RESUMO
During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.
Assuntos
Relógios Biológicos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glicosiltransferases/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/embriologia , Receptor Notch1/metabolismo , Somitos/embriologia , Animais , Proteínas de Ligação ao Cálcio , Desenvolvimento Embrionário , Hibridização in Situ Fluorescente , Mesoderma/metabolismo , Camundongos , Transdução de Sinais/fisiologiaRESUMO
Embryonic stem cells (ESCs) are cell lines derived from the mammalian pre-implantation embryo. Here we assess the impact of derivation and culture conditions on both functional potency and ESC transcriptional identity. Individual ESCs cultured in either two small-molecule inhibitors (2i) or with knockout serum replacement (KOSR), but not serum, can generate high-level chimeras regardless of how these cells were derived. ESCs cultured in these conditions showed a transcriptional correlation with early pre-implantation embryos (E1.5-E3.5) and contributed to development from the 2-cell stage. Conversely, the transcriptome of serum-cultured ESCs correlated with later stages of development (E4.5), at which point embryonic cells are more restricted in their developmental potential. Thus, ESC culture systems are not equivalent, but support cell types that resemble distinct developmental stages. Cells derived in one condition can be reprogrammed to another developmental state merely by adaptation to another culture condition.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Camundongos Endogâmicos C57BLRESUMO
During somitogenesis, epithelial somites form from the pre-somitic mesoderm (PSM) in a periodic manner. This periodicity is regulated by a molecular oscillator, known as the 'segmentation clock', that is characterised by an oscillatory pattern of gene expression that sweeps the PSM in a caudal-rostral direction. Key components of the segmentation clock are intracellular components of the Notch, Wnt and FGF pathways, and it is widely accepted that intracellular negative-feedback loops regulate oscillatory gene expression. However, an open question in the field is how intracellular oscillations are coordinated, in the form of spatiotemporal waves of expression, across the PSM. In this study, we provide a potential mechanism for this process. We show at the mRNA level that the Notch1 receptor and Delta-like 1 (Dll1) ligand vary dynamically across the PSM of both chick and mouse. Remarkably, we also demonstrate similar dynamics at the protein level; hence, the pathway components that mediate intercellular coupling themselves exhibit oscillatory dynamics. Moreover, we quantify the dynamic expression patterns of Dll1 and Notch1, and show they are highly correlated with the expression patterns of two known clock components [Lfng mRNA and the activated form of the Notch receptor (cleaved Notch intracellular domain, NICD)]. Lastly, we show that Notch1 is a target of Notch signalling, whereas Dll1 is Wnt regulated. Regulation of Dll1 and Notch1 expression thus links the activity of Wnt and Notch, the two main signalling pathways driving the clock.
Assuntos
Relógios Biológicos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/embriologia , Receptor Notch1/metabolismo , Transdução de Sinais/fisiologia , Somitos/embriologia , Algoritmos , Animais , Western Blotting , Proteínas de Ligação ao Cálcio , Embrião de Galinha , Glicosiltransferases/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Hibridização in Situ Fluorescente , Mesoderma/metabolismo , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Notch/metabolismoRESUMO
A segmented body plan is fundamental to all vertebrate species and this bestows both rigidity and flexibility on the body. Segmentation is initiated through the process of somitogenesis. This article aims to provide a broad and balanced cross-species overview of somitogenesis and to highlight the key molecular and cellular events involved in each stage of segmentation. We highlight where our understanding of this multifaceted process relies on strong experimental evidence as well as those aspects where our understanding still relies largely on models.
Assuntos
Desenvolvimento Embrionário/fisiologia , Somitos/citologia , Animais , Relógios Biológicos/genética , Relógios Biológicos/fisiologia , Padronização Corporal/genética , Padronização Corporal/fisiologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Somitos/embriologia , Somitos/metabolismoRESUMO
The relationship between membrane protein structure and thermal stability has been examined in the reaction centre from the bacterium Rhodobacter sphaeroides, a complex membrane protein comprising three polypeptide chains and 10 cofactors. The core of this protein exhibits an approximate twofold symmetry, the cofactors being held in two membrane-spanning branches by two polypeptides, termed L and M, that have very similar folds. In assays of the thermal stability of wild-type and mutant reaction centres embedded in the native bilayer membrane, replacement of a Phe at position 197 of the M polypeptide by His produced an increase in stability, whereas an opposing replacement of His by Phe at the symmetrical position 168 of the L-polypeptide produced a decrease in stability. In light of the known X-ray crystal structures of wild-type and mutant variants of this protein, and further mutagenesis, it is concluded that these stability changes result from the introduction or removal, respectively, of a hydrogen bond between the side-chain of the His and that of an Asn located two positions along the M or L polypeptide chain, in addition to a hydrogen bond between the His side-chain and an adjacent bacteriochlorophyll cofactor.