Abstract
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.
Highlights
During somitogenesis, epithelial spheres called somites bud off from the most rostral end of the pre-somitic mesoderm (PSM) in a rostral-to-caudal direction and, later in development, these give rise to the vertebral column, most of the skeletal musculature and much of the dermis (Dequeant and Pourquie, 2008)
Delta-like 1 (Dll1) and Notch1 mRNA expression is dynamic across the mouse PSM To investigate whether the expression of Dll1 and Notch1 displays an oscillatory pattern in the PSM, we initially examined nascent Dll1 and Notch1 mRNA expression [using in situ hybridisation with antisense probes that hybridise to intronic regions of these nascent transcripts, which are referred to here as Dll1(i) and Notch1(i)]
Using FISH, we found that the expression of mature Dll1 and Notch 1 mRNA varies considerably across different E10.5 embryos
Summary
Epithelial spheres called somites bud off from the most rostral end of the pre-somitic mesoderm (PSM) in a rostral-to-caudal direction and, later in development, these give rise to the vertebral column, most of the skeletal musculature and much of the dermis (Dequeant and Pourquie, 2008) This occurs with a remarkable periodicity that is regulated by a molecular oscillator (Cooke and Zeeman, 1976), which drives cyclic waves of gene expression caudo-rostrally through the PSM with the same. These three pathways interact reciprocally within the mechanism of the mouse segmentation clock (Dequeant and Pourquie, 2008; Gibb et al, 2010; Maroto et al, 2012; Niwa et al, 2007) It is widely accepted, on a single cell level in the vertebrate PSM, that oscillatory gene expression is established through negative-feedback loops of unstable clock gene products (Hirata et al, 2004; Lewis, 2003; Monk, 2003). In the case of the Notch pathway, it seems relatively clear how intracellular negative-feedback loops, involving Lfng and Hes proteins, contribute to the regulation of Notch target gene expression in a cell-autonomous manner
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