Fig 1: LRIG1 facilitates BMP and TGFβ signaling at the cell surface by controlling Smad activation in a ligand-dependent manner.a Control (Ctrl) and Lrig1 KO N2a clonal cell lines, serum starved overnight and then treated with (+) or without (-) BMP4 for 30 min at 37 °C and western blotted for LRIG1 and pSMAD1/5/9 stripped and re-probed for SMAD1 and GAPDH (loading control). See also Supplementary Fig. S3a. b Quantification of pSMAD1/5/9 levels relative to SMAD1 and normalized to the Ctrl -BMP4 condition. c Control (Ctrl) and Lrig1 KO N2a clonal cell lines, serum starved overnight and then treated with (+) or without (-) TGFβ1 for 30 min at 37 °C and western blotted for LRIG1 and pSMAD2 stripped and re-probed for SMAD2/3 and GAPDH (loading control). See also Supplementary Fig. S3b (d) Quantification of pSMAD2 levels relative to SMAD2/3 and normalized to the Ctrl - TGFβ1 condition. e Control (Ctrl) and Lrig1 KO N2a clonal cell lines transfected with both TGFβR1 and TGFβR2, serum starved overnight, and then treated with (+) or without (-) TGFβ1 for 30 min at 37 °C and western blotted for LRIG1 and pSMAD2 stripped and re-probed for SMAD2/3 and GAPDH (loading control). See also Supplementary Fig. S3c (f) Quantification of pSMAD2 levels relative to SMAD2/3 and normalized to the Ctrl -TGFβ1 condition. Source data for graphs are included in Supplementary Data 2.
Fig 2: Lrig1 KO results in decreased numbers of pSMAD-positive cells along the lateral wall of the V-SVZ.a, b Immunostaining of sections from the lateral ventricular wall (LW) with an antibody for phosphorylated SMAD2 (pSMAD2) from 15- and 24- week old Lrig1 KO and WT mice merged with Hoechst. c Quantification of the number of pSMAD2-positive cells along the (LW) from (a). d Quantification of the number of phosphorylated SMAD1/5/9 (pSMAD1/5/9) positive cells along the LW in 24-week old Lrig1 KO and WT mice. e Location of the area imaged in representative images of immunostaining for pSMAD1/5/9 and Hoechst. Created with BioRender.com (f, g). Scale bars represent 25 um. Error bars indicate S.E.M. For (c), N = 4 per group and for (d), N = 3 per group. Source data for graphs are included in Supplementary Data 2.
Fig 3: Lrig1 KO results in increased proliferation of NSCs in the V-SVZ.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 25 µm. Error bars indicate S.E.M. For (a–c, N = 5 per group, and for d–f, N = 3 per group. Source data for graphs are included in Supplementary Data 2.
Fig 4: LRIG1 interacts directly with TGFβRI, TGFβRII, BMPRI and TGFβ1 in vitro.a Immunoprecipitation of FLAG-tagged TGFβR1 and TGFβR2 and probed using antibodies for LRIG1 (long and short exposures) and FLAG by western blot. Load indicates a sample removed following cell lysis and prior to loading on the anti-FLAG magnetic beads. Eluted indicates sample retained on the anti-FLAG magnetic beads. Data are representative of N = 3 independent experiments. See also Supplementary Fig. S2a, b (b) Immunoprecipitation of FLAG-tagged TGFβR1 and BMPR1B and probed using antibodies for LRIG1 and FLAG by western blot. Data are representative of N = 3 independent experiments. See also Supplementary Fig. S2c (c) Proximity Ligation Assay (PLA) using antibodies to LRIG1 and BMPR1B on Lrig1 WT (with and without primary antibodies (Red) in addition to Lrig1 KO mice along the lateral wall of the lateral ventricle (LV), scale bar indicates 10 µm. d Biolayer interferometry using biotinylated TGFβ1 as the bait and the LRIG1 extracellular domain (ECD) in increasing concentrations as the analyte. Fitted curves used to measure KD are shown as grey lines. e Far-western assay using decreasing amounts of LRIG1 ECD bound to the nitrocellulose membrane and detected by incubating with biotinylated TGFβ1 and streptavidin HRP and quantification (right). For (c, d), data are representative of N = 2 independent experiments. Source data for graphs are included in Supplementary Data 2.
Fig 5: Enhanced proliferation in the context of Lrig1 KO does not impact neuronal progeny.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 50 µm for (a), 100 µm for (c) and 20 µm for (g). Error bars represent S.E.M. For (a, b, N = 5 per group and for c–f, N = 3 per group. Source data for graphs are included in Supplementary Data 2.
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