, Rnf43 and Dusp4. Surprisingly, we found that OPCs in lesions of hypoxic human neonatal white matter injury upregulated markers of high Wnt activity and lacked expression of APC. We also found that lack of Wnt repressor tone promoted permanent white matter injury after mild hypoxic insult. These findings suggest a state of pathological high-activity Wnt signaling in human disease tissues that lack predisposing genetic mutation.
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Change history
30 July 2014
In the version of this article initially published, the wrong image was included as Figure 5g, top left panel. The image that appeared was the bottom left image from Supplementary Figure 9a; the correct image is the top right one from Supplementary Figure 9a. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank R. Beddington (National Institute for Medical Research Mill Hill; deceased) and T. Yamaguchi (US National Institutes of Health) for SP5-null mice. We thank R. Fodde (Leiden University) for Apc-floxed mice. We thank C. Stiles (Harvard University) for antibody to Olig2. Procurements of human brain tissues have been supported by the University of California Multicampus Research Programs and Initiatives grant #142657. This work was supported by grants from the National Multiple Sclerosis Society (to D.H.R.), the US National Institutes of Health (to D.H.R. and E.J.H.) and the Medical Scientist Training Program at the University of California, San Francisco (to E.P.H.). S.E.B. is a Harry Weaver Neuroscience Scholar of the National Multiple Sclerosis Society. D.H.R. is a Howard Hughes Medical Institute Investigator.
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S.P.J.F. conceived of and performed all experiments and analyses with the exception of the following: E.P.H. performed and analyzed all experiments related to in vitro OPC cultures. J.C.S., T.J.Y. and L.R.S. helped analyze Wnt pathway activation in mouse hypoxic injury. S.E.B. performed bioinformatics. E.J.H. procured human brain developmental tissue. S.L. provided advice on ChIP protocol and analysis. D.H.R. conceived the experiments and oversaw all aspects of the analysis. The paper was written by S.P.J.F. and D.H.R.
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Integrated supplementary information
Supplementary Figure 1 APC expression in oligodendrocyte lineage.
In the adult mammalian CNS, Adenomatous polyposis coli (APC) expression is specific to mature oligodendrocytes, and APC protein co-localizes with mature oligodendrocyte markers Nogo-A and MBP. During murine developmental myelination, APC is one of the earliest markers that an OPC is beginning to differentiate, and can be seen to co-localize on occasion with PDGFRα in OPCs.
Supplementary Figure 2 Loss of APC causes significant deficits in developmental myelination.
(a) Generation of Olig2cre: APCfl/fl mice resulted in ~85% efficiency of APC protein loss in OPCs and ~15% “escapees” that failed to delete APC in P15 developing spinal cord (SC) which lead to a severe hypomyelination as seen on dark field (DF) compared to control Olig2cre: APCfl/+ littermates. (b) While numbers of OPCs expressing PDGFRα were normal in spinal cord (SC) of Olig2cre: APCfl/fl at P15, APC-deficient OPCs showed differentiation arrest and failed to express mature oligodendrocyte marker PLP in SC or corpus callosum (CC) at P15.
Supplementary Figure 3 Loss of APC causes OPC differentiation failure.
APC-deficient OPCs showed maturation arrest and failed to express the mature oligodendrocyte genes, myelin regulatory factor (MRF), or the mature oligodendrocyte markers Cnp, Mag, Mal and Fa2h at P15 in spinal cord of Olig2cre: APCfl/fl mice (b). (a) Only “escapees” of Olig2-cre activity that expressed APC were capable of differentiation to NOGO-A-positive mature oligodendrocytes.
Supplementary Figure 4 APC-deficient OPCs expressed greatly elevated levels of the Wnt transcriptional target Axin2.
This can be seen at P15 in spinal cord (SC) and corpus callosum (CC) in Olig2cre: APCfl/fl mice compared to Olig2cre: APCfl/+ littermates.
Supplementary Figure 5 Lef1 expression is activated in OPCs in a state of high-activity Wnt signaling.
(a) High-threshold Wnt signaling in colon cancer involves a switch from a TCF4-b-catenin to a LEF1-b-catenin complex. Although Lef1 is not expressed during normal oligodendrocyte development, we observed robust Lef1 expression in the spinal cord of Olig2-cre, APCfl/fl animals at P9, where it co-localizes with the highly upregulated Axin2 mRNA. (b) Such cells expressed high levels of Axin2 and remained persistently undifferentiated (failing to express mature oligodendrocyte marker PLP) at postnatal day 30 (P30), P120, and P650.
Supplementary Figure 6 High-activity Wnt signaling in OPCs does not lead to hyperproliferation.
Unlike Wnt-driven cancers of gut and hematopoetic systems, which respond to mitogenic Wnt signaling, we do not observe hyperproliferation of OPCs in Olig2cre:APC fl/fl mice. There is no observable difference in (a) the number of Nkx2.2+ OPCs, (b) the number of Ki67+ dividing cells, or (c) the number of PDGFRα+ OPCs in spinal cords of wildtype or Olig2cre:APC fl/fl mice at postnatal day 15.
Supplementary Figure 7 Predicted Sp5 binding sites in multiple mature oligodendrocyte gene promoters.
There are predicted binding sites for the transcription factor SP5 in the promoter regions of multiple mature oligodendrocyte/ myelin genes, including myelin basic protein (MBP), myelin associated glycoprotein (MAG), fatty acid-2-hydroxylase (FA2H), 2'3'cyclic-nucleotide 3'-phosphodiesterase (CNP), myelin and lymphocyte protein (MAL) and myelin regulatory factor (MRF).
Supplementary Figure 8 Oligodendrocyte development proceeds normally in SP5-null animals.
(a) Oligodendrocyte development proceeds normally in SP5-/- animals with normal white matter thickness on dark field (DF) in spinal cord (SC) at P9 and normal onset of expression of mature oligodendrocyte gene MAG at P1, and normal numbers of mature oligodendrocytes expressing mature marker PLP at P9 (b,c) and P15 (c) in SC.
Supplementary Figure 9 Precocious OPC differentiation in SP5-null remyelination.
(a, b) SP5-/- animals showed acceleration of OPC differentiation in the mouse model of remyelination using focal injection of lysolecithin into adult spinal cord white matter, with significant increases in number of cells expressing PLP mRNA at 7 days post lesioning (7dpl) and 14dpl (* p< 0.01, ** p<0.05).
Supplementary Figure 10 Loss of APC in OPCs leads to significant remyelination deficits.
(a) Loss of APC in OPCs during remyelination (lysolecithin spinal cord focal injection) leads to a failure of differentiation and onset of mature oligodendrocyte marker PLP in lesions at 14dpl. (b) Loss of APC in OPCs during remyelination results in a massive upregulation of the Wnt pathway target Axin2 mRNA, signaling a loss of Wnt repressor tone. (c) A small percentage of OPCs escape the Olig2-cre floxing activity and express APC in remyelinating lesions (a), and these are the only OPCs which do not express the Wnt target Axin2 and are capable of differentiating to NOGO-A mature remyelinating oligodendrocytes.
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Supplementary Table 1
Comparison of transcripts upregulated in Wnt-activated OPCs and colon cancer (XLSX 16 kb)
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Fancy, S., Harrington, E., Baranzini, S. et al. Parallel states of pathological Wnt signaling in neonatal brain injury and colon cancer. Nat Neurosci 17, 506–512 (2014). https://doi.org/10.1038/nn.3676
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DOI: https://doi.org/10.1038/nn.3676
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