Dppa4 (developmental pluripotency-associated 4) has been identified in several high-profile screens as a gene that is expressed exclusively in pluripotent cells. It encodes a nuclear protein with an SAP-like domain and appears to be associated preferentially with transcriptionally active chromatin. Its exquisite expression pattern and results of RNA interference experiments have led to speculation that Dppa4, as well as its nearby homolog Dppa2, might play essential roles in embryonic stem (ES) cell function and/or germ cell development. To rigorously assess suggested roles, we have generated Dppa4-deficient and Dppa4/Dppa2 doubly deficient ES cells, as well as mice lacking Dppa4. Contrary to predictions, we find that Dppa4 is completely dispensable for ES cell identity and germ cell development. Instead, loss of Dppa4 in mice results in late embryonic/perinatal death and striking skeletal defects with partial penetrance. Thus, surprisingly, Dppa4-deficiency affects tissues that apparently never transcribed the gene, and at least some loss-of-function defects manifest phenotypically at an embryonic stage long after physiologic Dppa4 expression has ceased. Concomitant with targeted gene inactivation, we have introduced into the Dppa4 locus a red fluorescent marker (tandem-dimer red fluorescent protein) that is compatible with green fluorescent proteins and allows noninvasive visualization of pluripotent cells and reprogramming events.

Although the properties of embryonic stem (ES) cells make these cells very attractive in the field of replacement therapy, the molecular mechanisms involved in the maintenance of their pluripotency are not fully characterized. Starting from the observation that most pluripotent markers are also expressed by spermatogonia stem cells, we identified Tex19 as a new potential pluripotency marker. We show that Tex19 is a mammalian-specific protein duplicated in mouse and rat, renamed Tex19.1 and Tex19.2, whereas only one form is found in human. In mouse, both forms are localized on chromosome 11 and transcribed in opposite directions. Tex19 proteins are well conserved, showing two highly conserved domains that do not present any similarity with any other known domains. We show that Tex19.2 is specifically detected in the male somatic gonad lineage, whereas Tex19.1 expression is very similar to that of Oct4. Transcripts are maternally inherited, and expression starts as soon as the early embryo and later is limited to the germ line. Tex19.1 transcripts were also detected in mouse pluripotent stem cells, and expression of Tex19.1, like that of Oct4, decreases after murine embryonic stem and germ cell differentiation. Human TEX19 was more closely related to murine Tex19.1 and was also detected in adult testis and in undifferentiated ES cells. By immunofluorescence, we found that Tex19.1 protein localizes to the nucleus of mouse ES and inner cell mass cells. All these results suggest that Tex19.1, as well as human TEX19, could be a new factor involved in the maintenance of self-renewal or pluripotency of stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19–30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development.