Molecular role of Pelota (PELO) in differentiation of embryonic and germ stem cells

Pelo ist ein evolutionär konserviertes Gen, das in diversen Spezies charakterisiert wurde. In der Maus führt der Verlust von Pelo zu embryonaler Letalität in frühen Postimplantationsstadien. In vitro Studien mit Pelo-null Blastocysten haben gezeigt, dass PELO möglicherweise eine Rolle bei der Regulation des Zellzyklus oder der Selbsterneuerung der pluripotenten Embryonaler Stammzellen (Embryonic Stem Cells, ESCs) spielt. In der vorliegenden Arbeit sollte die molekulare Rolle von PELO bei der Selbsterneuerung und bei der Differenzierung von ESCs und Keimbahnstammzellen mit Hilfe eines konditionalen Pelo Knockout-Mausmodels in Kombination mit in vitro sowie in vivo Experimenten untersucht werden. Im ersten Teil der Arbeit konnten wir zeigen, dass PELO für die Selbsterneuerung von ESCs oder deren Differenzierung in die drei Keimblätter nicht notwendig ist, jedoch unabdingbar ist für die Differenzierung des extraembryonalen Endoderms (ExEn). Im Umkehrschluss wird durch die Überexpression von Pelo in ESCs das Programm zur Differenzierung des ExEn`s aktiviert. Auf molekularer Ebene konnten wir zeigen, dass die beeinträchtigte Differenzierung des ExEn`s in Pelo-defizienten Embryoidkörpern (Embryonic Bodies, EBs) aus einer reduzierten Aktivität des Bone Morphogenetic Proteins (BMP) resultiert. Dieses Ergebnis wurde durch weitere Experimente bestätigt, die gezeigt haben, dass Pelo-defiziente Zellen durch Behandlung mit BMP4 in das ExEn differenzieren können. In vivo Studien haben gezeigt, dass Pelo-null Embryonen am Tag 6.5 (E6.5) das ExEn besitzen, jedoch an E7.5 versterben. Dies lässt vermuten, dass PELO nicht für die Induktion der Entwicklung des ExEn`s notwendig ist, sondern vielmehr für dessen Erhaltung oder abschließende Differenzierung in das funktionelle viszerale Endoderm, das den Embryo mit Wachstumsfaktoren für die weitere Entwicklung versorgt. Zudem ist PELO notwendig für die BMP-Aktivierung zu Beginn der somatischen Zellreprogrammierung. Der Verlust von PELO beeinträchtigt die Reprogrammierung zur induzierten Pluripotenz. Außerdem konnten wir die konservierte Funktion von PELO im Qualitätskontrollmechanismus der RNA in murinen ESCs feststellen. Im zweiten Teil der Arbeit haben wir demonstriert, dass die Pelo-Expression essentiell für die Erhaltung der männlichen Fertilität und Spermatogenese ist. Der Verlust von Pelo während der Entwicklung von männlichen Keimzellen in Mäusen hat gezeigt, dass PELO für die Selbsterneuerung und Erhaltung der Spermatogonialen Stammzellen (Spermatogonial Stem Cells, SSCs) notwendig ist, jedoch für die Entwicklung der späteren Spermatogenesestadien sowie die Spermienfunktion erlässlich ist. Insgesamt zeigen unsere Studien die molekulare Rolle(n) von PELO in der frühen Embryonalentwicklung der Maus und bei der männlichen Fertilität auf. Unsere Ergebnisse geben Hinweise auf Ursachen von Defekten, die mit dem PELO Verlust zusammenhängen

Adhesion Protein VSIG1 Is Required for the Proper Differentiation of Glandular Gastric Epithelia

VSIG1, a cell adhesion protein of the immunoglobulin superfamily, is preferentially expressed in stomach, testis, and certain gastric, esophageal and ovarian cancers. Here, we describe the expression patterns of three alternatively spliced isoforms of mouse Vsig1 during pre- and postnatal development of stomach and potential function of Vsig1 in differentiation of gastric epithelia. We show that isoforms Vsig1A and Vsig1B, which differ in the 39untranslated region, are expressed in the early stages of stomach development. Immunohistochemical analysis revealed that VSIG1 is restricted to the adherens junction of the glandular epithelium. The shorter transcript Vsig1C is restricted to the testis, encodes an N-terminal truncated protein and is presumably regulated by an internal promoter, which is located upstream of exon 1b. To determine whether the 59 flanking region of exon 1a specifically targets the expression of Vsig1 to stomach epithelia, we generated and analyzed transgenic mice. The 4.8-kb fragment located upstream of exon 1a was sufficient to direct the expression of the reporter gene to the glandular epithelia of transgenic stomach. To determine the role of VSIG1 during the development of stomach epithelia, an X-linked Vsig1 was inactivated in embryonic stem cells (ESCs). Although Vsig1 2/Y ESCs were only able to generate low coat color chimeric mice, no male chimeras transmitted the targeted allele to their progeny suggesting that the high contribution of Vsig1 2/Y cells leads to the lethality of chimeric embryos. Analysis of chimeric stomachs revealed the differentiation of VSIG1-null cells into squamous epithelia inside the glandular region. These results suggest that VSIG1 i

Pelota regulates the development of extraembryonic endoderm through activation of bone morphogenetic protein (BMP) signaling

Pelota (Pelo) is ubiquitously expressed, and its genetic deletion in mice leads to embryonic lethality at an early post-implantation stage. In the present study, we conditionally deleted Pelo and showed that PELO deficiency did not markedly affect the self-renewal of embryonic stem cells (ESCs) or their capacity to differentiate in teratoma assays. However, their differentiation into extraembryonic endoderm (ExEn) in embryoid bodies (EBs) was severely compromised. Conversely, forced expression of Pelo in ESCs resulted in spontaneous differentiation toward the ExEn lineage. Failure of Pelo-deficient ESCs to differentiate into ExEn was accompanied by the retained expression of pluripotency-related genes and alterations in expression of components of the bone morphogenetic protein (BMP) signaling pathway. Further experiments have also revealed that attenuated activity of BMP signaling is responsible for the impaired development of ExEn. The recovery of ExEn and down-regulation of pluripotent genes in BMP4-treated Pelo-null EBs indicate that the failure of mutant cells to down-regulate pluripotency-related genes in EBs is not a result of autonomous defect, but rather to failed signals from surrounding ExEn lineage that induce the differentiation program. In vivo studies showed the presence of ExEn in Pelo-null embryos at E6.5, yet embryonic lethality at E7.5, suggesting that PELO is not required for the induction of ExEn development, but rather for ExEn maintenance or for terminal differentiation toward functional visceral endoderm which provides the embryos with growth factors required for further development. Moreover, Pelo-null fibroblasts failed to reprogram toward induced pluripotent stem cells (iPSCs) due to inactivation of BMP signaling and impaired mesenchymal-to-epithelial transition. Thus, our results indicate that PELO plays an important role in the establishment of pluripotency and differentiation of ESCs into ExEn lineage through activation of BMP signaling

Modulation of NCAM/FGFR1 signaling suppresses EMT program in human proximal tubular epithelial cells.

Neural cell adhesion molecule (NCAM) and fibroblast growth factor receptor 1 (FGFR1) cross-talk have been involved in epithelial-to-mesenchymal transition (EMT) process during carcinogenesis. Since EMT also contributes to maladaptive repair and parenchymal damage during renal fibrosis, we became encouraged to explore the role of NCAM/FGFR1 signaling as initiating or driving forces of EMT program in cultured human proximal tubular epithelial cells (TECs). TECs stimulated with TGF-β1 (10ng/mL) was used as an established in vitro EMT model. TGF-β1 downstream effectors were detected in vitro, as well as in 50 biopsies of different human kidney diseases to explore their in vivo correlation. NCAM/FGFR1 signaling and its modulation by FGFR1 inhibitor PD173074 (100nM) were analyzed by light microscopy, immunolabeling, qRT-PCR and scratch assays. Morphological changes associated with EMT appeared 48h after TGF-ß1 treatment and was clearly apparent after 72 hours, followed by loss of CDH1 (encoding E-Cadherin) and transcriptional induction of SNAI1 (SNAIL), SNAI2 (SLUG), TWIST1, MMP2, MMP9, CDH2 (N-Cadherin), ITGA5 (integrin-α5), ITGB1 (integrin-β1), ACTA2 (α-SMA) and S100A4 (FSP1). Moreover, at the early stage of EMT program (24 hours upon TGF-β1 exposure), transcriptional induction of several NCAM isoforms along with FGFR1 was observed, implicating a mechanistic link between NCAM/FGFR1 signaling and induction of EMT. These assumptions were further supported by the inhibition of the EMT program after specific blocking of FGFR1 signaling by PD173074. Finally, there was evidence for an in vivo TGF-β1 pathway activation in diseased human kidneys and correlation with impaired renal excretory functions. Collectively, NCAM/FGFR1 signaling appears to be involved in the initial phase of TGF-ß1 initiated EMT which can be effectively suppressed by application of FGFR inhibitor

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

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

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

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