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First published online 28 February 2007
doi: 10.1242/dev.02828

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Shroom family proteins regulate -tubulin distribution and microtubule architecture during epithelial cell shape change

Department of Molecular Cell and Developmental Biology, and Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.

View larger version (48K): [in this window] [in a new window]   Fig. 1. Apical constriction and apicobasal cell elongation in the neural epithelium during neural tube closure. (A) Projection of confocal optical transverse sections through the anterior neural plate of neurulating Xenopus embryo. (a') High-magnification view of a neural epithelial cell from A. (B) A schematic diagram of the section in A, illustrating the neural epithelial cells (red) and other surrounding tissues (grey). (b') A schematic diagram of the section in a'. Cells in hingepoint regions constrict and elongate (arrow), cells in intermediate regions elongate, but constrict only a little (arrowhead). (C) Graph illustrating changes in apical constriction (blue; ratio of apical surface to basal surface measured from cross-sections) and apicobasal cell elongation (red; cell height in µm) of neuroepithelial cells during neural tube closure (stages 14-19; n=10 cells per stage). (D) Schematic illustrating the change in neural epithelial cell shape during neural tube closure.

View larger version (83K): [in this window] [in a new window]   Fig. 2. Shroom3 is required for apical constriction and apicobasal cell elongation in neural epithelial cells during neural tube closure. (A) Cross-section through the closing anterior neural tube of an embryo injected on one side with Shroom3-MO. Anti--tubulin staining reveals cell cortices and DAPI reveals nuclei. (a') Schematic of neuroepithelial cell shape in A. (B) Graph of apicobasal cell height and the distance between the basal cell surface and the basal limit of the nucleus for hingepoint cells in control and Shroom3-MO-injected regions at stage 18 (mean ± s.e.m.; ctl, n=12; Mo, n=11). (C) Graph of apical cell surface areas of control and Shroom3-MO-injected sides of fixed embryos in hingepoint (hp) and intermediate (int) regions of fixed embryos at stage 18 (mean ± s.e.m.; n=25). Hingepoint and intermediate regions are defined in Fig. S1 in the supplementary material. (D) Dorsal projection of stage 18 embryo to visualize apical cell surface areas. (E) Dorsal projection of stage 16 Xenopus embryo stained with phalloidin to visualize cell shapes. Cells defined as the hp and int regions are indicated by red outlines and labeled at the bottom of the panel (see also Fig. S1 in the supplementary material). Embryo has been injected on one side with Shroom3-MO.

View larger version (54K): [in this window] [in a new window]   Fig. 3. Shroom3 is required for assembly of discrete arrays of parallel MTs in elongating neural epithelial cells. (A) High-magnification view of MTs in control hingepoint cells (green, anti--tubulin; red, propidium iodide, nuclei). (a') Schematic of a cell shown in A. Arrow indicates parallel MT arrays. (B) High-magnification view of MTs in dorsolateral hingepoint (DLHP) cells lacking Shroom3 function. (b') Schematic of a cell shown in B. (C) High-magnification view of control hingepoint cells (green, ZO-1; red, anti--tubulin). (D) High-magnification view of hingepoint cells lacking Shroom3 function. Shape changes are eliminated, but ZO-1 remains apically localized at cell-cell junctions.

View larger version (85K): [in this window] [in a new window]   Fig. 4. Shroom3 controls the distribution of -tubulin in neuroepithelial cells during neural tube closure. (A) High-magnification projection of a stack of optical sections showing elongating neural epithelial cells stained for -tubulin (white lines indicate cell outline). (a') -tubulin staining for cells shown in A. (B) Schematic of the closing neural tube indicating location of section shown in A (red box). (C) -tubulin staining in a projection of optical sections reveals centrosome-like foci at the apical surfaces of mature neuroepithelial cells. (c') Acetylated--tubulin staining reveals cell outlines in the ventral region of the closed Xenopus neural tube. (D) Schematic of the neural tube indicating location of section shown in C (red box). (E) Optical section showing -tubulin in an embryo unilaterally injected with Shroom3-MO (right side is injected, left side is control). (e') -tubulin staining for cells shown in E.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Shroom3 is sufficient to drive apicobasal cell elongation in naïve epithelial cells. (A) Schematic depicting the epithelial character of the superficial blastomeres in Xenopus (see also Chalmers et al., 2005; Dollar et al., 2005; Haigo et al., 2003; Roberts et al., 1992). (B) Cross-section through superficial blastomeres of a control embryo. Phalloidin staining reveals cell cortices (green) and propidium iodide reveals nuclei (red). (C) Cross-section through superficial blastomeres of an embryo ectopically expressing Shroom3. Cells with accumulated apical actin also display increased cell height. (D) Graph of apicobasal cell height and the distance between the basal cell surface and the basal limit of the nucleus for superficial blastomeres in control and Shroom3-injected regions (mean ± s.e.m.; ctl, n=26; Shroom3, n=19).

View larger version (83K): [in this window] [in a new window]   Fig. 6. Shroom3 is sufficient to drive apical accumulation of -tubulin in naïve epithelial cells. (A) Control embryo showing surface view of the superficial blastomere epithelium stained with anti--tubulin. (B) Shroom3-expressing embryo showing surface view of the superficial blastomere epithelium stained with anti--tubulin. (C) Cross-section through superficial blastomeres of a control embryo at early stages stained with anti--tubulin. Merged image is shown in c' (green, -tubulin; red, propidium iodide). (D) Cross-section through superficial blastomeres of an embryo ectopically expressing Shroom3 at early stages stained for anti--tubulin. Merged image is shown in d' (green, -tubulin; red, propidium iodide). (E) Cross-section through superficial blastomeres of an embryo ectopically expressing Shroom3 at later stages. Embryo has been stained to detect Myc-Shroom3 (E; red) and -tubulin (e'; green). Merged image is shown in e''.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Expression of Shroom3 in epidermal cells recapitulates the events underlying neuroepithelial cell shape change. (A) Control epidermis stained for -tubulin. (B) Epidermis expressing Shroom3 stained for -tubulin. Cells expressing Shroom3 (marked by anti-Myc; b') constrict and elongate. Parallel arrays of MTs emanate from the apical cell surface. (C) Control epidermis stained for -tubulin. (D) Epidermis expressing Shroom3 stained for -tubulin. Cells expressing Shroom3 in this panel (marked by anti-Myc, red; d') are only slightly constricted, but are elongated and contain a broad, diffuse accumulation of -tubulin. (E) A single epidermis cell expressing high levels of Shroom3 apically constricts and also elongates. (e') Anti--tubulin staining of the cell shown in E.

View larger version (92K): [in this window] [in a new window]   Fig. 8. Shroom3 coordinates the polarized assembly of both actin and -tubulin cytoskeletons. (A) Control embryo at stage 6 showing surface view of the superficial blastomere epithelium stained with phalloidin (a') and anti--tubulin (a''). (B) Shroom3-expressing embryo at stage 6 showing surface view of the superficial blastomere epithelium stained with phalloidin (b') and anti--tubulin (b''). (C) Control embryo at stage 7.5 showing surface view of the superficial blastomere epithelium stained with phalloidin (c') and anti--tubulin (c''). (D) Shroom3-expressing embryo at stage 7.5 showing surface view of the superficial blastomere epithelium stained with phalloidin (d') and anti--tubulin (d''). Bracket indicates accumulated -tubulin with little actin; arrow indicates accumulated actin with little -tubulin.

View larger version (75K): [in this window] [in a new window]   Fig. 9. Shroom family proteins and cell elongation in non-neural cells. (A) In situ hybridization reveals Shroom1 expression in the cement gland. (B) In situ hybridization reveals Shroom3 expression in the cement gland, and also the invaginating nasal and otic placodes. (C) Cross-section through the cement gland, stained with phalloidin to reveal cell shapes. (c') A schematic diagram of the section in C, illustrating the cement gland epithelial cells (red) and other surrounding tissues (grey). (D) Graph of changes in apical constriction (blue) and apicobasal cell elongation (red) of cement gland cells over time (stages 19-25). (E)A schematic diagram illustrating the change in cement gland epithelial cell shape. (F) Projection of optical sections of -tubulin staining of cement gland epithelial cells. (f') -tubulin staining shown in F merged with propidium iodide staining to show nuclei. (G) Robust apical MT arrays in the cement gland are associated with a broad apical accumulation of -tubulin. (g') -tubulin staining shown in G merged with propidium iodide staining to show nuclei.

View larger version (27K): [in this window] [in a new window]   Fig. 10. Shroom1 expression is sufficient to elicit apical accumulation of -tubulin in naïve epithelial cells. (A) Surface view of blastomeres stained for -tubulin. Shroom1-expressing cells (right) accumulate high levels of -tubulin at cell-cell junctions. (B) Section view of blastomeres stained for -tubulin. (b') Shroom1-expressing cells [indicated by anti-Myc staining (red)] accumulate excess -tubulin at cell-cell boundaries. (C) Model for Shroom3-mediated cell shape changes in neural epithelial cells. Model is based on data from the current work and also on the work of Hildebrand (Hildebrand, 2005) and Haigo et al. (Haigo et al., 2003).

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