Developmental Biology

Bidirectional translocation of actomyosin drives epithelial invagination in ascidian siphon tube morphogenesis

Jinghan Qiao
Pengyu Yu
Hongzhe Peng
Wenjie Shi
Bo Li author has email address
Bo Dong author has email address

Fang Zongxi Center, MoE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China
Institute of Biomechanics and Medical Engineering, Applied Mechanics Laboratory, Department of Engineering Mechanics, Tsinghua University, Beijing, China
Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao, China

https://doi.org/10.7554/eLife.108588.1

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Figures and data

Cellular processes of Ciona atrial siphon tube invagination
(A) Representative images of atrial siphon morphogenesis in Ciona embryos from 13.5 hpf to 18 hpf. Scale bar: 10 μm (B) Measurement parameters of the Ciona atrial siphon. The cell undergoing the most prominent apical constriction at the center of invagination was defined as the center cell (0). The adjacent cells on the left and right were defined sequentially as −1, −2, −3, −4 and +1, +2, +3, +4, respectively. (C) Quantification of the invagination depth in the atrial siphon of Ciona embryos. Red lines indicate linear regression fits of invagination depth during the initial (13.5-16 hpf, k = 0.2617) and accelerated (16-18 hpf, k = 2.7920) stages. n = 20. (D, E) Quantification of the center cell height and apical-to-basal area ratio at the atrial siphon of Ciona embryos. The blue-shaded region represents the initial stage, while the orange-shaded region indicates the accelerated stage. n= 20.

Bidirectional translocation of actomyosin between apical and lateral domains during atrial siphon tube invagination
(A) Representative images of Ciona embryos stained for active myosin II (anti-pS19 MRLC,red) and F-actin (green) at different stages of atrial siphon invagination. The heatmap color scale represents the fluorescence intensity of myosin II signal (8-bit grayscale range: 0-255), with red indicating the highest intensity. Scale bar: 10 μm. (B) Normalized fluorescence intensity of active myosin II (anti-pS19 MRLC) and F-actin at the apical and lateral regions of center cells during different stages of atrial siphon invagination (basal level set to 1). **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 20. (C) Schematic model illustrating the mechanical forces driving atrial siphon primordium invagination. Brown arrows indicate the direction of contractile forces; red areas depict active myosin II localization.

Modulation of atrial siphon invagination by overexpression of myosin mutants
(A) Representative images of Ciona atrial siphon primordium expressing wild-type and mutant MRLC constructs. Scale bar: 10 μm. (B) Quantification of invagination depth and the center cell height in MRLC (T18ES19E), MRLC (T18AS19A), and MRLC groups. The blue-shaded region represents the initial stage, while the orange-shaded region indicates the accelerated stage. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Disruption of contractile forces during rapid invagination of the Ciona atrial siphon using an optogenetic system
(A) Schematic diagram depicting the structure and mechanism of the MLCP-BcLOV4 system. The PP1C::MYPT169::BcLOV4::mCherry::NES fusion protein is initially dispersed in the cytoplasm. Upon exposure to blue light, BcLOV4 undergoes a conformational change, allowing it to interact electrostatically with the plasma membrane. This leads to the recruitment of the PP1C and MYPT169 components, subunits of myosin light chain phosphatase (MLCP), to the membrane, where they reduce myosin activity. (B) Representative images of developmental progression in the MLCP-BcLOV4 expression group exposed to blue light for 1 h, and in the dark control group maintained in darkness for 1 h. Scale bar: 10 μm (C, D) Quantification of invagination depth and the center cell height in the MLCP-BcLOV4 expression group, the dark control group and MLCP control group (Figure 4—figure supplement 1). *p < 0.05, ***p < 0.001, ****p < 0.0001.

Simulations and analysis based on vertex model
(A) Schematic of the cell-based vertex model. The epithelial tissue is constructed by the apical, basal, and lateral vertices and edges. The effective energy U takes into cell area constraint (modulus K_A), passive cortical contraction (coefficient K_C), tissue surface bending (modulus K_B), basal interaction (coefficient k_bm), and apical, basal and lateral active tensions ( Γ^a,Γ^l and Γ^b, respectively). r_i = (r_xi, r_yi) denotes the Cartesian coordinates of vertex i.A_J, L_J, and are the area, perimeter, and length of apical, basal, lateral edges of cell J. θ_i represents the angle between adjacent apical or basal edges connected to vertex i. A₀ is the reference area and h₀ is the initial cell height. (B) Temporal evolution of actomyosin intensities at the apical, lateral, and basal domains of the center cell in the model, parameterized based on experimentally measured F-actin distribution. The blue-shaded and orange-shaded regions represent the apical constriction stage, and the invagination stage, respectively. (C) Evolution curves of invagination depth (blue) and center cell height (orange) in simulations. Dots represent corresponding experimentally measured values. (D) Snapshots at specific timepoints during simulation progression. The colors of edges represent actomyosin intensity. (E, F) Invagination depth (E) and center cell height (F) regulated by changes in actomyosin intensities under the scaling factor α at both apical and lateral regions of all cells. The data points are from the mutant experiments in Figure 3B. Figure 5—video 1. Vertex model simulation. Edge colors represent actomyosin intensity. Bottom row (left to right):
Evolution curves of invagination depth;
Evolution curves of center cell height;
Temporal evolution of actomyosin intensities at the apical, lateral, and basal domains of the center cell in the model, parameterized based on experimentally measured F-actin distribution.

Simulations and analysis based on vertex model
(A) Schematic of the cell-based vertex model. The epithelial tissue is constructed by the apical, basal, and lateral vertices and edges. The effective energy U takes into cell area constraint (modulus K_A), passive cortical contraction (coefficient K_C), tissue surface bending (modulus K_B), basal interaction (coefficient k_bm), and apical, basal and lateral active tensions ( Γ^a,Γ^l and Γ^b, respectively). r_i = (r_xi, r_yi) denotes the Cartesian coordinates of vertex i.A_J, L_J, and are the area, perimeter, and length of apical, basal, lateral edges of cell J. θ_i represents the angle between adjacent apical or basal edges connected to vertex i. A₀ is the reference area and h₀ is the initial cell height. (B) Temporal evolution of actomyosin intensities at the apical, lateral, and basal domains of the center cell in the model, parameterized based on experimentally measured F-actin distribution. The blue-shaded and orange-shaded regions represent the apical constriction stage, and the invagination stage, respectively. (C) Evolution curves of invagination depth (blue) and center cell height (orange) in simulations. Dots represent corresponding experimentally measured values. (D) Snapshots at specific timepoints during simulation progression. The colors of edges represent actomyosin intensity. (E, F) Invagination depth (E) and center cell height (F) regulated by changes in actomyosin intensities under the scaling factor α at both apical and lateral regions of all cells. The data points are from the mutant experiments in Figure 3B. Figure 5—video 1. Vertex model simulation. Edge colors represent actomyosin intensity. Bottom row (left to right):
Evolution curves of invagination depth;
Evolution curves of center cell height;
Temporal evolution of actomyosin intensities at the apical, lateral, and basal domains of the center cell in the model, parameterized based on experimentally measured F-actin distribution.

Mechanics of epithelial invagination revealed by vertex model simulations
(A, C) Invagination depth (A) and center cell height (C) under varying apical actomyosin intensity represented by a scaling factor α_a, with the lateral intensity unchanged. A Schematic illustration of the bending mode induced by apicobasal imbalance is presented. A snapshot of a specific inversed evagination modeling under α_a = 0.1 is shown. (B, D) Invagination depth (B) and center cell height (D) under varying lateral actomyosin intensity represented by a scaling factor α_l, with the apical intensity unchanged. A Schematic illustration of the contraction mode induced by lateral tension is presented. (E) Temporal evolution of the sum S_exp and ratio R_exp of apical and lateral actomyosin intensities in the center cell in experiments. (F) Varying sum S_c and ratio R_c of actomyosin intensities regulated by α and β in simulations. (G, H) Invagination depth (G) and center cell height (H) under varying α and β.

Quantification of F-actin intensity and intercellular distance during Ciona atrial siphon morphogenesis
(A) Normalized F-actin intensity at the apical and lateral regions of center cells during Ciona atrial siphon morphogenesis (basal level set to 1). (B) Quantification of the linear distance between the −3/-4 and +3/+4 cell junctions at the apical or basal surface in the atrial siphon of Ciona embryos. The blue-shaded region represents the initial stage, while the orange-shaded region indicates the accelerated stage. Representative images are shown in Figure 1A. n = 20.

EdU and TUNEL staining during Ciona atrial siphon morphogenesis
(A) Representative images of EdU staining at 14-15 hpf. Orange: EdU-positive nuclei indicating cell proliferation. Blue: DAPI. No EdU signal was detected in the atrial siphon primordium (white dashed outline). Scale bar: 10 μm. n = 10. (B1-3) Representative images of TUNEL staining at 15 hpf. (B1) Positive control: DNase I pretreatment (20 U/mL, 10 min) induced DNA fragmentation. (B2) Negative control: staining performed without terminal deoxynucleotidyl transferase (TdT) enzyme. (B3) Experimental group: no detectable TUNEL signal in the atrial siphon primordium (white dashed outline). Scale bar: 10 μm. n = 10.

MRCL control group in the optogenetic experiment
(A) Schematic diagram depicting the structure and mechanism of the MLCP control system. The PP1C::MYPT169::mCherry::NES fusion protein remained diffuse in the cytoplasm under light exposure and failed to function. (B) Representative images of developmental progression in the MLCP control group exposed to blue light for 1 h. Scale bar: 10 μm. Figure 4—video 1. The developmental processes of the MLCP control group under blue light illumination for 1 h. Scale bar: 10 μm. Figure 4—video 2. The developmental processes of MLCP-BcLOV4-expressed group under blue light illumination for 1 h. Scale bar: 10 μm.

Key resource table

Primer sequences used in this study

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