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First published online May 16, 2007
doi: 10.1242/10.1242/dev.000885

Development 134, 2007-2016 (2007)
Published by The Company of Biologists 2007
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The FERM protein Epb4.1l5 is required for organization of the neural plate and for the epithelial-mesenchymal transition at the primitive streak of the mouse embryo

Jeffrey D. Lee1, Nancy F. Silva-Gagliardi2, Ulrich Tepass3, C. Jane McGlade2 and Kathryn V. Anderson1,*

1 Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA.
2 The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumor Research Center and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1X8, Canada.
3 Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.


Figure 1
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  		Fig. 1. Disruption of morphogenesis and maintenance of patterning in lulu mutants. Expression patterns at E8.5 assayed by immunofluorescence (A,B) or in situ hybridization (C-H). (A,B) 3D reconstructions from confocal z-stacks of wild-type (WT; A) and lulu (B) embryos stained with anti-Sox2 antibodies. (C,D) Krox20 is expressed in rhombomeres 3 and 5 of the wild-type hindbrain (C), and in the lulu neural plate (D, arrow). (E,F) Brachyury (T) expression in the notochord is discontinuous in lulu (F, arrows). (G,H) Meox1 expression in the paraxial mesoderm is unsegmented in lulu (H). All views are dorsal, except F, which is ventral. Anterior is up in all panels. Scale bars: 200 µm in A-D; 150 µm in E,F,H; 120 µm in G.

 

Figure 2
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  		Fig. 2. Molecular identification of lulu and expression of Lulu protein. (A) Domain structure of Epb4.1l5, showing the lulu mutation site and the gene trap insertions. The FERM domain consists of amino acids (aa) 43 to 350. The cladogram shows FERM-domain relatedness among Lulu homologs and related mouse ERM proteins. (B) E8.5 lulu/Epb4.1l5GT1 transheterozygous mutant embryo (right; abnormal allantois, bracket), with its wild-type sibling (left). (C) lulu/Epb4.1l5GT2 transheterozygote (right) and wild-type sibling (left) at E9.5. (D-F) Anti-Lulu immunofluorescence on transverse sections of wild-type (D,E) and Epb4.1l5GT1 homozygous (F) embryos. Lulu is apically enriched in the E7.5 epiblast (D, arrowheads in inset) and E8.5 neural tube (E). Lulu is found at the periphery of cells ingressing in the primitive streak (D, bracket; inset is 2x magnification of the streak). (F) E8.5 neural plate from an Epb4.1l5GT1 homozygous embryo lacks detectable Lulu. (G) E7.5 transverse section of an Epb4.1l5GT2/+ embryo, showing apical GT2-ß-galactosidase activity. Anterior is up in B and left in C,D; dorsal is up in E,F. Scale bars: 150 µm in B,C; 50 µm in D,F,G; 20 µm in E.

 

Figure 3
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  		Fig. 3. Impaired paraxial mesoderm production in lulu. (A-D) Expression of Tbx6 RNA in E8.5 wild-type (WT; A,C) and lulu (B,D) embryos, shown in whole mount (dorsal view in A, ventral in B) and transverse sections through the streak (C,D). (A,B) Tbx6 expression flanks the streak and node in wild type (A); this domain is reduced in lulu mutants (B). (C,D) Cells accumulate under the streak in lulu mutants, producing a bulge of Tbx6-negative cells (D, arrow). (E,F) Immunofluorescence on transverse sections through the streak at E8.5. E-cadherin (red) and Sox2 (green) are maintained in cells underlying the streak in lulu mutants (F, arrow), but are lost in cells exiting the streak in wild type (E, arrow). Anterior is to the left in A,B; dorsal is up in C-F. Scale bars: 50 µm in A,B; 30 µm in C-F.

 

Figure 4
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  		Fig. 4. Defective EMT in the lulu primitive streak. E7.5 (A,B) and E7.75 (C-L) transverse sections of wild-type (WT; A,C,E,G,I,K) and lulu (B,D,F,H,J,L) embryos. (A,B) In wild-type and lulu embryos, E-cadherin is expressed in the epiblast but is downregulated in the mesodermal wings (arrowheads). (C-H) Phalloidin labels F-actin. Arrows in C,D mark the primitive streak; boxes are magnified in E,F; bars mark the regions magnified in G,H. Mesodermal wings are thicker in wild type than in lulu mutants (bars; E,F). F-actin is evenly distributed around delaminating cells in the wild-type streak (arrowheads, G), whereas cells in the lulu streak show spots of bright punctate F-actin staining (arrowheads, H). (I,J) Anti-laminin staining marks the epiblast basal lamina and highlights the increased width of the lulu streak compared with wild type (brackets). The epiblast near the streak is also thicker in lulu mutants (double-headed arrows). (K,L) Crumbs proteins are apically concentrated in the wild-type (K) and lulu (L) epiblast but are absent in involuting mesoderm. Anterior is to the left in A-D; apical is up in E,F and to the left in G-L. Scale bars: 50 µm in A-D; 10 µm in E-L.

 

Figure 5
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  		Fig. 5. Morphogenetic defects in the lulu neural plate. (A-D) Transverse sections of E8.5 wild-type (WT; A,C) and lulu mutant (B,D) embryos. (A,B) Sox2 antibody (green) labels the pseudo-stratified neural plate (dorsal) and the columnar gut endoderm (ventral). E-cadherin (red) labels the gut endoderm and the cuboidal surface ectoderm lateral to the neural plate. The neural plate in lulu mutants appears thickened and broader than in wild type, and the foregut fails to close. (C,D) Phospho-histone H3 (green)-labeled mitotic nuclei are apical in wild type (C); some mitotic nuclei are not apical in lulu (arrows, D). Phalloidin (red) shows cell shape. (E,F) 3D reconstructions of DAPI-stained nuclei (representatives false-colored) in wild-type (E) and lulu (F) anterior neural plates; the apical surface (asterisks) and apical-basal axis (double-headed arrows) are indicated. Wild-type nuclei (E) are ellipsoid and align with the apical-basal axis (cyan nucleus). lulu nuclei (F) vary in shape (spherical nucleus, yellow) and appear to align randomly (magenta nucleus). Dorsal is up in all panels. Scale bars: 20 µm in A-D; 5 µm in E,F.

 

Figure 6
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  		Fig. 6. Apical markers are correctly localized in the lulu neural plate. (A-H) Immunofluorescence on transverse sections of E8.5 wild-type (WT; A,C,E,G) and lulu mutant (B,D,F,H) neural plates. (A,B) ZO-2 marks the tight junctions at the apical-basal border of the neuroepithelium. (C,D) N-cadherin marks adherens junctions. (E,F) Crumbs is restricted to the apical surface. (G,H) Pals1 is localized to the apical and apico-lateral domain. Dorsal is up in all panels. Scale bar: 10 µm in A,B; 5 µm in C-H.

 

Figure 7
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  		Fig. 7. Cytoskeletal alterations in the lulu neural plate. (A-H) Transverse sections of E8.5 wild-type (WT; A,C,E,G) and lulu (B,D,F,H) anterior neural plates; boxes in A,B show the equivalent positions of the high-magnification images in C,E,G and D,F,H, respectively. (A-D) Phalloidin reveals F-actin localization and tissue shape; ectopic concentrations of F-actin appear away from the apical surface of the lulu neuroepithelium (arrowheads, D). (E,F) Myosin IIB is concentrated at ectopic sites in the lulu neural plate (arrowheads, F). (G,H) Anti-phospho-ERM antibody recognizes activated (actin-binding) ERM proteins; lulu neural plates show ectopic phospho-ERM staining away from the apical surface. Scale bars: 25 µm in A,B; 5 µm in C-F; 10 µm in G,H.

 

Figure 8
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  		Fig. 8. Expression of Lulu alters the actin organization of HeLa cells. HeLa cells transfected with either pEGFP alone (A-C) or pEGFP-lulu (D-F) were grown on glass coverslips, fixed, permeabilized and stained with phalloidin (red). pEGFP-Lulu localized to the plasma membrane and colocalized with increased phalloidin staining at the cell cortex. Scale bars: 20 µm.

 

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© The Company of Biologists Ltd 2007