Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Celsr2 expression is enriched in mouse and human spinal motor neurons. Using Celsr2 LacZ transgenic mice, Celsr2 expression is detected by anti-β-gal immunostaining. ( A – E ) E12.5 spinal sections were immunostained for Isl1 (red), β-gal (green) and counterstained for DAPI (blue). The merged image showed that all Isl1-positive cells in the ventral horn (VH) co-labelled by β-gal immunoreactivity ( E ). ( F – J ) In adult spinal sections, all ChAT-positive (red) spinal motor neurons co-expressed β-gal (green) indicated by arrows ( J ). ( K – O ) In WPC8 human spinal sections, ISL1 and CELSR2 double immunofluorescent staining showed that CELSR2 was expressed in ISL1-positive neurons in the ventral horn (VH) indicated by arrows ( O ). B – E , G – J and L – O correspond to boxed areas in A , F and K , respectively. Nuclei were counterstained by DAPI (blue).

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Expressing, Transgenic Assay, Immunostaining, Staining

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Celsr2 knockout improves axon growth in mouse spinal motor explant culture . ( A ) Schema of the experimental procedure for explant culture and analysis. ( B – G ) Spinal motor neuron explants from E13.5 Celsr2 +/+ ( B – D ) and Celsr2 −/− mouse embryos ( E–G ) were cultured for 6 DIV and then immunostained for ChAT ( B and E ; red) and Tuj1 ( C and F ; green). Both signals co-localize ( D and G ; yellow). Representative axonal bundles are indicated in the insets of C and F . ( H – J ) Quantification of the maximal area covered by growing axons in 10 7 µm 2 ( H ; control: 0.86 ± 0.03, mutant: 1.02 ± 0.05; P < 0.05), maximal axon length in 10 3 µm ( I ; control: 1.68 ± 0.09, mutant: 2.04 ± 0.07; P < 0.001) and number of large axon bundles (>8 µm in diameter; J ; control: 3.11 ± 0.45, mutant: 6.77 ± 0.74; P < 0.001). These parameters are increased significantly in the mutant compared to the control. *** P < 0.001; ** P < 0.01; * P < 0.05; Student’s t -test; n = 19 in the control and n = 22 in the mutant.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Knock-Out, Cell Culture, Control, Mutagenesis

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Celsr2 knockout contributes to neurite growth in primary spinal motor neuron culture. ( A and B ) E13.5 spinal motor neurons from Celsr2 +/+ ( A ) and Celsr2 –/– ( B ) mouse embryos were cultured for 6 DIV and immunostained for Tuj1 (red). DAPI counterstained nuclei (blue). ( C and D ) Double immunostaining of cultured neurons for F-actin (blue) and Tuj1 (red) disclosed the axon shafts and growth cones. ( E and F ) Statistical analysis of total neurite length ( E ; control: 165.25 ± 10.52 μm, mutant: 354.19 ± 24.89 μm; P < 0.0001, n = 41 in the control and n = 36 in the mutant) and growth cone areas ( F ; control: 17.48 ± 0.91 µm 2 , mutant: 84.89 ± 7.75 µm 2 ; P < 0.0001, n = 58 in the control and n = 40 in the mutant). ( G ) Protein extracts from E13.5 ventral horns of cervical spinal segments was subjected to western blots using anti-EB3 and β-III tubulin (tubulin). There was a dramatic increase of EB3 in the mutant compared to the control (control: 0.99 ± 0.09, mutant: 1.35 ± 0.01; P < 0.05, n = 3 animals in each group). * P < 0.05; **** P < 0.0001; Student’s t -test.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Knock-Out, Cell Culture, Double Immunostaining, Control, Mutagenesis, Western Blot

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Celsr2 cKO in spinal motor neurons improves functional recovery and NMJ formation after root avulsion/reimplantation. ( A and B ) After root avulsion/reimplantation, the function of the affected forelimb was assessed using the grooming ( A ) and climbing test ( B ). Scores were significantly higher in Celsr2 cKO ( Isl1-Cre; Celsr2 f/– ) compared to littermate controls ( Celsr2 f/– ) at Days 14, 21, 28, 35, 42, 49 and 56 post-injury. During the climbing test, usage of injured (R) and intact (L) forelimbs was compared; the R/L ratio was increased in the Celsr2 cKO. * P < 0.05; ** P < 0.01; Student’s t -test. ( C and D ) Biceps collected 56 days after injury were more atrophic on the injured than the intact side, in both mutant and control mice ( C ). Muscle wet weight on the intact side was comparable in both groups, whereas it was higher on the injured side in mutants versus controls, as reflected by the increased the R/L ratio (injured side to intact side) ( D ). Wet weight, control: 0.0339 ± 0.0008 g, mutant: 0.0339 ± 0.0011 g on the intact side, P < 0.05; control: 0.0237 ± 0.0024 g, mutant: 0.0300 ± 0.0010 g on the injured side, P < 0.05; the R/L ratio: 0.69 ± 0.06 and 0.89 ± 0.02 in the control and the mutant, respectively, P < 0.01. * P < 0.05; ** P < 0.01; Student’s t -test; n = 7 in the control and n = 8 in the mutant. ( E and F ) NMJs were examined using anti-NF200 and anti-ɑ-BT double staining 56 days post-surgery. On intact sides ( E ), several cholinergic receptor clusters showed axonal terminals overgrowth in Celsr2 cKO (17/32; indicated by arrows), but rarely in the control (4/52). On injury sides ( F ), there were more numerous growing axons and NMJs in the mutant than in the control (control: 56.33 ± 3.53, mutant: 213.00 ± 16.56 NMJs/muscle, P < 0.001, n = 3 animals in each group). ** P < 0.01; *** P < 0.001; Student’s t -test for NMJ number comparison and chi-square for receptor cluster comparison. ( G – I ) EMG of biceps was recorded 56 days post-surgery ( G ). The latencies were increased after injury, with no difference between both groups ( H ; control: 0.514 ± 0.014, mutant: 0.600 ± 0.033 ms on the intact side; 1.129 ± 0.170 ms for control and 1.632 ± 0.083 ms for mutant on the injured side; ratio of latency: 2.195 ± 0.335 and 1.632 ± 0.083 in the control and the mutant, respectively; P > 0.05 in all comparisons). Denervation resulted in a significant decrease of the peak–peak amplitude in both groups, but the amplitudes and the ratio of the injured to the intact muscle was significantly higher in the mutant compared to the control ( I ; control: 11.182 ± 0.923 mV, mutant: 11.269 ± 1.055 mV on the intact side, P > 0.05; control: 2.740 ± 0.523 mV, mutant: 5.572 ± 0.873 mV on the injured side, P < 0.05; ratio: 0.244 ± 0.044 and 0.492 ± 0.067 in the control and the mutant, respectively, P < 0.05). * P < 0.05; ** P < 0.01; *** P < 0.001; n.s, not significant; Student’s t -test; n = 7 in the control and n = 9 in the mutant.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Functional Assay, Mutagenesis, Control, Double Staining, Comparison

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Improved axon regeneration in Celsr2 cKO mice after root avulsion/reimplantation. ( A ) Toluidine Blue staining of musculocutaneous nerves from intact and injured sides 56 days after root avulsion/reimplantation. ( B ) Electron microscope images of musculocutaneous nerves from intact and injured sides. On the intact side, axons were more closely packed in the mutant than in the control. ( C and D ) Statistical analysis showed that axon number was comparable on the intact side in the two genotypes but higher in the mutant on the injured side ( C ; control: 734.0 ± 24.8, mutant: 696.3 ± 20.2 axons/section on the intact side, P > 0.05; control: 201.0 ± 21.5, mutant: 300.3 ± 25.8 axons/section on the injured side, P < 0.05; the ratio: 0.244 ± 0.044 and 0.492 ± 0.067, P < 0.05; n = 7 in the control and n = 9 in the mutant). The ratio of the injured to the intact side (R/L) was higher in the mutant ( C ; control: 0.27 ± 0.02, mutant: 0.43 ± 0.03, P < 0.05; n = 3 in each group). The distribution of axons according to their diameter showed no differences in the two genotypes on the intact side and an increased number of axons with 2–6 µm diameter on the operated side in the mutant relative to the control ( D ). * P < 0.05; ** P < 0.01; *** P < 0.001; n.s, not significant; Student’s t -test.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Staining, Microscopy, Mutagenesis, Control

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: CELSR2 knockdown increases axonal regeneration in human embryonic spinal motor explant culture . ( A – D ) Cultured spinal motor neuron explants from WPC7 human embryos were transfected with a CELSR2 scrambled shRNA as control ( A and B ) and with CELSR2-shRNA ( C and D ). After 5 DIV, cultured explants were immunostained for Tuj1. A′ , B′ , C′ and D′ are enlarged areas from A , B , C and D , respectively. Upon CELSR2-shRNA knockdown, growing axons grew in circles (one example indicated in C ) and formed large axonal bundles (arrows in C′ and D′ ). ( E – H ) The maximal area in 10 7 µm 2 ( E ; control: 0.82 ± 0.12, CELSR2-shRNA: 3.93 ± 0.40; P < 0.0001, n = 23 in each group), maximal axon length in 10 3 µm ( F ; control: 1.20 ± 0.05, and CELSR2-shRNA: 3.30 ± 0.25; P < 0.0001, n = 23 in each group), explants with axons growing into circles ( G ; 1/26 in the control and 11/27 in the CELSR2-shRNA) and the number of large axon bundles ( H ; control : 7.83 ± 1.37, mutant: 53.96 ± 3.98, P < 0.0001, n = 23 in each group) were significantly increased in the CELSR2-shRNA knockdown explants compared to control. **** P < 0.0001; Student’s t -test ( E , F and H ). ** P < 0.01; chi-square test ( G ).

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Knockdown, Cell Culture, Transfection, shRNA, Control, Mutagenesis

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: CELSR2 knockdown promotes axonal growth in primary human spinal motor neuron culture. ( A – C ) CELSR2 scrambled shRNA ( A , control) and CELSR2-shRNA ( B ) were used to transfect cultured primary spinal motor neurons from WPC7 and WPC8 human embryos. Transfected neurons were visualized by virus-encoded GFP (green). After 5 DIV, neurons were immunostained for Tuj1 (red). GFP- and Tuj1-immunoreactivity overlapped in the somas and neurites as shown in the merged images. A significant increase of total neurite length was observed in CELSR2-shRNA transfected neurons ( C ; in µm, control: 118.70 ± 5.66, CELSR2-shRNA: 321.28 ± 21.90, P < 0.0001, n = 43 in the control and n = 36 in the CELSR2-shRNA). ( D – F ) Double immunostaining for F-actin (blue) and Tuj1 (red) reveal axon shafts and growth cones in control ( D ) and CELSR2-shRNA ( E ) transfected neurons (GFP labelling, green). The growth cone area was significantly increased in CELSR2-shRNA versus control transfected neurons ( F ; control: 16.92 ± 1.40 µm 2 , and CELSR2-shRNA: 109.00 ± 9.12, P < 0.0001, n = 52 in the control and 55 in the CELSR2-shRNA). ( G ) Western blot analysis of 5-DIV cultured neurons with antibodies to EB3 and β-III tubulin (tubulin, reference) showed an increase of EB3 levels in CELSR2-shRNA transfected neurons (control: 0.99 ± 0.09, CELSR2-shRNA: 1.34 ± 0.02, P < 0.05, n = 3 independent experiments). ( H ) Intracellular calcium influx was evaluated in 19 DIV-cultured neurons by measuring Fluoro-4 AM fluorescence intensity. The curves were drawn from captured images before and after potassium application. There was an increase of the fluorescent peaks in CELSR2-shRNA transfected neurons (control: 0.84 ± 0.13, and CELSR2-shRNA: 2.22 ± 0.09, P < 0.05, n = 3 independent experiments). * P < 0.05; **** P < 0.0001; Student’s t -test.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Knockdown, shRNA, Control, Cell Culture, Transfection, Virus, Double Immunostaining, Western Blot, Fluorescence

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Celsr2 knockout increases Cdc42/Rac1 and JNK/c-Jun signalling in injured ventral horns. ( A – D ) Western blot analysis of GST-pulldown proteins and samples from spinal ventral columns 3 days after root avulsion, using anti-Cdc42 ( A ), anti-Rac1 ( B ), anti-JNK ( C ), anti-c-Jun ( D ) antibodies. Anti-GADPH antibody was used as reference. ( E – H ) GTP-bound Cdc42 ( E ; control: 1 ± 0, mutant: 2.22 ± 0.17, P < 0.05) and Rac1 ( F ; control: 1 ± 0, mutant: 1.90 ± 0.13, P < 0.05) proteins, as well as concentrations of JNK ( G ; control: 2.01 ± 0.12, mutant: 3.65 ± 0.44, P < 0.05) and c-Jun ( H ; control: 0.48 ± 0.02, mutant: 0.84 ± 0.07, P < 0.05) were increased in mutant compared to control samples. * P < 0.05; Student’s t -test; n = 3 animals in each group.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Knock-Out, Western Blot, Control, Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Generation of Celsr1 and Celsr2 loss-of function mutant mice by CRISPR/Cas9. (A) Schematic representation of Celsr1 and Celsr2 protein domains. The two proteins are 55% identical in amino acid sequence and have the same overall domain organization. (B) CRISPR-Cas9 targeting of Celsr1 and Celsr2 genomic loci. Guide RNAs were targeted to the sequence encoding the signal peptide for each of Celsr1 and Celsr2 . The resulting targeted alleles are shown with the ATG and signal sequence in purple font and deleted sequences highlighted in yellow. (C) Celsr1 −/− and wild type ( WT ) littermate at P12. Note curly tail and whorled hair pattern on the head of Celsr1 −/− homozygote. (D) Left and right paws of Celsr1 −/− and WT littermate at P12. Celsr1 −/− homozygotes exhibit prominent hair whorl on each paw. (E) Celsr1 −/− and WT littermate embryos at E15.5. Celsr1 −/− homozygotes display curly tail. (F) Western blot of epidermal lysates from WT and Celsr1 −/− P0-P3 backskins with anti-Celsr1 antibody. (G) Western blot of epidermal lysates from three individual WT and two individual Celsr2 −/− P0-P3 pups with anti-Celsr2 antibody. (H) Confocal immunofluorescence image of whole mount epidermis from E15.5 WT and Celsr1 −/− mutant embryos labeled with Celsr1 antibodies. Scale bars: 10 µm. (I) Quantification of Celsr1 mean fluorescence intensity in WT and Celsr1 −/− mutant epidermis (n = 3 skin regions from 4 different WT embryos and n = 3 skin regions from 3 different Celsr1 −/− embryos).

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Mutagenesis, CRISPR, Sequencing, Western Blot, Immunofluorescence, Labeling, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Celsr1, but not Celsr2, is necessary for correct asymmetric orientation of developing hair follicles. (A) Average intensity projection of WT embryonic back skin at E15.5, labelled for P-cadherin (green) and Sox9 (magenta). White box denotes zoomed in region shown below, left. Average intensity projection of a typical WT hair follicle imaged at higher mag (below, right). Scale bars: 1000, 200, and 25 µm, respectively. Anterior is to the left. (B–D) As for (A) , except Celsr1 −/− , Celsr2 −/− and Celsr1 −/− ;Celsr2 −/− respectively. (E) Bar chart showing cumulative percentage of polarized (grey bar) vs. non-polarized (white bar) hair follicles in n = 3 E15.5 back skins from 3 different embryos. n in figure represents total number of follicles analyzed. Error bars = SEM. (F–H) As for (E) , except Celsr1 −/− , Celsr2 −/− and Celsr1 −/− ;Celsr2 −/− respectively. (I) Rose plot of polarized follicles in (E) showing the angle of orientation, with anterior = 0° and posterior = 180°. Shaded areas in bars represent relative contribution of each replicate (n = 3 backskins from 3 different embryos), with n in figure representing total number of polarized hair follicles analyzed. (J–L) As for (I) , except Celsr1 −/− , Celsr2 −/− and Celsr1 −/− ;Celsr2 −/− respectively.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Loss of core PCP protein asymmetry in the epidermis of Celsr1 −/− and Celsr1 −/− ; Celsr2 −/− double mutants. (A–H) Representative planar views of the basal layer of the interfollicular epidermis at E15.5 showing Celsr1, Fz6 and Vangl2 distribution as detected by immunofluorescence. Anterior is to the left. Scale bar: 20 µm. Magnified areas below are overlaid with colored lines representing the axis (line angle) and magnitude (line length) of polarity. Quantification of polarity distributions are displayed below on circular histograms. (A-B′) Celsr1 (A) , Fz6 (B) and Vangl2 (B′) in WT embryos, n = 11,951 basal cells, 3 embryos. (C-D′) Celsr1 (C) , Fz6 (D) , and Vangl2 (D′) in Celsr1 −/− embryos, n = 11,629 basal cells, 3 embryos. (E-F′) Celsr1 (E) , Fz6 (F) and Vangl2 (F′) in Celsr2 −/− embryos, n = 12,099 basal cells, 3 embryos. (G-H′) Celsr1 (G) , Fz6 (H) and Vangl2 (H′) in Celsr1 −/− ; Celsr2 −/− embryos, n = 9,064 basal cells, 3 embryos.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Celsr2 enriches at cell-cell junctions by homotypic interactions less efficiently than Celsr1. (A) Representative images of cell pairs expressing Celsr1-GFP, Celsr2-GFP or GFP-CAAX as indicated. Bottom panels show zoomed in junctional regions. Note the stronger enrichment of Celsr1-GFP at junctions compared to Celsr2-GFP and both isoforms are significantly more enriched at junctions compared to a non-junctional plasma membrane marker GFP-CAAX. Scale bars 20 µm (top panel) and 10 µm (bottom panel). (B) Plot of the junctional enrichment score (ratio of junctional mean intensity to the mean intensity of the cell pair). n = 32 Celsr1-GFP junctions, n = 43 Celsr2-GFP junctions, n = 60 GFP-CAAX junctions. Kolmogorov-Smirnov test p < 0.0001. Data pooled from two independent experiments where each experiment reflects the represented trend. (C) Fluorescence Recovery After Photobleaching (FRAP) of junctional Celsr1-GFP and Celsr2-GFP. Shown are representative images of the junctional region between cell pairs expressing Celsr1-GFP or Celsr2-GFP before and after bleaching as indicated. Bleached ROIs are marked by yellow arrowheads. (D) FRAP recovery plots. Shown is the normalized mean intensity with standard deviations of the bleach and recovery profiles plotted versus time for Celsr1-GFP (blue) and Celsr2-GFP (magenta) at junctions (in bold) and free cell edges that are not juxtaposed to a transfected cell (in lighter shade). (n = 36 ROIs for Celsr1 edge, 38 ROIs for Celsr2 edge, 78 ROIs for Celsr1 junctions and 75 ROIs for Celsr2 junctions). Data pooled from two independent experiments for cell edge measurements and three independent experiments for junction measurements. (E) Cell mixing experiment between cells expressing Celsr1-3xFLAG and Celsr2-GFP (top panels) or Celsr1-3xFLAG and Celsr1-GFP (bottom panels). Images show cell pairs forming heterotypic junctions. Celsr1-3xFLAG appears to enrich with both Celsr1-GFP and Celsr2-GFP, in trans , across cell-cell junctions. (F) Histogram depicting the frequency of Celsr1-3xFLAG: Celsr1-GFP and Celsr1-3xFLAG::Celsr2-GFP junctions across the range of junction enrichment ratios obtained for Celsr1-GFP and Celsr2-GFP, respectively. Inset shows box plot for the junction enrichment values of Celsr1-GFP and Celsr2-GFP. n = 56 Celsr1-GFP junctions and n = 64 Celsr2-GFP junctions. Kolmogorov-Smirnov test, p = 0.0004. Data pooled from two independent experiments.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Expressing, Clinical Proteomics, Membrane, Marker, Fluorescence, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Celsr2 recruits Fz6 and Vangl2 to keratinocyte junctions, similar to Celsr1. (A–C) Representative cell pair co-expressing Celsr1-GFP and Fz6-tdTomato (A) , tdTomato-Vangl2 (B) or a non-junctional membrane marker tdTomato-CAAX (C) . Arrowheads mark the junction between 2 cells and a magnified view of the junction is represented below the respective images. Scale bars = 20um. (D–F) Representative cell pair co-expressing Celsr2-GFP and Fz6-tdTomato (D) , tdTomato-Vangl2 (E) and tdTomato-CAAX (F) . Arrowheads mark the junction between 2 cells and a magnified view of the junction is represented below the respective images. Scale bars = 20 um. (G) Box plots depicting junction enrichment ratios for Fz6-tdTomato compared to tdTomato-CAAX when co-expressed with Celsr1-GFP or Celsr2-GFP (n = 33 for Celsr1-tdTomato CAAX, n = 49 for Celsr2-tdTomato-CAAX, n = 36 for Celsr1-Fz6-tdTomato, n = 66 for Celsr1-Fz6-tdTomato). (H) Box plots depicting junction enrichment values of Celsr1-GFP versus Celsr2-GFP in cells co-expressing Fz6-tdTomato. (I) Box plots depicting junction enrichment ratios for tdTomato-Vangl2 compared to tdTomato-CAAX when co-expressed with Celsr1-GFP or Celsr2-GFP (n = 66- Celsr1-tdTomatoCAAX, n = 87-Celsr2 tdTomatoCAAX, n = 63-Celsr1 tdTomatoCAAX, n = 67-Celsr2-tdTomatoCAAX). (J) Box plots depicting junction enrichment of Celsr1-GFP and Celsr2-GFP in cells expressing tdTomato-Vangl2. Data pooled from two independent experiments for Fz6-tdTomato and three independent experiments for tdTomato-Vangl2. Kolmogorov-Smirnov tests, **** p < 0.0001, ** p = 0.009.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Expressing, Membrane, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Product information of key antibodies and reagents used in this study.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Knock-Out

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Genotyping details for Celsr1 and Celsr2 including primer sequences and product sizes for WT and knockout animals.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Knock-Out, Western Blot, Plasmid Preparation, Software