During the past year, we identified the biological roles of three genes that are unique to vertebrates and that are expressed at the very beginning of mammalian development. All three genes were discovered by our group. One of these genes, DKKL1, is unique to mammals. We discovered that DKKL1 is expressed specifically during implantation of the embryo and during development of spermatocytes into sperm. Moreover, genetic inactivation of DKKL1 in mice resulted in production of sperm that are defective in fertilization. The second gene, TEAD2, is one of a highly conserved family of four transcription factors that share a common DNA binding domain. Surprisingly, TEAD2 is not required until after implantation has occurred and the nervous system begins to form. When we genetically inactivated TEAD2, mice had serious difficulty in forming a neural tube. In developing vertebrates, the neural tube is the embryo's precursor to the central nervous system, which comprises the brain and spinal cord. Failure to close the neural tube in mice is called exencephaly, which is related to anencephaly, a common human birth defect that can be prevented by folic acid. The third gene is TEAD4. We discovered that TEAD2 and TEAD4 are the only TEAD genes that are expressed in preimplantation mouse embryos, and that genetic inactivation of TEAD4 (but not TEAD2) arrests development prior to formation of a blastocyst. In fact, we discovered that TEAD4 is required for specification of the trophectoderm linage, thus making it the earliest gene known so far on the pathway to formation of the placenta. It now appears that the totipotent blastomeres in mouse morula that express the transcription factor TEAD4 become trophectoderm and eventually placenta, whereas those that express the transcription factor OCT4 become the inner cell mass and eventually the embryo. Thus, TEAD4 appears to be a master gene that sets into motion the first round of cell differentiation during mammalian development. ? ? Another gene that is expressed at the beginning of mammalian development is LA, a protein involved in the processing of RNA. In a collaborative effort with Dr. Richard Maraia and his colleagues, we helped to demonstrate that La is required for mammalian development by virtue of the fact that it is required for establishment of embryonic stem cells.? ? Specifics:? ? 1) TEAD2, one of the first transcription factors expressed at the beginning of mammalian development, appears to be required during neural development. For example, Tead2 expression is greatest in the dorsal neural crest where it appears to regulate expression of Pax3, a gene essential for brain development. Consistent with this hypothesis, we found that inactivation of the Tead2 gene in mice significantly increased the risk of exencephaly (a defect in neural tube closure). However, none of the embryos exhibited spina bifida, the major phenotype of Pax3 nullizygous embryos, and expression of Pax3 in E11.5 Tead2 nullizygous embryos was normal. Thus, Tead2 plays a role in neural tube closure that is independent of its putative role in Pax3 regulation. In addition, the risk of exencephaly was greatest with Tead2 nullizygous females, and could be suppressed either by folic acid or pifithrin-α. These results reveal a maternal genetic contribution to neural tube closure, and suggest that Tead2 deficient mice provide a model for anencephaly, a common human birth defect that can be prevented by folic acid.? ? 2) Specification of cell lineages in mammals begins shortly after fertilization with formation of a blastocyst consisting of trophectoderm, which contributes exclusively to the placenta, and inner cell mass (ICM), from which the embryo develops. Here we report that ablation of the mouse Tead4 gene results in a preimplantation lethal phenotype, and TEAD4 is one of two highly homologous TEAD transcription factors that are expressed during zygotic gene activation in mouse 2-cell embryos. Tead4-/- embryos do not express trophectoderm specific genes, such as Cdx2, but do express ICM specific genes, such as Oct4/Pou5f1. Consequently, Tead4-/- morulae do not produce trophoblast stem cells, trophectoderm, or blastocoel cavities, and therefore do not implant into the uterine endometrium. However, Tead4 /- embryos can produce embryonic stem cells, a derivative of ICM, and if the Tead4 allele is not disrupted until after implantation, then Tead4-/- embryos complete development. Thus, Tead4 is the earliest gene shown to be uniquely required for specification of the trophectoderm lineage.? ? 3) The La protein is a target of autoantibodies in patients suffering from Sjogren's syndrome, systemic lupus erythematosus, and neonatal lupus. Ubiquitous in eukaryotes, La functions as a RNA-binding protein that promotes the maturation of tRNA precursors and other nascent transcripts synthesized by RNA polymerase III as well as other noncoding RNAs. La also associates with a class of mRNAs that encode ribosome subunits and precursors to snoRNAs involved in ribosome biogenesis. Thus, it was surprising that La is dispensable in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the organisms from which it has been characterized most extensively. To determine whether La is essential in mammals and if so, at which developmental stage it is required, mice were created with a disrupted La gene, and the offspring from La+/-intercrosses were analyzed. La-/- offspring were detected at the expected frequency among blastocysts prior to implantation, whereas no nullizygotes were detected after implantation, indicating that La is required early in development. Blastocysts derived from La+/- intercrosses yielded 38 La+/+ and La+/- embryonic stem (ES) cell lines but no La-/- ES cell lines, suggesting that La contributes a critical function toward the establishment or survival of ES cells. Consistent with this, La-/- blastocyst outgrowths revealed loss of the inner cell mass (ICM). The results indicate that in contrast to the situation in yeasts, La is essential in mammals and is one of a limited number of genes required as early as the development of the ICM.? ? For additional information, visit our web site at (http://depamphilislab.nichd.nih.gov/).
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