Generation and expression analysis of the <i>Vsig1-EGFP</i> transgenic allele.

<p>(A) Schematic representation of the <i>Vsig1-EGFP</i> transgenic construct. The <i>Vsig1-EGFP</i> construct consists of the 4.5-kb genomic fragment located upstream of exon 1a of the <i>Vsig1</i> gene (black box) and the EGFP gene (green box). (B) Expression of the <i>Vsig1-EGFP</i> transgenic allele in adult stomachs and testes of different transgenic lines and wild-type (WT) mice was determined by Northern-blot hybridization using the <i>EGFP</i> probe. Integrity of RNA samples was documented by images of the corresponding agarose gel. (C) Immunoblot of EGFP expression in cellular extracts from different tissues of 3-month-old transgenic mouse. The protein blot was subsequently probed with anti-α-tubulin antibody. (D) Expression of the <i>Vsig1-EGFP</i> transgenic allele during pre- and postnatal stomach development was examined by immunoblotting using total lysates obtained from transgenic stomachs of embryos at E15.5 and E18.5, and from P10, P20 and P60 mice. Protein extract from wild-type stomach (WT) was used as controls. (E) Temporal expression of VSIG1 during prenatal and postnatal development of stomach was examined by immunoblotting. (F) Fluorescent micrographs of stomachs from transgenic embryos at E18.5 and 60-day-old mice show EGFP epifluorescence in the posterior stomach (P) but not in the anterior stomach (A). (G) Expression of Vsig1-EGFP in the glandular epithelium was confirmed by immunofluorescence in paraffin sections of E15.5, P0.5, P10 and P20 with anti-EGFP (green fluorescence) and anti-VSIG1 (red fluorescence) antibodies. DAPI (blue fluorescence) was used for nuclear staining. Scale bar (F) = 500 µm; (G) = 200 µm.</p

Transdifferentiation of the <i>Vsig1^−/Y</i> cells into squamous epithelia inside the gastric corpus of chimeric <i>Vsig1^−/Y↔Vsig1^+/Y</i> stomachs.

<p>(A–F) Serial sections of stomach prepared from 5-month-old chimeric mice were stained with H&E (A and B) or immunohistologically analyzed for expression of glandular and squamous epithelia-specific markers (C–F). The bracket in H&E-stained sections (A) marks the area containing squamous epithelia, which are present inside the glandular epithelia of the gastric corpus and is magnified in B. Cells of this lesion do not express VSIG1 (C) or glandular epithelium-specific markers H⁺,K⁺-ATPase (D) and GATA4 (E), but do express cytokeratin K5 (F), which is normally expressed in squamous epithelia of the forestomach (G). Inserts show higher magnification. (H and I) Dissected stomachs from <i>Vsig1^−/Y↔Vsig1^+/Y</i> chimeras at E17.5 were longitudinally sectioned and immunological stained with anti-VSIG1 antibody. The transition zone between glandular mucosa and stratified squamous epithelia of the forestomach is indicated by an arrow. The box in H is magnified in I and denotes a patch of atypical squamous epithelium that lacks the primordial gastric units of the glandular epithelium as well as cells that express VSIG1. Scale bar (A and C–H) = 500 µm; (B and I) = 100 µm; inserts = 200 µm.</p

Characterization and expression analysis of <i>Vsig1</i> splice variants.

<p>(A) Schematic diagram of the <i>Vsig1</i> gene. Boxes and lines represent the exons and introns, respectively. Positions of both polyA signals and probes used in Northern blot analysis are shown. (B) Schematic representation of exonic sequences present in the different <i>Vsig1</i> mRNA isoforms. Black boxes represent the coding exon, while white boxes represent the 5′ and 3′UTRs of the <i>Vsig1</i> splice variants. (C) Northern blot with total RNA from different tissues of 3-month-old mice was hybridized with probe 1 (top panel) and probe 2 (middle panel). Integrity and variation of loaded RNA samples were assessed by rehybridization with a probe for human elongation factor 2 (EF-2). (D) Restricted expression of <i>Vsig1C</i> isoform in testis was confirmed by RT-PCR analysis using primers containing the sequence of exons 1b and 4. The used primers only amplify the 396-bp cDNA fragment in testis RNA. Production of the control <i>Hprt</i> products was observed throughout tissues, demonstrating the presence of intact loaded RNA. (E) Immunoblot with cellular extracts from different tissues was probed using polyclonal anti-VSIG1 antibodies and subsequently reprobed with monoclonal anti- α-tubulin antibodies (α-Tub). (F) Immunoblot with untreated and N-glycosidase F-treated stomach extracts was probed with anti-VSIG1 antibodies.</p

Targeting disruption of the <i>Vsig1</i>.

<p>(A) Structure of the wild-type, targeting vector and recombinant allele are shown together with the relevant restriction sites. A 2.5-kb genomic fragment containing exon 1a was replaced by a <i>pgk-neo</i> selection cassette (NEO). The probe used and predicted length of the <i>Eco</i>RI restriction fragment in Southern blot analysis are shown. TK, thymidine kinase cassette; E, <i>Eco</i>RI; X, <i>Xba</i>I; X*, disrupted <i>Xba</i>I site; Xh, <i>Xho</i>I. (B) Blot with <i>Eco</i>RI-digested genomic DNA of recombinant ESC clones was probed with the 3′ probe shown in panel A. The external probe recognized only a 10.7-kb fragment of recombinant allele in <i>Vsig1^−/Y</i> ESCs and a 12.2-kb fragment of the wild-type allele in <i>Vsig1^+/Y</i> ESCs. (C) PCR assay using microsatellite markers was performed to determine the degree of chimerism in the stomachs of chimeric male mice. The 129- and C57-specific fragments were amplified using DNA of the 129/Sv and C57BL/6J mouse strains, and stomach isolated from different chimeric males (Ch).</p

Expression analysis of VSIG1 during stomach development.

<p>(A) RNA blot of total RNA isolated from the stomach of different stages of pre- (E) and postnatal (P) development was hybridized with <i>Vsig1</i> (probe 2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025908#pone-0025908-g001" target="_blank">Fig. 1A</a>) and the <i>hEF-2</i> cDNA probe. (B) Immunohistochemistry of paraffin sections with anti-VSIG1 antibody shows the restricted expression of VSIG1 in the glandular epithelium of the stomach at E12.5 (B), E13.5 (C) and E17.5 (D). (E) Expression of GATA4 in glandular epithelia of the stomach at E17.5. Arrows in C–E mark the transitional junction between the glandular and squamous epithelia. In 3-month-old stomachs, VSIG1 is located at the adhesion junctions between epithelial cells of the gastric unit (F). The box in F is magnified in G and shows restricted localization of VSIG1 to the basolateral membrane of pit cells (G). Scale bar (B–E) = 500 µm; (F) = 100 µm; (G) = 20 µm.</p

Apoptosis-Related Gene Expression Profiles of Mouse ESCs and maGSCs: Role of Fgf4 and Mnda in Pluripotent Cell Responses to Genotoxicity

<div><p>Stem cells in the developing embryo proliferate and differentiate while maintaining genomic integrity, failure of which may lead to accumulation of mutations and subsequent damage to the embryo. Embryonic stem cells (ESCs), the <em>in vitro</em> counterpart of embryo stem cells are highly sensitive to genotoxic stress. Defective ESCs undergo either efficient DNA damage repair or apoptosis, thus maintaining genomic integrity. However, the genotoxicity- and apoptosis-related processes in germ-line derived pluripotent cells, multipotent adult germ-line stem cells (maGSCs), are currently unknown. Here, we analyzed the expression of apoptosis-related genes using OligoGEArray in undifferentiated maGSCs and ESCs and identified a similar set of genes expressed in both cell types. We detected the expression of intrinsic, but not extrinsic, apoptotic pathway genes in both cell types. Further, we found that apoptosis-related gene expression patterns of differentiated ESCs and maGSCs are identical to each other. Comparative analysis revealed that several pro- and anti-apoptotic genes are expressed specifically in pluripotent cells, but markedly downregulated in the differentiated counterparts of these cells. Activation of the intrinsic apoptotic pathway cause approximately ∼35% of both ESCs and maGSCs to adopt an early-apoptotic phenotype. Moreover, we performed transcriptome studies using early-apoptotic cells to identify novel pluripotency- and apoptosis-related genes. From these transcriptome studies, we selected <em>Fgf4</em> (Fibroblast growth factor 4) and <em>Mnda</em> (Myeloid cell nuclear differentiating antigen), which are highly downregulated in early-apoptotic cells, as novel candidates and analyzed their roles in apoptosis and genotoxicity responses in ESCs. Collectively, our results show the existence of common molecular mechanisms for maintaining the pristine stem cell pool of both ESCs and maGSCs.</p> </div

Expression profiling of apoptosis-related genes in differentiated ESCs and maGSCs.

<p>(<b>A</b>) OligoGEArray blot showing the expression pattern of apoptosis-related genes in ESCs and maGSCs of the 129Sv background that had been differentiated for 21 days with retinoic acid. (<b>B</b>) Scatterplot analysis of differentiated ESCs and maGSCs revealing similar gene expression patterns in both differentiated cell types with upregulation of <i>Nfkb1</i> and downregulation of <i>Pycard</i> and <i>Bnip3</i> in differentiated maGSCs. (<b>C</b>) Heatmap analysis of undifferentiated ESCs and maGSCs (from both the 129Sv and the Stra8-EGFP backgrounds) as well as differentiated ESCs and maGSCs, revealing all undifferentiated cell types in one cluster, while differentiated cell types are distant and clustered together. (<b>C′</b>) Specific and strong expression of several anti-apoptotic and pro-apoptotic genes in undifferentiated ESCs and maGSCs relative to differentiated cells was highlighted.</p

Role of <i>Fgf4</i> and <i>Mnda</i> during genotoxic stress response of ESCs.

<p>(<b>A</b>) Stacked bar graphs showing the percentage of cells at various stages of cell cycle (SubG1/Apoptotic, G1, S and G2/M) in untreated, Control ESCs – wildtype ESCs; Fgf4-OE – Fgf4 overexpression cells; Fgf4-KO – Fgf4 knockout cells; Mnda-OE – Mnda overexpression cells; Mnda-DN – Mnda downregulation cells. (<b>B–F</b>) Genotoxic stress was induced by treatment with NCS for 30 min followed by recovery for indicated time points and analyzed for cell cycle parameters in Control ESCs – wildtype ESCs; Fgf4-OE – Fgf4 overexpression cells; Fgf4-KO – Fgf4 knockout cells; Mnda-OE – Mnda overexpression cells; Mnda-DN – Mnda downregulation cells. The cell cycle data of three or more independent biological replicates were calculated and represented as a mean ±SD. The values which are statistically significant are indicated with asterisks (∗p<0.05).</p

List of GO terms associated with upregulated genes in early-apoptotic cells.

<p>List of GO terms associated with upregulated genes in early-apoptotic cells.</p

List of GO terms associated with downregulated genes in early-apoptotic cells.

<p>List of GO terms associated with downregulated genes in early-apoptotic cells.</p