Krneta-Stankic V , Corkins ME , Paulucci-Holthauzen A , Kloc M , Gladden AB , Miller RK .

K01 DK092320 NIDDK NIH HHS , R01 DK115655 NIDDK NIH HHS , R03 DK118771 NIDDK NIH HHS , P30 CA016672 NCI NIH HHS

	Graphical Abstract
	Figure 1Daam1 co-localizes with junctional F-actin and E-cadherin during early nephron development. (A) Confocal maximum image projections of whole-mount immunostaining of Xenopus nephric primordium labeled by Lhx1 (white) and GFP to visualize Daam1 (green) in conjunction with phalloidin staining to visualize F-actin (magenta); scale bar, 50 μm. a’–a’’’’ are close-up images of region a; scale bars, 20 μm. The first panel consists of the entire z stack to show the cell positions within the entire kidney. In contrast, images displayed in a’–a’’’’ contain a subset of the z slices to exclude the intense signal from phalloidin within the Xenopus skin. The white dotted box in a’’–a’’’’ marks the junction shown enlarged in the insets (scale bars, 10 μm) and analyzed in (B). (B) A line plot showing the junctional intensity of F-actin (magenta) and GFP-Daam1 (green) of the junction highlighted in (A), a’’–a’’’’, by the white dotted box. (C) Confocal maximum image projections of whole-mount immunostaining of Xenopus nephric primordium labeled by Lhx1 (white) and GFP to visualize Daam1 (green) in conjunction with E-cadherin (magenta); scale bar, 50 μm. c’–c’’’’ are close-up images of region c; scale bars, 20 μm. The first panel consists of the entire z stack to show the cell positions within the entire kidney. In contrast, images displayed in c’–c’’’’ contain a subset of the z slices to exclude the intense signal from E-cadherin within the Xenopus skin. The white dotted box in c’’–c’’’’ marks the junction shown enlarged in the insets (scale bars, 10 μm) and analyzed in (D). (D) A line plot showing the junctional intensity of E-cadherin (magenta) and GFP-Daam1 (green) of the junction highlighted in c’’–c’’’’ by the white dotted box.
	Figure 2. Daam1 localizes to newly formed cell-cell contacts (A) Schematic showing the steps involved in preparing “windowed kidney” embryos and primary cultures expressing GFP-Daam1. For clarity, the 8-cell GFP-Daam1-injected Xenopus blastomere is fate-mapped strictly to the nephric primordium, and blastomere also contributes to the epidermis, ventral and dorsal somites, hindgut, proctodeum, and trunk neural crest cells (DeLay et al., 2016; Moody and Kline, 1990). (B) Time-lapse imaging montage of the nephric primordium expressing GFP-Daam1 in “windowed kidney” embryos. Elapsed time is indicated at the top; scale bars, 10 μm. The white arrows point to cellular protrusions shown enlarged in the insets (scale bars, 5 μm); see Video S1. (C) Time-lapse imaging montage showing cells isolated from a developing nephron expressing GFP-Daam1 mRNA adhering to each other. Elapsed time is indicated at the top; scale bars, 5 μm. The orange dotted line delineates the border arising between two cells, and the white arrows point to filopodium-like cellular protrusions; see Video S2.
	Still image from Video S1. Time-lapse of the nephric primordium expressing GFP-Daam1, related to Figure 2
	Still image from Video S2. Imaging of GFP-Daam1 localization during de novo contact formation in cells isolated from the nephric primordium expressing GFP-Daam1, related to Figure 2
	Figure 3Effects of Daam1 depletion on the nephrogenic primordium. Kidney-targeted microinjections using control or Daam1 MO combined with the membrane-GFP mRNA (used as a lineage tracer) were carried out to analyze the effect of Daam1 depletion on Xenopus developing kidney. (A) Maximum-projection confocal images of control or Daam1 KD embryos stained whole mount with antibodies against GFP (to detect the membrane-GFP tracer, green) and Lhx1 (to label nephric progenitors, white), in conjunction with phalloidin (to visualize F-actin, magenta). The top panels consist of the entire z stacks to show the cell positions within the entire kidney; scale bars, 50 μm. a’–a’’ and b’–b’’ are close-up images of the corresponding regions in white boxes consisting of a subset of the z slices to exclude the intense F-actin signal within the Xenopus skin; scale bars, 20 μm. The “seam” of enriched actin in the nephron center corresponds to the region where the lumen will form; see Videos S3 and S4. (B) Western blot and graph of densitometry measures showing Daam1 and GAPDH protein levels for uninjected wild-type (WT), control (standard MO)-injected, and Daam1 KD (Daam1 MO)-injected embryos. Embryo lysates were pooled from 10–20 V2-cell injected embryos at stages NF 28–32, and approximately 1/2 embryo was loaded per lane. Individual band intensities are normalized to the uninjected band and plotted in arbitrary units (a.u.) for uninjected (black), control (orange), and Daam1 KD (blue). The results are expressed as means ± SEM from four independent experiments. nsp > 0.05, ∗∗∗p ≤ 0.001, analyzed by one-way ANOVA. (C) The graph showing the mean relative fluorescence intensity levels in a.u. of junctional F-actin in the nephric primordia of control (orange) and Daam1 KD (blue) embryos. nControl = 40 junctions and nDaam1 KD = 40 junctions on 2 embryos, denoted by open and closed circles, respectively. ∗∗∗∗p ≤ 0.0001, analyzed by unpaired t test. (D–G) Morphometric analyses of control and Daam1-depleted nephric primordia. The thick bars represent the mean for control (orange) and Daam1-depleted (blue) embryos. ∗∗∗∗p ≤ 0.0001, analyzed by unpaired t test. Graphs show comparison of control and Daam1-depleted nephric primordia of (D) the average number of Lhx1-positive nephron progenitors, where nControl = 5 embryos and nDaam1KD = 5 embryos; (E) the relative distance between nearest neighbors of Lhx1-positive nuclei; (F) the relative cell area; and (G) the relative circularity, where 1 represents a perfect circle. (E–G) nControl = 40 junctions on 2 embryos and nDaam1 KD = 40 junctions on 2 embryos, denoted by open and closed circles, respectively.
	Still image from Video S3. 3D projection of sections from nephric primordium of control embryos stained for F-actin with phalloidin, related to Figure 3
	Still image from Video S4. 3D projection of sections from nephric primordium of Daam1 KD embryos stained for F-actin with phalloidin, related to Figure 3
	Figure 4 Daam1 regulates assembly of junctional F-actin in developing nephrons F-actin dynamics at cell junctions of control and Daam1 KD developing nephrons expressing mCherry-Utrophin were assessed using FRAP. (A–E) Control (black, ntotal = 27 junctions, 1–5 junctions/embryo) and Daam1 KD (purple, 16 junctions; orange, 11 junctions; ntotal = 27 junctions, 1–5 junctions/embryo). (A) Percentage of junctions showing recovery of fluorescence after bleaching in control and Daam1 KD nephrons. (B) Typical time-lapse images of control and Daam1 KD cell junctions before and after photobleaching. In each image, the bleached region is highlighted with a circle (black, control junction showing recovery; purple, Daam1 KD junction showing recovery; orange, Daam1 KD junction showing no recovery of fluorescence after photobleaching). Scale bars, 10 μm. (C) Graph showing average recovery curves in a.u. obtained from individual best-fit plots for control (black) and Daam1 KD junctions with (purple) and without (orange) recovery of fluorescence after photobleaching. (D and E) Bar graphs comparing control and Daam1 KD profiles calculated from individual best-fit curves for control (black) and Daam1 KD junctions with recovery of fluorescence after photobleaching (purple). The dots represent analyzed junctions. Data represent the mean ± SEM from three independent experiments. The p values were analyzed by unpaired t test. (D) Bar graph of the relative half-times for F-actin. (E) Bar graph of the relative mobile fraction for F-actin.
	Figure 5 Daam1 promotes localization of junctional E-cadherin (A) Maximum-projection confocal images of whole-mount immunostaining of Xenopus nephric primordium labeled by Lhx1 (magenta) and E-cadherin (cyan) in control and Daam1 KD embryos; scale bars, 50 μm. a’–a’’ and b’–b’’ are close-up images of the corresponding regions in the white boxes; scale bars, 20 μm. The top panels consist of the entire z stacks to show nephric progenitors’ cell positions within the developing kidney. In contrast, images displayed in a’–a’’ and b’–b’’ contain a subset of the z slices to exclude the intense signal from E-cadherin expressed in the epithelium of the Xenopus skin. a’–b’, nephric cell progenitors labeled by Lhx1 (magenta) and E-cadherin (cyan); a’’–b’’, E-cadherin (cyan). (B) Violin plots depicting the relative fluorescence intensity in a.u. of junctional E-cadherin in the nephric primordia of control (orange) and Daam1 KD (blue). nControl = 88 junctions on 4 embryos and nDaam1 KD = 84 junctions on 4 embryos. Centerlines represents the median; limits show the first and third quartiles. ∗∗∗∗p ≤ 0.0001, analyzed by unpaired t test. (C) Western blot and graph of densitometry measures in a.u. showing Daam1, E-cadherin, and GAPDH protein levels in uninjected WT, control (standard MO)-injected, and Daam1 KD (Daam1 MO)-injected embryos. Embryo lysates were pooled from 10–20 1-cell injected embryos at stages NF 11–12, and approximately 1/2 embryo was loaded per lane. Individual band intensities are normalized to the uninjected band and plotted in a.u. for uninjected (black), control (orange), and Daam1 KD (blue). The results are expressed as means ± SEM from four independent experiments. nsp > 0.05, ∗∗∗∗p ≤ 0.0001, analyzed by one-way ANOVA.
	Still from Video S5. 3D projection of sections from the mature control nephron stained for E-cadherin, related to Figures 5 and S5
	Still from Video S6. 3D projection of sections from a mature Daam1-depleted nephron stained for E-cadherin, related to Figures 5 and S5
	Figure 6The FH2 domain of Daam1 mediates E-cadherin localization to cell junctions. (A) Maximum-projection confocal images of whole-mount immunostaining showing E-cadherin (cyan) in Xenopus nephric primordium labeled by Lhx1 (magenta) expressing GFP-Daam1 or GFP-Daam1 FH2 mutant mRNA (labeled by anti-GFP, green); scale bars, 50 μm. a’–a’’ and b’–b’’ are close-up images of the corresponding regions in the white boxes; scale bars, 20 μm. The top panels consist of the entire z stacks to show nephric progenitors’ cell positions within the developing kidney. In contrast, images displayed in a–a’ and b–b’ contain a subset of the z slices to exclude the intense signal from E-cadherin expressed in the epithelium of the Xenopus skin. a’–b’, nephric cell progenitors labeled by Lhx1 (magenta), anti-GFP (green), and E-cadherin (cyan); a’’–b’’, E-cadherin (cyan). (B) Violin plots depicting the relative fluorescence intensity of junctional E-cadherin in nephric primordia expressing GFP-Daam1 (orange) and GFP-Daam1 FH2 mutant (blue) mRNA. nDaam1 = 60 junctions on 3 embryos and nDaam1FH2mutant = 55 junctions on 3 embryos. Centerlines show the median; limits show the first and third quartiles. ∗∗∗p ≤ 0.001, analyzed by unpaired t test. (C) Western blot and the graph of densitometry measures showing the exogenous and endogenous protein levels of Daam1 in uninjected WT embryos and embryos injected with 1 ng Daam1 or Daam1 FH2 mutant mRNA. The non-specific band confirms equal loading. Embryo lysates were pooled from 10–20 1-cell injected embryos at stages NF 11–12, and approximately 1/2 embryo per lane. The results are expressed as means ± SEM from three independent experiments. Individual band intensities are normalized to the uninjected band and plotted in a.u. for uninjected (black), Daam1 (orange), and Daam1 FH2 mutant (blue). nsp > 0.05, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, analyzed by one-way ANOVA.
	Figure 7 Daam1-depleted MDCK cells display compromised localization of E-cadherin at cell-cell (A) Maximum-projection confocal images of E-cadherin (green), F-actin (magenta), and DAPI (cyan) in subconfluent MDCK control and shDaam1 KD cells. The first column shows merged images of individual color channels. Images of individual color channels for F-actin and E-cadherin are shown in the second and third columns, respectively. Scale bars, 50 μm. E-cadherin localization in nascent cell-cell contacts (marked by white brackets and shown enlarged in the corresponding white boxes; scale bars, 10 μm) is impaired in shDaam1 KD cells. (B) Western blot showing Daam1 and GAPDH protein levels in MDCK control and shDaam1 KD cells. Individual band intensities are normalized to the control band and plotted in a.u. for control (orange) and Daam1 KD (blue). The graph of densitometry measures represents the means ± SEM of two independent experiments. ∗p ≤ 0.05, analyzed by unpaired t test. (C–F) Summary of the wound healing experiments for MDCK control and Daam1 KD cells; see Video S7. (C) Daam1 depletion impairs wound closure. The graph represents the percentage of the wound surface area over time for control (purple) and Daam1 KD (orange) cells. Error bars indicate SE of the mean of 4 assays. (D–F) Manually tracking single cells’ migration paths during wound closure demonstrates that Daam1 organizes collective movement of MDCK epithelial monolayers by modulating the speed and directionality of individual cells. Daam1 results in increased velocity and random migration. nControl = 52 cells from 4 assays and nDaam1 KD = 42 cells tracked from 4 assays. Cells were tracked in 15-min increments for 12 h. (D) Violin plots represent migration velocity calculated from tracking traveled distances of single cells for control and Daam1 KD cells. Centerlines show the median; limits show the first and third quartiles. ∗∗∗p ≤ 0.001, analyzed by unpaired t test. (E) Wind rose plot showing migration tracks of individual control and Daam1 KD cells. and impaired cohesion during collective movement
	Still image from Video S7. Confocal time lapse of wound healing assay in MDCK control (left) and Daam1 KD (right) cells, related to Figure 7 Superimposed tracks show the movement of individual cells during wound healing. Daam1 deficient cells exhibit a delay in wound closure, uncoordinated movement, and random detachment from migrating epithelial sheets compared to Control cells. Elapsed time is shown in hours at the top of the upper panels. Scale bars equal 50 microns.
	Figure S1. Daam1 is absent from cell-cell junctions in the fully developed embryonic nephron, Related to Figures 1 and 2. Maximum confocal projections of the mature Xenopus embryonic nephron labeled with 3G8 and 4A6 antibodies (green), RFP antibody to visualize Daam1(magenta), and DAPI staining labeling nuclei (blue); scale bar equals to 50 microns. a’-a’’’ close-up images of the white box; scale bars equal to 20 microns.
	Figure S2. Daam1 regulates the morphology of intercellular boundaries in developing nephrons, Related to Figures 3 and 4. Confocal time-lapse imaging of F-actin in Control and Daam1KD developing nephrons. (A-B) Stills from time-lapse movies of "kidney-windowed" embryos. Elapsed time is indicated at the top. Scale bars equal to 5 microns. mCherry tagged Utrophin marks F-actin (magenta), and membrane tagged GFP (green) indicates cell boundaries in (A) Control and (B) Daam1 KD cells in developing nephrons.
	Figure S3. FRAP analysis of Daam1 KD nephric junctions, Related to Figure 4. Daam1 KD nephrons exhibited both junctions with and without recovery of F-actin fluorescence signal following photobleaching. We compared the pre-bleach fluorescence levels of F-actin and the bleaching depths between Daam1 KD junctions with and without recovery of F-actin fluorescence signal (magenta and orange, respectively) in these nephrons. Measures normalized for background and general photobleaching [IN] were used to estimate the mean fluorescence intensity of each junction before bleaching [IN pre-bleach] and to plot the percentage of recovery after photobleaching, which was determined as follows: % recovery rate = (IN / IN pre-bleach) x 100. The bleaching depth for each junction was determined based on its % recovery rate plot as follows: % bleaching depth = %recovery rate pre- bleach - % recovery rate post-bleach. Each dot represents one embryo with 1-3 junctions analyzed per nephron, per condition (ntotal=8 embryos across 3 experiments; 27 junctions comprising 16 recovered, and 11 no-recovery junctions). (A) The bar graph represents the quantification of F-actin pre-bleach fluorescence levels and bleaching depths for Daam1 KD junctions with recovery and no-recovery of fluorescence signal following photobleaching. Significance analyzed by Student's t-test; mean +/- S.D. The distribution of recovery and no-recovery junctions within individual embryos and across different trials is shown in a summary table to the right. Representative % recovery plot of Daam1 KD junctions with and without recovery of F-actin fluorescence signal following FRAP is shown within the yellow box.
	Figure S4. In-vivo imaging of E-cadherin localization in Daam1-depleted developing nephrons, Related to Figures 5 and 6. Confocal time-lapse imaging of GFP-E-cadherin (green) in Control and Daam1KD developing nephrons. Stills from time-lapse movies of "kidney-windowed" embryos. Elapsed time is indicated at the top. Scale bars equal to 5 microns. GFP-E-cadherin in (A) Control and (B) Daam1 KD pronephric progenitor cells.
	Figure S5. Loss of Daam1 results in cell shape and tissue architecture abnormalities in mature nephrons, Related to Figures 5 and 6. Kidney-targeted morpholino microinjections were carried out to manipulate the expression levels of Daam1. Control (Standard) or Daam1 antisense morpholinos were co-injected with membrane tagged RFP (mRFP) mRNA as a linage tracer. Analyses of mature nephrons by confocal and Transmission Electron Microscope (TEM) imaging show that a decrease in Daam1 signaling levels affects the size and shape of nephric cells. (A) Maximum projection confocal images of mature nephrons labeled with lectin (green) and antibodies against RFP (magenta) and E-cadherin (cyan); scale bars equal to 50 microns. a-a’ - close-up images corresponding to regions in the white boxes; scale bars equal to 20 microns. The top panels consist of the entire z-stacks to show the overall morphology. a-a’’ and b’-b’’ represent close-up images of the corresponding regions in white boxes consisting of a subset of the z slices within the projections as the intense E-cadherin signal within the Xenopus skin, see Videos S5 and S6. (B) Western blot and the graph of densitometry measures showing Daam1 and GAPDH protein levels for uninjected wild type (WT), Control (Standard morpholino), and Daam1 KD (Daam1 morpholino) injected embryos. Embryo lysates pooled from 10-20 NF stages 39/40 embryos (2 cell V2 embryo injections, approximately 2 embryos per lane). Individual band intensities plotted in arbitrary units (au) for Control (orange) and Daam1 KD (blue). The results are expressed as means ± S.E.M. from three independent experiments. nsP > 0.05, **P ≤ 0.01, analyzed by one way ANOVA. (C) TEM images show cross-section samples of the Control and Daam1-depleted nephrons; scale bars equal to 10 microns. b-b’ - close-up images corresponding to regions in the gray boxes; scale bars equal to 2 microns. The orange and blue dotted lines outline the morphology of intercellular junctions.
	Figure S6. The FH2 domain of Daam1 mediates nephron morphology, Related to Figure 6. Nephric progenitors expressing full-length Daam1 mRNA ultimately develop structurally normal nephrons compared to nephric progenitors expressing Daam1 FH2 mutant mRNA. Maximum projection confocal images of mature nephrons (NF stages 39/40) visualized by 3G8 and 4A6 antibodies (green) in embryos injected with 1ng Daam1 or 1ng Daam1FH2 mutant mRNA. Embryo microinjections were carried out at the 8 cell-stage into V2 blastomere fate-mapped to pronephric primordium. Scale bars equal to 50 microns. (A) Representative image of mature nephron from embryos injected with 1ng Daam1 mRNA. (B) Representative images of mature nephrons from embryos injected with 1ng Daam1 FH2 mutant mRNA. (C) The graph represents the quantification of the phenotypic severity of mature nephrons in embryos from A and B. Bars represent the mean percentage of nephrons in each phenotypic category for Daam1 control injected (orange) and Daam1 FH2 mutant (blue) embryos. nDaam1=54 embryos across 3 experiments and nDaam1FH2mutant = 66 embryos across 3 experiments. Error bars indicate S.E. of the mean. nsP > 0.05, **P ≤ 0.01, analyzed by unpaired t-test.
	Figure S7. E-cadherin localizes to shDaam1 depleted mature cell-cell junctions, Related to Figure 7. Maximum confocal projections showing E-cadherin (green), F-actin (magenta), and DAPI (cyan) in confluent the MDCK Control and shDaam1 KD cells. The first column shows merged images of individual color channels. Scale bars equal to 10 microns. The second column shows images of individual color channels for F-actin and E-cadherin. a'-a’’’ and b’-b’’’ are close-up images corresponding to regions shown in a and b, respectively. Scale bars equal to 5 microns.
	Figure 3. Effects of Daam1 depletion on the nephrogenic primordiumKidney-targeted microinjections using control or Daam1 MO combined with the membrane-GFP mRNA (used as a lineage tracer) were carried out to analyze the effect of Daam1 depletion on Xenopus developing kidney.(A) Maximum-projection confocal images of control or Daam1 KD embryos stained whole mount with antibodies against GFP (to detect the membrane-GFP tracer, green) and Lhx1 (to label nephric progenitors, white), in conjunction with phalloidin (to visualize F-actin, magenta). The top panels consist of the entire z stacks to show the cell positions within the entire kidney; scale bars, 50 μm. a’–a” and b’–b” are close-up images of the corresponding regions in white boxes consisting of a subset of the z slices to exclude the intense F-actin signal within the Xenopus skin; scale bars, 20 μm. The “seam” of enriched actin in the nephron center corresponds to the region where the lumen will form; see Videos S3 and S4.(B) Western blot and graph of densitometry measures showing Daam1 and GAPDH protein levels for uninjected wild-type (WT), control (standard MO)-injected, and Daam1 KD (Daam1 MO)-injected embryos. Embryo lysates were pooled from 10–20 V2-cell injected embryos at stages NF 28–32, and approximately 1/2 embryo was loaded per lane. Individual band intensities are normalized to the uninjected band and plotted in arbitrary units (a.u.) for uninjected (black), control (orange), and Daam1 KD (blue). The results are expressed as means ± SEM from four independent experiments. nsp > 0.05, ***p ≤ 0.001, analyzed by one-way ANOVA.(C) The graph showing the mean relative fluorescence intensity levels in a.u. of junctional F-actin in the nephric primordia of control (orange) and Daam1 KD (blue) embryos. ncontrol = 40 junctions and nDaam1 KD = 40 junctions on 2 embryos, denoted by open and closed circles, respectively. ****p ≤ 0.0001, analyzed by unpaired t test.(D–G) Morphometric analyses of control and Daam1-depleted nephric primordia. The thick bars represent the mean for control (orange) and Daam1-depleted (blue) embryos. ****p ≤ 0.0001, analyzed by unpaired t test. Graphs show comparison of control and Daam1-depleted nephric primordia of (D) the average number of Lhx1-positive nephron progenitors, where ncontrol = 5 embryos and nDaam1KD = 5 embryos; (E) the relative distance between nearest neighbors of Lhx1-positive nuclei; (F) the relative cell area; and (G) the relative circularity, where 1 represents a perfect circle.(E–G) ncontrol = 40 junctions on 2 embryos and nDaam1 KD = 40 junctions on 2 embryos, denoted by open and closed circles, respectively.
	Figure 4. Daam1 regulates assembly of junctional F-actin in developing nephronsF-actin dynamics at cell junctions of control and Daam1 KD developing nephrons expressing mCherry-Utrophin were assessed using FRAP. (A–E) Control (black, ntotal = 27 junctions, 1–5 junctions/embryo) and Daam1 KD (purple, 16 junctions; orange, 11 junctions; ntotal = 27 junctions, 1–5 junctions/embryo).(A) Percentage of junctions showing recovery of fluorescence after bleaching in control and Daam1 KD nephrons.(B) Typical time-lapse images of control and Daam1 KD cell junctions before and after photobleaching. In each image, the bleached region is highlighted with a circle (black, control junction showing recovery; purple, Daam1 KD junction showing recovery; orange, Daam1 KD junction showing no recovery of fluorescence after photobleaching). Scale bars, 10 μm.(C) Graph showing average recovery curves in a.u. obtained from individual best-fit plots for control (black) and Daam1 KD junctions with (purple) and without (orange) recovery of fluorescence after photobleaching.(D and E) Bar graphs comparing control and Daam1 KD profiles calculated from individual best-fit curves for control (black) and Daam1 KD junctions with recovery of fluorescence after photobleaching (purple). The dots represent analyzed junctions. Data represent the mean ± SEM from three independent experiments. The p values were analyzed by unpaired t test.(D) Bar graph of the relative half-times for F-actin.(E) Bar graph of the relative mobile fraction for F-actin.
	Figure 5. Daam1 promotes localization of junctional E-cadherin(A) Maximum-projection confocal images of whole-mount immunostaining of Xenopus nephric primordium labeled by Lhx1 (magenta) and E-cadherin (cyan) in control and Daam1 KD embryos; scale bars, 50 μm. a’–a” and b’–b” are close-up images of the corresponding regions in the white boxes; scale bars, 20 μm. The top panels consist of the entire z stacks to show nephric progenitors’ cell positions within the developing kidney. In contrast, images displayed in a’–a” and b’–b” contain a subset of the z slices to exclude the intense signal from E-cadherin expressed in the epithelium of the Xenopus skin. a’–b’, nephric cell progenitors labeled by Lhx1 (magenta) and E-cadherin (cyan); a”–b”, E-cadherin (cyan).(B) Violin plots depicting the relative fluorescence intensity in a.u. of junctional E-cadherin in the nephric primordia of control (orange) and Daam1 KD (blue). ncontrol = 88 junctions on 4 embryos and nDaam1 KD = 84 junctions on 4 embryos. Centerlines represents the median; limits show the first and third quartiles. ****p ≤ 0.0001, analyzed by unpaired t test.(C) Western blot and graph of densitometry measures in a.u. showing Daam1, E-cadherin, and GAPDH protein levels in uninjected WT, control (standard MO)-injected, and Daam1 KD (Daam1 MO)-injected embryos. Embryo lysates were pooled from 10–20 1-cell injected embryos at stages NF 11–12, and approximately 1/2 embryo was loaded per lane. Individual band intensities are normalized to the uninjected band and plotted in a.u. for uninjected (black), control (orange), and Daam1 KD (blue). The results are expressed as means ± SEM from four independent experiments. nsp > 0.05, ****p ≤ 0.0001, analyzed by one-way ANOVA.
	Figure 6. The FH2 domain of Daam1 mediates E-cadherin localization to cell junctions(A) Maximum-projection confocal images of whole-mount immunostaining showing E-cadherin (cyan) in Xenopus nephric primordium labeled by Lhx1 (magenta) expressing GFP-Daam1 or GFP-Daam1 FH2 mutant mRNA (labeled by anti-GFP, green); scale bars, 50 μm. a’–a” and b’–b” are close-up images of the corresponding regions in the white boxes; scale bars, 20 μm. The top panels consist of the entire z stacks to show nephric progenitors’ cell positions within the developing kidney. In contrast, images displayed in a–a’ and b–b’ contain a subset of the z slices to exclude the intense signal from E-cadherin expressed in the epithelium of the Xenopus skin. a’–b’, nephric cell progenitors labeled by Lhx1 (magenta), anti-GFP (green), and E-cadherin (cyan); a”–b”, E-cadherin (cyan).(B) Violin plots depicting the relative fluorescence intensity of junctional E-cadherin in nephric primordia expressing GFP-Daam1 (orange) and GFP-Daam1 FH2 mutant (blue) mRNA. nDaam1 = 60 junctions on 3 embryos and nDaam1FH2mutant = 55 junctions on 3 embryos. Centerlines show the median; limits show the first and third quartiles. ***p ≤ 0.001, analyzed by unpaired t test.(C) Western blot and the graph of densitometry measures showing the exogenous and endogenous protein levels of Daam1 in uninjected WT embryos and embryos injected with 1 ng Daam1 or Daam1 FH2 mutant mRNA. The non-specific band confirms equal loading. Embryo lysates were pooled from 10–20 1-cell injected embryos at stages NF 11–12, and approximately 1/2 embryo per lane. The results are expressed as means ± SEM from three independent experiments. Individual band intensities are normalized to the uninjected band and plotted in a.u. for uninjected (black), Daam1 (orange), and Daam1 FH2 mutant (blue). nsp > 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, analyzed by one-way ANOVA.
	Figure 7. Daam1-depleted MDCK cells display compromised localization of E-cadherin at cell-cell contacts and impaired cohesion during collective movement(A) Maximum-projection confocal images of E-cadherin (green), F-actin (magenta), and DAPI (cyan) in subconfluent MDCK control and shDaam1 KD cells. The first column shows merged images of individual color channels. Images of individual color channels for F-actin and E-cadherin are shown in the second and third columns, respectively. Scale bars, 50 μm. E-cadherin localization in nascent cell-cell contacts (marked by white brackets and shown enlarged in the corresponding white boxes; scale bars, 10 μm) is impaired in shDaam1 KD cells.(B) Western blot showing Daam1 and GAPDH protein levels in MDCK control and shDaam1 KD cells. Individual band intensities are normalized to the control band and plotted in a.u. for control (orange) and Daam1 KD (blue). The graph of densitometry measures represents the means ± SEM of two independent experiments. *p ≤ 0.05, analyzed by unpaired t test.(C–F) Summary of the wound healing experiments for MDCK control and Daam1 KD cells; see Video S7.(C) Daam1 depletion impairs wound closure. The graph represents the percentage of the wound surface area over time for control (purple) and Daam1 KD (orange) cells. Error bars indicate SE of the mean of 4 assays.(D–F) Manually tracking single cells’ migration paths during wound closure demonstrates that Daam1 organizes collective movement of MDCK epithelial monolayers by modulating the speed and directionality of individual cells. Daam1 results in increased velocity and random migration. ncontrol = 52 cells from 4 assays and nDaam1 KD = 42 cells tracked from 4 assays. Cells were tracked in 15-min increments for 12 h.(D) Violin plots represent migration velocity calculated from tracking traveled distances of single cells for control and Daam1 KD cells. Centerlines show the median; limits show the first and third quartiles. ***p ≤ 0.001, analyzed by unpaired t test.(E) Wind rose plot showing migration tracks of individual control and Daam1 KD cells.

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