Taube nuss is a novel gene essential for the survival of pluripotent cells of early mouse embryos.

The cells of the inner cell mass constitute the pluripotent cell population of the early embryo. They have the potential to form all of the tissues of the embryo proper and some extra-embryonic tissues. They can be considered a transient stem cell population for the whole of the embryo, and stem cells maintaining the same capacity can be isolated from these cells. We have isolated, characterised and mutated a novel gene, taube nuss (Tbn), that is essential for the survival of this important cell population. The taube nuss protein sequence (TBN) was highly conserved between human, mouse, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana, particularly in a domain that is not present in any published proteins, showing that TBN is the founding member of a completely new class of proteins with an important function in development. The Tbn gene was expressed ubiquitously as early as E2. 5 and throughout embryonic development. It was also expressed in adult brain with slightly higher levels in the hippocampus. The Tbn mutant embryos developed normally to the blastocyst stage and contained inner cell masses. They hatched from the zonae pellucidae, implanted and induced decidual reactions, but failed to develop beyond E4.0. At this time the trophoblast cells were viable, but inner cell masses were not detectable. At E3.75, massive TUNEL-positive DNA degradation and chromatin condensation were visible within the inner cell masses, whereas the cell membranes where intact. Caspase 3 was expressed in these cells. In vitro, the inner cell mass of mutant embryos failed to proliferate and died after a short period in culture. These results indicate that the novel protein, taube nuss, is necessary for the survival of the inner cell mass cells and that inner cell mass cells died of apoptosis in the absence of the taube nuss protein. As cell pruning by apoptosis is a recognised developmental process at this stage of development, the taube nuss protein may be one of the factors regulating the extent of programmed cell death at this time point.

Expression pattern of Oct-4 in preimplantation embryos of different species.

POU transcription factors are involved in transcriptional regulation during early embryonic development and cell differentiation. Oct-4, a member of this family, has been shown to be under strict regulation during murine development. The expression of Oct-4 correlates with the undifferentiated cell phenotype of the mouse preimplantation embryo. In this study, expression of a gene construct consisting of selected parts of the region upstream from the murine Oct-4 gene as promoter/enhancer, enhanced green fluorescent protein (EGFP) as reporter and the five exons of the murine Oct-4 gene (GOF18-delta PE EGFP) was evaluated in murine, porcine, and bovine preimplantation embryos. For comparison, expression of the endogenous Oct-4 gene was also analyzed in all three species by immunocytochemistry. The transgene construct was microinjected into zygotes cultured in vitro to various developmental stages. The EGFP fluorescence was visualized in developing embryos by excitation with blue light at different days following microinjection and showed similar expression patterns in all three species. Most embryos displayed a mosaic pattern of transgene expression. The EGFP fluorescence was not restricted to the inner cell mass (ICM) but was also seen in trophoblastic cells. An affinity-purified polyclonal antibody specific to Oct-4 was used for immunocytochemical analysis of in vivo- and in vitro-derived bovine and porcine blastocysts and also of in vivo-derived murine blastocysts. In the in vivo-derived murine embryos, Oct-4 protein was detectable in the ICM but not the trophectoderm, whereas in porcine and bovine blastocysts, derived in vivo or in vitro, Oct-4 protein was detected in both the ICM and the trophectoderm. Thus, in the two large animal species, Oct-4 expression from the endogenous gene was clearly not restricted to the pluripotent cells of the early embryo. These results show that Oct-4 regulation differs between these species and that the presence of Oct-4 protein may not be sufficient for selection of undifferentiated cell lines in domestic animals.

Oct-4: lessons of totipotency from embryonic stem cells.

The Oct-4 transcription factor is expressed in embryonic stem cells and germ cells of the mouse embryo. Cells that are driven into embryonic and extraembryonic somatic differentiation lose Oct-4 expression. Oct-4 is crucial for the maintenance of the totipotency of embryonic cells at early developmental stages as demonstrated recently by homologous recombination. Since during embryogenesis Oct-4 is restricted to germ cells, it is possible that it plays a key role for the germline lineage by preventing differentiation of these cells during gastrulation. Thus, Oct-4 appears to be a key regulator of totipotency during the life cycle of mammals.

Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.

Oct4 is a mammalian POU transcription factor expressed by early embryo cells and germ cells. We report that the activity of Oct4 is essential for the identity of the pluripotential founder cell population in the mammalian embryo. Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent. Instead, they are restricted to differentiation along the extraembryonic trophoblast lineage. Furthermore, in the absence of a true inner cell mass, trophoblast proliferation is not maintained in Oct4-/- embryos. Expansion of trophoblast precursors is restored, however, by an Oct4 target gene product, fibroblast growth factor-4. Therefore, Oct4 also determines paracrine growth factor signaling from stem cells to the trophectoderm.

A mouse model for hereditary thyroid dysgenesis and cleft palate.

Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathke's pouch. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2-/- results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.