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First published online 29 September 2004
doi: 10.1242/dev.01415

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Distinct GATA6- and laminin-dependent mechanisms regulate endodermal and ectodermal embryonic stem cell fates

¹ Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel
² Department of Cell and Molecular Biology, Lund University, Box 94, S-221 00, Lund, Sweden
³ Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool L69 3G3E, UK

View larger version (26K): [in a new window]   Fig. 1. FGF signalling is required for early EB differentiation. (A) EB differentiation is most sensitive to the Fgfr inhibitor SU5402 when added at the start or before (day –2) ES cell aggregation. (B) Western blot. -Fetoprotein expression shows the effect of SU5402 on visceral endoderm differentiation. Differentiation was defined as the percent of EBs exhibiting two epithelia and cavitation, as seen in plastic-embedded Toluidine Blue stained sections.

View larger version (49K): [in a new window]   Fig. 2. GATA4 and GATA6 are under FGF control and overexpress BM proteins. (A) Effect of the dnFgfr mutation on Fgf, Fgfr and GATA factor expression; data from a DNA microarray experiment. Units are kDa. (B) GATA6 or GATA4 greatly enhances synthesis of laminin 1 and collagen IV even in dnFgfr and Lamc1 mutant ES cells (western blot).

View larger version (90K): [in a new window]   Fig. 3. GATA6 transfected endoderm-like derivatives of ROSA11 ES cells grown as adherent cultures (A-C) and as cysts in suspension cultures (D-L) (confocal images). (A-C) During the first day of culture, the cells accumulate laminin 1 in the cytoplasm (A), which, by day 3 (B) to 4 (C), is secreted to the extracellular space. (D,G,J) Fluoresceinated phalloidin; (E,H,K) BM proteins (Cy3), see left side for antibody used; (F,I,L) merged images.

View larger version (70K): [in a new window]   Fig. 4. Genetic evidence for the role of laminin 1 in the cell-to-matrix interaction required for epiblast polarization. Upon co-cultivation, GATA4 transformed endoderm-like ES cells rescue the dnFgr mutant (A-D). The GATA6 transferred homozygous Lamc1 mutant, in contrast to the heterozygote (E-H), does not accumulate BM proteins and cannot rescue dnFgfr cells. (A-D) Plastic-embedded sections; (E-H) ß-galactosidase and Neutral Red staining. (E-H) Whole-mount confocal microscopy: green, phalloidin (a marker for F-actin); red, laminin 1. (A) AB2.2, wild-type EBs; (B) dnFgfr EB expressing ß-galactosidase; (C) GATA4 transformed AB2.2 cells; (D) co-cultivation of dnFgfr (1C6) ES cells with GATA4 transformed AB2.2. (E) GATA6-transformed Lamc1-/- cells. Lamc1-/- GATA6 cells form aggregates with minimal BM protein expression. (F) Co-cultivation of Lamc1-/- GATA6 cells with dnFgr cells does not lead to phenotypic rescue. (G) Lamc1+/- GATA6 cells form cysts containing BM proteins. (H) Lamc1+/- GATA6 cells rescue differentiation of 1C6. Scale bars: 50 µm.

View larger version (30K): [in a new window]   Fig. 5. Cells of the dnFgfr ES cell line1C6 colonize most lineages of the early embryo when introduced as chimera aggregated with wild-type cells. (A-C) ß-Galactosidase staining indicates derivatives of the 1C6 line. (A) 7.5 dpc embryo, whole mount. (B) 7.5 dpc crosssection at the level of line in A. (C) 9.5 dpc embryo sagittal section. (B,C) Plastic embedded sections. Scale bars: 50 µm.

View larger version (141K): [in a new window]   Fig. 6. Pluripotent stem cells and their ectoderm derivatives exhibit laminin receptors. (A-D) Confocal images. (A) Day 4, 1C6 dnFgfr ES cells activated with Lamc1+/- GATA6 culture supernatant. Stained with antibody to laminin 1. The single layer of columnar cells covered by laminin 1 (red). (B) 1C6 dnFgfr ES cells incubated with Lamc1-/- GATA6 culture supernatant harvested between day four and day five of culture and stained with antibody to laminin 1. Differentiation and laminin 1 staining are both absent. (C) Wild-type ROSA11 ES cells on feeder layer stained with antibody to laminin 1 and phalloidin (green). See fibroblast in left upper quarter. There is a lack of laminin staining. (D) Wild-type ROSA11 ES cells on feeder layer treated with ROSA11-GATA6 culture supernatant for 6 hours and stained with antibody to laminin 1 (red) and phalloidin (green). There is laminin deposition in the membrane of all ES cells.

View larger version (119K): [in a new window]   Fig. 7. RhoC and ROCK are involved in epiblast differentiation. (A) Results of DNA microarray analysis reveals that Rac1 and Cdc42 are expressed throughout EB differentiation and may not be dependent on FGF signalling. However, expression of RhoC is higher in the wild type than in the dnFgfr mutant and has its maximum at day 4, when epiblast differentiation is at its peak. (B-F) Transcription electronmicroscopy. (B-D) Wild type. (B) Overview; (C) adhesion structure in endoderm; (D) adhesion structure in the epiblast. (E,F) Y-27632 treatment of wild type. (E) Overview; (F) adhesion structure in endoderm. Abbreviations: end, endoderm; ect, ectoderm or epiblast. Scale bars: in E, 10 µm for B,E; in F, 0.5 µm for C,D,F. Arrows in C,D,F indicate adhesion structures. White square in B indicates the position of the adhesion structure in D.

View larger version (74K): [in a new window]   Fig. 8. ROCK activity is required for epiblast differentiation and polarization. Confocal microscopy. Green, phalloidin staining detecting F-actin. Red, specific protein signal. (A) Wild-type (untreated) BM is detected by antibody to laminin 1. There is F-actin accumulation in the apical domain of the epiblast. (B) Y-27632 treatment, note the lack of columnar ectoderm and partial cavitation without F-actin accumulation. BM detected by laminin 1 antibody. (C) H-1152 treatment, antibody to collagen IV, (D) EB formed by ES cells expressing dnROCKII. Note flat internal cells without significant F-actin accumulation in the apical domain. (E,F) ZO-1 staining. The pattern is similar in the wild-type endoderm (E) and in H-1152-treated EB (F). (G,H) ROCKII staining. (G) Wild type; (H) EB formed by dnFgfr derived GATA supernatant treated ES cells. There is F-actin accumulation in the epical epiblast domain and a lack of endoderm with strong ROCKII staining in H.