In Vivo Function and Evolution of the Eutherian-Specific Pluripotency Marker UTF1

Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. UTF1 is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the UTF1 gene during mouse development. We found that homozygous UTF1 mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of UTF1 expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of UTF1 expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the UTF1 gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the UTF1 genes.This study was supported in part by the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), and mostly by the Support Program for the Strategic Research Foundation at Private Universities, 2008–2012. This study was performed as a part of the Core Research for Evolutional Science and Technology (CREST) Agency. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Striking Similarity in the Gene Expression Levels of Individual Myc Module Members among ESCs, EpiSCs, and Partial iPSCs

<div><p>Predominant transcriptional subnetworks called Core, Myc, and PRC modules have been shown to participate in preservation of the pluripotency and self-renewality of embryonic stem cells (ESCs). Epiblast stem cells (EpiSCs) are another cell type that possesses pluripotency and self-renewality. However, the roles of these modules in EpiSCs have not been systematically examined to date. Here, we compared the average expression levels of Core, Myc, and PRC module genes between ESCs and EpiSCs. EpiSCs showed substantially higher and lower expression levels of PRC and Core module genes, respectively, compared with those in ESCs, while Myc module members showed almost equivalent levels of average gene expression. Subsequent analyses revealed that the similarity in gene expression levels of the Myc module between these two cell types was not just overall, but striking similarities were evident even when comparing the expression of individual genes. We also observed equivalent levels of similarity in the expression of individual Myc module genes between induced pluripotent stem cells (iPSCs) and partial iPSCs that are an unwanted byproduct generated during iPSC induction. Moreover, our data demonstrate that partial iPSCs depend on a high level of c-Myc expression for their self-renewal properties.</p> </div

Comparison of the expression of Core and Myc module genes in EpiSCs and ESCs.

<div><p>(A) Average gene expression values (log₂) of Core, Myc, and PRC module genes in EpiSCs using values from ESCs as references. Data from 99 Core, 426 Myc, and 474 PRC module genes deposited in GEO under GSE30056 were used for the analyses. Data from 12 Core (111 genes), 77 Myc (503 genes), and 86 PRC (560) module genes are not available in the deposited data sets.</p> <p>(B) Comparison of the expression of individual Core, Myc, and PRC module genes between ESCs and EpiSCs. Left, middle, and right scatter plots show the expression values of individual Core, Myc, and PRC module genes, respectively, in ESCs and EpiSCs. Red and blue spots indicate genes with expression levels that are higher or lower by more than 2-fold in EpiSCs compared with those in ESCs, respectively. Gene symbols corresponding to red and blue are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone.0083769.s011" target="_blank">Table S2</a>. The variance value was calculated and is shown for each scatter plot.</p> <p>(C) Left, middle, and right scatter plots show the expression values of the selected Core, Myc, and Core module genes (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone.0083769.s012" target="_blank">Table S3</a>), respectively, in ESCs and EpiSCs. Red and blue spots indicate as described in B. The variance value was calculated and is shown for each scatter plot.</p></div

Highly specific activation of Core and Myc modules and repression of the PRC module in pluripotent cells.

<p>Publicly available DNA microarray data for 20 different tissue/somatic cell and stem cell types were obtained from the NCBI GEO database. To compare the same sets of genes used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone-0083769-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone-0083769-g002" target="_blank">2</a>, data obtained using the same DNA microarray platform (Mouse Expression Array 430 platform, Affymetrix) by Hayashi et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#B29" target="_blank">29</a>] were selected from the database. Average gene expression values (log₂) of Core (upper panel), Myc (meddle panel), and PRC (lower panel) modules in each sample were calculated using those in ESCs as references. The data were aligned in an ordered fashion based on the value of average Myc module gene expression in which a sample showing the highest score, i.e., gPSC, was put at the left end of graph. The accession numbers of the obtained DNA microarray data are listed in the Materials and Methods. Data from germline stem cells and their derivatives, somatic stem cells, tissues, terminally differentiated hematopoietic cells and EpiSCs/EpiLCs are indicated by pink, blue, green, red, and gray bars, respectively, in the graph.</p

Most Myc module members maintain constant levels of expression in naïve and primed human iPSCs.

<div><p>(A) Average gene expression values (log₂) of Core, Myc, and PRC module genes in primed human iPSCs using those in human iPSCs converted to a naïve state as references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#B33" target="_blank">33</a>. Data from 69 Core, 321 Myc, and 423 PRC module genes deposited in GEO under GSE21222 were used for the analyses. Data from six Core, 34 Myc, and 28 PRC module genes are not available in the deposited data sets.</p> <p>(B) Comparison of the expression of individual Core, Myc, and PRC module genes between naïve and primed human iPSCs. Left, middle, and right scatter plots show the expression values of individual Core, Myc, and PRC module genes, respectively, in naïve and primed human iPSCs. Red and blue spots indicate genes with expression levels that are higher or lower by more than 2-fold in primed human iPSCs compared with those in naïve human iPSCs, respectively. Gene symbols corresponding to red and blue are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone.0083769.s014" target="_blank">Table S5</a>. The variance value was calculated and is shown for each scatter plot.</p> <p>(C) Scatter plot analyses of the selected genes from Core (left), Myc (middle), and PRC (right) modules. The same sets of genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone.0083769.s012" target="_blank">Table S3</a>) used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone-0083769-g001" target="_blank">Figure 1C</a> were used for the analyses. The data lacked information for 15, 33, and 13 genes of the selected Core (50), Myc (98), and PRC (115) module genes, respectively. Red and blue spots indicate as described in B.</p></div

<i>In Vivo</i> Function and Evolution of the Eutherian-Specific Pluripotency Marker UTF1

<div><p>Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. <i>UTF1</i> is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the <i>UTF1</i> gene during mouse development. We found that homozygous <i>UTF1</i> mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of <i>UTF1</i> expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of <i>UTF1</i> expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the <i>UTF1</i> gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the <i>UTF1</i> genes.</p></div

Role of UTF1 in the placenta.

<p>(A) Immunohistochemical analyses of UTF1 protein expression in wild-type placentas at 12.5 and 14.5 dpc. LB, labyrinth layer. (B) H&E-stained sections of <i>UTF1</i> heterozygous and homozygous mutant placentas. The boundary between the LB region and others is indicated by the dotted line. Each column of bar graph represents the mean of placenta weight with standard deviation (SD) (n = 3). *, p<0.05; **, p<0.01. (C) Cells in the mitotic phase in <i>UTF1</i> homozygous and heterozygous mutant placentas at 12.5 dpc visualized by immunostaining with an anti-phospho-histone H3 (Ser10) antibody. Phospho-histone H3-positive mitotic cells are marked by white arrowheads. Cells were counterstained with 4′,6′ diamidino-2-phenylindole (DAPI). Each column of bar graph represents the mean of number of phosphorylated histone H3 in 0.1 mm square with SD (n = 3). *, p<0.05. (D) Magnified view of H&E-stained sections of placentas. Placentas with the indicated genotypes were sectioned and stained. Dc, deciduas; Gly, glycogen trophoblast; TGC, trophoblast giant cell; Sp, spongiotrophoblast.</p