Transgenic Mice and procedures
Animal husbandry and procedures were performed according to UCSF guidelines under IACUC approved protocols.
The Axin2-LacZ mouse has been described previously (Lustig, B, et al. Mol Cell Biol 22:1184–1193 (2002)). Insertion of β-galactosidase gene into Axin2 locus (Axin2-LacZ) provides a useful tool for visualizing cells that are actively responding to Wnt in vivo. LacZ insert mimics the expression pattern of Axin2 but does not lead to detectable phenotype in heterozygous state. In homozygous state, this effectively acts as an Axin2 null animal.
A multi-functional mouse line was constructed previously (Schüller, U, et al. Cancer Cell 14:123-134 (2008)). Olig2-cre allowed for cre mediated activity in oligodendrocyte lineage cells.
The DA-Cat mouse was produced previously (Harada, N, et al. EMBO J. 18(21):5931-5942 (1999)) and has exon 3 of mouse β-catenin gene located between loxP sequences. Cre recombinase mediated deletion of exon 3 produces dominant stable mutant β-catenin protein.
Induction of demyelination with lysolecithin in mouse spinal cord
Demyelinated lesions were produced in ventrolateral spinal cord white matter of 8-10 week old Axin2 null and wild-type littermate mice. The method has been described previously (Fancy, SPJ, et al. Genes and Development 23, 1571-1585 (2009)). Animals were euthanased at three survival time points: 5 days post lesion (dpl), representing peak OLP recruitment, 10 dpl representing onset of OLP differentiation and 14 dpl representing new myelin sheath formation (n=4 for each survival time).
The method has been described previously (Fancy, SPJ, et al. Genes and Development 23, 1571-1585 (2009)). The primary antibodies were: Tcf4 (mouse monoclonal 6H5-3, Upstate), Olig2 (rabbit polyclonal from CD Stiles, Harvard), Nkx2.2 (mouse monoclonal, Developmental Studies Hybridoma Bank), PDGFRα (rat 558774, BD Biosciences), APC (CC1)(mouse monoclonal OP80, Calbiochem), NOGO-A (rabbit, Millipore), β-gal (rabbit 55976, MP), Tankyrase (mouse monoclonal 19A449, Abcam), Iba1 (rabbit, Wako), CD3 (rabbit, Dako), GFAP (mouse, Sigma), Naked1 (rabbit, Cell Signaling Technology) and Notum (rabbit, Abcam). Secondary fluorescent antibodies from Alexa were used.
Cell counts and statistical measures
For all cell counts, at least four animals per genotype were used for each time point indicated. Cells were counted on three or more non-adjacent sections per mouse and presented as an average +/− standard deviation. Statistical comparisons of cell counts were established using Student t test.
Human MS tissue
Human post-mortem tissue blocks were provided by the UK Multiple Sclerosis Tissue Bank at Imperial College London. All tissues were collected following fully informed consent by the donors via a prospective donor scheme following ethical approval by the London Multicentre Research Ethics committee (MREC 02/2/39). MS lesions were characterized according to Lock et al. Nat. Med. 8: 500-508 (2002), using Luxol Fast Blue to assess demyelination, SMI-31 immunohistochemistry to assess preservation of axons, and LN3 immunohistochemistry to assess inflammatory cell activity. Lesions with florid parenchymal and perivascular inflammatory cell infiltration, myelin fragmentation, and demyelination with indistinct margins were classified as active plaques (AP). Chronic active plaques (CAP) were classified as those with extensive demyelination well demarcated borders and abundant inflammatory cells at the lesion edge. Chronic plaques (CP) were classified as those with extensive demyelination, well demarcated borders but few or no inflammatory cells in any part of the demyelinated area.
Human Developmental Tissue
All human tissue was collected in accordance with guidelines established by the University of California San Francisco Committee on Human Research (H11170-19113-07). Immediately after procurement, all brains, except Case 1, were immersed in 4% phosphate buffered formalin for one week at room temperature. In Case 1, the brain was immersed in phosphate buffered saline with 4% paraformaldehyde for three days. On day 3, brains were cut in coronal plane at level of Mamillary Body and immersed in fresh 4% paraformaldehyde/PBS for additional three days. Post fixation, all tissue samples were equilibrated in PBS with 30% sucrose for at least 2 days, before OCT embedding. The diagnosis of hypoxic ischemic encephalopathy (HIE) requires clinical and pathological correlations. With respect to the pathological features, all HIE cases in study showed consistent evidence of diffuse white matter injury, including astrogliosis and macrophage infiltration. These findings were confirmed by increase in number and staining intensity of GFAP- or CD68-positive cells, respectively. The HIE cases also showed evidence of neuronal injury, including presence of ischemic neurons and variable degrees of neuronal loss, in cerebral cortex, hippocampus and basal ganglia. Representative images in Supplementary Figure 2
show GFAP immunoreactivity most intense in the Layer 1 of cerebral cortex and in the subcortical white matter.
Case 1, 2, 3, 4 and 5 demonstrated clinical evidence of HIE and pathological evidence of HIE and low output state in multiple organs on post-mortem examination. More specific findings in Case 1 (8 week-old, born at full term) included hypoplastic left heart status-post procedure and diffuse white matter injury in brain with focal neuron dropout in cerebral and cerebellar cortices. Case 2 (five-day old, born full term) with a clinical diagnosis of severe HIE who underwent therapeutic hypothermia showed diffuse white matter injury on post-mortem evaluation. Case 3 (five month old, born preterm at 26 weeks gestation) clinically showed failure to thrive, enterocolitis, hypoxemia and ventriculomegaly. Decreased myelination relative to the patient’s age, diffuse white matter gliosis, and focal loss of cortical neurons were noted in this patient. Case 4 (6 day old male born at 35 and 5/7 weeks) with in utero polyhydramnios showed neuropathological findings of hypoxic ischemic encephalopathy and intraventricular hemorrhage. Case 5 (6 day old female born at 37 and 4/7 weeks) with intrauterine growth retardation showed hypoxic ischemic encephalopathy characterized by widespread neuronal death in the putamen, thalamus, hippocampus, diffuse hypoxic white matter changes, and diffuse cerebral edema. Case 6 (12 week old, born at 35 3/7 weeks) with congenital diaphragmatic hernia, hypoplastic heart, and coarctation of aortic arch showed periventricular leukomalacia adjacent to the lateral ventrical. Case 7 (one-day old term infant) with midgut volvulus with extensive hemorrhagic necrosis of small bowel was not found to have significant neuropathological findings of HIE. Case 8 (1 day old female, born at 37 weeks) with hypoplastic left heart complex died shortly after birth and showed no evidence of white matter damage. Case 9 (four month old male born at 38 5/7 weeks) with congenital diaphragmatic hernia and hypoplastic aortic arch status post procedure was complicated by uncontrolled respiratory syncytial virus (RSV) pneumonia. Neuropathological evaluation of case 9 did not show diffuse white matter injury.
In vitro Oligodendrocyte Progenitor cell culture
OLPs were isolated by immunopanning postnatal day 7 CD-1 mouse cerebral cortices as previously described (Emery, B, et al. Cell 138(1): 172-185 (2009); Harrington, E, et al. Ann Neurol 68:703-16 (2010)). OLPs were plated on poly-D-lysine coated plates and coverslips and maintained in proliferation media in a 10% CO2 37°C incubator with 50% media changes every two days. Proliferation media has been described previously (Harrington, E, et al. Ann Neurol 68:703-16 (2010)). For differentiation, OPC proliferation media was replaced with differentiation media: OPC mitogens in proliferation media replaced with CNTF (10ng/ml) and T3 (40ng/ml).
XAV939 treatment of OLP cultures and differentiation assays
After immunopanning OLPs were plated at a density of 5,000 cells/coverslip in proliferation media and allowed to recover for 24 hours. XAV939 (Tocris) or DMSO was added to media for 24 hours. Differentiation was induced by replacement of media with differentiation media including pre-treatment XAV939 concentration or DMSO. For westerns OPCs were plated at a density of 0.5×106 cells/15cm plate and maintained until 75% confluent ~4 days. XAV939 or DMSO was added to media for indicated time period. For differentiation, cell counts of at least 3 coverslips and 3 separate experiments (n=9 and n=300-1000 cells for each test group) were imaged and counted. Comparison of means between two groups were analyzed with unpaired T-test and statistical significance was set at p<0.01.
Western Blot of OLP
Western blot was performed as previously described (Harrington, E, et al. Ann Neurol 68:703-16 (2010)). The primary antibodies were: beta-actin 1:5000 (Cell Signaling 4970), axin1 1:250 (Cell Signaling 2087), axin2 1:250 (Cell Signaling 2151), phospho-beta catenin 1:1000 (Cell Signaling 9561), tankyrase 1:1000 (Abcam ab13587), MBP 1:10,000 (Sternberger SMI-94).
XAV939 treatment in vivo in mouse spinal cord demyelination model
XAV939 (Tocris) was dissolved in DMSO to a concentration of 10mM, then diluted in sterile water to a concentration of 10μM. This stock was then added to 1% lysolecithin immediately before lesioning to a final concentration of 0.1μM XAV939. DMSO alone was added for control animals. Wild-type young adult mice (8-10 week old) were then co-injected with the mixture in dorsal or ventral funiculus spinal cord white matter as described above, and harvested at the times described post lesioning.
Expression profiling of Olig2cre/DA-cat vs WT spinal cord at P4
Whole spinal cords were harvested, homogenized in Trizol and then RNA was extracted using RNeasy kit (Qiagen). Microarray analysis was performed at the Genetics Core Facility, Gladstone Institute, San Francisco. For each of P4 WT and Olig2cre/DA-cat, RNA from three animals was pooled for use in each array chip, and three Affymetrix Mouse Genome 430 2.0 chips were run for each group, for a total of 9 animals for each group of WT and Olig2cre/DA-cat.
Ex vivo cerebellar slice cultures
Following decapitation, the brains of newborn CD1 wild-type mice (postnatal day 0-1) were dissected out into cold MEM (MEM supplemented with penicillin/streptomycin, Invitrogen) and L-Glutamine (4mM). Excess tissue was trimmed around the brain, ensuring that the cerebellum remained attached to the underlying piece of hindbrain. Using a McIlwain tissue chopper, 350μm sagittal slices of the cerebellum were cut and plated on Millicell-CM™ organotypic culture inserts (Millipore, 0.4μm) in slice culture medium containing 50% MEM with Earle’s salts, 35% Earle’s balanced salt solution, 25% heat-inactivated horse serum, glutamax, fungizone, penicillin-streptomycin (each from Invitrogen), and glucose (Sigma).
For the myelinating slice cultures, XAV or DMSO was added at the time of plating. Cultures were maintained at 37°C and 7.5% CO2. Membranes were transferred into fresh medium supplemented with tested factors every two days. The hypoxic slice cultures were exposed to 2% O2 conditions for 24h between 2-3 days in culture and then returned to normal culture conditions. XAV or DMSO was added following hypoxia exposure. Both the myelinating and hypoxic slices were fixed at 12 days in vitro. Remyelinating slice cultures were adapted from previous described methods (Birgbauer et al., 2004). After culturing slices in medium with no factors added for 14 days, demyelination was induced by the addition of 0.5% lysolecithin (Sigma) to the slice culture medium for 18 hours. Following demyelination, slices were then transferred to medium containing XAV or DMSO and maintained for an additional 14 days to allow remyelination to occur.
Immunostaining of slice cultures
Slices were immersed in 4%PFA for 1h while attached to membranes at the desired timepoints. Slices were then rinsed in PBS, blocked with 3% heat-inactivated horse serum, 2% bovine serum albumin, and 0.25% Triton X-100 in PBS, then incubated overnight at 4°C in primary antibody diluted in block solution. Primary antibodies were: chicken polyclonal anti-neurofilament 200kDa (NFH, Encor Biotech), rat monoclonal anti-MBP (Serotec), and rabbit polyclonal anti-Caspr (AbCam, 1:500).
Slice Culture imaging and quantification
Confocal z-stacks were acquired (12 slices) using Leica SP5 confocal microscope. Upon examination of staining, sections were chosen based on their intact cytoarchitechture and the formation of parallel myelinated tracts in the cerebellum. Any slices that did not meet these criteria were not analyzed. Imaging was focused on the areas of the cerebellum that contained parallel tracts of myelinated axons. Obtained images were processed and analyzed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on Internet at http://rsb.info.nih.gov/nih-image/
). Following compression of images into one z-stack, NIH Image was used to threshold area of staining for Caspr and NFH. Myelination was then quantified by a ratio of percent area stained for Caspr to percent area stained for NFH. All imaging and subsequent data analysis was done blinded to conditions being tested. Three independent experiments were conducted and analyzed. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test (GraphPad Prism).