Analysis of the functional divergence of the Notch ligands Delta1 and Delta4 in vitro and in vivo

• Applicant: Professor Dr. Achim Gossler
• Subject Area: Developmental Biology ; Cell Biology
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 272080564

• Final Report Year: 2019

Final Report Abstract

The highly conserved Notch signaling pathway mediates cell-to–cell communication between adjacent cells and thereby regulates numerous developmental processes and tissue homeostasis Notch genes encode large transmembrane proteins that act as receptors at the surface of the cell. They interact with transmembrane proteins on neighboring cells that act as ligands and are encoded by the Delta and Serrate (called Jagged in vertebrates) genes. The mouse genome encodes four Notch (Notch1-4) receptors and four activating ligands, DLL1, DLL4, JAG1 and JAG2. Notch receptors and ligands are co-expressed in various tissues and developmental contexts. However, little is known about how different ligands interact with various Notch receptors, and whether the signals elicited by these interactions are quantitatively or qualitatively different. DLL1 and DLL4 have similar amino acid sequences (47% identical plus 14% similar amino acids), the same number of EGF-like repeats in the extracellular domain and an identical domain architecture. The Dll1 and Dll4 genes are expressed both in unique and overlapping patterns during mouse embryonic development and in adult tissues, and depending on the context DLL1 and DLL4 act either redundantly or have unique functions. The aims of this project were to further analyze the functional divergence of these two NOTCH ligands and factors that contribute to their different activities as Notch ligands. Towards these aims we analysed the influence of fringe (FNG) and JAG1 proteins on DLL1 and DLL4 activity, the significance of extracellular and intracellular regions of DLL1 and DLL4 chimeric proteins on ligand function in cell culture assays, and for selected chimeras, in biochemical binding assays and in vivo in mice. Major findings of our analyses were: i) modification of Notch1 by LFNG or MFNG had no distinct effect on Notch1 activation in HeLaN1 cells by DLL1 or DLL4; ii) JAG1, which can act as inhibitor of DLL1-mediated activation of MFNG-modified NOTCH does not differently affect DLL1 or DLL4 in the CHO-HeLaN1 co-culture system; iii) the extracellular domains determine ligand function in vivo in the context of somitogenese and myogenesis. During somitogenesis the whole ECD of DLL1 was functional, whereas during myogenesis a chimeric ligand consisting of the N-terminal MNNL and DSL domains and EGF repeats 1-3 fused to EGF4 and the remaining C-terminal portion of DLL4 was sufficient for normal DLL1 function; iv) DLL1 and DLL4 show selectivity for NOTCH1 and NOTCH2: DLL4 preferentially activates NOTCH1 over NOTCH2, whereas DLL1 is equally effective in activating NOTCH1 and NOTCH2; v) the N-terminal regions up to and including EGF3 determine receptor selectivity, with a clear contribution of EGF3 vi) contact amino acids and binding affinity are no major factors for ligand activity in vivo.

Publications

• 2015. Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo. PLoS Genet 11, e1005328
  Preusse, K., Tveriakhina, L., Schuster-Gossler, K., Gaspar, C., Rosa, A.I., Henrique, D., Gossler, A., Stauber, M.
  
• 2018. The ectodomains determine ligand function in vivo and selectivity of DLL1 and DLL4 toward NOTCH1 and NOTCH2 in vitro. Elife 7, 25477
  Tveriakhina, L., Schuster-Gossler, K., Jarrett, S.M., Andrawes, M.B., Rohrbach, M., Blacklow, S.C., Gossler, A.