vangl2 morphant embryos  (VANGL2 LTD)

Journal: The Journal of Biological Chemistry

Article Title: A vertebrate Vangl2 translational variant required for planar cell polarity

doi: 10.1016/j.jbc.2024.106792

Figure Lengend Snippet: Vangl2 and Vangl2-Long knockdown disrupts core PCP protein asymmetric distribution. A , synthetic mRNAs encoding Dvl1-GFP and mRFP were injected at the 16-cell stage in one ventral animal blastomere, with control, Vangl2 or Vangl2-Long morpholinos. Fluorescent protein localization in epidermal cells was recorded in live embryos at stage 31. In all panels, the scale bars represent 10 μm. B , graph displaying the ratio of Dvl1-GFP signal intensity between dorsoanterior and ventroposterior domains of the cell membrane. Note that the dorsoanterior enrichment of Dvl1-GFP in control morphant cells (n = 59) is reduced in Vangl2 (n = 28) and Vangl2-Long (n = 65) morphant cells. C , synthetic mRNAs encoding GFP-Pk2 and mRFP were injected at the 16-cell stage in one ventral animal blastomere, with control, Vangl2 or Vangl2-Long morpholinos. Fluorescent protein localization in epidermal cells was recorded in live embryos at stage 31. In all panels, the scale bars represent 10 μm. D , graph displaying the ratio of GFP-Pk2 signal intensity between dorsoanterior and ventroposterior domains of the cell membrane. Note that the ventroposterior enrichment of GFP-Pk2 in control morphant cells (n = 23) is reduced in Vangl2 (n = 28) and Vangl2-Long (n = 48) morphant cells. Statistical analyses were done using GraphPad Prism software, with Student’s t tests to evaluate significance. PCP, planar cell polarity.

Article Snippet: However, many Vangl2-Long morphant MCCs projected outside the ventroposterior quadrant, consistent with a decrease of tissue-level coordination of orientation, typical of deficient PCP ( , ).

Techniques: Knockdown, Injection, Control, Membrane, Software

Journal: The Journal of Biological Chemistry

Article Title: A vertebrate Vangl2 translational variant required for planar cell polarity

doi: 10.1016/j.jbc.2024.106792

Figure Lengend Snippet: Centriole polarization is impaired by Vangl2 and Vangl2-Long knockdown. A , rational of the experiment. The flow generated by the coordinated beating of myriads of cilia in epidermal MCCs is represented by arrows along the flanks of a stage 31 Xenopus embryo (the scale bars represent 1 mm). Centriole/cilium rotational polarity is evaluated through double IF staining of the basal body (BB, Centrin) and the basal foot (γ-Tubulin) (the scale bar represents 1 μm). For each MCC, a rose histogram is generated by the Oriana software to plot the angles of orientation of unambiguously doubly stained BBs. The coordinates of the rose plot are aligned on those of the embryo. B , rose plots displaying mean BB orientation in control, Vangl2, and Vangl2-Long morphant MCCs. Each arrow represents one MCC. The length of the arrow is proportional to the degree of alignment of BBs with the mean vector. The two colors represent MCCs from two independent experiments. Note that Vangl2 and Vangl2-Long knockdown cause the dispersion of angles of orientation of individual MCCs. C , graph displaying the circular SD (CSD) of the angles of orientation of BBs, within MCCs analyzed in B . A low CSD indicates that BBs are well aligned along the mean vector in a given MCC, whereas a high CSD indicates a poor degree of alignment. Note that CSD values were higher than control in Vangl2 but not in Vangl2-Long morphant MCCs, indicating poor BB alignment in the former case. Statistical analyses were carried out with GraphPad Prism software with a Student’s t test to evaluate significance. IF, immunofluorescence; MCC, multiciliated cell.

Article Snippet: However, many Vangl2-Long morphant MCCs projected outside the ventroposterior quadrant, consistent with a decrease of tissue-level coordination of orientation, typical of deficient PCP ( , ).

Techniques: Knockdown, Generated, Staining, Software, Control, Plasmid Preparation, Dispersion, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: Distinct spatiotemporal contribution of morphogenetic events and mechanical tissue coupling during Xenopus neural tube closure

doi: 10.1242/dev.200358

Figure Lengend Snippet: NP anterior movement requires CE of the posterior tissue. (A) Stills from time-lapse recordings of representative control and Vangl2 morphant embryos. Dotted lines delineate NP boundaries. NP narrowing and neural fold movement towards the midline is defective in the Vangl2 morphant embryo. (B) Quantification of ML junction length over time in control ( n =4) and Vangl2 morphant ( n =3) embryos. **** P <0.0001 (two-sided, unpaired Student's t -test); ns, not significant; mean±s.e.m.; n =210 and 148 junctions from control and Vangl2 morphant embryos, respectively. (C) Representative examples of anterior neuroepithelial cell displacement within a 60 min time period in a control and Vangl2 morphant embryo. Boxed areas are shown at higher magnification below. (D) Representative examples of anterior neuroepithelial cell displacement within a 60 min time period in an anterior NP explant, in an embryo with wound-induced physical separation of anterior and posterior NP and in embryos lacking the posterior NP. (E) Quantification of anterior neuroepithelial cell velocity. **** P <0.0001 (one-way ANOVA); mean±s.e.m.; n =187 cells from three control embryos 209 cells from three Vangl2 morphant embryos; 202 cells from three anterior NP explants; 64 cells from an embryo with wound-induced physical separation of anterior and posterior NP; and 120 cells from three embryos lacking the posterior NP. Scale bars: 100 µm. A, anterior; a.u., arbitrary units; P, posterior.

Article Snippet: NP narrowing and neural fold movement towards the midline is defective in the Vangl2 morphant embryo. (B) Quantification of ML junction length over time in control ( n =4) and Vangl2 morphant ( n =3) embryos.

Techniques:

Journal: Development (Cambridge, England)

Article Title: Distinct spatiotemporal contribution of morphogenetic events and mechanical tissue coupling during Xenopus neural tube closure

doi: 10.1242/dev.200358

Figure Lengend Snippet: SE medial movement requires forces generated by NP morphogenesis. (A) Stills from a time-lapse recording of deep SE cells plated on an FN-coated coverslip. Tracks (spots) are time colour-coded. (B) Displacement map from a 26 min time window from the time-lapse recording used for cell tracking in A, indicating absence of cell movement. Boxed area is shown at higher magnification in inset. (C) Stills from a tracked time-lapse recording of a control embryo before (left) and after (right) AC. (C′) Zoomed tracks from the recording shown C showing the increase of cell displacement and velocity (V) during AC. (D) Quantification of the average cell velocity of SE cells during distinct phases of NTC. **** P <0.0001 (two-sided, unpaired Student's t -test); mean±s.e.m. n =46, 53 and 54 cells for the three different time periods. (E) Stills from a tracked time-lapse recording showing SE cells during NTC. As NTC progresses, the surface area of SE cells increases, as shown by the schematics above. (F) Quantification of the apical surface area of SE cells during NTC. **** P <0.0001 (two-sided, unpaired Student's t -test); mean±s.e.m. n =100 cells for each time point. (G) Stills from a tracked time-lapse recording of a control embryo (left) and a Vangl2 morphant embryo (right). (G′) Zoomed tracks from the recording shown in C showing decrease of cell displacement and velocity (V) in the absence of CE in Vangl2 morphants. (H) Quantification of the average cell velocity from control and Vangl2 morphant embryos. **** P <0.0001 (two-sided, unpaired Student's t -test); mean±s.e.m., n =63 SE cells from a control and 97 SE cells from a Vangl2 morphant embryo. (I) Stills from a tracked time-lapse recording of a control embryo (left) and a Shroom3 morphant embryo (right). The displacement and velocity (V) of SE cells is decreased in the absence of AC. (J) Quantification of the average cell velocity from control and Shroom3 morphant embryos. **** P <0.0001 (two-sided, unpaired Student's t -test); mean±s.e.m., n =59 SE cells from a control and 49 SE cells from a Shroom3 morphant embryo. Scale bars: 100 µm. A, anterior; P, posterior.

Article Snippet: NP narrowing and neural fold movement towards the midline is defective in the Vangl2 morphant embryo. (B) Quantification of ML junction length over time in control ( n =4) and Vangl2 morphant ( n =3) embryos.

Techniques: Generated, Cell Tracking Assay

Journal: Development (Cambridge, England)

Article Title: Distinct spatiotemporal contribution of morphogenetic events and mechanical tissue coupling during Xenopus neural tube closure

doi: 10.1242/dev.200358

Figure Lengend Snippet: Normal SE development is necessary for NTC. (A) Schematic showing the process for SE targeted microinjections. D, dorsal; V, ventral. (B) Representative example from a cross-section (inset) of a stage 15 embryo with SE-targeted unilateral injection of Itgβ1 MO. Itgβ1 MO was co-injected with histone-GFP (hGFP). n =21 embryos. (C) High-magnification images of the SE shown in B. The thickness of the SE (double-headed arrows) is increased on the Itgβ1 morphant side. (D) Quantification of SE thickness in embryos unilaterally injected with Itgβ1 MO. **** P <0.0001 (two-sided, paired Student's t -test); mean±s.e.m., n =21 embryos. (E) 3D image of a representative example of a stage 16 embryo unilaterally injected with Itgβ1 MO. White dotted line indicates the midline. Double-headed arrows indicate the length of the NP in the control and Itgβ1 MO-injected side. n =18 embryos (F) Quantification of NP length at the control side and the Itgβ1 MO side in embryos unilaterally injected with Itgβ1 MO. **** P <0.0001 (two-sided, paired Student's t -test); mean±s.e.m., n =28 embryos. A, anterior; P, posterior. Scale bars: 100 μm.

Article Snippet: NP narrowing and neural fold movement towards the midline is defective in the Vangl2 morphant embryo. (B) Quantification of ML junction length over time in control ( n =4) and Vangl2 morphant ( n =3) embryos.

Techniques: Injection

Journal: Development (Cambridge, England)

Article Title: Distinct spatiotemporal contribution of morphogenetic events and mechanical tissue coupling during Xenopus neural tube closure

doi: 10.1242/dev.200358

Figure Lengend Snippet: AC and CE are defective in embryos with defective SE development. (A) Representative example of an embryo unilaterally injected with Itgβ1 MO. White dotted line indicates the midline. Double-headed arrows indicate the length of the NP in the control and Itgβ1 MO-injected side. n =20 embryos. (B) High magnification image of the NP from A. White dotted line demarcates the midline. Red lines mark the ML junctions. ML junctions at the NP side adjacent with Itgβ1 morphant SE fail to shrink. (C) Representative example of an embryo expressing PK2-GFP and unilaterally injected with Itgβ1 MO (inset shows H2B-GFP). Boxed area is shown at higher magnification to the right. PK2 displays enriched localization at ML junctions of the control NP side (white arrows) and the NP side adjacent with Itgβ1 morphant SE (yellow arrows). (D) Quantification of ML junction length in the control NP side and the adjacent NP side with Itgβ1 morphant SE. **** P <0.0001 (two-sided, unpaired Student's t -test); mean±s.e.m., n =50 junctions. (E) Quantification of ML PK2-GFP localization in control NP side and the adjacent NP side with Itgβ1 morphant SE. ns, not significant (two-sided, unpaired Student's t -test); mean±s.e.m., n =60 junctions. (F) Representative neuroepithelial cell surface area in control NP and the adjacent NP with Itgβ1 morphant SE. (G) Quantification of apical cell surface area of neuroepithelial cells. **** P <0.0001 (two-sided, unpaired Student's t -test); mean±s.e.m., n =300 cells from six control embryos and 300 from six Itgβ1 morphant embryos. Scale bars: 100 µm. A, anterior; P, posterior.

Article Snippet: NP narrowing and neural fold movement towards the midline is defective in the Vangl2 morphant embryo. (B) Quantification of ML junction length over time in control ( n =4) and Vangl2 morphant ( n =3) embryos.

Techniques: Injection, Expressing

Journal: Development (Cambridge, England)

Article Title: Distinct spatiotemporal contribution of morphogenetic events and mechanical tissue coupling during Xenopus neural tube closure

doi: 10.1242/dev.200358

Figure Lengend Snippet: SE medial movement requires forces generated by NP morphogenesis. (A) Stills from a time-lapse recording of deep SE cells plated on an FN-coated coverslip. Tracks (spots) are time colour-coded. (B) Displacement map from a 26 min time window from the time-lapse recording used for cell tracking in A, indicating absence of cell movement. Boxed area is shown at higher magnification in inset. (C) Stills from a tracked time-lapse recording of a control embryo before (left) and after (right) AC. (C′) Zoomed tracks from the recording shown C showing the increase of cell displacement and velocity (V) during AC. (D) Quantification of the average cell velocity of SE cells during distinct phases of NTC. **** P <0.0001 (two-sided, unpaired Student's t -test); mean±s.e.m. n =46, 53 and 54 cells for the three different time periods. (E) Stills from a tracked time-lapse recording showing SE cells during NTC. As NTC progresses, the surface area of SE cells increases, as shown by the schematics above. (F) Quantification of the apical surface area of SE cells during NTC. **** P <0.0001 (two-sided, unpaired Student's t -test); mean±s.e.m. n =100 cells for each time point. (G) Stills from a tracked time-lapse recording of a control embryo (left) and a Vangl2 morphant embryo (right). (G′) Zoomed tracks from the recording shown in C showing decrease of cell displacement and velocity (V) in the absence of CE in Vangl2 morphants. (H) Quantification of the average cell velocity from control and Vangl2 morphant embryos. **** P <0.0001 (two-sided, unpaired Student's t -test); mean±s.e.m., n =63 SE cells from a control and 97 SE cells from a Vangl2 morphant embryo. (I) Stills from a tracked time-lapse recording of a control embryo (left) and a Shroom3 morphant embryo (right). The displacement and velocity (V) of SE cells is decreased in the absence of AC. (J) Quantification of the average cell velocity from control and Shroom3 morphant embryos. **** P <0.0001 (two-sided, unpaired Student's t -test); mean±s.e.m., n =59 SE cells from a control and 49 SE cells from a Shroom3 morphant embryo. Scale bars: 100 µm. A, anterior; P, posterior.

Article Snippet: The movement of the anterior NP towards the future rostral site was blocked in Vangl2 morphants, suggesting that its repositioning during NTC is passive ( C,E, Fig. S3A-C , Movie 7 ).

Techniques: Generated, Cell Tracking Assay