EMBO/SourceData huggingface dataset:
Figure 1B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (B) Representative bright-field images of spheroids treated with dmPGE2. Scale bars, 200 µm.B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM dmPGE2 (average area was 3761 µm2 for 0 nM group; n = 4 images from two independent experiments).B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (D) Representative histological sections of spheroids treated with 1 µM dmPGE2 or an equivalent volume of DMSO and stained for hematoxylin. Scale bars, 100 µm.B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (E) Quantification of the average expression ± s.e.m. of Cldn4mRNA relative to the 0 nM treatment group (n = 3 independent experiments).B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (F) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM Iloprost (n = 4 images from two independent experiments).
Figure 3B Heatmap showing unsupervised hierarchical clustering of genes that were significantly (FDR-adjusted p<0.05) and differentially expressed between the three groups (n = 4 samples per treatment from two independent experiments). Each column is one sample and each row is one gene. Red and blue indicate genes with enriched or de-enriched expression in a particular group, respectively. The dendrogram for the gene clustering is colored to highlight major gene clusters 1-6. The number of probes in each cluster is in parentheses.E, F Spheroids were cultured as indicated for 24 hours followed by a 1-hr pulse with EdU to mark the cells undergoing DNA synthesis. (E) Representative images of EdU staining (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm. (F) Quantification of EdU-positive nuclei as a percent of the total nuclei (n = minimum of 15 spheroids counted per sample from 2 independent experiments). ****p<0.0001 by 1-way ANOVA and Tukey's post-test.G Graph of the fold change in background-subtracted luminescence ± s.e.m. (relative to 0 hr measurement) of Cdc25A-CBRLuc spheroids (n = 3 independent experiments with 4 technical replicates). ****p<0.0001 for dmPGE2- and EP4i-treated spheroids compared to stem cells by repeated measures 2-way ANOVA (variable = treatment). p<0.001 at the 16 hr, 20 hr and 24 hr time points by Dunnett's post-test comparing dmPGE2-treated and EP4i-treated spheroids to the stem cell control.H Quantification of the average expression ± s.e.m. of Lgr5 and Mki67 mRNAs in jejunal spheroids cultured in stem cell (Stem) or in differentiation medium with the indicated supplements relative to the stem cell group (n = 3 independent experiments). **p<0.01, ****p<0.0001 by one-way ANOVA and Dunnett's post-test. n.d., not detected.
Figure 4D Representative images of spheroids stained for Cldn4 (red). Nuclei are visualized with bisbenzimide (blue) (n = 2 independent experiments). Scale bars, 20 µm.E, F Jejunal spheroids were cultured as in Figure 2B-D. Quantification of the average expression ± s.e.m. of Dpcr1 (E) and Cd55b mRNAs relative to DMSO group (n = 3 independent experiments). *p<0.05, ****p<0.0001 by one-way ANOVA and Dunnett's post-test.G-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mousecolon. (G) The basal plasma membranes are outlined in orange solid lines, lateral plasma membranes are indicated with orange arrowheads, and nuclei are outlined with wide yellow dashed lines. Insets show a magnified view of the apical cell surface.G-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mousecolon. Quantification of cytoplasmic:nuclear ratio (H) and microvillar length (I) from the TEM images (n = minimum of 5 images per group). *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test.G-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mousecolon. (J) Higher power image of the cytoplasm. Single mitochondria are outlined with a narrow blue dashed line, vacuole structures are indicated with red asterisks. Scale bars, 1 µm.K Representative image of a spheroid stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). A similarly stained section of a small intestinal ulcer is shown for comparison. Arrowheads indicate the apical cell membrane. Scale bars, 50 µm.
Figure 5A Representative image of a spheroid stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). A similarly stained section of a mouse villus is shown for comparison. Arrowheads indicate the apical cell membrane. Scale bars, 50 µm.B, C Jejunal spheroids were cultured as indicated. (B) Quantification of the average expression ± s.e.m. of Fabp1, Ace2, and Maoa mRNAs (n = 3 independent experiments). *p<0.05, **p<0.01 by one-way ANOVA and Tukey's post-test.B, C Jejunal spheroids were cultured as indicated. (C) Representative images of spheroids stained for Ace2 (green) and β-catenin (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm.F, G Representative TEM images of EP4i-treated spheroids. (F) The basal plasma membranes are outlined in orange solid lines, lateral plasma membranes are indicated with orange arrowheads, and nuclei are outlined with wide yellow dashed lines. Insets show a magnified view of the apical cell surface. (G) Higher power image of the cytoplasm. Single mitochondria are outlined with a narrow blue dashed line, vacuole structures are indicated with red asterisks. Scale bars, 1 µm.H Graph of spheroid oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) ratio expressed as mean ± s.e.m. (n = 3 independent experiments). **p<0.01by 1-way ANOVA and Tukey's post-test.
Figure 6A-D Quantification of the average expression ± s.e.m. of Dpcr1 and Cd55b mRNAs (A, C, E) or Fabp1 and Ace2 mRNAs (B, D, F) in jejunal spheroids cultured in differentiation medium containing EP4i or dmPGE2. (A, B) Gene expression analyzed 2, 6, 12 or 24 hours after the start of treatment (n = 3 independent experiments). ***p<0.001, ****p<0.0001 comparing the two treatment groups and †p<0.05, †††p<0.001, ††††p<0.0001 compared to the 2 hr time point of the same medium by 2-way ANOVA and Sidak's multiple comparisons test. (C, D) Gene expression was analyzed after culturing spheroids in EP4i (E) or dmPGE2 (P) for the first 12 hours followed by wash out and re-feeding with EP4i or dmPGE2 for the second 12 hours as shown (n = 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by one-way ANOVA and Dunnett's post-test.E, F Gene expression was analyzed in Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δspheroids and expressed as fold change relative to EP4i-treated Ptger4flox/flox (n = 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA and Tukey's post-test comparing the genotypes within a treatment group. †††p<0.001, ††††p<0.0001 by two-way ANOVA and Sidak's post-test comparing the effects of the treatments within a genotype.
Figure 7A, B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO. (A) Representative bright-field images. Scale bars, 200 µm.A, B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO. (B) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to the DMSO treatment group (n = 3 independent experiments). *p<0.05 compared to DMSO group by unpaired t test.C, D Representative Western blots for β-catenin detected in nuclear (Nuc) and cytoplasmic (Cyt) protein lysates. Lamin A/C and Actin were used as loading controls for the nuclear and cytoplasmic fractions, respectively (n = 3 independent experiments). (C) Spheroids were cultured in stem, enterocyte (EP4i) or WAE (dmPGE2) media. (D) Spheroids were cultured in enterocyte medium with DMSO or 10 µM CHIR 99021.E, F Spheroids were cultured in enterocyte medium with the indicated concentrations of CHIR 99021. Quantification of the average expression ± s.e.m. of Cldn4 (E) and Fabp1 (F) mRNAs shown as fold change relative to 0 µM group (n = 3 independent experiments). **p<0.01, ****p<0.0001 as determined by one-way ANOVA and Dunnett's post-test.G Representative images of Ptger4Δ/Δspheroids cultured in differentiation medium with DMSO or 10 µM CHIR 99021 and stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm.H Graph of TOPflash (TOP) to FOPflash (FOP) luciferase reporter ratios ± s.e.m. (n = 3 independent experiments) in transfected 293FT cells treated with conditioned mediums collected from L-WRN cells and diluted as indicated. L-WRN CM had been produced in the presence or absence of the porcupine inhibitor C59 (10-fold dilutions, 10 µM to 1 pM). *p<0.05, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Dunnett's post-test compared to 0% L-WRN CM group.I, J Spheroids were cultured in enterocyte media, WAE media or WAE media containing 100 pM C59. Quantification of the average expression ± s.e.m. of Cldn4 (I) and Fabp1 (J) mRNAs shown as fold change relative to EP4i group (n = 3 independent experiments). ***p<0.001, ****p<0.0001 as determined by one-way ANOVA and Tukey's post-test.K Quantification of the average expression ± s.e.m. of Axin2 mRNA in spheroids cultured in stem cell or in differentiation medium with the indicated supplements relative to stem (n = 3 independent experiments). ****p<0.0001 compared to stem cell group as determined by one-way ANOVA and Dunnett's post-test.
Figure 8A Representative whole-mount images of wounds from Ptger4flox/flox (Ptger4fl/fl) and Villin-Cre Ptger4flox/flox (VilCrePtger4fl/fl) mice four days post-biopsy injury. Scale bars, 750 µm. Wounds are outlined with a black dashed line. Asterisk indicates fibrin clot.B, C Quantification of percent healing (1 - [Day 4 wound area/original wound area] x 100) (B) and fibrin clot areas (C) ± s.e.m. (n = 7-8 mice per genotype with 3-4 wounds each, three independent experiments). *p=0.0270, **p=0.0057 by two-tailed unpaired t-test.D, E Representative images of serial wound tissue sections from the indicated genotypes. (D) Fluorescent images of sections stained for Claudin 4 (red). Nuclei are visualized with bisbenzimide (blue). Epithelial crypts adjacent to the wound bed are indicated by dashed yellow lines. Layer of adherent WAE cells indicated by white arrowheads. Non-adherent residual WAE cells indicated by orange arrows.D, E Representative images of serial wound tissue sections from the indicated genotypes. (E) Grayscale images of hematoxylin and eosin-stained sections. Insets of the boxed regions show a higher magnification of the region typically containing WAE cells (red arrowheads, dashed line indicates border with wound bed). Orange arrow indicates region of non-adherent residual WAE cells. Scale bars, 100 µm.
Figure 1(A-D) Immunostaining with dopamine antibody at late 3rd instar (96h AEL) in control background (domeMESO-GFP/+). The inset shows DomeGFP+ cells (B') and the corresponding dopamine levels (C') in them. (E-L) Similar expression analysis of dopamine at early 3rd (72h AEL, E-H) and early 2nd instar (48h AEL, I-L). (D, H and L) The spectral mode of iDopamine representation with blue pixels indicating low and red pixels indicating high intensity. (M) Quantifications of dopamine levels in DomeGFP+ progenitors (domeMESO-Gal4, UAS-GFP/+, n= 14 at 48h, n=13 at 72h and n=16 at 96h AEL). Data information: DNA is stained with Hoechst in blue, intracellular Dopamine (iDopamine) in red, domeMESO expression in green (representative of progenitors in the medullary zone, MZ). The iDopamine channel has been converted to spectral mode in panels D, H and L. All image panels show a 20µm scale bar. The inset represents a 2X zoom of region of interest (ROIs) marked in white dashed square. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line for segregation between Dome+ and Dome- cells. The quantification in M shows median with whiskers indicating the maximum and minimum values,. box indicates the lower and upper quartiles. The statistical analysis applied is One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.
Figure 6(A-I'') Representative lymph gland images at 48, 72 and 96h AEL depicting the lymph gland growth trajectory along with dopamine level status in the PSC and remaining lymph gland. (A-C'') The control profile of lymph gland and PSC iDopamine levels at 48h (A-A''), 72h (B-B'') and 96h (C-C''). (D-I'') Downregulation of the dopamine synthesising enzymes TH and Ddc in the PSC at 48h (D-D'' and G-G''), 72h AEL (E-E'' and H-H'') and at 96h AEL (F-F'' and I-I''). (J) Quantification of mean dopamine intensity in the PSC at upon PSC perturbation of dopamine synthesis at 48h AEL (Antp-Gal4; UAS-GFP/+, n=33, Antp-Gal4; UAS-GFP; THRNAi, n=8, Antp-Gal4; UAS-GFP; DdcRNAi, n=14), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=26, Antp-Gal4; UAS-GFP; THRNAi, n=25, Antp-Gal4; UAS-GFP; DdcRNAi, n=20) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=17). (K) Quantification of mean dopamine levels in the lymph gland upon PSC perturbation of dopamine synthesis at 48h AEL (Antp-Gal4; UAS-GFP/+, n=28, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=18), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=25, Antp-Gal4; UAS-GFP; THRNAi, n=24, Antp-Gal4; UAS-GFP; DdcRNAi, n=22) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=16, Antp-Gal4; UAS-GFP; DdcRNAi, n=18). Data information: DNA is stained with Hoechst in blue, Antp-Gal4; UAS-GFP (representative of Posterior Signaling Center, PSC) in green, iDopamine in red. All image panels show a 20µm scale bar. AEL indicates After Egg Laying. The quantification in J, K represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in J, K is Two-way Anova with Tukey's multiple comparisons test. The control animals Antp-Gal4; UAS-GFP/+ were analysed by One-way Anova with Tukey's multiple comparisons test across time points. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines and the PSC in yellow dashed line.(L) Analysis of the total cell counts upon loss of TH and Ddc in the PSC at 48h AEL (Antp-Gal4; UAS-GFP/+, n=19, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=17), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=17, Antp-Gal4; UAS-GFP; THRNAi, n=18, Antp-Gal4; UAS-GFP; DdcRNAi, n=22) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=14, Antp-Gal4; UAS-GFP; DdcRNAi, n=15) Data information: The quantification in L represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in L is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Figure 1(A) Substrate-dependent nitrogenase activity, measured through reduction of acetylene into ethylene, in isolated B. diazoefficiens bacteroids upon supplementation of malate, succinate, arginine, and nodule extract. Data represents the mean and standard error of the mean of at least eight independent replciates.(B) Fold change in intracellular ATP level in isolated B. diazoefficiens bacteroids upon supplementation of succinate, arginine, and nodule extract. The values obtained for each of the 4 individual measurements (points) together with the regression trajectory of each experiment is shown.
Figure 3(A) The aerial part of M. truncatula upon infection with S. meliloti ∆argI1, ∆argI2, ∆argI1,2 and the dicarboxylate transport mutant ∆dctAB were smaller than those inoculated with the wild type strain, highlighting the importance of arginine catabolism for nitrogen fixation(B) Cross sections of nodules bearing S. meliloti WT or arginine catabolism mutant ∆argI1, ∆argI2 reveals the yellowish color of non-functional nodules induced by the ∆argI1 ∆argI2 double deletion mutant.(C) Bacteroid ultrastructure across nodule sections determined by scanning electron microscopy indicates the presence of hollow nodules with aberrant cell morphology in the ∆argI1 ∆argI2 double deletion mutant defective in arginine catabolism.(D) Nitrogenase activity in M. truncatula nodules inoculated with S. meliloti strains defective in arginine catabolism. Data points are the mean of at least 30 plants measured after 8 weeks post inoculation; error bars indicate standard error of the mean.
Figure 4(B) Heat map representing enzymatic activities participating in the arginine transamination network with relative enzyme activities highlighted as a color map Rows are annotated on the left with the abbreviated names of the substrates and on the right with the corresponding EC numbers. Columns are annotated by corresponding enzyme names.(C) Recombinant enzymes involved in arginine catabolism were combined into a single reaction mixture using arginine and pyruvate as substrates. Substrate (S) consumption into the products (P) succinate and alanine, including their intermediates (I1,I2), was determined along the time series using LC-MS/MS.(D) The relative metabolite abundance along the reaction time-course is highlighted as a color map
Figure 1Representative Ca2+ signals in sperm from a patient with deafness-infertility syndrome lacking functional CatSper channels (CATSPER2-/-) (red) and a healthy donor (black), evoked by progesterone, PGE1, NH4Cl, or ionomycin. NH4Cl increases the intracellular pH. Bar graph: Amplitudes (n = 4; mean ± SD) of Ca2+ signals in CATSPER2-/- spermRepresentative monovalent CatSper currents in CATSPER2-/- sperm (blue, green, orange, purple, brown) and in sperm from a healthy donor (black) and corresponding current-voltage relationship (right). The membrane voltage was stepped from -100 mV to +100 mV in increments of 10 mV from a holding potential of -80 mV. Outward and inward current amplitudes (mean ± SD) at +100 mV and -100 mV, respectively, in CATSPER2-/- sperm (color code: panel B) and sperm from healthy donors (black).Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2-/- sperm form DIS patients using antibodies directed against CatSper 3 (D) or CatSper 4 (E); DNA was labelled with DAPI (blue). Scale bars represent 10 µm.3D-STORM image in xy-projection of sperm from a healthy donor labelled with the anti-CatSper 3 antibody (left). Axial projection of the boxed region (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.3D-STORM images in xy-projection of CATSPER2-/- sperm (left) labelled with the anti-CatSper 3 (upper panel) and anti-CatSper 4 (lower panel) antibody. Axial projection of the boxed regions (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.
Figure 2Dark-field microscopy of a single sperm; shown are single frames obtained at t = 0, 48, 96, and 136 ms. Scale bar = 25 µmDark-field imaging of a sperm population; left: single frames at t = 185, 363, 540, and 718 ms. Sperm selected for analysis are highlighted (1-4). Right: Temporal change in the brightness (blinking) of sperm heads. The blue lines correspond to the time-points of the single frames. Scale bar = 25 µm.Representative distribution of rotation frequencies of freely swimming non-capacitated (0 mM bicarbonate; black; n = 218) and capacitated sperm (25 mM bicarbonate; red; n = 232) determined by dark-field imaging.Rolling frequency (mean ± SD) of non-capacitated (0 mM bicarbonate, n = 1,455) and capacitated sperm (25 mM bicarbonate, n = 1,097).Rolling frequency (mean ± SD) of freely swimming sperm in 0 (n = 1,175), 0.2 (n = 832), and 1 % (w/v) (n = 599) methyl cellulose. .Bright-field images of an optically trapped sperm cell, obtained at t = 0, 55, 155, and 185 ms. Scale bar represents 10 µm.Representative time-course of the rotation frequency of trapped sperm (each sperm cell is represented by a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis..Distribution of rotation frequencies in trapped sperm (n = 32; mean frequency ± SD 6.0 ± 2.1 Hz).Image series of a sperm cell trapped parallel to the optical axis; images were obtained at t = 0, 95, 165, and 205 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 µm.Representative distribution of rotation frequencies of freely swimming CatSper-deficient sperm incubated under non-capacitating (0 mM bicarbonate; black; n = 73) and capacitating conditions (25 mM bicarbonate; red; n = 272).Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm incubated under non-capacitating (0 mM bicarbonate, n = 1009) and capacitating (25 mM bicarbonate, n = 946) conditions.Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm in 0 (n = 457), 0.2 (n = 389), and 1 % (w/v) (n = 187) methyl cellulose.Image series of a CATSPER2-/- sperm cell optically trapped perpendicular to the optical axis; images were obtained at t = 0, 75, 150, and 225 ms. Scale bar represents 10 µm. Image series of a CATSPER2-/- sperm cell optically trapped parallel to the optical axis; images were obtained at t = 0, 60, 195, and 320 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 µm. Representative time courses of the rotation frequencies of optically trapped CATSPER2-/- sperm (each sperm is represented in a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis.
Figure 3Rotation frequency of a trapped sperm cell before and after dragging across the barrier stream. The green trace indicates the fluorescence of fluorescein included in the barrier stream. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm cells before and after dragging across the barrier stream.Rotation frequency of a trapped sperm cell before and after dragging from the control stream containing 0 mM bicarbonate into the stimulus stream containing 25 mM bicarbonate. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm at 0 and 25 mM bicarbonate.Rotation frequency of a trapped sperm cell in the presence and absence of extracellular Ca2+. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies in the presence and absence of extracellular Ca2+.Fraction of motile sperm (mean ± SD) in a sperm population incubated in the presence (black) and absence (at t = 0) of extracellular Ca2+ (red) (n ≥ 5).Distribution of rotation frequencies in populations of freely swimming sperm in the presence (black, n = 335) and absence (red, n = 224) of extracellular Ca2+ (red).