Mechano-gradients drive morphogen-noise correction to ensure robust patterning

Abstract

Morphogen gradients instruct cells to pattern tissues. Although the mechanisms by which morphogens transduce chemical signals have been extensively studied, the roles and regulation of the physical communication between morphogen-receiver cells remain unclear. Here, we show that the Wnt/β-catenin-morphogen gradient, which patterns the embryonic anterior-posterior (AP) axis, generates intercellular tension gradients along the AP axis by controlling membrane cadherin levels in zebrafish embryos. This "mechano-gradient" is used for the cell competition-driven correction of noisy morphogen gradients. Naturally and artificially generated unfit cells, producing noisy Wnt/β-catenin gradients, induce local deformation of the mechano-gradients that activate mechanosensitive calcium channels in the neighboring fit cells, which then secrete annexin A1a to kill unfit cells. Thus, chemo-mechanical interconversion-mediated competitive communication between the morphogen-receiver cells ensures precise tissue patterning.

Fig. 1.. Wnt/β-catenin gradient generates mechano-gradient.

(A) Wnt/β-catenin activity gradient formation. (B) Membrane β-catenin, E-cadherin, F-actin, and p-MLC protein levels. Optical sagittal cross sections (dorsal side) of 8-hpf embryos. Scale bar, 50 μm. Right: Means ± SD; n = 10 embryos; across anterior-posterior (AP) axis. Relative fluorescent intensity in the anterior (A), central middle (M), and posterior (P) regions shown in the images on the left was quantified. (C) Wnt signaling and cortical actomyosin contraction underneath adherence junction. (D) RhoA activity gradients. Tg (HS:GFP-βcatCA) or Tg (HS:dkk1b-GFP) and E-cadherin–mRNA–injected zebrafish embryos and sibling embryos at 9 hpf. Scale bar, 50 μm. Right: Means ± SD; n = 10 embryos. Relative fluorescent intensity in the anterior (A), central middle (M), and posterior (P) regions was quantified. n.s., not significant. (E) Left: Laser dissection method. Bottom: Embryos injected with Ruby-Lifeact mRNA (magenta) before and after laser dissection. White arrows: Maximum recoil distances in (right) anterior, central middle, and posterior tissues. Scale bar, 20 μm. EVL, epithelial envelope layer (orange); deep cells (purple). Means ± SD (n = 31, 29, and 28 experiments). (F) d2EGFP in Wnt/β-catenin reporter (OTM:d2EGFP)–transgenic embryos (dorsal view) treated with DMSO (control) or 2.5 μM blebbistatin or injected with bcl-2 mRNA. Red arrowheads: Abnormally high or low Wnt/β-catenin activity. Scale bar, 200 μm. (G) otx2 and cdx4 markers in embryos treated with DMSO (control) or 2.5 μM blebbistatin. Scale bar, 200 μm. Left: AP tissue marker expression. (H) Percentage of embryos with normal or abnormal expression patterns (n = 100). Two-tailed one-way analysis of variance (ANOVA). (I) Zebrafish larvae (32 hpf) treated with DMSO (control) or 2.5 μM blebbistatin from 6 to 9 hpf. Red arrowheads: Abnormal cell proliferation. Scale bar, 500 μm. Right: Percentages of embryos with normal or abnormal morphology. The total number of embryos analyzed are shown above the graph (Fisher’s exact test). (J) Naturally generated “noise” cells are eliminated via E-cadherin gradient formation.

Fig. 2.. Unfit Wnt/β-catenin activity generates local tension change.

(A) Introduction of Wnt/β-catenin–unfit cells mosaically in embryos. (B) Active caspase-3 (green) in mosaic embryos expressing mKO2 alone (control) or with βcatCA or Axin1 (magenta) and treated with DMSO (control) or 2.5 μM blebbistatin. White arrowheads: Caspase-3–active cells. Scale bar, 50 μm. Right: Means ± SD (n = 20 embryos; three independent experiments) of GFP⁺caspase-3–active cell frequencies; unpaired two-tailed t test. (C) Embryos injected with RhoA-biosensor mRNA (RhoA-bio, green) and mosaically introduced with mKO2⁺ alone (control) or with βcatCA⁺ or Axin1⁺ cells (magenta). Right: RhoA-bio fluorescence intensities (means ± SD; n = 50 cells); two-tailed one-way ANOVA. Scale bars, 20 μm. (D) Left: Active caspase-3 (green) in mosaic embryos expressing either mKO2⁺RhoACA⁺ or mKO2⁺RhoADN⁺ (magenta). White arrowheads: Caspase-3–active cells. Scale bar, 50 μm. Right graphs: Means ± SD of caspase-3–active cell frequencies within the divided range along the AP axis of embryos mosaically expressing mKO2⁺RhoACA⁺ or mKO2⁺RhoADN⁺ (n = 5 embryos). (E) Quantification of angle and distance of neighboring cell movements. (F and G) Directions and distances of neighboring cell movements relative to βcatCA⁺ cells in the anterior region (F) or GSK3β⁺ cells in the posterior region (G) were quantified (top). Centroid of neighboring cell approaching unfit cells: negative direction (−D); centroid of cell that moved away: positive direction (+D). Bottom left: mKO2⁺βcatCA⁺ (F) or mKO2⁺GSK3β⁺ (G) cells (magenta). Plasma membrane (green). Lines: Distance between centroids of mKO2⁺ (yellow spot) and neighboring cells (white spot) at time points t (white lines) and t + 1 (red lines). Images were acquired in the anterior region (mKO2⁺βcatCA⁺) or posterior region (mKO2⁺GSK3β⁺). Scale bar, 20 μm. Bottom right: Means ± SD (n = 102 cells; three independent experiments) of change in distance between the centroid of mKO2⁺ cells and neighboring cells during 10 s, separated according to the movement direction; unpaired two-tailed t test. (H) Actomyosin contractile force changes around cells with abnormal Wnt/β-catenin signaling activity.

Fig. 3.. Local tension change activates mechanosensitive calcium channels.

(A) Mechanosensitive calcium channel activation by membrane stretching or curvature changes. (B) Active caspase-3 (green) in mosaic embryos expressing mKO2⁺ alone (control) or with βcatCA⁺ or Axin1⁺ (magenta), treated with or without 2 μM GsMTx4. White arrowheads: Caspase-3–active cells. Scale bar, 50 μm. Right: Means ± SD (n = 20 embryos; three independent experiments) of mKO2⁺caspase-3–active cell frequencies; unpaired two-tailed t test. (C) Inhibition of mechanosensitive channels blocks GFP-Smad2 translocation into unfit Wnt/β-catenin–active cell nuclei. Embryos injected with GFP-Smad2 mRNA (200 pg; green) and mosaically introduced with mKO2⁺ alone (control) or βcatCA⁺ cells (magenta), treated with or without 2 μM GsMTx4. Scale bar, 20 μm. Right: Nuclear/cytoplasmic GFP-Smad2 ratio. Whiskers: Minimum and maximum values. Dot: One cell (means ± SD; n = 50 cells; three independent experiments). Unpaired two-tailed t test. (D) Schematic illustration of the time course to observe calcium sparks. (E) Schematic illustration of quantification of calcium sparks. (F) Rapid calcium sparks in neighboring cells around unfit cells. mKO2⁺ alone (control) or βcatCA⁺, Axin1⁺, or E-cadherin–overexpressing (E-cad⁺; magenta) mosaic embryos were injected with GCaMP7 mRNA. The timing relative to the first image: White text. Three independent experiments. Scale bar, 50 μm. (G) Means ± SD (n = 20 embryos; three independent experiments) of the number of calcium sparks around unfit cells. All unfit cells were induced and quantified under the same conditions. Quantification: Anterior (control, βcatCA⁺, and E-cad⁺) or posterior (Axin1⁺) region. Two-tailed one-way ANOVA. (H) mKO2⁺βcatCA⁺-overexpressing (magenta) mosaic embryos were injected with GCaMP7 mRNA and treated with or without 2 μM GsMTx4. The timing relative to the first image: White text. Scale bar, 50 μm. (I) Means ± SD (n = 20 embryos; three independent experiments) of the number of calcium sparks in the anterior region. Quantified under the same conditions as (G). Two-tailed one-way ANOVA.

Fig. 4.. ANXA1 mediates unfit cell killing.

(A) Left: Fluorescence-activated cell sorting (FACS) of embryos. Right: Differentially expressed genes (DEGs) in GFP⁻ cells from GFP⁺ or GFP⁺βcatCA⁺-mosaic embryos. Red: Up-regulated DEGs. Blue: Down-regulated DEGs. Gray: Non-DEGs. (B) Endogenous annexina1a (anxa1) mRNA levels in mosaic embryos expressing mKO2⁺alone (control) or with βcatCA⁺ or Axin1⁺ (magenta) and treated with DMSO (control), 2.5 μM blebbistatin, or 2 μM GsMTx4. Black arrowheads: Neighbors expressing anxa1. Scale bars, 50 μm. (C) Number of anxa1-expressing cells around the mKO2⁺cells. Graph: Means ± SD (n = 20 unfit cells; three independent experiments) of anxa1(⁺) cells around unfit cells. Quantification: Anterior (control and βcatCA⁺) or posterior (Axin1⁺) region. (D) Embryos injected with GFP-Smad2 mRNA and mosaically introduced with mKO2⁺ alone (control) or with βcatCA⁺cells with control or anxa1 morpholino oligonucleotides (MO). Scale bar, 50 μm. Black arrowheads: mKO2⁺ cells. Right: Nuclear/cytoplasmic GFP-Smad2 ratio. Means ± SD; n = 50 cells; three independent experiments. (E) Active caspase-3 (green) in mosaic embryos expressing mKO2⁺ alone (control) or with βcatCA⁺ co-injected with control or anxa1 MO. Black arrowheads: Caspase-3–active cells. Scale bars, 50 μm. Right: Means ± SD (n = 20 embryos; three independent experiments) of mKO2⁺caspase-3–active cell frequencies. (F) Cells expressing mKO2⁺ annexin A1 (ANXA1⁺) in GFP-Smad2 mRNA–injected embryos. Scale bar, 50 μm. Right: Nuclear/cytoplasmic ratio of GFP-Smad2 intensity in neighboring cells around mKO2⁺ alone (control) and ANXA1⁺ cells. n = 10 embryos from three independent experiments. (G) Embryos mosaically transfected with mKO2⁺ alone (control) or with ANXA1⁺ cells. White arrowheads: ANXA1⁺ cells. Black arrowheads: Caspase-3–active cells. Scale bar, 50 μm. Right: Means ± SD (n = 20 embryos; three independent experiments) of mKO2⁺caspase-3–active cell frequencies. Unpaired two-tailed t test. (H) Left: Active caspase-3 around mKO2⁺ANXA1⁺ cells induced very few numbers. Right: Means ± SD (n = 20 ANXA1⁺ cells; three independent experiments) of the number of mKO2⁺caspase-3–active cells around ANXA1⁺ cells according to the distance from ANXA1⁺ cells. [(C) to (F) and (H)] Two-tailed one-way ANOVA.

Fig. 5.. Chemo-mechanical interconversion–driven cell competition ensures robust formation of morphogen gradients.

(A) d2EGFP in Tg (OTM:d2EGFP) embryos (dorsal view) treated with or without 2 μM GsMTx4 (left) and injected with control or anxa1 MO (right). Red arrowheads: Abnormally high Wnt/β-catenin activity. Blue arrowheads: Abnormally low Wnt/β-catenin activity. Scale bars, 200 μm. (B) otx2 and cdx4 expression in embryos treated with or without 2 μM GsMTx4 (left) and injected with control MO or anxa1a MO (right). Scale bars, 200 μm. Bottom: Brain AP marker expression patterns. (C) Percentage of embryos with normal or abnormal expression patterns. (D and E) GsMTx4 treatment or anxa1a KD induces abnormal morphogenesis. Images show 32-hpf zebrafish larvae treated with or without 2 μM GsMTx4 from 6 to 9 hpf (D) or injected with control MO or anxa1a MO (E). Red arrow indicates abnormal cell proliferation. Scale bars, 500 μm. Percentages of embryos with normal or abnormal morphology are shown in the graph on the right. The numbers shown above the graph indicate the total number of embryos analyzed (Fisher’s exact test). (F) Mechano-gradient–mediated correction of noisy morphogen gradients.