Ets2 is required for trophoblast stem cell self renewal

The Ets2 transcription factor is essential for the development of the mouse placenta and for generating signals for embryonic mesoderm and axis formation. Using a conditional targeted Ets2 allele, we show that Ets2 is essential for trophoblast stem (TS) cells self renewal. Inactivation of Ets2 results in slower growth, increased expression of a subset of differentiation associated genes and decreased expression of several genes implicated in TS self renewal. Among the direct TS targets of Ets2 is Cdx2, a key master regulator of TS cell state. In addition other Ets2 responsive genes include Pace4, Errb, Socs2 and Bmp4. Thus Ets2 contributes to the regulation of multiple genes important for maintaining the undifferentiated state of TS cells and as candidate signals for embryonic development. Keywords: cell type comparison We analysed RNA with Illumina MouseRef-8 and MouseRef-6 Expression BeadChips using the manufacturers BeadArray Reader and collected primary data using the supplied Scanner software. Data analysis was done in three stages. First, expression intensities were calculated for each gene probed on the array for all hybridizations (24 in total) using illumina’s Beadstudio#1 software. Second, intensity values were quality controlled and normalized: quality control was carried out by using the illumina Beadstudio detection P-value set to <0.01 as a cutoff. This removed genes which were effectively absent from the array (that is, were not detected). After this step, the initial gene set was reduced to 14888 genes. All the arrays were then normalized using the normalize.quantiles routine from the Affy R-package in Bioconductor. This procedure accounted for any variation in hybridization intensity between the individual arrays. An assessment of several different normalization techniques using the Bioconductor maCorrPlot routine suggested that normalize.quantiles was the most appropriate for the data. Finally, these normalized data were imported into GeneSpring and analysed for differentially expressed genes. The groups of biological replicates were described to the software and significantly differentially expressed genes determined on the basis of t-tests and fold difference changes in expression level. GroupA (8 replicates): AdenoGFP Ets2 f/f. GroupB (7 replicates) Adeno Cre Ets2 f/f. GroupC (3 replicates) TS cells Ets2 f/f. GroupD (4 replicates) TS differ Ets2 f/f. DM medium GroupE (2 replicates) SB teated DM cells Ets2 f/f . Here we describe the generation of an Ets2 conditional allele and show that Cre-mediated Ets2 inactivation causes a defect in the self renewal of TS cells. Inactivation of Ets2 results in changes in gene expression normally associated with differentiation caused by growth factor withdrawal. Among the genes sensitive to Ets2 inactivation are Pace4, Cdx2 and BMP4. Ets2 is essential to maintain expression of multiple genes necessary for TS cell self renewal. To facilitate study of Ets2 function in different cell types, we generated by gene targeting a conditional Ets2 allele that could be inactivated by Cre-mediated recombination. Two loxP sites were engineered into the targeting vector to flank exon 9 and 10, a major part of the conserved DNA binding domain that is essential for Ets2 function. Additionally, a Pgk-neo cassette flanked by Flip recombinase recognitions sites (frt) was inserted into intron 8, which together with a thymidine kinase gene inserted upstream of exon 6, provided both positive and negative selection for homologous recombination. Twenty-seven targeted ES cell clones were identified from a total of 132 clones screened by Southern blot analysis with a 3’ flanking probe and PCR screening for the presence of both loxP sites. After karyotyping, two clones were selected for injection into blastocysts and one transmitted the targeted allele, designated Ets2flox-neo, through the germ line. The Ets2flox-neo mice were phenotypically wild type and transmitted the targeted allele at the expected Mendelian frequency. An epiblast-specific Cre line, Mox2-Cre (MORE), was used to recombine the Ets2flox allele . The resulting deletion mutant, designated Ets2db2 due to its similarity to the previously described Ets2db1, caused recessive embryonic lethality around E8 . Ets2db2/db2 embryos at E7.5 were very similar to the previously described Ets2db1/db1 mutants, including much smaller concepti and unusual cone-shaped yolk sacs. We further tested the viability of embryos containing a combination of the Ets2A72 hypomorphic knock-in allele and Ets2db2. As expected, Ets2A72/db2 embryos died at mid-gestation like previously described Ets2A72/db1 embryos while their Ets2A72/flox littermates developed normally (Table 1). The combination of Ets2flox and the Cre recombinase of MORE mice can be used as an alternative to tetraploid embryo aggregation because wild-type function will remain in primitive endoderm and trophectoderm derivatives that are devoid of Cre activity. We tested whether the recessive lethality of Ets2db2 could be rescued when one allele was generated in the MORE background. Indeed, the wild-type function provided by one parental Ets2flox allele in the extraembryonic tissues was sufficient to support development of the otherwise largely homozygous Ets2db2 embryos. The near complete inactivation of Ets2 in the somatic tissues of these mice was confirmed by PCR analysis that was capable of determining the Ets2flox:Ets2db2 ratio in genomic DNA. Interestingly, these mice recapitulated the “waved” hair phenotype of Ets2db1/db1 mice rescued by tetaploid embryo aggregation. The young animals were distinguished by wavy hair and curly whiskers, which became less apparent over time. Skin sections revealed misaligned and dysplastic hair follicles, consistent with the observation in Ets2db1/db1 mice. Both sexes of these MORE;Ets2db2/flox mice were fertile, and without obvious abnormality. Gene expression changes associated with Ets2 inactivation. RNA expression profiles of Ets2flox/flox TS cells infected with Adenovirus expressing Cre recombinase were compared with those of TS cells infected with control virus and to TS cells induced to differentiate by growth factor withdrawal. The analysis of replicate samples from two different TS cell isolates confirmed reliable reproduction of gene expression changes. Unsupervised cluster analysis of individual samples revealed that inactivation of Ets2 under normal growth conditions caused only moderate changes in gene expression most closely related to control cultures either not infected or infected with virus not expressing Cre. By contrast RNAs from differentiated TS cells (DM) clustered together and were distinguished from mouse embryo fibroblast RNAs (MEF) and undifferentiated TS cells (GM). To identify individual candidate Ets2 target genes, grouped samples were analyzed. While the changes in gene expression due to differentiation were greater than those due to inactivation of Ets2, most of the changes associated with Ets2 inactivation were in genes that also changed when TS cells were induced to differentiate. 89 of the top 100 changes in gene expression resulting from AdenoCre infection of Ets2flox/flox TS cells also changed significantly upon growth factor withdrawal induced differentiation. Thus Ets2 inactivation caused changes in gene expression associated with differentiation but these genes represented only a subset of differentiation dependent gene expression changes. Gene expression changes associated with infection with either virus were modest and did not overlap with either Ets2-dependent TS genes or differentiated dependent genes. The fold changes in gene expression due to inactivation of Ets2 were less than cells induced to differentiate by growth factor withdrawal. . Among the Ets2 sensitive TS genes are: Errb, that has been previously implicated in ES cell self renewal ; Socs2, an inhibitor of intracellular signaling; Cdx2, a master regulator of trophoblast specification and self renewal and Pace4, an extraembryonic protease that activates Nodal to promote TS cell maintenance. In contrast to Pace4, Furin, a second protease implicated in Nodal processing , is unaffected by Ets2 inactivation (Figure 7C). Two Ets transcription factors (Etv4 and Elf5) are down regulated upon TS cell differentiation. Ets2 inactivation resulted in decreased Etv4 RNA but had no effect on Elf5 RNA. Four other members of the Ets family either changed little or increased upon differentation of TS cells Fewer significant increases in gene expression were associated with Ets2 inactivation. RNAs for Wnt7a and Cldn23, a junctional protein, were increased in AdenoCre infected Ets2flox/flox TS cells.