LRIG1 controls proliferation of adult neural stem cells by facilitating TGF and BMP signalling pathways – Nature.com

Mice

Lrig1 constitutive knockout mice were generated by crossing Lrig1CreERT2 (Jackson Strain No. 018418, Lrig1tm1.1(cre/ERT2)Rjc/J) heterozygous mice on the 129-Elite strain background (Charles River Canada, strain code 476) together to produce homozygous knockout mice as described in ref. 14. Genotypes were confirmed post-weaning from ear notch genomic DNA (isolated with DirectPCR Lysis Reagent (Ear, Cat # 401-E)) using PCR primers and Jackson Protocol 26202 exactly as described. For all experiments female and male mice were randomly assigned to experimental groups and analyzed at ages of 8-weeks, 15-weeks and 24-weeks. Specific timepoints are noted in the figure legends and captions. Mice used in this work all followed a 12h light/dark cycle and had ad libitum access to water and rodent chow. Mice did not demonstrate any visible signs of being immunocompromised or display any obvious behavioural phenotypes. All animal work conducted followed policies formed by the Canadian Council of Animal Care and approved by the Local Animal Care Committee at the University of Toronto and The Hospital for Sick Children.

Frozen tissue sections were dried for 1h at room temperature or 30min at 37C, then rehydrated in phosphate buffered saline (PBS, 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4) for 10min. Subsequently, sections were blocked and permeabilized in a solution of 5% BSA and 0.3% Triton X-100 in PBS. Following blocking, sections were incubated in primary antibody diluted in 1:1 PBS to blocking solution overnight, at 4C in a humidified chamber. The following day sections were washed in PBS three times for 5min, then incubated in diluted secondary antibody for 2h at room temperature. Afterwards, sections were washed three times in PBS for 5min per wash. Finally, sections were counterstained for 5min at room temperature in 0.5g/mL Hoechst 33258 (Sigma-Aldrich), washed with PBS and then mounted with PermaFluor mountant (Thermo Scientific).

5-ethynyl-2-deoxyuridine (EdU, Toronto Research Chemicals Cat #: E932175) was dissolved in PBS and injected intraperitoneally three times two hours apart at a dose of 50mg/kg to 15-week-old Lrig1 WT and KO mice. After 3 weeks brains were collected and prepared for IHC as described below. EdU+ cells were detected using the Click-it EdU Alexa 488 kit (Invitrogen, Cat #: C10337) following the manufacturers instructions and then stained with Hoechst 33258. EdU+ cells were counted from five sections per brain.

Tissue sections from 15-week-old Lrig1 WT and KO mice were prepared as described below, blocked and incubated overnight with 1:100 anti-LRIG1 and anti-BMPR-1B (1:100 see Supplementary TableS3) antibodies as described above for IHC. The next day sections were washed three times for 10min with Wash Buffer A (0.01M Tris, 0.15M NaCl and 0.05% Tween-20) and then incubated with the Duolink In Situ PLA Probe Anti-Goat

PLUS (Sigma-Aldrich) and Duolink In Situ PLA Probe Anti-Mouse MINUS Affinity purified Donkey anti-mouse IgG (H+L) (Sigma-Alrich, See Supplementary TableS4) for 1hour at 37 C in a humidified chamber. Following washing of the PLA probes, Ligation and Amplification was carried out as described by the manufacturer using the Duolink In Situ Detection Reagents kit (Sigma-Aldrich) and nuclei stained for Hoechst 33258.

The periventricular area containing the V-SVZ from N=2 female and N=2 male Lrig1 KO and N=3 female and N=1 male WT 8-week old brains was dissected as described in ref.18 from both hemispheres of each brain and stored at 80C until needed. One hemisphere sample was used to isolate RNA as described below. The other hemisphere sample was lysed and western blotted for LRIG1 and pEGFR as described previously14.

Lrig1 KO and WT mice were anesthetized with 23% inhaled isoflurane and then perfused transcardially with PBS followed by 4% paraformaldehyde (PFA). Brains were then dissected, post-fixed overnight in 4% PFA and then cryoprotected in 30% sucrose in PBS for 24h. Tissues were then embedded with O.C.T (Fisher Healthcare Tissue-Plus O.C.T. Compound, Cat # 23-730-571) and sections were cut 18m thick in the coronal plane using a Thermo Fisher Scientific HM525 NX cryostat at 20C. Sections were collected on glass slides (Fisherbrand Superfrost Plus Microscope Slides, Cat #1255015) coated with gelatin and stored frozen until use.

All primary and secondary antibodies used for western blot are listed in Supplementary TablesS1 and S2, while those used for IHC/PLA are listed in Supplementary TablesS3 and S4.

For all experiments, plasmid constructs were used following endotoxin-free maxipreps using a Qiagen EndoFree Plasmid Maxi Kit or a ZymoPURE II Plasmid Maxiprep Kit. Plasmid DNA concentration was determined using a NanoDrop 2000 (ThermoFisher). All plasmids used in this study are listed in Supplementary TableS5.

Images were collected using a Zeiss Spinning Disk confocal microscope system or a Zeiss AxioImager M2 microscope system with a Calibri LED light source. Images were acquired using Z-stacks (with the apotome engaged in the case of the AxioImager) and staked tiles imaging set-up using Zen Blue software. All images were acquired with Z-stack sizes ranging between 15-25 slices depending on the dataset analyzed. Images shown were produced using the orthogonal projection feature implemented in Zen Blue.

For all cell count analysis on acquired images from V-SVZ, counting was done using ImageJ. For cell counts in the V-SVZ, only visibly immunostained (positive) cells along the dorsal and ventral portions of the lateral wall (LW), closest to the ventricle (periventricular area) were counted. To differentiate between the dorsal and ventral areas of the LW, these regions were measured using the line drawing and measurement tools in ImageJ along the length of the entire LW. To assess proliferation, neuronal progeny and pEGFR-positive cells in the V-SVZ, the top 1/3rd of the length of the LW was considered the dorsal portion. The bottom 2/3 measurement of the LW was considered the ventral portion. To obtain a cell count value for each individual brain, positive cells counted from three anatomically matched 18 m thin coronal sections containing the V-SVZ LW region were totaled and averaged. For the proportion of proliferating (Ki67+) cells, a percent cell count was used to represent the data by dividing the number of SOX2+GFAP+Ki67+/SOX2+GFAP+ cells. For the number of DCX-positive and pEGFR-positive cells the total number of cells counted per ventricle area was used to represent these data. For cell counts in the OB, positive cells in the GCL, GL and MCL were counted using three anatomically 18 m thick coronal sections of the whole OB from each mouse brain. To create a cell count value for each individual brain, counts from all three sections were totaled and averaged. For the number of CalB or CalR-positive interneurons, the total number of cells in each layer were counted and used to represent these data. pSmad2 and pSmad1/5/9 positive cells only in the V-SVZ were counted on the entire ventral portion of the LW of the LV. Three coronal sections from each brain which were anatomically matched in order of contain the LW area of the V-SVZ and were used for cells count analysis, where the total positive cells from each coronal section were then averaged.

Following RNA isolation using a RNeasy Plus Mini Kit (Qiagen, Cat#:74134) from tissue isolated as described above, polyA selected mRNA next generation sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit and sequenced on one lane of an Illumina Novaseq SP flow cell achieving ~4050 million reads per sample. Following sequencing, FASTQ files were generated with bcl2fastq2 v2.20. Library preparation, sequencing and FASTQ file production were done by The Centre for Applied Genomics (TCAG) at the Hospital for Sick Children. FASTQ files were then used as input for Salmon19 for alignment to the mouse genome (Gencode m29) and for read quantification. Deseq2 as implemented in R and was used for normalization and differential gene expression analysis20.

Immunoprecipitation (IP) assays were done similar to those described in ref. 21. Neuro-2a cells (N2a) (ATCC Cat No: CCL-131) were used for all IP experiments which were cultured in Dulbeccos modified Eagles medium (DMEM) containing high levels of glucose supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). Transfections of N2a cells were carried out using Poly-Jet reagent (SignaGen Laboratories; Cat# SL100688) 24h after plating 400,000 cells per well of 6 well plate. Flag-tagged pCMV expression vectors containing either the TGFR1, TGFRII and BMPRI were used to co-transfect N2a cells with Lrig1 pCMV-overexpression plasmid using PolyJet In Vitro DNA Transfection Reagent (SignaGen Laboratories; Cat# SL100688). A mock condition with no plasmids was used as a negative control and BMPRI was used as a positive control21. N2a cells were lysed after 48h in TNTE buffer containing 0.5% Triton-X-100 (150mM NaCl, 50mM Tris pH 7.4, and 1mM EDTA)21. Anti-Flag Magnetic beads (Selleckchem Cat#: B26101) were used to carry out all immunoprecipitations. The beads and sample mixtures were incubated on a tube rotator at 4C for 2h. Supernatant was then discarded and the beads were washed three times with wash buffer (TNTE buffer containing 0.1% Triton-X100) on a magnetic separation rack. Following the final wash, magnetic beads were eluted using 1x SDS-PAGE loading buffer heated at 70C for 10min. 10% of the samples prior to immunoprecipitation were collected for a load condition in 5x SDS Sample Loading Buffer (10% SDS, 500mM DTT, 50% Glycerol, 250nM Tris-HCl, 0.5% bromophenol blue dye, pH 6.8). Western blotting was performed as described previously14 using anti-flag antibody (DYKDDDDK Tag Antibody Cell Signalling Technology Cat#: 2368) to detect the TGFR1, TGFRII and BMPRI receptors and LRIG1 antibody (R&D Systems, Cat#:3688) to detect LRIG1. GAPDH antibody was used as an internal reference to normalize protein expression levels.

LRIG1 ectodomain (ECD, Cat#: 3688-LR-050 from R&D Systems) concentrations of 2.5g/mL, 0.25g/mL, and 0.025g/mL were immobilized on a nitrocellulose membrane in duplicate. 2.5% Bovine serum albumin and 1xPBS were used as negative controls. Once dried, the blot was incubated at room temperature (RT) in blocking buffer (2% BSA in PBS-T (1xPBS containing 0.1% Tween)) for 30min. 1.2 ug of biotinylated human TGF1 (Avi- Tag, Biotin-Labeled, BPS Bioscence Cat #. 100843) was then added to blocking buffer on the blot and incubated for one hour at RT. A 1:5000 dilution of Streptavidin-HRP in blocking buffer was added for 30min followed by 35min and 315-minute washes with PBS-T. The blot was then developed with Bio-Rad Clarity Western enhanced chemiluminescence (ECL) Detection Reagent on a Syngene G:Box chemiluminescence imager.

The binding affinity of LRIG1 extracellular domain (ECD) to TGF1 was measured using a BLItz instrument. LRIG1 ECD in 1xPBS was used as the analyte and biotinylated-TGF1 was used as the bait. Streptavidin-coated sensors were hydrated in BLI rehydration buffer (PBS, 0.5mg/mL BSA, and 0.02% (v/v) Tween-20). The biotinylated bait protein was diluted in kinetics buffer (PBS, 0.5mg/mL BSA, 0.02% (v/v) Tween-20) to a final concentration of 12.4g/mL. The bait was immobilized on a streptavidin-coated biosensor tip for 30s. Next, the analyte concentrations were diluted in kinetic buffer in order to obtain concentration of LRIG1 ECD of 1M, 750nM, 500nM, 100nM. The binding association was measured over a period of 120s with subsequent dissociation measured after immersion of the biosensor tip into kinetic buffer for another 120s. The data were analyzed and sensorgrams were step corrected, reference corrected and globally fit to a 1:1 binding model. Dissociation constants (KD) were calculated using BLItz Pro Version 1.1.0.16 and the average of two independent determinations is reported here.

N2a cells were transfected with guide RNAs #1, #2, or LacZ control contained in the pU6-(Bbsl)_CBh_Cas9_T2A_mCherry plasmid (as described previously14 in using Lipofectamine STEM, according to manufacturers protocol in 6 well plates. Two days following transfection, cells were replated into 96 well plates at a concentration of 0.5 cells/200l media in each well. Cells were allowed to grow for 3 weeks and wells containing individual colonies that expressed red fluorescence from mCherry were selected and propagated further to produce clonal cell lines. Three control lines and four LRIG1 KO lines were selected following western blotting for LRIG1 to confirm loss of the LRIG1 protein and used for the experiments described here.

In the case of TGF1 and BMP4 treated non-transfected Ctrl N2a clonal cell lines and Lrig1 KO N2a clonal cell line experiments, cells were plated at 90,000 cells/well in DMEM media (described above) in 24-well plates. Two days later the media was changed to DMEM containing 0.1% FBS for serum starvation overnight. The following day, 10ng/ml TGF1 (Cedarlane; Cat# 781802) or BMP4 (Peprotech Cat#315-27) ligands suspended in DMEM containing 0.1% FBS. The later of which on its own was used as the control. Following 30min at 37C, the cells were collected using 50L of lysis buffer containing, tris-buffered Saline, pH 7.4, 0.5% b-octyl-D-glucopyranoside, 0.5% Triton X-100, 1mM NaF, 1mM -glycerophosphate, 1mM Na3VO4 and 1 cOmplete, mini, EDTA-free protease inhibitor tablet per 10ml. Protein assay was done with the samples using the DC Assay (Bio-Rad) and then equal amounts of protein were used for western blots to assess the pSmad2/3, total Smad2, pSMAD1/5/9 and total SMAD1 along with GAPDH (as a loading control) and LRIG1 (to confirm genotypes).

Since N2a cells express low levels of TGFRII, we used TGFRI and TGFRII constructs to express these receptors in Ctrl and Lrig1 KO clonal N2a cells followed by treatment with TGF1 to assess the response of this signalling pathway in the context of loss of LRIG1. To do so, 1g of DNA per well, using PolyJet of TGFRI and TGFRII were transfected one day after plating 90,000 cells per well of each of the Ctrl and Lrig1 KO clonal N2a cell lines. 24h after transfection, the cells were transitioned into DMEM containing 0.1% FBS media. The following the Lrig1 KO and WT clonal cell lines were treated with 10ng/ml per well of TGF1 for 30min at 37C, lysed, and protein concentrations determined as above. Western blotting for pSmad2/3, total Smad2 and GAPDH were then performed.

Neurospheres were grown essentially as described in ref. 18 using NeuroCult Mouse&Rat Basal Medium containing NeuroCult Proliferation Supplement (STEMCELL Technologies), FGF2 (Corning), EGF (STEMCELL Technologies), 0.2% heparin (Sigma-Aldrich, Cat #: H4784) and 1% penicillin-streptomycin from brains of 15 week old Lrig1 WT and KO mixed male and female mice (N=2 male and N=1 female KO mice, N=2 male and N=2 female WT mice). Following seven days of growth of secondary spheres, spheres from each brain were gently distributed into two wells of a 24-well plate and treated with BMP4 as described above without changing the culture media. Cells were then collected by centrifugation and western blots were performed as described above.

Data previously published by Mizrak et al.22, from lateral wall cells of 810 week old mice was obtained from NCBI GEO accession number: GSE109447. Specifically, the 13,055 cell data set was reduced to just the lateral wall cells and then was processed using Seurat (version 4.4.0) as implemented in R (version 4.2.1). Cells with less than 200 genes expressed in at least three cells were removed. The data were then normalized and scaled, principal components were computed and clustering was performed with 25 principal components and a resolution of 0.8. UMAP plots were created using the DimPlot and FeaturePlot functions as implemented in Seurat.

GraphPad Prism Version 9.3.1 software was used to perform all statistical analyses and create graphical representations. For statistical analyses in Figs.13, a two-way ANOVA and Tukeys Honestly Significant Difference (HSD) post-hoc test were used to compare data between the different experimental groups present in each paradigm (ex. KO vs. WT), and the area of the LW being analyzed (ex. dorsal vs ventral). In Fig.5, a two-tailed Students t-test in order to compare the differences between Lrig1 KO and WT groups. In Fig.7 and Supplementary Fig.S3, the results from the four conditions were averaged from two independent experiments and normalized to total SMAD2/3 for the TGFR transfections or total SMAD1 for the BMP experiments. Subsequently, a one-way ANOVA followed by a dk post-hoc test was used to compare groups. In all figures, values were reported as means. To create error bars, means were represented as the standard error of the mean (S.E.M). Experimental data was denoted as statistically significant at P values less than 0.05 (P<0.05). In all the figures, *p<0.05, **p<0.01, ***p<0.001 and ns = not significant.

a Immunostaining of the lateral wall of 15-week old Lrig1 KO and WT mice for GFAP, SOX2, Ki67 and merged with Hoechst to label nuclei. Sox2/GFAP-double positive cells allows identification of NSCs. Ki67/SOX2/GFAP-triple positive cells represent proliferating NSCs. Arrowhead indicate triple positive cells. Quantification of the number of SOX2/GFAP-double positive NSCs (b) and proportion of proliferating Ki67-positive NSCs (c) along the dorsal and ventral portions of the lateral wall in 15-week old Lrig1 KO and WT mice. d Immunostaining of the lateral wall of 24-week old Lrig1 KO and WT mice for GFAP, SOX2, Ki67 and Merged with Hoechst. Quantification of the number of SOX2/GFAP-double positive NSCs (e) and proportion of proliferating Ki67-positive NSCs (f) along the dorsal and ventral portions of the lateral wall in 24-week old Lrig1 KO and WT mice. Scale bars represents 25m. Error bars indicate S.E.M. For (ac, N=5 per group, and for df, N=3 per group. Source data for graphs are included in Supplementary Data2.

a Immunostaining of coronal sections through the lateral ventricle of 15-week old Lrig1 KO and WT mice for DCX to mark new-born neurons/neuroblasts and GFAP to label ventricular cells merged with Hoechst. b Quantification of the number of DCX-positive cells in the dorsal and ventral portions of the lateral wall. c Representative immunostaining of Olfactory Bulb (OB) sections with antibodies for Calretinin (CalR) and Calbindin (CalB) and merged with Hoechst at low magnification (left, indicating the Granule Cell Layer (GCL), the Mitral Cell Layer (MCL) and the Glomerular Layer (GL)) and at high magnification (right) from Lrig1 and KO mice. d Quantification of the number of CalB-positive cells in each of the granule cell (GCL), mitral cell (MCL) or glomerular layers (GL), (e) Quantification of the number of CalB-positive cells in the GCL, MCL and GL of the OB. f, g Quantification of the number of EdU-positive cells in the olfactory bulb (f) and representative staining of EdU-positive cells with Hoechst from an Lrig1 WT brain. Scale bars represent 50m for (a), 100m for (c) and 20m for (g). Error bars represent S.E.M. For (a, b, N=5 per group and for cf, N=3 per group. Source data for graphs are included in Supplementary Data2.

a Immunostaining of coronal sections along the lateral wall of Lrig1 KO and WT mice with antibodies for phosphorylated EGFR (pEGFR) and merged with Hoechst. b Quantification of the number of pEGFR-positive cells in the dorsal and ventral portions of the lateral wall of the V-SVZ. c Western blots using antibodies for LRIG1, pEGFR and GAPDH (loading control) from periventricular tissue dissected from one hemisphere per animal from 8-week old Lrig1 KO and WT mice See also Supplementary Fig.S2d. d Quantification of the western blot data from (c) for pEGFR relative to GAPDH. Scale bar represents 50um. Error bars represent S.E.M. For (a, b, N=5 per group and for c, N=4 per group. Source data for graphs are included in Supplementary Data2.

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LRIG1 controls proliferation of adult neural stem cells by facilitating TGF and BMP signalling pathways - Nature.com