Lfng and Dll3 cooperate to modulate protein interactions in cis and coordinate oscillatory Notch pathway activation in the segmentation clock

In mammalian development, oscillatory activation of Notch signaling is required for segmentation clock function during somitogenesis. Notch activity oscillations are synchronized between neighboring cells in the presomitic mesoderm (PSM) and have a period that matches the rate of somite formation. Normal clock function requires cyclic expression of the Lunatic fringe (LFNG) glycosyltransferase, as well as expression of the inhibitory Notch ligand Delta-like 3 (DLL3). How these factors coordinate Notch activation in the clock is not well understood. Recent evidence suggests that LFNG can act in a signal-sending cell to influence Notch activity in the clock, raising the possibility that in this context, glycosylation of Notch pathway proteins by LFNG may affect ligand activity. Here we dissect the genetic interactions of Lfng and Dll3 specifically in the segmentation clock and observe distinctions in the skeletal and clock phenotypes of mutant embryos showing that paradoxically, loss of Dll3 is associated with strong reductions in Notch activity in the caudal PSM. The patterns of Notch activity in the PSM suggest that the loss of Dll3 is epistatic to the loss of Lfng in the segmentation clock, and we present direct evidence for the modification of several DLL1 and DLL3 EGF-repeats by LFNG. We further demonstrate that DLL3 expression in cells co-expressing DLL1 and NOTCH1 can potentiate a cell's signal-sending activity and that this effect is modulated by LFNG, suggesting a mechanism for coordinated regulation of oscillatory Notch activation in the clock by glycosylation and cis-inhibition. [Display omitted] •Axial skeletal phenotypes after loss of DLL3 or LFNG are similar except in the sacrum.•Loss of DLL3 in the mouse PSM counterintuitively leads to a reduction in Notch signaling.•Both EGF repeats 2 and 5 of DLL3 are efficiently modified by LFNG.•When cells co-express NOTCH1 and DLL1, induction of DLL3 expression potentiates signal-sending activities.•LFNG blocks DLL3's effect on signal-sending activity, providing cyclic regulation of cis-interactions during somitogenesis.