RESUMO
The separation of the first two lineages - trophectoderm (TE) and inner cell mass (ICM) - is a crucial event in the development of the early embryo. The ICM, which constitutes the pluripotent founder cell population, develops into the embryo proper, whereas the TE, which comprises the surrounding outer layer, supports the development of the ICM before and after implantation. Cdx2, the first transcription factor expressed specifically in the developing TE, is crucial for the differentiation of cells into the TE, as lack of zygotic Cdx2 expression leads to a failure of embryos to hatch and implant into the uterus. However, speculation exists as to whether maternal Cdx2 is required for initiation of TE lineage separation. Here, we show that effective elimination of both maternal and zygotic Cdx2 transcripts by an RNA interference approach resulted in failure of embryo hatching and implantation, but the developing blastocysts exhibited normal gross morphology, indicating that TE differentiation had been initiated. Expression of keratin 8, a marker for differentiated TE, further confirmed the identity of the TE lineage in Cdx2-deficient embryos. However, these embryos exhibited low mitochondrial activity and abnormal ultrastructure, indicating that Cdx2 plays a key role in the regulation of TE function. Furthermore, we found that embryonic compaction does not act as a 'switch' regulator to turn on Cdx2 expression. Our results clearly demonstrate that neither maternal nor zygotic Cdx2 transcripts direct the initiation of ICM/TE lineage separation.
Assuntos
Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Fator de Transcrição CDX2 , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Oócitos/citologia , Gravidez , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genéticaRESUMO
The large micromeres of the 32-cell stage of sea urchin embryos are autonomously specified and differentiate into primary mesenchyme cells (PMCs), giving rise to the skeletogenic cells. We previously demonstrated that HpEts, an ets-related transcription factor, plays an essential role in the specification of PMCs in sea urchin embryos. In order to clarify the function of HpEts in the gene regulatory network involved in PMC specification, we analyzed the zygotic expression pattern and the cis-regulatory region of HpEts, and examined the activity of the HpEts protein as a transcription factor. Intron-based PCR reveals that zygotic expression of HpEts starts at the cleavage stage, and that the rate of transcription reaches maximum at the unhatched blastula stage. A series of progressive deletions of the fragments from -4.2 kbp to +1206 bp of the HpEts, which directs PMC-specific expression, caused a gradual decrease in the specificity, implying that coordination of several cis-regulatory elements regulates the expression in PMCs. A minimum cis-element required for the temporal expression is located within a 10 bp from -243 bp to -234 bp. The HpEts protein remains in the cytoplasm of entire embryonic cells in the cleavage stage. At the unhatched blastula stage, the HpEts protein translocates into the nucleus in presumptive PMCs. Transactivation assays demonstrate that the HpEts protein activates a promoter of Spicule Matrix Protein 50 (SM50), which is a target of HpEts, which binds to the regulatory region of SM50.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Hemicentrotus/citologia , Células-Tronco Mesenquimais/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Dados de Sequência Molecular , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Zigoto/fisiologiaRESUMO
Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.