Christodoulou N , Skourides PA .

             neural tube closure
             mesoderm development

	FIGURE 1. Neural plate and somitic mesoderm morphogenesis are temporally linked during neurulation. (A) Transverse cross sections of representative neurula stage Xenopus embryos. Numbering follows neural tube closure progression judged by neural plate mediolateral length. White dotted outline: Somitic mesoderm. Blue dotted line: Neural plate. Scale bars: 100 μm. (B) Quantification of neural plate width relative to somitic mesoderm convergent extension index (Somitic mesoderm dorsovetral/mediolateral length). Pearson correlation; ****p < .0001; n = 27 embryos. Anti-correlation between neural plate width and somitic mesoderm convergent extension index shows that neural tube closure progression, reduced neural plate width, is highly correlated with progression of somitic mesoderm convergent extension.
	FIGURE 2. Somitic mesoderm convergent extension occurs independently from neural plate morphogenetic program and surface ectoderm development. (A) Representative images of transverse cross sections from control and Vangl2 neural plate morphant stage 16 embryos. Control and Vangl2 morphant embryos are siblings raised side by side. Orange outline: Somitic mesoderm. Purple Outine: neural plate. Double headed arrows: neural plate width. Scale bar: 100 μm (B) Quantification of somitic mesoderm convergent extension in control and Vangl2 neural plate morphant embryos. Two-sided unpaired Student’s t-test; ns, p = .591; mean ± SEM; n = 6 embryos. (C) Representative images of transverse cross sections from Shroom3 unilateral morphant neural plate stage 16 embryo. Orange outline: Somitic mesoderm. Purple arrows: neural plate hinge point at the control neural plate side. Red arrows: absence of neural plate hinge point at the Shroom3 morphant side. Scale bar: 100 μm (C’) Magnified image from neural plate control and Shroom3 morphant side. Orange outlines: neural plate cells. Cells at the control side acquire a wedge shape, characteristic of apical constriction. Cells at the Shroom3 morphant side fail to undergo apical constriction and remain cuboidal. (D) Quantification of somitic mesoderm convergent extension in control and Shoom3 morphant neural plate. Two-sided paired Student’s t-test; ns, p = .516 n = 9 embryos (E) Representative images of transverse cross sections from a stage 16 embryo expressing FF-GFP at the one side of the surface ectoderm. Orange outline: Somitic mesoderm. Purple arrows: neural plate hinge point at the control neural plate side. Red arrows: absence of neural plate hinge point at the FF-GFP surface ectoderm side. Scale bar: 100 μm (E’) Zoomed images of control and FF-GFP surface ectoderm. Double arrowheads: surface ectoderm thickness. Control surface ectoderm is a double layered tissue while the FF-GFP surface ectoderm is composed of multiple cell layers. Phalloidin staining in panel E is weak due to high autofluorescence of yolk platelets within the cells. (F) Quantification of somitic mesoderm convergent extension in control and FF-GFP side from embryos displaying unilateral surface ectoderm FF-GFP expression. Two-sided paired Student’s t-test; ns, p = 0,683; n = 8 embryos SM, somitic mesoderm; CE, convergent extension.
	FIGURE 3. Somitic mesoderm convergent extension in necessary for neural tube closure. (A) Transverse cross section a representative Wee2 morphant stage 16 embryo. Yellow double arrowhead: somite dorsoventral (D/V) length. Purple double arrowhead: mediolateral (M/L) somite length. Asterisk indicated the Wee2 morphant side. Red arrows: hinge point at the neural plate side overlying normal somitic mesoderm. (D) dorsal. V, ventral. (A’) Zoomed images of control and Wee2 morphant somitic mesoderm. Scale bar:100 μm (B) Quantification of somitic mesoderm convergent extension index in control Wee2 morphant somitic mesoderm. Two-sided paired Student’s t-test; ****p < .0001; n = 14 embryos. (C) Stills showing the dorsal side of the embryo from a time lapse recording of a neurula embryo with unilateral Wee2 morphant somitic mesoderm. Asterisk: Wee2 morphant side. Red dotted line: neural plate midline. Green arrowheads: Neural plate boundary at the side overlying control somitic mesoderm. Orange arrowheads: Neural plate boundary at the side overlying Wee2 morphant somitic mesoderm. NTC is delayed at the neural plate side overlying Wee2 morphant somitic mesoderm (purple arrowheads). Scale bar: 100 μm (D) Quantification of neural plate mediolateral width extension. Neural plate is wider when the underlying somitic mesoderm is targeted with Wee2 morpholino. Two-sided paired Student’s t-test; ****p < .0001; n = 32 embryos. (E) Quantification of neural plate width over time. 3 regions along the anteroposterior axis of a neurula embryo were followed over time. Neural plate width at the side overlying Wee2 morphant somitic mesoderm remains wider as neurulation progresses. (F) Dorsal view from a tracked time lapse recording for 1 h time period, showing surface ectoderm boundary cells movement during neural tube closure. Asterisk: Wee2 morphant somitic mesoderm side. (A) Anterior; P: Posterior. Scale bar: 100 μm (G) Quantification of surface ectoderm boundary cells velocity as a proxy for neural plate hinge point movement. The velocity of surface ectoderm boundary cells is significantly reduced when somitic mesoderm convergent extension is impaired. Two-sided unpaired Student’s t-test; ****p < .0001; mean ± SEM; n = 15 cells.
	FIGURE 4. Hinge point formation depend on proper somitic mesoderm morphogenesis. (A) Dorsal view of a representative neurula stage 16 embryo stained with Phalloidin (F-actin). Red arrows: F-actin accumulation at the neural plate boundary denotes hinge point formation. Purple arrows: Hinge point formation is impaired at the neural plate side overlying Wee2 morphant somitic medoderm. Asterisk: Side overlying Wee2 morphant somitic mesoderm. Scale bar: 100 μm (B) Normalized F-actin intensity profile along the mediolateral neural plate axis. n = 10 embryos. Peak intensity indicates the position of the hinge point. Note that F-actin accumulation at the side overlying somitic mesoderm injected with Wee2 morpholino is impaired. Inset: Representative example of neural plate of an embryo with unilateral Wee2 morphant somitic mesoderm. Asterisk: Wee2 morphant side. Purple box was used to plot F-actin intensity profile along the neural plate mediolateral axis. Scale bar: 100 μm (C) Quantification of hinge point formation. χ2 test; ****p < .0001 n = 15 embryos. (D) Stills from a time lapse recording of a neurula embryo expressing Utrophin GFP. Green dotted line: midline. Cyan arrows: Neural plate surface ectoderm boundary at the side overlying control somitic mesoderm. Red arrows: Neural plate/surface ectoderm boundary at the neural plate side overlying Wee2 morphant somitic mesoderm. F-actin accumulation characteristic of hinge point formation occurs only at the neural plate/surface ectoderm boundary overlying control somitic mesoderm Scale bar: 50 μm.
	FIGURE 5. Apical constriction within the neuroepithelium is defective when somitic mesoderm morphogenesis is impaired. (A) Maximum intensity projection image of a representative stage 16 embryo. Asterisk: Wee2 somitic mesoderm morphant side. Scale bar: 100 μm (A’) Zoomed images of control neuroepithelial cells and neuroepithelial cells overlying Wee2 morphant somitic mesoderm. The apical surface area of neural plate cells overlying the morphant somitic mesoderm side is larger compared to that of control cells. (B) Quantification of apical cell surface area. Two-sided unpaired Student’s t-test; ****p < .0001; mean ± SEM; n = 150 cells. (C) Quantification of apical cell surface area over time from neuroepithelial cells in Movie 4 and panel (D); Two-sided unpaired Student’s t-test; ****p < .0001; n = 20 cells for each time point. Neuroepithelial cells overlying Wee2 morphant somitic mesoderm fail to undergo apical constriction (D) Stills from a time lapse recording of a neurula embryo with unilateral Wee2 morphant somitic mesoderm. Red dotted line: neural plate midline. Asterisk: Wee morphant side. Scale bar: 50 μm.
	FIGURE 6. Somitic mesoderm morphogenesis affects the ratchetting of contraction pulses during apical constriction. (A) Stills from a time lapse recording of a control neuroepithelial cell during apical constriction. Red arrowheads: pulsed medioapical actin accumulation. Scale bar: 20 μm. (A’) Outline of the apical cell surface area at 0 and 34 min showing reduction of apical cell surface area. (B) Stills from a time lapse recording of a neuroepithelial cell overlying Wee2 morphant somitic mesoderm during apical constriction. Red arrowheads: pulsed medioapical actin accumulation. Scale bar: 20 μm. (B’) Outline of the apical cell surface area at 0min and 34 min showing defective reduction of apical cell surface area. (C,D) Quantification of medioapical actin intensity and apical cell surface area over time. (C) 4 control neuroepithelial cells. (D) 4 neuroepithelial cells overlying morphant somitic mesoderm. Medioapical actin accumulation (red line) is followed by reduction of the cell surface area (black line) in control cells. The cell surface area is not reduced upon apical F-actin accumulation in neuroepithelial cells overlying morphant somitic mesoderm.
	FIGURE 7. Defects in neural plate and somitic mesoderm morphogenesis have additive impact on neural tube closure. (A) Dorsal views of representative Control, Vangl2 morphant, Wee2 morphant and Vangl2/Wee2 double morphant embryos. Blue outline: neural plate. Double headed arrow: neural plate width. Scale bars: 100 μm. Control and morphant embryos are siblings raised side by side (B) Quantification of neural plate width in Control, Vangl2 morphant, Wee2 and Vangl2/Wee2 double morphant embryos. Neural tube closure is severely affected in Vangl2/Wee2 double morphant embryos. One-way Anova; ns: no significant; ****p < .0001; mean ± SEM n = 9 embryos. (C) Dorsal views of representative Control, Shroom3 morphant, Wee2 morphant and Shroom3/Wee2 double morphant embryos. Blue outline: neural plate. Double headed arrow: neural plate width. Scale bars: 100 μm. Control and morphant embryos are siblings raised side by side (D) Quantification of neural plate width in Control, Shroom3 morphant, Wee2 morphant and Shroom3/Wee2 double morphant embryos. Neural tube closure is severely affected in Shroom3/Wee2 double morphant embryos. One-way Anova; ns: no significant; *p = .106: ****p < .0001; mean ± SEM n = 11 control, 9 Shroom3 and Wee2 morphant and 10 Wee2/Shroom3 double morphant embryos.

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