Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

In demyelinating diseases including multiple sclerosis (MS), neural stem cells (NSCs) can replace damaged oligodendrocytes if the local microenvironment supports the required differentiation process. Although chitinase-like proteins (CLPs) form part of this microenvironment, their function in this differentiation process is unknown. Here, we demonstrate that murine Chitinase 3-like-3 (Chi3l3/Ym1), human Chi3L1 and Chit1 induce oligodendrogenesis. In mice, Chi3l3 is highly expressed in the subventricular zone, a stem cell niche of the adult brain, and in inflammatory brain lesions during experimental autoimmune encephalomyelitis (EAE). We find that silencing Chi3l3 increases severity of EAE. We present evidence that in NSCs Chi3l3 activates the epidermal growth factor receptor (EGFR), thereby inducing Pyk2-and Erk1/2- dependent expression of a pro-oligodendrogenic transcription factor signature. Our results implicate CLP-EGFR-Pyk2-MEK-ERK as a key intrinsic pathway controlling oligodendrogenesis.

A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated

Clearly defined error bars
State explicitly what error bars represent (e.g. SD, SE, CI) Our web collection on statistics for biologists may be useful.

Software and code
Policy information about availability of computer code Data collection n/a Data analysis n/a For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. All studies must disclose on these points even when the disclosure is negative.

Sample size
No sample size calculation was performed. Sample size was chosen based on previous experiments and significance levels determined as described in the individual Figure legends.
Data exclusions No data were excluded.

Replication
In vitro experiments were repeated at least two times with at least n=3 independent technical replicates or three times with at least n=2 independent technical replicates. In vivo experiments included at least n=3 mice. Individual repeats and sample sizes as well as significance levels are indicated in the Figure

Blinding
The investigators were blinded as to experimental groups during data collection and analysis. Specifically, for in vivo experiments, EAE scoring and subsequent tissue analysis was performed by an independent researcher, who was blinded to experimental treatment groups.
Reporting for specific materials, systems and methods  Authentication -293T cells were authenticated by Life Technologies.
-H9 hESC derived NSCs (H9, N7800-100) were authenticated by the vendor and additional human genome sequencing and microarray analysis was performed (data not shown) -BV2 cells were positively tested for microglial markers, however no further authentication was performed.

Mycoplasma contamination
Testing for mycoplasma contamination was not performed.
Commonly misidentified lines (See ICLAC register) n/a

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
Female SJL/J/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All animals were housed in pathogenfree facilities. All experiments were performed with the approval of the local animal welfare committees (Harvard Medical Area Standing Committee on Animals, and LAGeSo, Berlin) and in accordance with national and international guidelines to minimize discomfort to animals (NIH and 86/609/EEC).

Wild animals are not included
Field-collected samples was not performed.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
Mice were perfused under anesthesia with cold PBS (without Mg+2 and Ca+2), the brain and spleen were extracted and kept in RPMI medium on ice. Each brain hemisphere was processed separately. In brief, the brain tissue was mechanically homogenized with a syringe plug through a 70μm cell strainer (Corning) and washed with RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS) and antibiotics. The myelin rich cell suspension was resuspended in 37% Percoll (Sigma-Aldrich), the myelin-free non-neuronal cells were collected from the pellet after centrifugation at 2800g, and washed two times in medium. Single cell suspension of spleen was obtained by homogenizing the tissue though a 100μm cell strainer; erythrocytes were lysed for 10 minutes with 0.15 M ammonium chloride and washed two times. Flow cytometry staining was performed at 4°C in 1ml Micronic tubes in PBS without calcium and magnesium (Gibco) containing 0.5% BSA (Serva). First, the cells were incubated for 15 minutes with anti-mouse CD16/CD32 (clone 2.4G2, BD Biosciences) to