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

<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

The <i>UTF1</i> gene is present only in the genomes of eutherian (placental) mammals.

<p>(A) Genomic organizations surrounding the <i>UTF1</i> locus in mammals and their corresponding genomic regions in other organisms. Black boxes indicate regions without available genomic sequences. (B) VISTA Browser (VGB2.0) plot of the 65.5 kb interval (ch10∶134,506,934-134,572,432) containing <i>KNDC1</i>, <i>UTF1</i> and <i>VENTX</i> genes in the human genome. Conservation plots for elephant (top panel) mouse (second panel), opossum (third panel) and wallaby (bottom panel), with respect to human, are shown in the coordinates of the human sequence (horizontal axis). Dark blue boxes indicate portions with unavailable DNA sequences in the database.</p

Acquisition of pluripotency by <i>UTF1</i>-knockout homozygotes.

<p>(A) Comparison of the frequency between knockouts of first and second alleles in serial <i>UTF1</i> gene targeting. A <i>UTF1-</i>targeting vector carrying the puromycin resistance gene was introduced into wild-type and <i>UTF1</i> heterozygous mutant ESCs bearing the blasticidin resistance gene in one of the <i>UTF1</i> loci by electroporation, and were then selected with medium containing puromycin only or puromycin and blasticidin, respectively. Genomic DNAs were prepared from drug-resistant clones and used as templates for PCR to distinguish between homologous recombination and random integration of the vector. Frequencies of targeted disruption in wild-type and <i>UTF1</i> heterozygous mutant ESCs were 2.08% and 0.88%, respectively. Because the probability of targeted disruption in wild-type ESCs is considered to be twice as high as that in heterozygous mutant ESCs, we concluded that the second allele knockout in serial gene targeting of the <i>UTF1</i> loci is not a rare event compared with the first allele knockout. (B) Western blot analyses of UTF1 and other pluripotency marker proteins in <i>UTF1</i> homozygous mutant ESCs (UTF1 KO ES) generated in A and iPSCs generated from <i>UTF1</i> homozygous mutant MEFs (UTF1 KO iPS). Wild-type ESCs were used as references. (C) H&E-staining of sections of a teratoma containing differentiated cells of all three germ layers generated by injection of <i>UTF1</i>-null iPSCs into nude mice. Representative portions of the three germ layers are marked by brackets.</p