RESEARCH ARTICLE 1029

Development 136, 1029-1038 (2009) doi:10.1242/dev.028415 Foxa2 regulates polarity and epithelialization in the endoderm germ layer of the mouse embryo Ingo Burtscher and Heiko Lickert*

In the mouse, one of the earliest events in the determination of cell fate is the segregation of cells into germ layers during gastrulation; however, the cellular and molecular details are not well defined due to intrauterine development. We were able to visualize a clear sequence of events occurring in the process of germ-layer formation, using immunohistochemistry and time-lapse confocal imaging. The T-box (T) and the Forkhead transcription factor Foxa2 specify mesoderm and endoderm in the posterior epiblast. Fate-specified epiblast cells lose their polarity and undergo epithelial-mesenchymal transition to invade into the primitive streak region, where these cell populations quickly separate and differentiate into morphologically and molecularly distinct Foxa2-positive endoderm and T-positive mesoderm populations. The endoderm cells flatten and acquire apical- basal polarity during intercalation into the outside epithelium in order to establish proper intracellular junctions with pre-existing cells. By contrast, the mesodermal cells become spherical during migration and acquire a mesenchymal fate. Interestingly, axial mesodermal cells are descended from Foxa2-positive epiblast cells that upregulate T in the anterior primitive streak region. These cells, as well as Foxa2-positive endoderm cells, are highly polarized and epithelialized, suggesting that Foxa2 promotes an epithelial fate and suppresses a mesenchymal fate. This observation is supported by the fact that Foxa2 mutant endodermal cells fail to maintain polarity and do not establish proper cellular junctions, and are thus unable to functionally integrate into the endoderm epithelium. We propose that Foxa2 regulates a molecular program that induces an epithelial cellular phenotype.

KEY WORDS: Foxa2, Brachyury, Epithelial-mesenchymal transition, Mesenchymal-epithelial transition, Morphogenesis, Cell polarity, Cell adhesion, Epithelialization, Gastrulation, Germ-layer formation, Time-lapse imaging

INTRODUCTION lineages that are destined for different parts of the body (Kinder et During gastrulation the multilayered body plan of the mouse embryo al., 1999; Kinder et al., 2001). Therefore, allocation of mesoderm is established through differentiation and highly coordinated and endoderm in the embryo takes place in an anteroposterior (AP) morphogenetic events. By the start of gastrulation, at embryonic day manner determined by the timing and order of recruitment through (E) 6.5, the embryonic cup-shaped epiblast is surrounded by a the PS. The majority of definitive endodermal (DE) cells ingress single-layered epithelium of visceral endoderm (VE) that will give through the anterior end of the primitive streak (APS) at the mid- rise to the endodermal component of the yolk sac (Wells and Melton, streak (MS) stage and intercalate into the overlying VE to give rise 1999). Pluripotent epiblast cells constitute the progenitor cells for to the foregut (Kwon et al., 2008); however, a small population of all cell lineages in the embryo proper and differentiate to form the DE cells might directly delaminate into the VE from the epiblast three principal germ layers: endoderm, mesoderm and ectoderm (Tam and Beddington, 1992). Taken together, these studies clearly (Beddington and Robertson, 1999; Tam and Loebel, 2007). Clonal indicate that mesoderm and endoderm are specified in a analysis of epiblast cell fate revealed that in the early-streak embryo spatiotemporal manner during gastrulation; however, it is not clear at E6.5, the proximal one-third of the posterior epiblast contains the if these cells become specified in the epiblast or PS region and when precursors of the extra-embryonic mesoderm and the primordial these cells differentiate into morphological and molecular distinct germ cells. By contrast, the distal region of the epiblast contains the cell populations. precursors of the entire neural ectoderm, and the intermediate The T-box transcription factor brachyury (T) was shown to mark posterior epiblast contains the precursors for the anterior mesoderm progenitor cells for mesoderm and endoderm in ES cell and definitive endoderm (Lawson et al., 1991; Lawson and differentiation cultures, suggesting that these cells originate from a Pedersen, 1992; Tam and Beddington, 1992; Lawson and Hage, common progenitor (Kubo et al., 2004). In the mouse embryo, T 1994). Clonal descendants were not necessarily confined to a single protein is localized in the posterior epiblast at the early-streak stage germ layer, indicating that these lineages are not separated at the and is detected in nascent mesoderm in the PS region during beginning of gastrulation. In support of this notion are embryonic gastrulation, as well as in the node and notochord from the late- stem (ES) cell differentiation experiments (Kubo et al., 2004), as streak (LS) stage onwards (Inman and Downs, 2006). T localization well as conditional targeting results, indicating that bipotential in the mesoderm and notochord suggests that abnormalities in these mesendodermal progenitor cell populations exist (Lickert et al., cell populations are responsible for the homozygous mutant 2002) (for a review, see Rodaway and Patient, 2001). At various phenotype (Wilkinson et al., 1990). By contrast, Foxa2 is also stages of gastrulation, the primitive streak (PS) has been shown to expressed in the posterior epiblast from the early stage onwards and contain precursor cells of different mesodermal and endodermal is then confined to anterior definitive endoderm (ADE) and axial mesoderm, which consists of the head process, prechordal plate, Helmholtz Zentrum München, Institute of Stem Cell Research, Ingolstädter notochord and node (Sasaki and Hogan, 1993; Monaghan et al., Landstrasse 1, 85764 Neuherberg, Germany. 1993). Foxa2 is a member of the Forkhead transcription factor family, which includes three related transcription factors: Foxa1, *Author for correspondence (e-mail: [email protected]) Foxa2 and Foxa3, first identified by their ability to regulate -

Accepted 15 January 2009 specific (Lai et al., 1990; Lai et al., 1991). A null DEVELOPMENT 1030 RESEARCH ARTICLE Development 136 (6) mutation of the Foxa2 gene leads to absence of ADE and axial GAG; Tomato rev (5Ј-SpeI), 3Ј-NNNACTAGTTTACTTGTACAGC- mesoderm (Ang and Rossant, 1994; Weinstein et al., 1994). Foxa1 TCGTCCATGCCG; YFP fwd, 5Ј-GCGGCCGCATCTAGAATGGTGAG- and Foxa3 are expressed from E7.5 onwards in the definitive CAAGGGCGAGGAGCTGTTC; YFP rev, 3Ј-ACTAGTTTACTTGTAC- endoderm and can compensate for the loss of Foxa2 in the null AGCTCGTCCATGCCGAGAG. NotI/SpeI-digested PCR products were mutants, which allows hindgut, but not fore- and midgut formation cloned into the pBKS vector. (Sasaki and Hogan, 1993; Monaghan et al., 1993; Ang and Rossant, For generation of Lyn-Tomato, an oligonucleotide was subcloned between the NotI and XbaI sites in the pBKS vector in front of the 1994; Weinstein et al., 1994; Dufort et al., 1998). These results td-Tomato: Lyn-Oligo fwd, 5Ј-GGCCGCATAACTTCGTATAGCATA - collectively demonstrate that T and Foxa2 are functionally important CATTATACGAAGTTATGCCACCATGGGATGTATTAAATCAAAAA- for mesoderm and endoderm development; however, it is not clear GGAAAGACGGGGCCCGGTACT; Lyn-Oligo rev, 5Ј-CTAGAGTA- how these transcription factors regulate a molecular and cellular CCGGGCCC CGTCTTT CCTTTTTGATTTAATACATCCCATGGTG G C- program for the differentiation of these cell populations. ATAACTTC GTATAATGTATGCTATACGAAGTTATGCTTATGC. The In addition to cellular differentiation, the gastrulating embryo also NotI/SpeI-digested fluorescent markers were subcloned into the NotI/NheI undergoes dramatic morphological changes to form the three sites of the eukaryotic expression vector pCAGGS (Niwa et al., 1991). principal germ layers and the basic body plan. One of the first Generation of fluorescent reporter ES cell lines morphogenetic events is the formation of the PS when signals and The fluorescent ES cell and mouse lines used in this study were generated factors trigger epithelial-mesenchymal transition (EMT) of epiblast by electroporation of ScaI-linearized pCAGGS vector DNA containing cells to give rise to mesoderm and endoderm (Thiery and Sleeman, dsRed, YFP or Lyn-Tomato into wild-type IDG3.2 ES cells (Hitz et al., 2006). During this process, epiblast cells lose their apical-basal (AB) 2007) or Foxa2–/– R1 ES cells (Ang et al., 1994). Cells were selected with 1 epithelial polarity, downregulate the cell-cell adhesion molecule E- μg/ml puromycin, and resistant clones were screened for uniform and cadherin (cadherin 1 – Mouse Genome Informatics) and break ubiquitous reporter expression in cell culture and in vivo using embryos through the basement membrane (BM) to invade into the PS region. derived from ES cells. The interstitial mesodermal cells acquire a mesenchymal cellular Generation of chimeras and mouse lines fate and migrate over long distances between the endoderm and the Diploid or tetraploid chimeras were generated according to standard ectoderm germ layer before they re-aggregate to form distinct organs protocols (Nagy, 2003). Embryos were collected from dsRed- (Vintersten et such as the heart or . By contrast, cells that are fate-specified al., 2004) and YFP- (Hadjantonakis et al., 2002) expressing mouse lines, to become DE appear in the APS region from MS to LS stage both maintained on mixed genetic backgrounds (CD1/129Sv/C57/Bl6). T- (Lawson et al., 1991; Tam et al., 1997; Kinder et al., 2001; Tam and GFP targeting construct was used to generate ES cells and a mouse line as Beddington, 1992). These cells acquire an epithelial fate and previously described (Fehling et al., 2003). intercalate into the outside epithelium, but it is not clear if these cells Time-lapse live imaging undergo EMT followed by mesenchymal-epithelial transition or Embryos were dissected in DMEM containing 10% FCS and 20 mM alternatively maintain epithelial polarity and just transiently HEPES. Embryos were cultured on glass-bottom dishes using 200 μl downregulate cell-cell adhesion molecules to leave the epiblast embryo culture medium (50% rat serum, 40% DMEM without Phenol Red, epithelium. By the end of gastrulation the germ layers have formed 2 mM glutamine, 100 μM 2-mercaptoethanol and 1 mM sodium pyruvate in and have already acquired AP, dorsoventral (DV) and left-right (LR) a 37°C incubator with 5% CO2 and 5% O2). To avoid evaporation the patterning information through signals from the embryonic medium was covered with mineral oil. Image acquisition was performed on organizer tissues, which include anterior VE, ADE, axial mesoderm, a Leica DMI 6000 confocal microscope and image analysis was carried out using Leica LAS AF software. node, notochord and floorplate (Tam and Loebel, 2007). Functional analysis of in mouse has greatly contributed to Statistical analysis the understanding of germ-layer formation in the mouse embryo; Cell measurements were carried out using Leica LAS AF software. Average however, the phenotypic analysis has been hampered by static and standard deviation are shown in the graphs. P-values were determined techniques that often only describe end points, as well as the fact using a two-tailed Student’s t-test with unequal variance with the number of that embryogenesis in all placental mammals occurs in utero and is cells and embryos stated in the figure legends. not easily amenable to ex vivo observation. The establishment of Whole-mount in situ hybridization static embryo culture systems and the genetic introduction of Whole-mount in situ hybridization was performed as previously described fluorescent marker in transgenic animals has now allowed (Lickert at al., 2002). The following probes were used: Eomes (Ciruna and for direct imaging of mouse embryogenesis (Yamanaka et al., 2007; Rossant, 1999), Hex (Hhex – Mouse Genome Informatics) (Thomas et al., Kwon et al., 2008). In this study, we established an ex utero static 1998) and claudin 4 (RZPDp981G04226D). Embryos were photographed embryo culture system to continuously monitor the cellular using a Zeiss Stereo Lumar V12 microscope. processes occurring during germ-layer formation. The generation Antibodies and immunohistochemistry of genetic mosaics using aggregation chimera allowed us to Immunofluorescence whole-mount stainings were performed as distinguish embryonic and extra-embryonic lineages using previously described (Nakaya et al., 2005). Briefly, embryos were isolated, fluorescent labels in order to follow mesoderm and endoderm fixed for 20 minutes in 2% PFA in PBS, and then permeabilized in 0.1% formation at cellular resolutions. We present evidence for a specific Triton X-100 in 0.1 M glycine pH 8.0. After blocking in 10% FCS, 3% role of Foxa2 in the formation of polarized and epithelialized cell goat serum, 0.1% BSA, 0.1% Tween 20 for 2 hours, embryos were types, namely the definitive endoderm and axial mesoderm (node incubated with the primary antibody o/n at 4°C in blocking solution. After and notochord). several washes in PBS containing 0.1% Tween-20 (PBST) embryos were incubated with secondary antibodies (donkey anti-mouse 594, donkey anti-rabbit 488, donkey anti-goat 594 Alexa fluor, Molecular Probes) in MATERIALS AND METHODS blocking solution for 3 hours. During the final washes with PBST, Generation of expression vectors embryos were stained with 4Ј,6-diamidino-2-phenylindole, Genes encoding fluorescent proteins (td-Tomato, YFP) were amplified by dihydrochloride (DAPI), transferred into 40% glycerol and embedded PCR using the following primers: Tomato fwd (5Ј-NotI-Kozak-XbaI), 5Ј- between two coverslips using 120 μm Secure-Seal spacers (Invitrogen,

GCGGCCGCAGCCACCATGTCTAGAATGGTGAGCAAGGGCGAG- S24737) and ProLong Gold antifade reagent (Invitrogen, P36930). DEVELOPMENT Foxa2 regulates mesendoderm formation RESEARCH ARTICLE 1031

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.

Antibodies: Foxa2 (Abcam, Ab408749), brachyury (N-19, Santa Cruz), material). Proximal epiblast cells upregulate T protein after EMT GFP (A11122, Invitrogen), E-cadherin (610181, BD), ZO-1 (Tjp1 – and show a round, mesenchymal cellular phenotype in the PS region Mouse Genome Informatics) (33-9100, Zymed). (Inman and Downs, 2006), whereas distal epiblast cells upregulate Foxa2 protein after EMT and show a flattened cell morphology (see RESULTS Fig. S2 in the supplementary material). Interestingly, Foxa2- Specification and differentiation in the gastrula- expressing flattened cells appeared anterior to the anatomical end of stage embryo the PS, forming a two-cell-diameter row of polarized cells (see Fig. Foxa2 is a Forkhead transcription factor required for anterior axial S2D, white arrows, in the supplementary material). From fate maps mesoderm and definitive endoderm formation (Ang and Rossant, (Tam and Beddington, 1992) and our imaging results (see Fig. 2 1994; Weinstein et al., 1994), whereas the T-box transcription factor below), this seems to be the region in which the first DE cells appear brachyury (T) is necessary for posterior, but not anterior, mesoderm at the surface, suggesting that a small population of DE cells might formation (Wilkinson et al., 1990). As previously reported, the directly delaminate into the outside VE. At the LS stage, only mRNAs for both genes are expressed during gastrulation in the epiblast cells underlying the APS were Foxa2-positive and showed posterior epiblast, but it was not clear whether the proteins are signs of EMT (Fig. 1C; see Fig. S1B in the supplementary material). synthesized in the same cells of the epiblast and epiblast-derived We could clearly distinguish three cell populations in the PS region: mesoderm and endoderm descendants (Sasaki and Hogan, 1993; T-positive posterior mesoderm; Foxa2- and T-double-positive axial Monaghan et al., 1993; Herrmann, 1991). To investigate the cellular mesoderm; and Foxa2-positive VE and DE populations. At the LS distribution of these two transcription factors during gastrulation, stage, we could rarely detect single Foxa2-positive cells in the APS we used whole-mount immunohistochemistry (IHC) with antibodies region. This implies that Foxa2+ epiblast cells quickly upregulate T to T and Foxa2 and laser scanning microscopy (LSM) of fixed protein after EMT, which suggests that cells for the axial mesoderm embryos. Surprisingly, in pre-streak-stage embryos the double are recruited from the APS region (Kinder et al., 2001). These results immunofluorescent antibody staining revealed that T and Foxa2 collectively indicate that endoderm and mesoderm is specified in the protein was synthesized in two intermingled, but mutually exclusive, epiblast and differentiates after EMT, which can be distinguished by cell populations in the posterior epiblast (Fig. 1A; see Fig. S1A in morphology and marker gene expression. Interestingly, Foxa2 the supplementary material). At MS stage, these two cell epiblast precursor cells gave rise to polarized and epithelialized populations segregated into proximal and distal domains of the endoderm and axial mesoderm, including the polarized and

posterior epiblast (Fig. 1B; see Fig. S2 in the supplementary epithelialized cells of the node and notochord. DEVELOPMENT 1032 RESEARCH ARTICLE Development 136 (6)

Fig. 2. Time-lapse imaging of endoderm formation. (A) Generation of diploid (2n) or tetraploid (4n) embryo } 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 } 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 } 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.

Time-lapse imaging reveals characteristic mutant cells in an otherwise wild-type environment. For the time- morphogenetic behavior of mesodermal and lapse imaging we used an inverse confocal microscope in endodermal cell populations combination with a static embryo culture system (Fig. 2B). To gain further insight into the morphogenetic mechanisms Analyzing single optical sections using LSM at the mid-sagittal and underlying mesoderm and endoderm formation during gastrulation, surface level of fixed 4n chimeras revealed that the epiblast was we developed a static embryo culture system using time-lapse always completely derived from the ES cells at the pre-streak stage confocal imaging (Fig. 2A-D). One major difficulty in the analysis (E6.5) and was covered by a single-layered epithelium of VE from of endoderm development is the lack of appropriate marker genes the 4n embryo (Fig. 2C) (n>100). As predicted from fate map that can distinguish the embryonic DE from the extra-embryonic VE studies of the mouse embryo (Lawson et al., 1991; Lawson and (Lewis and Tam, 2006). To this end, we analyzed germ-layer Pedersen, 1992; Tam and Beddington, 1992), the first DE cells were formation using aggregation chimera, which allowed us to label the recruited from the epiblast and intercalated into the surface VE in embryonic and extra-embryonic lineages by means of different the APS region at MS stage (Fig. 2C). By the LS stage, the fluorescent marker genes (Fig. 2A). In tetraploid (4n) or diploid (2n) recruitment of DE was almost finished and the VE was mostly embryo } wild-type 9 (wt) ES cell aggregation chimera (hereafter displaced by DE (Fig. 2C). From these results we concluded that called 2n/4n } wt chimera), the ES cells can only contribute to the fluorescent-lineage tagging using aggregation chimeras generates embryonic epiblast, whereas the extra-embryonic lineages are useful genetic mosaics to monitor cellular processes and lineage always formed by the cells of the 2n or 4n embryo (Tam and allocation in the pre- to LS-stage embryo. Next we performed time- Rossant, 2003). Therefore, using 2n and 4n chimera allowed us to lapse live imaging analysis using LSM of static immobilized 4n distinguish between embryonic lineages, namely ectoderm, dsRed } wt YFP chimera during gastrulation (Fig. 2D). As already mesoderm and DE, and extra-embryonic lineages, specifically indicated by our analysis of fixed MS chimera (Fig. 2C), DE cells trophectoderm and VE. Generating 2n chimera allowed us formed in the APS region and intercalated into the overlying VE at

additionally to generate genetic mosaics in the epiblast to study this developmental stage (Fig. 2D; see Movie 1 in the supplementary DEVELOPMENT Foxa2 regulates mesendoderm formation RESEARCH ARTICLE 1033

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 } 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 } 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 } 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.

material). The time-lapse analysis revealed that the DE and the the distal tip of the embryo even at the end of LS stage, indicating mesoderm populations are morphologically distinct cell populations that the node and definitive endoderm cells are either not formed or in the PS region, even before the DE cells intercalate into the outside that these cells do not reach the surface epithelial layer (Fig. 3A) VE (Fig. 2D) (time 0:00–0:39). The DE cells showed flat (n>30). We noticed that cells accumulated in the APS region and morphology and had an average length-width ratio of 4:1 frequently led to an indentation of posterior epiblast epithelium into (l=13.1±1.7 μm; w=3.24±0.67 μm; l/w=4.21±0.9; n=50), whereas the amniotic cavity from E7.5 onwards (Fig. 3A; see Movie 3 in the the mesoderm cells showed a characteristic round morphology with supplementary material; data not shown). We next performed time- an approximate length-width ratio of 1.4:1 (l=7.73±1.54 μm; lapse imaging using LSM of Foxa2–/– ES 2n chimeras to analyze the w=5.63±1.16 μm; l/w=1.41±0.36; n=50) at LS stage. Furthermore, behavior of Foxa2 mutant cells in an otherwise wild-type T-positive mesoderm cells and Foxa2-positive endoderm cells environment (Fig. 3B; see Movie 2 in the supplementary material) showed distinct morphology at the MS stage (see Fig. S2 in the (n=9). As shown earlier in this study, Foxa2-positive epiblast cells supplementary material), indicating that mesoderm and endoderm reside in the APS region (Fig. 1), leave the epiblast epithelium and can be distinguished by marker gene expression and morphology. form DE, which intercalates into the overlying VE (Fig. 2). Imaging This observation is consistent with results previously obtained in Foxa2 null cells from MS stage onwards clearly revealed that APS zebrafish (Warga and Nüsslein-Volhard, 1999), demonstrating that cells leave the epiblast and ingress into the APS region (Fig. 3B) mesoderm and endoderm cell populations can also be distinguished (t=0:00-1:45 h, white asterisk). In contrast to wild-type DE cells, by morphological criteria in higher vertebrates and that these cell Foxa2–/– ‘endoderm-like’ cells showed endoderm morphology (Fig. populations are specified in the epiblast (Fig. 1) and differentiate and 3B) (time 0:00; l=13.3±2.6 μm; w=3.8±0.7 μm; l/w: 3.6±0.9; n=50), segregate in the PS region (Fig. 2). made contact and partially integrated into the outside VE, but failed to epithelialize (Fig. 3B) (time 0:00-1:15, black asterisks). We Foxa2 regulates epithelialization of the endoderm wondered whether in 2n } wt chimeras wild-type cells had a germ layer competitive advantage and substituted or rescued DE formation; To analyze how the Foxa2 transcription factor regulates definitive thus this might have been the reason that Foxa2–/– cells were not endoderm development on the cellular level, we took advantage of integrated in the outside epithelium. Therefore we analyzed the the Foxa2 knockout ES cell line (Ang and Rossant, 1994) and cellular behavior of mutant cells in 4n } Foxa2–/– chimeras (Fig. analyzed genetic mosaics using LSM. In 4n } wt chimeras, DE cells 3C; see Movie 3 in the supplementary material). We clearly intercalated into the outside VE in the APS region from MS stage observed cells, which were intercalated but left the outside onwards and displaced and dispersed the VE by the LS stage (Fig. epithelium (Fig. 3C) (time 0:00-0:36). This indicates that Foxa2 is 3A) (n>20). In striking contrast, all 4n } Foxa2–/– chimeras showed necessary for functional integration of DE cells into the VE

no sign of DE intercalation and failed to form an anatomical node at epithelium. DEVELOPMENT 1034 RESEARCH ARTICLE Development 136 (6)

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 } 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 } 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. 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 } wt Lyn-Tomato chimera at LS stage. Next we analyzed the identity of endoderm-like cells, which Sagittal confocal section in the posterior PS region taken from Movie 4 formed and intercalated into the outside VE in the absence of Foxa2. at the indicated time points (see Movie 4 in the supplementary We performed whole-mount in situ hybridization to detect material). During intercalation, endoderm cells extend filiopodia (dotted endoderm-specific genes that regulate EMT and mesendoderm line, T=0) and aquire AB polarity, as indicated by the apical fluorescent formation [Eomes (Arnold et al., 2008)], transcription and endoderm marker protein Lyn-Tomato (white arrowheads, T=0:15-0:45). (B) (Top) } formation [Hex (Thomas et al., 1998; Martinez Barbera et al., DE cells show apical localization of Lyn-Tomato in 4n YFP wt Lyn- 2000)], as well as cell-matrix adhesion [integrin alpha3 (Tamplin et Tomato chimera at MS to LS stage. Arrowheads indicate polarized (white) and non-polarized (red) cells. (Bottom) Foxa2 mutant cells fail to al., 2008)] and tight junction formation (claudin 4). In completely } –/– –/– localize Lyn-Tomato in 4n YFP Foxa2 Lyn-Tomato chimera. Foxa2 ES-cell-derived MS-stage embryos, Eomes was expressed (Middle) There is a statistically significant difference (*P<0.01) in apical at normal levels in the PS, confirming that Foxa2 mutant cells Lyn-Tomato localization between wild-type DE cells (78.9±4.6%; underwent EMT and formed mesendoderm (Fig. 4A; Fig. 3). n=109; three embryos) and Foxa2 mutant cells (54.9±5.7%; n=111; Moreover, ADE formation was clearly induced in Foxa2 mutant four embryos). cells, as indicated by the expression of the endoderm marker gene Hex (Fig. 4B). We previously used gene expression profiling of 4n } wt and Foxa2–/– chimeras to identify differentially expressed genes at the gastrulation stage (Tamplin et al., 2008). This analysis These results clearly indicate that endoderm-like cells are formed in revealed that the tight junction markers claudin 4 and the cell-matrix Foxa2 mutants, but accumulate in the APS region, fail to induce adhesion molecule integrin alpha3 (Itga3) are potential target genes claudin 4 and do not functionally integrate into the outside VE for Foxa2 in the DE. Strikingly, we found that whereas Itga3 was (compare with Fig. 3). strongly expressed in the APS region of 4n } Foxa2–/– chimeras at the head-fold stage (Tamplin et al., 2008) (Fig. 2C,F), the tight Foxa2 is important to establish AB polarity and junction marker claudin 4 was not detectable in the anterior cell-cell adhesion endoderm region of 4n } Foxa2–/– chimera (Fig. 6C). To further To better understand the cellular and molecular defects of Foxa2 characterize the identity of Foxa2–/– cells on a cellular level, we mutant endoderm cells, we analyzed the process of endoderm performed whole-mount IHC to detect the mesoderm marker protein intercalation in greater detail. For this purpose, we made use of a T. As expected, Foxa2–/– endoderm-like cells, which where partially ubiquitous Lyn-Tomato-expressing ES cell line (Fig. 5). The 10 N- integrated into the outside VE, were negative for T protein (Fig. 4C), terminal amino acids of the Lyn-kinase containing a consensus N- indicating that Foxa2–/– endoderm cells did not switch to a myristoylation and S-palmitoylation sites were fused to the mesodermal fate, but still remained endoderm-like, expressing N-terminus of the Tomato protein to target the fusion protein to

Eomes, Hex and Itga3, but not the tight-junction marker claudin 4. the plasma membrane. Surprisingly, the ubiquitously expressed DEVELOPMENT Foxa2 regulates mesendoderm formation RESEARCH ARTICLE 1035

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 } 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 } 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.

Lyn-Tomato protein accumulated on the apical membrane surface detect E-cadherin and ZO-1 in the endoderm epithelium. Comparing of the epiblast and DE epithelium (Fig. 3A, red asterisks). In the PS MS to LS stage 4n } wt and Foxa2–/– chimeras clearly demonstrated region, where epiblast cells lose polarity and undergo EMT, the that the adherens junction protein E-cadherin was not localized at continuous apical localization of the Lyn-Tomato protein was junctions between adjacent mutant cells, but surprisingly mutant-wt disrupted. Using Lyn-Tomato as a tool to analyze cell polarity, we cell junctions showed a similar extent of basolateral localization as performed time-lapse live imaging of intercalating DE cells in 4n wt-wt adherens junctions (Fig. 6A). We speculate that the correct YFP } wt Lyn-Tomato chimera (Fig. 5A; see Movie 4 in the positioning of E-cadherin in mutant-wt cell junctions is due to supplementary material). By the beginning of intercalation, DE cells homotypic molecular interactions of the E-cadherin protein in in contact with the outside VE were not polarized, but extended mutant cells with those correctly localized to the basolateral domain filopodia processes into the outside epithelium (Fig. 5A) (time 0:00). in wt cells. However, these interactions may be transient, as the During intercalation, DE cells became more and more polarized mutant cells failed to functionally integrate into the outside (Fig. 5A) (time 0:15–0:30) and by the end of the process clearly epithelium. Due to the fact that claudin 4 is not expressed in Foxa2 showed AB cell polarity by the means of Lyn-Tomato localization mutants (Fig. 6C), we investigated the localization of the tight- (Fig. 5A) (time 0:45). junction protein ZO-1. Comparing MS to LS stage 2n } wt and Using Lyn-Tomato as an apical membrane marker, we compared Foxa2–/– chimeras revealed that wt cells localized ZO-1 to the cellular polarization in 4n } wt or Foxa2–/– chimeras (Fig. 5B). basolateral junctions in a punctate manner, whereas most mutant Analyzing MS- to LS-stage embryos revealed that Foxa2 mutant cells ectopically localized ZO-1 to the apical surface (Fig. 6B). It is cells were able to localize Lyn-Tomato to the apical membrane (Fig. well known that Claudins are the major cell-adhesion molecules of 5B, white arrowheads). However, the analysis also showed a tight junctions (Tsukita et al., 2001; Furuse and Tsukita, 2006) and statistically significant difference in the cellular polarization bind specifically to ZO-1, ZO-2 (Tjp2 – Mouse Genome between wt and Foxa2 mutant cells. To investigate the cause of the Informatics) and ZO-3 (Tjp3 – Mouse Genome Informatics) via an cell polarity defects, we analyzed the formation of adherens and/or intracellular PDZ domain (Itoh et al., 1999). Therefore, failure to

tight junctions using whole-mount immunolocalization studies to induce claudin 4 or other Claudins expressed in the endoderm DEVELOPMENT 1036 RESEARCH ARTICLE Development 136 (6)

(Sousa-Nunes et al., 2003; Hou et al., 2007) might explain the posterior epiblast can be divided into a distal Foxa2-positive and ectopic localization of ZO-1 at the apical membrane of Foxa2 proximal T-positive precursor cell population, giving rise to anterior mutant endoderm-like cells. Alternatively, Foxa2 might regulate a and posterior mesendodermal cell populations, respectively. molecular program of cell polarity important to establish functional tight and adherens junctions. Foxa2 is upstream of T and initiates axial mesoderm development DISCUSSION How does axial mesoderm, namely the head process, prechordal In this study we analyzed germ-layer formation in wild-type and plate, notochord and node, develop? It was previously suggested that Foxa2 mutant embryos and chimera by immunohistochemistry and Foxa2 is on top of a developmental program for axial mesoderm time-lapse live imaging. We showed that T-positive mesoderm and formation (Yamanaka et al., 2007). At the MS stage we detected an Foxa2-positive axial mesoderm and endoderm cell populations are APS population, which was Foxa2-positive and was fate-mapped to already specified in the epiblast. These cells undergo EMT and give rise to the axial mesoderm and endoderm (Kinder et al., 2001). ingress into the PS region, where they differentiate and segregate At the LS stage, the epiblast cells generated three distinct cell into molecularly and morphologically distinct populations of populations by morphology and marker gene expression: a T- mesoderm and endoderm. Flat endoderm cells polarize and integrate positive posterior mesoderm population, a Foxa2-positive endoderm into the overlying epithelium by formation of adherens and tight population and an anterior Foxa2-positive and T-positive axial junctions. We showed that Foxa2 is translated in epiblast precursor mesoderm population. We noticed that Foxa2-positive epiblast cells cells of polarized and epithelialized cell types: namely endoderm at MS to LS stage upregulated T protein after EMT, indicating that and axial mesoderm (node and notochord). Axial mesodermal cells Foxa2 epiblast cells give rise to axial mesoderm. From knockout upregulate T protein after EMT, which suggests that Foxa2 is studies it is known that Foxa2 mutants do not form axial mesoderm upstream of T in this cell population. In Foxa2 mutants, an at all, whereas the T mutants initially form but fail to maintain anatomically characteristic node structure is not formed at the distal posterior notochord. We also showed in this study that no anatomical tip of the embryo, and although endoderm-like cells are formed and node structure is formed at the distal tip of Foxa2 mutant chimera. accumulate in the anterior PS region, they do not functionally This suggests that Foxa2 is on top of the axial mesoderm hierarchy integrate into the outside epithelium. These cells fail to polarize and (Yamanaka et al., 2007) and is consistent with loss of brachyury epithelialize, implicating that Foxa2 regulates a molecular program expression, specifically in the node and AME, but not PS, of important for these processes. tetraploid-derived Foxa2 null embryos at E7.5 (Dufort et al., 1998). Interestingly, axial mesodermal cells (node and notochord) did not Epiblast cells are specified and differentiate in the acquire a mesenchymal fate along with the rest of the T-positive PS region mesoderm population in the posterior PS, but rather constituted a An important question in embryology and stem cell biology is when population of cells that were highly polarized and connected through and how precursor cells are specified and differentiate. To our cell-cell adhesion. For example, node cells formed a characteristic surprise, the T-box transcription factor brachyury (T) and the anatomical structure in the surface endoderm layer at the distal tip Forkhead box transcription factor Foxa2 are specifically synthesized of the embryo. The cells showed clear AB polarity, were in specified mesoderm and endoderm precursor cells in the posterior monociliated and interconnected through E-cadherin-mediated cell- epiblast during gastrulation. Using time-lapse imaging and cell adhesion (Yamanaka et al., 2007). Also the notochord immunohistochemistry, we have shown that mesodermal and descendents of the node cells were highly polarized and formed a endodermal cells quickly segregate and differentiate after EMT. T- solid rod-like structure through cell-cell adhesion, between the positive epiblast cells differentiate into T-positive mesenchymal endoderm and the ectoderm epithelium. This suggests that Foxa2 cells in the PS, whereas Foxa2-positive epiblast cells differentiate progenitor cells in general give rise to polarized, interconnected cell into Foxa2-positive epithelial endodermal cells that integrate into types and that Foxa2 promotes an epithelial fate and suppresses a the overlying epithelium and Foxa2-positive, T-positive axial mesenchymal fate. mesodermal cells. Fate map analyses have revealed that the cells in the anterior end of the PS of the MS-stage embryo, which we have Foxa2 induces an epithelial cellular phenotype shown are Foxa2-positive, will give rise to anterior mesoderm and In this study, we have shown that Foxa2 mutant progenitor cells endoderm (Kinder et al., 2001), whereas cells in the posterior region leave the epiblast, but fail to integrate into the outside epithelium, of the PS, which we have shown are T-positive, will give rise to which leads to an accumulation of mesenchymal cells in the APS posterior as well as extra-embryonic mesoderm (Kinder et al., 1999). region. This is consistent with the idea that Foxa2 regulates a This is consistent with the gene functional analysis of either of these program necessary to acquire an epithelial cellular phenotype. This genes. T null mutants lack posterior mesoderm and notochord is also accordant with the lack of polarized and epithelialized cell (Wilkinson et al., 1990; Kispert and Herrmann, 1994), whereas types in the Foxa2 mutant embryos, i.e. node, notochord and anterior Foxa2 null mutants lack anterior mesoderm and endoderm, as well definitive endoderm (Ang and Rossant, 1994; Weinstein et al., as the node and notochord (Ang and Rossant, 1994; Weinstein et al., 1994), but how does Foxa2 regulate cell-cell polarity and 1994). Using a T-Cre and Foxa2-Cre genetic lineage tracing epithelialization in the endoderm germ layer? In our attempts to approach, we and others have recently shown that Foxa2 epiblast identify novel Foxa2 target genes at the gastrulation stage (Tamplin precursor cells give rise to anterior mesoderm and endoderm, et al., 2008), we have identified many potential target genes, whereas T epiblast precursors give rise to posterior mesoderm and including the transcription factors Hex and Otx2 endoderm (Uetzmann et al., 2008; Park et al., 2008; Kumar et al., (Kimura-Yoshida et al., 2007), the signaling molecules Cer1 and 2007; Verheyden et al., 2005). These results are consistent with the Shh (Epstein et al., 1999; Jeong and Epstein, 2003), the SRY-related idea that a bipotential mesendodermal progenitor cell population HMG box transcription factor Sox17 and the Forkhead box exists in mammals (Rodaway and Patient, 2001; Lickert et al., 2002; transcription factor Foxa1 (Duncan et al., 1998). Most of these

Kubo et al., 2004). Taken together, these results suggest that the endoderm-specific patterning factors are expressed in the endoderm DEVELOPMENT Foxa2 regulates mesendoderm formation RESEARCH ARTICLE 1037 germ layer, but not in Foxa2-positive epiblast precursor cells. This Epstein, D. J., McMahon, A. P. and Joyner, A. L. (1999). Regionalization of is consistent with the idea that Foxa2 is a pioneer factor, which opens Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3-dependent and -independent compact chromatin and acts in higher-order gene regulation to allow mechanisms. Development 126, 281-292. mesendoderm and endoderm specific transcription factors to specify Fehling, H. J., Lacaud, G., Kubo, A., Kennedy, M., Robertson, S., Keller, G. cell fate (Cirillo et al., 2002). But how does this molecular program and Kouskoff, V. (2003). Tracking mesoderm induction and its specification to translate into cellular changes that lead to the mesoderm or the hemangioblast during embryonic stem cell differentiation. Development 130, 4217-4227. endoderm lineage decisions? In this respect it was interesting to find Furuse, M. and Tsukita, S. (2006). Claudins in occluding junctions of humans and proteins involved in cell adhesion, such as the tight junction protein flies. Trends Cell Biol. 16, 181-188. claudin 4, the homotypic cell-cell adhesion molecules Flrt2, Flrt3 Hadjantonakis, A. K., Macmaster, S. and Nagy, A. (2002). Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter and Pcdh19, as well as the cell-matrix adhesion molecule Itga3, as usage within a single animal. BMC Biotechnol. 2, 11. potential Foxa2 endoderm target genes (Tamplin et al., 2008). It was Herrmann, B. G. (1991). Expression pattern of the Brachyury gene in whole- recently shown that hepatocyte nuclear factor 4a (HNF4a; Hnf1a – mount TWis/TWis mutant embryos. Development 113, 913-917. Hitz, C., Wurst, W. and Kuhn, R. (2007). Conditional brain-specific knockdown Mouse Genome Informatics), an important nuclear for of MAPK using Cre/loxP regulated RNA interference. Nucleic Acids Res. 35, e90. endoderm development (Lemaigre and Zaret, 2004), triggers Hou, J., Charters, A. M., Lee, S. C., Zhao, Y., Wu, M. K., Jones, S. J., Marra, formation of functional tight junctions and establishment of M. A. and Hoodless, P. A. (2007). A systematic screen for genes expressed in polarized epithelial morphology by specifically inducing Claudin definitive endoderm by Serial Analysis of Gene Expression (SAGE). BMC Dev. Biol. 7, 92. expression (Chiba et al., 2003; Satohisa et al., 2005). ZO-1 has been Inman, K. E. and Downs, K. M. (2006). Brachyury is required for elongation and proposed to be a scaffolding protein between transmembrane and vasculogenesis in the murine allantois. Development 133, 2947-2959. cytoplasmatic proteins, and possibly forms a link between the Itoh, M., Furuse, M., Morita, K., Kubota, K., Saitou, M. and Tsukita, S. (1999). Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, adherens and tight junctions, e.g. formation of the adherens junction and ZO-3, with the COOH termini of claudins. J. Cell Biol. 147, 1351-1363. through E-cadherin is associated with the formation and localization Jeong, Y. and Epstein, D. J. (2003). Distinct regulators of Shh transcription in the of tight junction proteins, particularly ZO-1 (Rajasekaran et al., floor plate and notochord indicate separate origins for these tissues in the mouse node. Development 130, 3891-3902. 1996; Siliciano and Goodenough, 1988). Taken together, we suggest Kimura-Yoshida, C., Tian, E., Nakano, H., Amazaki, S., Shimokawa, K., that Foxa2 mutant endoderm-like cells fail to initiate an endodermal Rossant, J., Aizawa, S. and Matsuo, I. (2007). Crucial roles of Foxa2 in mouse molecular program regulated by Foxa2 and different endoderm- anterior-posterior axis polarization via regulation of anterior visceral endoderm- specific patterning factors, which results in a change of cellular specific genes. Proc. Natl. Acad. Sci. USA 104, 5919-5924. Kinder, S. J., Tsang, T. E., Quinlan, G. A., Hadjantonakis, A. K., Nagy, A. and morphology dictated by cell-cell, cell-matrix adhesion and cell Ta m , P. P. (1999). The orderly allocation of mesodermal cells to the polarity molecules. extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo. Development 126, 4691-4701. We thank Wenke Barkey, Patrizia Giallonardo, Susanne Weidemann and Kinder, S. J., Tsang, T. 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