Figure 1. Scrib regulates mitotic spindle orientation and neural tube morphogenesis.

A) Immunostainings of neural keel progenitors showing that the orientation of the mitotic spindle changes over the course of mitosis. γ-tubulin (centrosomes; red), α-tubulin (spindle; green), and DAPI (DNA, 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 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). While 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,G) Representation of mitotic spindle rotation in live (F) wt (n=20) and (G) abberantly rotating scrib morphant (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 as solid line, midline as 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 hpf wt embryo have a bilateral distribution, but have a predominantly unilateral distribution in the posterior hindbrain/anterior spinal cord region of an mzscrib mutant. Arrowheads indicate the position of the first somite (left is hindbrain, spinal cord is to the right), ovals mark the otic vesicle, dotted line indicates the midline. In all panels, anterior is to the left and double arrowheads indicate the apico-basal axis of neuroepithelial progenitors. See also Supp.Fig.1.