RESUMO
Pluripotent cells in the inner cell mass (ICM) or epiblast of mammalian embryos exhibit the capacity to differentiate into all cells represented in the three germ layers. Embryonic stem (ES) cells can be derived from the ICM of preimplantation stage blastocysts, while epiblast stem cells (EpiSCs) can be derived from the epiblast of postimplantation embryos or preimplantation stage embryos. The ability to derive distinct types of pluripotent cells from blastocyst-stage embryos suggests that optimization of culture conditions can promote self-renewal of various stem cell populations. Moreover, because mouse EpiSCs resemble human pluripotent stem (hPS) cells, EpiSCs are a useful model to study common and divergent mechanisms of self-renewal between orthologous species. In addition, studies have demonstrated that haploid embryos and ES cells can be derived from chemically activated oocytes. Here, we describe a protocol for deriving maternal (parthenogenetic/gynogenetic) EpiSCs (maEpiSCs) from haploid blastocyst-stage embryos. This protocol is suitable to establish an experimental model for the study of mechanisms of EpiSC self-renewal and differentiation.
Assuntos
Técnicas de Cultura de Células/métodos , Camadas Germinativas/citologia , Oócitos/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Células Cultivadas , Meios de Cultura/química , Epigênese Genética , Feminino , Fator 4 de Crescimento de Fibroblastos/deficiência , Haploidia , CamundongosRESUMO
Embryonic morphogenesis of a complex organism requires proper regulation of patterning and directional growth. Planar cell polarity (PCP) signaling is emerging as a crucial evolutionarily conserved mechanism whereby directional information is conveyed. PCP is thought to be established by global cues, and recent studies have revealed an instructive role of a Wnt signaling gradient in epithelial tissues of both invertebrates and vertebrates. However, it remains unclear whether Wnt/PCP signaling is regulated in a coordinated manner with embryonic patterning during morphogenesis. Here, in mouse developing limbs, we find that apical ectoderm ridge-derived Fgfs required for limb patterning regulate PCP along the proximal-distal axis in a Wnt5a-dependent manner. We demonstrate with genetic evidence that the Wnt5a gradient acts as a global cue that is instructive in establishing PCP in the limb mesenchyme, and that Wnt5a also plays a permissive role to allow Fgf signaling to orient PCP. Our results indicate that limb morphogenesis is regulated by coordination of directional growth and patterning through integration of Wnt5a and Fgf signaling.
Assuntos
Padronização Corporal/fisiologia , Polaridade Celular/fisiologia , Fator 4 de Crescimento de Fibroblastos/fisiologia , Fator 8 de Crescimento de Fibroblasto/fisiologia , Proteína Wnt-5a/fisiologia , Animais , Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Extremidades/embriologia , Fator 4 de Crescimento de Fibroblastos/deficiência , Fator 4 de Crescimento de Fibroblastos/genética , Fator 8 de Crescimento de Fibroblasto/deficiência , Fator 8 de Crescimento de Fibroblasto/genética , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais , Proteína Wnt-5a/deficiência , Proteína Wnt-5a/genéticaRESUMO
The inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages.
Assuntos
Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/embriologia , Células-Tronco Pluripotentes/citologia , Ativinas/administração & dosagem , Animais , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Fator 4 de Crescimento de Fibroblastos/deficiência , Fator 4 de Crescimento de Fibroblastos/genética , Fator 4 de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Comunicação Parácrina , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Transcrição SOXF/genética , Tretinoína/administração & dosagemRESUMO
Neurons integrate and encode complex synaptic inputs into action potential outputs through a process termed "intrinsic excitability." Here, we report the essential contribution of fibroblast growth factor homologous factors (FHFs), a family of voltage-gated sodium channel binding proteins, to this process. Fhf1-/-Fhf4-/- mice suffer from severe ataxia and other neurological deficits. In mouse cerebellar slice recordings, WT granule neurons can be induced to fire action potentials repetitively (approximately 60 Hz), whereas Fhf1-/-Fhf4-/- neurons often fire only once and at an elevated voltage spike threshold. Sodium channels in Fhf1-/-Fhf4-/- granule neurons inactivate at more negative membrane potential, inactivate more rapidly, and are slower to recover from the inactivated state. Altered sodium channel physiology is sufficient to explain excitation deficits, as tested in a granule cell computer model. These findings offer a physiological mechanism underlying human spinocerebellar ataxia induced by Fhf4 mutation and suggest a broad role for FHFs in the control of excitability throughout the CNS.