Abstract
The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.
Highlights
COMMUNICATION between adjacent cells mediated by the evolutionary conserved Notch-signaling pathway regulates multiple developmental processes in different tissues and species
A fragment of human Jag1 encompassing the DSL domain and first three EGF repeats was shown to bind to fragments of human Notch1 encompassing EGF repeats 10–13 (Cordle et al 2008a), and deletion analyses of human DLL1 and DLL4 showed that the regions containing the MNNL to EGF3 were necessary and sufficient for full activation of Notch1 (Andrawes et al 2013)
Our analyses show that disrupting disulfide bridges in any EGF repeat impairs ligand activity and reveals context-dependent different sensitivity of developmental processes to reduced DLL1mediated Notch signaling, anterior–posterior patterning of somites being most sensitive
Summary
COMMUNICATION between adjacent cells mediated by the evolutionary conserved Notch-signaling pathway regulates multiple developmental processes in different tissues and species (reviewed in Andersson et al 2011). Notch receptors and their ligands encode type 1 transmembrane proteins with multiple EGF-like repeats in their extracellular domains. A fragment of human Jag encompassing the DSL domain and first three EGF repeats was shown to bind to fragments of human Notch encompassing EGF repeats 10–13 (Cordle et al 2008a), and deletion analyses of human DLL1 and DLL4 showed that the regions containing the MNNL to EGF3 were necessary and sufficient for full activation of Notch (Andrawes et al 2013). Mutations in Dll that affected somitogenesis showed vertebral column malformations resembling spondylocostal dysostosis of varying severity, a human condition known to be caused by abnormal Notch signaling during somitogenesis (reviewed in Penton et al 2012), but not yet associated with mutations in Dll
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