Johansson et al., 2019 - Dkk1 Controls Cell-Cell Interaction through Regulation of Non-nuclear β-Catenin Pools. Developmental Cell   51(6):775-786.e3 Full text @ Dev. Cell

Figure 1

Dkk1 Disrupts Collective Cell Migration Independently of Cell Fate

(A) Expression of dkk1 (green) and goosecoid (gsc, red) from 30% to 70% epiboly (EB), using RNAscope. Confocal imaging, dorsal view with anterior to the top. Maximum projection in the left panels, 5 μm z stacks at higher magnification on the right. Single-plane image at 80% EB shows dkk1 transcripts overlapping with gsc transcripts at the boundary of the gsc-expressing region. Scale bar, 10 μm.

(B) Progression of axial cells. Confocal maximum projections from shield to 80% epiboly stages in transgenic Tg(gsc:GFP) embryos, with or without injection of Dkk1 RNA. Embryos are shown in dorsal view with anterior to the top. Scale bar, 100 μm. See also Video S1.

(C) Maximum projections of anterior axial cells from time-lapse confocal imaging of Tg(gsc:GFP) embryos showing a lack of progression of the prechordal plate (green) in Dkk1-expressing embryos. Embryos were imaged at 75% epiboly for a time (t) period of 48 min. The dashed line shows the position of the anteriormost prechordal plate cells at the first and last time point (t = 0 min and t = 48 min, respectively). Scale bar, 20 μm. See also Video S2.

(D) Tracking of migrating axial cells from Tg(gsc:GFP) embryo time-lapse videos from 70% to 90% epiboly at 2-min intervals (n = 3 for each condition). Tracks from individual cells from one representative embryo for each condition are shown. Axial cells in Dkk1-expressing embryos move slower and exhibit reduced persistence and displacement. p < 0.05 and ∗∗p < 0.01; p values were calculated using linear mixed-effects models.

(E) Dkk1 cell migration behavior is independent of transcriptional regulation of β-catenin target genes. 24 h post-fertilization (hpf) embryos (left) and confocal maximum projections of Tg(gsc:GFP) embryos at 80% epiboly (EB) (right). The Dkk1-induced cell migration defect persists in embryos at 80% EB lacking Tcf3a (Tcf3 KD). See also Figures S1A and S1B.

Figure 2

Dkk1 Controls Organization of Filamentous Actin

(A) Single-plane confocal images of the axis in Tg(gsc:GFP) embryos (green axis) labeled with phalloidin (red) at the tail bud (TB) stage show disruption of filamentous actin organization in Dkk1-expressing embryos (n = 12). Expansion of axial cell fate induced by knock down (KD) of the nodal antagonists Lefty1 and Lefty2 (n = 6) or upregulation of Wnt target gene transcription by Tcf3 KD (n = 7) has no effect on axial polarity. Conversely, the loss of Dkk1 (n = 5) results in a compact hyperpolarized axis. Scale bar, 20 μm.

(B) The loss of filamentous actin boundary organization induced by Dkk1 is not dependent on planar cell polarity (PCP) signaling. Single-plane confocal images of Tg(gsc:GFP) embryos (green axis) labeled with phalloidin (red) at the late tail bud (LB) stage show a distinct convergence extension phenotype in Vangl2 knock down (Vangl KD) embryos (n = 8) compared to control embryos (n = 11), distinct from the Dkk1-induced phenotype (n = 9). Vangl KD embryos injected with low levels of Dkk1 RNA display an additive phenotype (n = 11). Scale bar, 20 μm.

(C) Axial boundary straightness was quantified in phalloidin-labeled Tg(gsc:GFP) embryos at the late tail bud (LB) stage. Downregulation of Vangl2 (n = 16 boundaries) results in a straighter axial boundary than control (n = 22 boundaries). Reduction of boundary straightness in Dkk1-RNA-injected embryos (n = 18 boundaries) is not rescued by downregulation of Vangl2 (n = 16 boundaries). p > 0.05, ns, not significant, p < 0.05, ∗∗∗p < 0.001. Scale bar: 20 μm.

Figure 3

Dkk1 Reduces Cell Adhesion

(A) Single-plane confocal imaging, dorsal view, of tail bud stage embryos (n = 6 of each condition) ubiquitously expressing EGFP-tagged myosin light chain (MyosinEGFP, green) stained with phalloidin (red) labeling filamentous actin. Scale bar, 20 μm.

(B) Single-plane confocal images of axial cells in Tg(gsc:GFP) embryos (green axis) acquired at the tail bud (TB) stage, showing lack of organized cell-cell adhesions by E-cadherin antibody staining (red) in Dkk1-expressing embryos (n = 8 for each condition). Scale bar, 20 μm.

(C) Distribution of endogenous β-catenin (red) in Tg(gsc:GFP) embryos (green axis). Dorsal view maximum projections of embryos were acquired at 80% epiboly (EB). Dashed squares indicate the area of higher magnification shown on the right panels in single-plane confocal images. Single-plane higher magnification confocal images reveal loss of cell-cell adhesion integrity in axial cells of Dkk1-RNA-injected embryos. Control (Ctrl) n = 14 and Dkk1 RNA n = 16. Scale bar, 20 μm.

(D) Tg(gsc:GFP) embryos stained with anti-E-cadherin (red). Dashed squares show one example of six neighboring cells in control and Dkk1-expressing embryos, used for quantification of E-cadherin distribution across boundaries. Dkk1-expressing embryos exhibit a diffuse distribution of E-cadherin (red) compared to the control, indicating a loss of cell-cell adhesion (n = 5 for each condition). Scale bar, 20 μm.

(E) Quantification of E-cadherin distribution across boundaries between neighboring axial (green) cells. Normalized intensity distributions across cell-cell boundaries show that Dkk1 expression reduces E-cadherin intensity at cell boundaries and broadens lateral distribution of E-cadherin intensity. Normalized intensity distributions were calculated for boundaries between six neighboring cells in five embryos for both control and Dkk1-expressing embryos. ∗∗∗p < 0.001. p value was calculated using the Kolmogorov-Smirnov test.

Figure 4

Dkk1 Impacts Cell Shape and Orientation

(A) Axial cell angles were measured in dorsally oriented tail bud stage Tg(gsc:GFP) embryos (green axis) expressing membrane mCherryCAAX (red) in control embryos (n = 6,227 cells) and embryos injected with Dkk1 RNA (n = 6,176 cells). p < 0.05. Scale bar, 20 μm.

(B) Dkk1-RNA-injected embryos (n = 6,176 cells) are less elongated and have a larger cell area than control embryos (n = 6,227 cells). Aspect ratio and cell area were quantified in Tg(gsc:GFP) embryos in (A) expressing membrane mCherry. ∗∗∗p < 0.001.

Figure 5

Loss of Dkk1 Increases Cell Adhesion and Polarization

(A and B) Single-plane confocal images of Tg(gsc:GFP) embryos (green axis) at 80% epiboly (EB) stained with phalloidin (red). (A) Prechordal plate leading edge. A stitch-like pattern of actin distribution in cells surrounding the prechordal plate is evident in Dkk1 knock down (KD) embryos (arrowheads). (B) Filamentous actin along the anterior half of the axis. Dashed rectangles indicate the area of higher magnification of the single-plane confocal images shown on the right panels. Filamentous actin is highly concentrated in discrete puncta at the plasma membrane in the paraxial cells of Dkk1 KD embryos (arrowheads) (n = 10–15 for each condition). Scale bar, 20 μm.

(C) Single-plane confocal images of β-catenin antibody-stained Tg(gsc:GFP) embryos at the tail bud (TB) stage show strong patches of endogenous β-catenin (red) along the plasma membrane between connected cells. Dashed squares indicate the area of higher magnification shown on the right side of single-plane confocal images. Control (Ctrl) n = 5 and Dkk1 KD n = 6. Scale bar, 20 μm.

EXPRESSION / LABELING:
Antibody:
Fish:
Knockdown Reagent:
Anatomical Term:
Stage: Bud

Figure 6

Ubiquitous Expression of GFP-Tagged Dkk1 Reveals Polarized Distribution of Dkk1 Protein at Tissue and Subcellular Levels

(A) Live Tg(hsp70l:dkk1b-GFP) embryos expressing Dkk1 (green) at different epiboly (EB) stages. On the right panel, higher magnification single-plane confocal image, of the area highlighted by the dashed frame, shows exclusion of Dkk1GFP from the axis, despite abundant extracellular Dkk1GFP in the surrounding non-axial tissue. The extracellular non-bound pool of Dkk1GFP is not detectable after fixation. See also Figure S2. Scale bar, 20 μm.

(B) Subcellular localization of Dkk1GFP in 80% epiboly (EB) and tail bud (TB) stage fixed embryos. Single-plane high magnification confocal images are shown of the region of interest in dashed white frames. Localization of Dkk1GFP to actin-rich membrane junctions was visualized by coexpression with red fluorescent protein-tagged actin-binding utrophin (RFP-UTR, red). Arrow highlights Dkk1GFP and RFP-UTR colocalization at a cell-cell junction. n = 6 for each condition. Scale bar, 20 μm.

(C) Dkk1GFP colocalizes with plasma membrane-associated β-catenin (red)-positive puncta. Embryos were fixed shortly after heat shock. Arrowheads highlight β-catenin positive puncta (red), which colocalize with Dkk1GFP (arrows).

(C’) Quantification of colocalization (n = 10). Mean Pearson’s R value is 0.60 for Dkk1-β-catenin and 0.03 for the randomized control. Scale bar, 20 μm.

Figure 7

Dkk1 Sequesters β-Catenin at the Plasma Membrane

(A–D) Sibling and axin1 mutant embryos at 24 h post-fertilization (hpf) (left column) and 80% epiboly (EB) with (B) and (D) or without (A) and (C) Dkk1 misexpression. 80% EB embryos were stained with an antibody against β-catenin (green) to assess endogenous β-catenin distribution in different subcellular compartments in various regions of the embryo. White-bordered squares in the second column indicate the location of single-plane confocal images in three regions of the embryo shown at a higher magnification in the three last columns. Scale bar, 20 μm. (A) Sibling embryos show patterned distribution of β-catenin (green) in different regions of the embryo at tissue and subcellular levels. In the anterior region β-catenin is detected exclusively at the plasma membrane, while high levels are seen in both nuclei and the plasma membrane, laterally. Axial cells show a low level of β-catenin in both nuclei and plasma membrane. (B) Dkk1-expressing sibling embryos display large heads and extension defects at 24 hpf and show a loss of nuclear β-catenin (green) in anterior, lateral, and axial regions of the embryo but maintain β-catenin at the plasma membrane despite the loss of cell-cell adhesion. Arrowheads point to diffuse membrane morphology. (C) axin1 null mutants show a loss of anterior fate at 24 hpf and β-catenin-positive (green) nuclei in the anterior region at 80% EB. Contrary to the current model, these gastrula embryos display the same relative subcellular distribution of β-catenin found in normal siblings across the three areas measured. (D) Despite the expected lack of telencephalon and eyes, the Dkk1-expressing axin1 mutants show an overall AP organization similar to Dkk1-expressing embryos at 24 hpf. Similar to Dkk1-RNA-injected siblings, β-catenin was retained at the plasma membrane, and cells displayed a loss of cell-cell adhesion. Arrowheads point to diffuse membrane morphology.

(E) Nucleus to plasma membrane (PM) β-catenin (green) fluorescence intensity ratios quantified in anterior, lateral, and axial cells in sibling (Sib) and axin1 mutant (axin−/−) embryos, with or without Dkk1 expression. A significantly higher proportion of β-catenin is present at the PM in Dkk1-expressing Sibs and mutants in the lateral and axial regions than control embryos. Fluorescence intensity ratios were measured in 10 cells per region in each embryo (Sib, n = 2; Sib + Dkk1 RNA, n = 2; axin−/−, n = 1; and axin−/− + Dkk1 RNA, n = 1). p > 0.05 ns,p < 0.05, and ∗∗∗p < 0.001.

(F) Absolute levels of β-catenin nuclear expression, calculated by fluorescence intensity in 10 cells in each of the three regions per embryo. A, anterior; L, lateral; and Ax, axial. p > 0.05 ns, ∗∗p < 0.01, ∗∗∗p < 0.001.

Acknowledgments:
ZFIN wishes to thank the journal Developmental Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Cell, 51(6), Johansson, M., Giger, F.A., Fielding, T., Houart, C., Dkk1 Controls Cell-Cell Interaction through Regulation of Non-nuclear β-Catenin Pools, 775-786.e3, Copyright (2019) with permission from Elsevier. Full text @ Dev. Cell