RESUMO
The first lineage differentiation in mammals gives rise to the inner cell mass and the trophectoderm (TE). In mice, TEAD4 is a master regulator of TE commitment, as it regulates the expression of other TE-specific genes and its ablation prevents blastocyst formation, but its role in other mammals remains unclear. Herein, we have observed that TEAD4 ablation in two phylogenetically distant species (bovine and rabbit) does not impede TE differentiation, blastocyst formation and the expression of TE markers, such as GATA3 and CDX2, although a reduced number of cells in the inner cell mass was observed in bovine TEAD4 knockout (KO) blastocysts. Transcriptional analysis in bovine blastocysts revealed no major transcriptional effect of the ablation, although the expression of hypoblast and Hippo signalling-related genes tended to be decreased in KO embryos. Experiments were conducted in the bovine model to determine whether TEAD4 was required for post-hatching development. TEAD4 KO spherical conceptuses showed normal development of the embryonic disc and TE, but hypoblast migration rate was reduced. At later stages of development (tubular conceptuses), no differences were observed between KO and wild-type conceptuses.
Assuntos
Blastocisto , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Animais , Bovinos , Feminino , Camundongos , Coelhos , Blastocisto/metabolismo , Blastocisto/citologia , Fator de Transcrição CDX2/metabolismo , Fator de Transcrição CDX2/genética , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ectoderma/metabolismo , Ectoderma/embriologia , Ectoderma/citologia , Embrião de Mamíferos/metabolismo , Fator de Transcrição GATA3/metabolismo , Fator de Transcrição GATA3/genética , Via de Sinalização Hippo , Fatores de Transcrição de Domínio TEA/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Trofoblastos/metabolismo , Trofoblastos/citologiaRESUMO
Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss, and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors mediate the signaling actions of prostaglandins and other lipids, and, between them, PPARG has been pointed out to play a relevant role in conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG knock-out embryos in vitro using two independent gene ablation strategies and assessed their developmental ability. In vitro development to Day 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass, and trophectoderm cell numbers were similar between wild-type and knock-out D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, embryonic disk formation, and hypoblast migration rates were unaffected by the ablation. The development of tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disk was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.
Assuntos
Desenvolvimento Embrionário , PPAR gama , Animais , Bovinos/embriologia , Desenvolvimento Embrionário/fisiologia , PPAR gama/metabolismo , PPAR gama/genética , Feminino , Blastocisto/metabolismo , Blastocisto/fisiologia , Embrião de Mamíferos , Técnicas de Cultura Embrionária , Técnicas de Inativação de GenesRESUMO
In brief: MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. This paper demonstrates that MEK is required for hypoblast specification in the inner cell mass of the ovine blastocyst and that it plays a role during the hypoblast migration occurring following blastocyst hatching. Abstract: Early embryo development requires the differentiation of three cell lineages in two differentiation events. The second lineage specification differentiates the inner cell mass into epiblast, which will form the proper fetus, and hypoblast, which together with the trophectoderm will form the extraembryonic membranes and the fetal part of the placenta. MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. The aim of this work was to analyse the role of MEK signalling on hypoblast specification at the blastocyst stage and on hypoblast migration during post-hatching stages in vitro in the ovine species. Using well-characterized and reliable lineage markers, and different MEK inhibitor concentrations, we demonstrate that MEK signalling pathway is required for hypoblast specification in the inner cell mass of the ovine blastocyst, and that it plays a role during the hypoblast migration occurring following blastocyst hatching. These results show that the role of MEK signalling pathway on hypoblast specification is conserved in phylogenetically distant mammals.