Pan, Q.; Mercker, M.; Klimovich, A.; Wittlieb, J.; Marciniak-Czochra, A.; Boettger, A.

The Notch-signalling pathway plays an important role in pattern formation in Hydra. Using pharmacological Notch inhibitors (DAPT and SAHM1), it has been demonstrated that HvNotch is required for head regeneration and tentacle patterning in Hydra. HvNotch is also involved in establishing the parent-bud boundary and instructing buds to develop feet and detach from the parent. To further investigate the functions of HvNotch, we successfully constructed NICD (HvNotch intracellular domain)-overexpressing and HvNotch-knockdown transgenic Hydra strains. NICD-overexpressing transgenic Hydra showed a pronounced inhibition on the expression of predicted HvNotch-target genes, suggesting a dominant negative effect of ectopic NICD. This resulted in a Y-shaped phenotype, which arises from the parent-bud boundary defect seen in polyps treated with DAPT. Additionally, multiple heads, two-headed and ectopic tentacles phenotypes were observed. The HvNotch-knockdown transgenic Hydra with reduced expression of HvNotch exhibited similar, but not identical phenotypes, with the addition of a two feet phenotype. Furthermore, approximately 20% of the HvNotch-knockdown polyps were unable to regenerate a new head after decapitation. We integrated these findings into a mathematical model based on long-range gradients of signalling molecules underlying sharply defined positions of HvNotch-signalling cells at the Hydra tentacle and bud boundaries.