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First published online December 31, 2008
doi: 10.1242/10.1242/jcs.037663

Journal of Cell Science 122, 233-242 (2009)
Published by The Company of Biologists 2009
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Integrins are required for the differentiation of visceral endoderm

Jie Liu1, Xiaowen He1, Siobhan A. Corbett1, Stephen F. Lowry1, Alan M. Graham1, Reinhard Fässler2 and Shaohua Li1,*

1 Department of Surgery, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA
2 Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany


Figure 1
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  		Fig. 1. Expression of integrin subunits in EBs and early mouse embryos. (A) Cell lysates of 5-day-old normal EBs were subjected to western blot analysis for integrin subunits β1, {alpha}1, {alpha}2, {alpha}3, {alpha}5, {alpha}6, {alpha}v, β3, β4 and β5. β1, {alpha}5, {alpha}6 and {alpha}v were expressed in differentiated EBs. (B) Normal EBs at different developing stages (days 2-7) were immunostained for integrins {alpha}5, {alpha}6 and {alpha}v, and for the BM components fibronectin and laminin. The insets are 2x magnifications of the boxed regions. en, endoderm; epi, epiblast; cv, cavitation. (C) Co-immunoprecipitation showed that integrin β1 formed a complex with the integrin {alpha}5, {alpha}6 and {alpha}v subunits. (D) Immunofluorescence analysis of E6.5 mouse embryos showed that integrin {alpha}5 was mainly expressed in the endoderm, whereas {alpha}6 was mainly expressed in the epiblast, and both were recruited to the embryonic BM (EBM) zone (arrows), as revealed by fibronectin and nidogen immunostaining. Integrin {alpha}v was not detected at the cell-EBM adhesion but at the trophoblast–Reichert's-membrane (RM) interface (arrowheads).

 

Figure 2
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  		Fig. 2. Ablation of integrin β1 in embryoid bodies (EBs) disrupts endoderm morphogenesis. Wild-type (WT) and integrin-β1-null (β1–/–) EBs were cultured in suspension for 2-7 days. (A) Live phase micrographs show endoderm (en) formation on day 3, and epiblast (epi) polarization and cavitation (cv) on days 5 and 7, respectively, in wild-type EBs (arrows). By contrast, endoderm cells detached from or loosely adhered to the EB surface (arrowheads) in most of the mutant EBs. BM, basement membrane. (B) 5-day-old EBs were immunostained for the endoderm markers {alpha}-fetoprotein ({alpha}-FP), cytokeratin EndoA, GATA4 and disabled-2 (Dab2). BM is identified by laminin {alpha}1/{gamma}1 chain (Lm {alpha}1/{gamma}1) or perlecan (perl) immunofluorescence. Arrowheads point to positively stained endoderm cells. (C) EBs that had developed a continuous {alpha}-FP-positive endoderm layer were counted and plotted as a percentage of total EBs examined. More than 85% of the WT EBs developed a continuous endoderm layer, whereas less than 4% of the mutant EBs had nearly normal endoderm. (D) Western analysis was performed for the expression of endoderm markers on 5-day-old normal and mutant EBs collected together with detached endoderm cells. Actin serves as loading control. (E) Normal endoderm cells were harvested by brief trypsinization and mechanical dissociation. The detached integrin-β1-null endoderm cells were collected from the conditioned medium. The expression of the BM proteins laminin-111, nidogen, collagen IV (Col IV) and fibronectin (Fn) was analyzed by western blotting.

 

Figure 3
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  		Fig. 3. GATA4 is mislocalized to the cytoplasm in integrin-β1-null endoderm cells. (A,B) Endoderm cells collected from normal (WT) and integrin-β1-null (β1–/–) EBs were immunostained for {alpha}-FP, cytokeratin EndoA, Dab2, laminin {alpha}1 and GATA4. Nuclei were counterstained with DAPI. Dab2 and laminin are not detected in the mutant cells. GATA4 is mainly localized to the nucleus in normal endoderm cells but is present in the cytoplasm in the mutants (arrowheads). (C) Endodermal cells with cytosolic (Cy) or nuclear (Nu) GATA4 staining were counted by fluorescence microscopy and plotted as a percentage of total cells examined. (D) Cytosolic or nuclear fractions of cell lysates were analyzed by western blotting for GATA4 expression. Actin serves as loading control.

 

Figure 4
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  		Fig. 4. Integrin-β1-null endoderm cells fail to adhere and/or spread on BM substrates but can spread on vitronectin; vitronectin upregulates integrin β3 and induces GATA4 nuclear translocation. (A) Live phase micrographs show normal (WT) and integrin-β1-null (β1–/–) endoderm cells cultured on fibronectin for 16 hours. The cells were fixed and immunostained for paxillin, laminin {alpha}1 chain (Lm {alpha}1) and Dab2. F-actin was stained with rhodamine-phalloidin. (B) Endoderm cells were plated on various ECM substrates for 1 hour and adherent cells were quantified by crystal-violet staining and spectrophotometry. n=4 for each group. (C) Endoderm cells collected from the conditioned medium were fixed and immunostained for the apoptosis marker cleaved caspase-3. Positive cells were counted and plotted as a percentage of total cells examined. [n=326 for the wild-type (WT) and n=308 for integrin-β1-null (β1–/–) cells]. (D) Integrin-β1-null endoderm cells were grown on vitronectin (Vn)-coated coverslips for 2 days and immunostained for integrin β3, integrin {alpha}v, paxillin, laminin {alpha}1 and Dab2. (E) The expression of integrin β3 was analyzed by FACS in integrin-β1-null endoderm cells before and after 2 days of culturing on vitronectin. (F) Integrin-β1-null endoderm cells were cultured on vitronectin, and GATA4 levels in cytosolic and nuclear fractions were assayed by western blotting at designated time points. The intracellular distribution of GATA4 was also analyzed by immunostaining. Vitronectin induced GATA4 nuclear translocation.

 

Figure 5
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  		Fig. 5. Forced expression of integrin β3 in integrin-β1-null EBs rescues endoderm morphogenesis. (A) EBs were cultured for 5 days using integrin-β1-null ES cells stably transfected with integrin-β3–GFP (β3-GFP) or GFP. The expression of integrin β3 was analyzed by western blotting. (B) Wild-type and integrin-β1-null EBs expressing integrin-β3–GFP or GFP were cultured for 5 days and endoderm formation was examined by live phase microscopy. The percentage of EBs with endoderm formation was plotted. n=344 for the wild-type, n=328 for the GFP and n=311 for the β3-GFP group. (C) The β3-GFP or GFP EBs were cultured for 5 days and immunostained for integrin β3 and the endoderm markers Dab2 and GATA4. BM was stained for laminin {alpha}1 or perlecan. Integrin-β3–GFP is recruited to the BM zone (arrowheads). The inset is a 2x magnification of the boxed region to show GATA4 nuclear staining. en, endoderm; epi, epiblast.

 

Figure 6
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  		Fig. 6. Knockdown of integrin {alpha}v in integrin-β1-null endoderm cells blocks vitronectin-induced FA formation, GATA4 nuclear translocation and laminin synthesis. (A) Integrin-β1-null ES cells were infected with retrovirus expressing short hairpin RNAs (shRNAs) targeting mouse integrin {alpha}v. Stable cell lines were established by puromycin selection. Western blots show that transduction of shRNA1 and shRNA4 inhibits the expression of integrin {alpha}v but not that of integrin {alpha}5. (B,C) Integrin-β1-null endoderm cells stably expressing integrin-{alpha}v shRNA or control vector pSM2 were cultured on vitronectin for 2 days. The cells were fixed and immunostained for integrin β3, paxillin, GATA4 and laminin {gamma}1. Nuclei were counterstained with DAPI. GATA4 (red) is mainly localized to the nucleus (purple when merged with DAPI staining) in control cells (arrowheads). By contrast, it is distributed primarily in the cytoplasm in integrin-{alpha}v knockdown ({alpha}v KD) cells (arrows). The GATA4 nuclear staining closely correlates with laminin expression (arrows and arrowheads). (D) Cells with positive laminin {gamma}1 and nuclear GATA4 staining were counted and plotted.

 

Figure 7
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  		Fig. 7. ERK1/2 and p38 MAPK activation is suppressed in integrin-β1-null endoderm cells, and their inhibition abrogates vitronectin-induced GATA4 nuclear translocation and cellular maturation. (A) Endoderm cells isolated from 5-day EBs were either analyzed directly or cultured on vitronectin (Vn) for 2 days and then analyzed by western blotting. The activation of ERK1/2, p38 MAPK and Akt was detected using kinase phosphorylation-specific antibodies. (B) Integrin-β1-null endoderm cells were cultured on vitronectin and treated with the MEK1/2 inhibitor U0126 (10 µM), the p38 MAPK inhibitor SB203580 (10 µM), the PI3K inhibitor wortmannin (1 µM) or Akt1/2 inhibitor (10 µM). After 2 days, the cells were fixed and stained for Dab2 and GATA4. (C) The cells treated with the above kinase inhibitors were subject to western blot analysis for laminin-111 and nidogen.

 

Figure 8
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  		Fig. 8. Expression of constitutively active MEK1 or p38 MAPK induces GATA4 nuclear translocation and cellular maturation in integrin-β1-null endoderm cells. (A,B) Integrin-β1-null endoderm cells were transfected with HA-tagged constitutively active (CA) MEK1 or control vector pcDNA3. The transfected cells were grown on fibronectin for 2 days and stained for HA-tag, GATA4 and Dab2. In untransfected cells, GATA4 was found mainly in the cytoplasm (red arrows). HA–CA-MEK1 expression led to GATA4 nuclear localization and increased Dab2 expression (white arrowheads). Some of the transfected cells spread on fibronectin and formed FAs containing HA–CA-MEK1 (white arrows). (C) Western blots show increased expression of laminin, nidogen and Dab2 in HA–CA-MEK1-transfected cells. (D) Integrin-β1-null endoderm cells were transfected with CA HA-tagged MKK6-p38{alpha} or the non-phosphorylatable mutant MKK6-p38{alpha}/AGF. The transfected cells were stained for HA-tag, GATA4 and Dab2. (E) Western blots show increased expression of laminin, nidogen and Dab2 in MKK6-p38{alpha}-transfected cells.

 

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© The Company of Biologists Ltd 2009