dipeptidyl peptidase IV, CD26, which code for a cell surface protease that cleaves the chemokine CXCL12 [21]. Interestingly, differentiating ES cells also showed an upregulation of CXCL12 transcription. CXCL12 has been shown to regulate many biological processes but also plays an important role in tumorigenesis [22,23]. CXCL12 binds to the widely expressed cell surface seven transmembrane domain G-protein coupled receptor CXCR4 [24,25] and to the recently identified receptor CXCR7/RDC1 [26]. Upon ligand binding, CXCR4 undergoes a conformational change that facilitates activation of heterotrimeric G proteins and signaling effectors at the plasma membrane [27]. This initiates a signaling cascade resulting in downstream phosphorylation of proteins such as ERK1/2 and AKT [28,29]. These activities are dependent on CXCR4 expression at the plasma membrane and cellular events that reduce the latter can abrogate the biological effects. Following activation, CXCR4 undergoes b-arrestin-medi- ated endocytotosis and although recycling of CXCR4 can occur this receptor can also be ubiquinated and directed to lysosomes where it is degraded [30,31]. Both CXCL12 and CXCR4 expression have been associated with tumorigenesis in many cancers including breast, ovarian, renal, prostate, and neuroblastoma [22,23,24]. These CXCR4 expressing tumors preferentially spread to tissues that highly express CXCL12, including lung, liver, lymph nodes and bone marrow [22,23,24]. Therefore, the inverse correlation between 5T4 and CXCL12 with CD26 transcript levels during mouse ES cell differentiation, and the known roles of these molecules in cell migration/motility, may suggest that particular regulatory processes are common to both ES cell differentiation and tumor metastasis. This article reports the unexpected discovery that 5T4 molecules are required for functional expression of CXCR4 at the cell surface of differentiating ES cells, and mouse embryonic fibroblasts (MEF). Results Microarray analysis of differentiating mES cells stratified by cell surface 5T4 phenotype Analysis of the microarray data from undifferentiated and differentiating E14 ES cells identified significant up or down regulation of transcripts in 148 and 277 genes respectively (GEO accession number GSE20372). There was a pattern of transcrip- tional changes relating to a loss of pluripotency in ES cells with significant (adjusted P value ,0.1) downregulation of Klf4 (18.4x), Oct4 (1.66) and Sox2 (1.96). Many genes known to bind these transcription factors and form part of the extended transcriptional network influencing pluripotency of ES cells were also found to be downregulated [32,33]. These are approximately 25% of the significantly downregulated genes and include 77 which can bind one or more of these transcription factors. The complex changes in transcription seen in the differentiating ES cells analyzed here reflect the several different pathways that can control ES cell pluripotency and self-renewal. In respect of this report, the microarray data indicated a significant downregulation of Dpp4 also called Cd26 (6.26), upregulation of Cxcl12 (3.46) but no change in the transcripts of Cxcr4. CD26 is a surface peptidase which cleaves CXCL12 chemokine and CXCR4 is the receptor of the latter. We decided to further investigate whether this was of functional significance. Differentiating mES cells show 5T4-dependent CXCL12 chemotaxis The latter microarray data were confirmed by qPCR (Figure 1A) and FACS analysis showed that as the ES cells differentiate cell surface expression of CD26 decreases while 5T4 increases; by 5T4 Regulates CXCR4 Chemotaxis contrast the pluripotent ES marker SSEA-1 did not significantly change over this time (Figure 1B). Consistent with the transcript analysis, western blotting shows there is no change in the total CXCR4 expression upon differentiation of either wild-type (WT) or 5T4 knock out (5T4KO)-ES cells (Figure 1C). A greater than 2 fold increase in CXCL12 was detected in the culture medium by ELISA after 3 days of differentiation of WT (7164 vs 17169 pg/ ml) or 5T4KO (4062 vs 8465 pg/ml) ES cells, (Figure 1D). These data demonstrate that in early differentiation of ES ells there is increased CXCL12 ligand production and decreased expression of CD26 dipeptidase that can destroy its activity but there is no change in either the CXCR4 transcript or protein levels, consistent with the microarray results. To examine biological response to CXCL12, WT and 5T4KO- ES cells were tested for CXCL12 chemotaxis before and after differentiation. Both WT and 5T4KO undifferentiated ES cells showed no chemotaxis towards CXCL12. Upon differentiation WT-ES cells showed a .2-fold response, while 5T4KO-ES cells remained unresponsive (Figure 1E). Following LIF withdrawal, both WT and 5T4KO-ES cells undergo an EMT with the tightly packed ES colonies becoming dispersed with the differentiating cells showing an arborized morphology. Although the differenti- ating 5T4KO-ES cells show reduced motility [16], their failure in chemotaxis was not a result of delayed kinetics in response since daily testing for up 6 days still provided no evidence for CXCL12 dependent chemotaxis (data not shown). The specificity of the chemotaxis was confirmed by showing that the differentiated WT- ES cell chemotaxis to CXCL12 was blocked by specific antibodies to the chemokine (Figure 1F) and by blocking the CXCR4 receptor with the inhibitor AMD3100 (Figure 1G). Further, the lack of chemotaxis of differentiating 5T4KO-ES cells was not the result of continued CD26 activity destroying CXCL12, since pre- incubation with the competitive CD26 inhibitor diprotin A did not restore chemotactic behavior (Figure 1H). To test whether 5T4 might play a role in CXCL12 dependent chemotaxis, undifferen- tiated and differentiating 5T4KO-ES cells were infected with recombinant adenoviral vector encoding mouse 5T4 (RAd-m5T4) or RAd-GFP control vector. There was no change in chemotaxis of either WT or 5T4KO undifferentiated ES cells infected with the different vectors (Figure 2A). Expression of m5T4 in differentiating 5T4KO-ES cells restores CXCL12 chemotaxis comparable to that of differentiating WT-ES cells (Figure 2B). A recombinant adenovirus encoding human 5T4 also restored chemotaxis (Figure 2C). These data suggest that 5T4 expression is a necessary cofactor for CXCR4 functional expression and CXCL12 chemotaxis in differentiating ES cells. One mechanism that might account for these results would be if 5T4 molecules facilitate stable cell membrane expression of CXCR4 molecules in differentiating ES cells. 5T4 expression influences plasma membrane expression of CXCR4 in differentiating ES cells The expression and cellular localization of 5T4 and CXCR4 molecules before and after differentiation of WT and 5T4KO-ES cells was determined by immunofluorescence of fixed cells grown on glass plates (Figure 3A). Undifferentiated WT-ES cells are 5T4- negative with CXCR4 expression low and intracellular however following differentiation both molecules can be detected at the cell surface with clear areas of co-localization. By contrast, differen- tiated 5T4KO ES cells show only intracellular CXCR4 expres- sion. Quantitatively, 98% of differentiating WT-ES cells showed cell surface CXCR4 expression and only 2% cytoplasmic while differentiating KO-ES had 1% cell surface and 89% cytoplasmic CXCR4 labeling; 10% were CXCR4 negative. It is apparent that PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e9982