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First published online February 20, 2009
doi: 10.1242/10.1242/dev.028415

Development 136, 1029-1038 (2009)
Published by The Company of Biologists 2009
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Foxa2 regulates polarity and epithelialization in the endoderm germ layer of the mouse embryo

Ingo Burtscher and Heiko Lickert*

Helmholtz Zentrum München, Institute of Stem Cell Research, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany.


Figure 1
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  		Fig. 1. Specification and differentiation in the gastrula-stage mouse embryo. Mid-sagittal confocal sections of a pre-streak (A), mid-streak (MS) (B) or late-streak (LS) (C) stage embryo, showing whole-mount immunofluorescent staining of brachyury (T, red), Foxa2 (green) and DAPI (blue), with bright-field images on the left. The boxed region is magnified in the panels showing the separate chanels and overlay. (A) Foxa2 and brachyury antibodies mark mutually exclusive precursor cell populations in the posterior epiblast of a pre-streak embryo. (B) At the MS stage, two epiblast domains (white line shows border), comprising Foxa2-positive (green asterisk) and T-positive (white asterisk) cells, are visible. These precursor cells give rise to T-positive (red arrowhead), T- and Foxa2-positive (yellow arrowhead) and Foxa2-positive (green arrowhead) cells in the primitive streak (PS). (C) At the LS stage, three cell populations can be distinguished: T-positive cells in the posterior PS (dotted line), Foxa2 and T double-positive cells in the anterior primitive streak (APS), and Foxa2-positive visceral (VE) and definitive (DE) endoderm cells. Note that Foxa2-positive progenitor cells are still found in the epiblast (green arrowheads in the Foxa2 panel), which undergo EMT (white arrowheads in the Foxa2 and T overlay panel) and upregulate T (red arrowhead in the Foxa2 and T overlay panel). mes, mesoderm.

 

Figure 2
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  		Fig. 2. Time-lapse imaging of endoderm formation. (A) Generation of diploid (2n) or tetraploid (4n) embryo {leftrightarrow} ES cell chimeras for lineage labeling and mosaic analysis. (B) Schematic of the static embryo culture system. Mouse embryos are immobilized on a glass-bottom dish in a lateral position and are imaged with an inverted confocal microscope. (C) Monitoring DE formation in tetraploid (4n) YFP {leftrightarrow} wt dsRed ES cell aggregation chimera. Mid-saggital and surface confocal sections of pre-streak (E6.5), MS (E7.0) and LS (E7.5) stage tetraploid chimera are shown. The epiblast and DE are derived from the dsRed-expressing ES cells. Extra-embryonic tissues, including VE and extra-embryonic ectoderm, are derived from the tetraploid embryo. (D) Time-lapse imaging sequence of DE formation in a 4n dsRed {leftrightarrow} wt YFP chimera at MS to LS stage. Sagittal confocal sections are taken from Movie 1 at the indicated time points (T=hours: minutes) (see Movie 1 in the supplementary material). YFP-positive DE progenitor cells with a slightly elongated morphology line the dsRed-positive VE epithelium (black asterisks, T=0:00) and start to intercalate into the visceral endoderm layer (blue asterisks, T=0:24-0:39). Mesoderm cells (red asterisks) have a round morphology and migrate between epiblast and VE. Note that all embryos are oriented with posterior to the right and distal to the bottom. EPI, epiblast; ExE, extra-embryonic ectoderm. Scale bars: 100 µm in C; 50 µm in D.

 

Figure 3
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  		Fig. 3. Foxa2 mutant chimera fail to form an anatomical characteristic node and definitive endoderm during gastrulation. (A) Sagittal confocal section of a 4n YFP {leftrightarrow} Lyn-Tomato wt (left panel, MS stage) or Foxa2–/– (right panel, LS stage) chimera. Wild-type DE intercalates into the outside VE at the MS stage. Foxa2 mutant cells accumulate in the PS region and do not intercalate into the overlying VE epithelium. A characteristic node is not formed at the distal tip of the embryo. Note that the anterior epiblast and intercalated DE cells show clear apical localization of Lyn-Tomato (red asterisks). (B) Time-lapse imaging sequence of DE formation in a 2n dsRed {leftrightarrow} Foxa2–/– YFP chimera at MS to LS stage. Sagittal confocal sections are taken from Movie 2 at the indicated time points (see Movie 2 in the supplementary material). Foxa2 mutant `endoderm-like' cells with an elongated morphology (black asterisks) line the VE, but fail to intercalate into the outside epithelium. Note the EMT of Foxa2 mutant cells (white asterisks) in the APS (T=0:00 to 1:45). (C) Time-lapse imaging sequence of an endoderm-like cell leaving the VE epithelium in a 4n dsRed {leftrightarrow} Foxa2–/– YFP chimera at MS to LS stage. Mid-sagittal section, anterior to the left and distal to the bottom at the indicated time points (see Movie 3 in the supplementary material). AP, apical; BAS, basal; EL, endoderm-like cell; EPI, epiblast.

 

Figure 4
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  		Fig. 4. Molecular identity of Foxa2 mutant cells. (A) Whole-mount in situ hybridization showing comparable expression of the mesendoderm and EMT marker Eomes in wild-type embryos (n=5) and 4n {leftrightarrow} Foxa2–/– chimeras (n=3) at the LS stage. (B) At the MS stage, Hex mRNA is highly expressed in the anterior VE (asterisks) and in the APS region in both the wild-type (n=3) and Foxa2 mutant chimeras (n=6). (C) Whole-mount immunostaining to detect T protein in 2n {leftrightarrow} Foxa2–/– YFP chimeras at LS stage. Foxa2–/– endoderm-like cells (labeled with an antibody to YFP, green) are detected in the endoderm epithelial layer (end), but do not synthesize the mesodermal marker protein T (red arrows). The epiblast (epi), mesoderm (mes) and endoderm (end) germ layers are separated by the dotted lines in the DAPI channel.

 

Figure 5
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  		Fig. 5. Foxa2–/– mutant cells fail to acquire apical-basal polarity during intercalation into the outside epithelium. (A) Time-lapse imaging sequence of a DE cell (asterisks) intercalating into the YFP-positive (green) VE in a 4n YFP {leftrightarrow} wt Lyn-Tomato chimera at LS stage. Sagittal confocal section in the posterior PS region taken from Movie 4 at the indicated time points (see Movie 4 in the supplementary material). During intercalation, endoderm cells extend filiopodia (dotted line, T=0) and aquire AB polarity, as indicated by the apical fluorescent marker protein Lyn-Tomato (white arrowheads, T=0:15-0:45). (B) (Top) DE cells show apical localization of Lyn-Tomato in 4n YFP {leftrightarrow} wt Lyn-Tomato chimera at MS to LS stage. Arrowheads indicate polarized (white) and non-polarized (red) cells. (Bottom) Foxa2 mutant cells fail to localize Lyn-Tomato in 4n YFP {leftrightarrow} Foxa2–/– Lyn-Tomato chimera. (Middle) There is a statistically significant difference (*P<0.01) in apical Lyn-Tomato localization between wild-type DE cells (78.9±4.6%; n=109; three embryos) and Foxa2 mutant cells (54.9±5.7%; n=111; four embryos).

 

Figure 6
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  		Fig. 6. Foxa2–/– mutant cells do not acquire apical-basal polarity and fail to localize adherens and tight-junction proteins. (A) Mid-sagittal section of a whole-mount LS chimeric mouse embryo (2n YFP {leftrightarrow} Lyn-Tomato). The wild type is shown in the pair of panels at the top, the Foxa2 mutant at the bottom. Sections are stained with anti-GFP antibodies to detect Foxa2–/– cells (YFP, green; blue asterisks) or wild-type cells (which are not stained; red asterisks), anti-E-cadherin antibodies (E-Cad, red), and DAPI (blue) to label all nuclei. Adherens junctions that stain for E-cadherin are found at the basolateral membrane between wild-type cells (red arrow; wt-wt in bar chart) and between wild-type and Foxa2–/– cells (green arrowheads; KO-wt), but not between two Foxa2–/– cells (yellow arrowheads; KO-KO). The bar chart illustrates the statistically significant difference (P<0.01) between E-cadherin localization to adherens junctions in wt-wt (87.7±6.2%; n=23) or KO-wt (88.3±4.8%; n=34) versus KO-KO (16±9.4%; n=21) cells from three different chimeric embryos. (B) Foxa2–/– mutant cells fail to localize the ZO-1 tight junction protein to the apical surface. Mid-sagittal section of a whole-mount immunostained LS chimeric mouse embryo (2n wt {leftrightarrow} Foxa2–/– YFP) stained with anti-GFP antibodies to detect Foxa2–/– cells (YFP, green; white asterisks), with anti-ZO-1 (yellow) and with DAPI (blue) to label all nuclei. In wild-type endoderm cells (YFP negative) the ZO-1 protein is localized in a dot-like pattern to basolateral tight junctions (blue arrows), whereas Foxa2–/– mutant cells show accumulation of ZO-1 at the apical surface (white arrows). Quantification reveals a statistically significant (P<0.01) difference in tight-junction ZO-1 localization between wild-type (85.4±5.1%; n=237) and Foxa2 mutant (15.4±6.7%; n=123) cells. (C) In situ hybridization of wild type (n=7) and Foxa2–/– chimeras (n=4) illustrates that claudin 4 mRNA is strongly reduced in the anterior definitive endoderm of Foxa2–/– mutants at the headfold stage.

 

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© The Company of Biologists Ltd 2009