Zigman et al., 2011 - Zebrafish Neural Tube Morphogenesis Requires Scribble-Dependent Oriented Cell Divisions. Current biology : CB   21(1):79-86 Full text @ Curr. Biol.

Fig. 1 Scrib Regulates Mitotic Spindle Orientation and Neural Tube Morphogenesis

(A and B) Immunostainings of neural keel progenitors (A) showing that the orientation of the mitotic spindle changes over the course of mitosis. γ-tubulin (centrosomes) is shown in red, α-tubulin (spindle) in green, and DAPI (DNA) in blue. This process results in the bilateral distribution of daughter cells as schematized in (B).

(C) Quantification of mitosis orientation at anaphase. Chi-square analysis shows that the three distributions shown are highly significantly different from wild-type (WT) (n = 269): 3 μg/ml cytochalasin D-treated (n = 28, χ2 = 51, 1 degree of freedom [df]; p < 0.001), scrib morphant (n = 235, χ2 = 306, 8 df; p < 0.001), mzscrib mutant (n = 76, χ2 = 201, 2 df; p < 0.001). Although all three distributions are more homogeneous than WT, only cytochalasin D treatment results in a statistically randomized distribution (χ2 = 2.7, 3 df; p = 0.44).

(D) Posterior hindbrain lumen morphology, defined by apical F-actin, with an abnormal, branched organization in the mzscrib mutant compared to WT.

(E) Pard3-GFP localization to subapical foci upon cytokinesis (arrows) occurs normally in WT and misoriented mzscrib mutant neural keel progenitors.

(F and G) Representation of mitotic spindle rotation in live WT (F; n = 20) and aberrantly rotating scrib morphant (G; n = 32) progenitors. Plots present the angle between inferred mitotic spindle axis and the midline over time.

(F′) Scheme of spindle rotation in a mitotic progenitor. Mitotic spindle axis is shown as a solid line; midline is shown as a dashed line.

(H) Inhibition of cell proliferation results in diminished neural tube morphogenesis defects in mzscrib embryos.

(I) Requirement of Scrib for cross-midline cell divisions in the neural keel. Labeled cells in a 22 hours postfertilization (hpf) WT embryo have a bilateral distribution, but mzscrib mutant embryos have a predominantly unilateral distribution in the posterior hindbrain/anterior spinal cord region. Arrowheads indicate the position of the first somite; ovals mark the otic vesicle (o.v.); dotted line indicates the midline.

In all panels, anterior is to the left and two-way arrows indicate the apicobasal axis of neuroepithelial progenitors. See also Figure S1.

Fig. 2 Neither Planar Cell Polarity Components nor the Par Complex Are Required for Proper Spindle Orientation in the Neural Keel

(A) GFP-Prickle (GFP-Pk)-positive foci (arrows) in posterior hindbrain neural keel progenitors of WT, vangl2-/- mutant, and scrib morphant embryos. In (A) and (D), anterior is to the top and two-way arrows indicate the apicobasal axis.

(B and C) Quantification of anaphase orientation without functional planar cell polarity and Par complex components. The distributions of the various mutant or morphant conditions shown are not significantly different from WT (n = 269): vangl2-/- mutant (n = 72, χ2 = 5.1, 4 df; p = 0.28), wnt11 morphant (n = 74, χ2 = 2.6, 3 df; p = 0.41), dsh2 morphant (n = 86, χ2 = 9.6, 4 df; p = 0.05), pk1a + pk1b morphant (n = 38, χ2 = 2.6, 3 df; p = 0.46), pard6gb-/- mutant (n = 28, χ2 = 2.7, 3 df; p = 0.43); the distribution of mitotic angles in aPKCζ + aPKCλ double morphants is slightly more biased toward apicobasal than WT (n = 139, χ2 = 14.4, 4 df; p = 0.006).

(D) The branched, disorganized neural tube lumen of mzscrib and mzscrib;pard6gb-/- double mutants. Dorsal optical sections at 18 hpf immunostained with γ-tubulin (red), ZO-1 (green), and DAPI (blue) are shown.

See also Figure S2.

Fig. 3 Reduction of α-Catenin Foci in the Neural Keel Correlates with Aberrant Mitotic Orientation and Neural Tube Architecture Defects

(A) Equatorially positioned cortical Ctnna-citrine foci (α-catenin, green, arrows) are stationary while chromosomes (H2B-RFP, purple) rotate in a time-lapse of a dividing neural keel progenitor. Upper panel shows merge; lower panel shows Ctnna-citrine alone.

(B) Scrib and Ncad/Cdh2 are required for Ctnna-citrine abundance and the localization of equatorial cortical Ctnna-citrine foci in neural keel mitotic progenitors. Posterior hindbrain is shown at 6–8 somites with Ctnna-citrine (left column), as Ctnna-citrine fluorescence intensity in pseudocolors (middle column), and in pseudocolors of single mitotic progenitors (right column). Localization of Ctnna-citrine is shown in WT (top row), cdh2 morphants (middle row), and scrib morphants (bottom row). The unchanged Ctnna-citrine signal in nonneural peridermal cells is marked by an asterisk.

(B′) Fluorescence intensity plots of Ctnna-citrine levels in WT, cdh2 morphant, and scrib morphant mitotic cells (shown in B), with the y axis displaying arbitrary gray values along a line across a mitotic cell at equatorial (white dashed arrow in pictures; red line in plots) and lateral (faint blue dashed arrow in pictures; blue line in plots) positions averaged over 8 pixels in width.

(C) Quantification of mitosis orientation at anaphase in cdh2 morphants. This distribution is highly significantly different from WT (n = 267, χ2 = 224, 8 df; p < 0.001). For WT controls, see Figure 1C.

(D) Disorganized, branched neural tube midline in the cdh2-/- neural tube in horizontal cryosections. F-actin/phalloidin is shown in green, aPKCζ in white, and DAPI (DNA) in purple.

(E and F) Representation of mitotic spindle rotation in live WT (E; n = 6) and cdh2 morphant (F; n = 16) neural keel progenitors. Spindle orientation is inferred from the orientation of the chromosomes marked with H2B-GFP.

Images in all panels are in dorsal view. Two-way arrows indicate the apicobasal axis. See also Figure S3.

Fig. 4 Nonautonomous Rescue of Oriented Cross-Midline Cell Division of Single cdh2-/- and mzscrib Mutant Cells

(A–E) Genetic mosaics at neural tube stage (21 hpf) in which donor-derived cells (purple) were transplanted into the presumptive posterior hindbrain of WT host embryos (F-actin in green).

(A) Bilateral distribution of WT cells (arrows) in a WT environment.

(B) cdh2-/- cells form unilateral aggregates in a WT environment.

(C) In contrast, isolated cdh2-/- progenitors show rescued bilateral distribution and normal cell shape.

(D) A large group of mzscrib mutant cells in a WT environment with unilateral cell distribution.

(E) Isolated mzscrib cells in a WT environment have rescued shape and bilateral distribution.

(F) Utr-CH-RFP reporter in mosaic embryos showing normal apical enrichment in WT control transplants, loss of apical enrichment in a cluster of transplanted cdh2-/- cells, and rescue in a single cdh2-/- cell that is surrounded by WT host cells. Images represent maximum-intensity projections of optical sections.

(G) Quantification of occurrence of abnormal F-actin organization in WT (black; n = 5 experiments, 13 embryos), mzscrib (blue; n = 3 experiments, 21 embryos), and cdh2-/- (red; n = 2 experiments, 15 embryos) mosaics.

Embryos are shown in dorsal view with anterior to the top. Dotted white line indicates the neural tube midline; two-way arrows indicate the apicobasal axis of the neuroepithelium. See also Figure S4.

Fig. S1 Requirement of Scrib in Zebrafish Posterior Hindbrain Organization
(A) Posterior hindbrain structure in mzscrib mutants as analyzed by aPKCζ staining. Labeled apical cell membranes are aligned at the midline of WT embryos and misaligned in mzscrib mutants.
(B) Alignment of the subapically localized tight junctional component Mpp5a at the midline in WT and aberrant alignment in mzscrib mutants. These are the same embryos as shown in Figure 1D, imaged in a different channel.
(C) Abnormal organization of the neural tube lumen in mzscrib compared to WT in transverse sections at posterior hindbrain level as analyzed by aPKCζ green), F-actin (red) and DAPI (blue) staining. Dorsal is to the top.
(D) mzscrib mutants display aberrant vagus motor neuron positioning visualized in tg(isl1:GFP) transgenic zebrafish. Dorsal view. Anterior is to the top in A,B,D.

Fig. S2 Scrib Is Not Essential for Proper Velocity of Hindbrain Neural Keel Cell Convergence, and Scrib Shows Different Effects on Zebrafish Hindbrain Organization from Vangl2
(A) Single optical slices of WT and scrib morphant hindbrain neural plate architecture in dorsal horizontal view (upper row) and transverse view (lower row) with membrane-bound GFP at 1-2 somites (10.5 hpf).
(B) Hindbrain neural keel convergence analyzed by multi-photon imaging of membrane-bound GFP in dorsal horizontal view starting at 4 somites (11.3 hpf). An image was acquired every 3 minutes for 105 minutes, ending at the 8 somite stage. Pictures were taken at all z-levels of the neural keel and the broadest keel positions were used for the analysis. Note abnormal convergence in the vangl2 morphant and only slightly slower convergence in scrib morphant compared to WT. Arrows indicate the same cells at different time points. Anterior is to the left in all panels. Average neural keel convergence velocity in the posterior hindbrain noted on top of each panel (n = 2 for each genotype).
(C) Medial positioning of mitotically dividing cells marked with anti phosphohistone H3 (Ser10) medially along the midline in the posterior hindbrain neural keel of WT as well as in mzscrib mutants at 14 hpf. Images present maximal intensity projections of 4.8 μm confocal optical sections in dorsal horizontal view, anterior is to the top.
(D) Quantification of the positioning of phosphohistone H3 positive mitotic cells relative to the midline in confocal sections of the dorsal 30 m of the neural keel plotted in μm on the horizontal axis. The y-axis displays distribution frequency. Mitotic cells were positioned at an average distance of 11.36 ± 7.16 μm (n = 196 cells, 9 embryos) in the WT (grey) and 12.28 ± 8.64 μm (n = 238 cells, 9 embryos) in the mzscrib mutants (green). The differences between the two populations were not significant (p > 0.2, t-test).
(E) Organization of hindbrain neural progenitors in horizontal dorsal view showing the abnormally branched midline in mzscrib compared to the double midlines of vangl2-/- mutants. aPKCζ (red), γ-tubulin (green) and DAPI (blue). Anterior to the top in all panels.

Fig. S3 GFP-Prickle Localizes to Cytoplasm, GFP-Scrib Shows Cortical Localization in WT Mitotic Neural Keel Progenitors, and Scrib Is Not Required for Maintenance of Subapical -Catenin Localization in the Mature Neural Tube Epithelium
(A) Optical sections from timelapse imaging (min’s“) revealing anterior polarization of GFP-Pk foci (arrows) in interphase and subsequent to cytokinesis (cells followed marked by stars), but absence of GFP-Pk foci in mitotic cells. Time of optical sections given in min’s“.
(B) Persistent cortical GFP-Scrib localization in WT neural keel progenitors with arrowhead noting a mitotic cell and arrows pointing to a cell undergoing cytokinesis. Dorsal horizontal view with anterior to the top in all panels. Double arrowheads indicate apicobasal axis.
(C) Ctnna-citrine fusion protein of Gt(ctnna-citrine) transgenic line colocalizes with the endogenous Ctnna protein in immunofluorescent stainings. Single confocal images taken in the region of nasal epithelium (n.e.) and neuroepithelium of the retina.
(D) Ctnna-citrine localization (arrows) in the mature neural tube reveals residual Ctnna-citrine foci in the disorganized hindbrain epithelium of scrib morphants. Double arrowheads indicate apicobasal axis. Anterior-posterior axis is horizontal.

Fig. S4 Rescue of Apicobasal Cell Division Orientation of a Single cdh2-/- Mutant Cell in a Wild- Type Environment
Selected time points from an in vivo imaging of a genetic mosaic in which a cdh2-/- donor derived cell (expressing H2B-GFP (green) and membrane bound mRFP1 (purple)) was transplanted into the presumptive posterior hindbrain of a WT host embryo. Double arrowhead indicates the apicobasal axis of the neuroepithelium, yellow arrows indicate the final orientation of cell division. Anterior is to the top. The times of the optical sections are given in min’s.

Acknowledgments:
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