In Hydra, head formation depends on Wnt/β-catenin signaling, which positively regulates Sp5 and Zic4, with Sp5 limiting Wnt3/β-catenin expression and Zic4 triggering tentacle formation. Using transgenic lines in which the HySp5 promoter drives eGFP expression in the epidermis or gastrodermis, we show that in intact animals, epidermal HySp5:GFP is expressed strongly apically and weakly along the body column, while gastrodermal HySp5:GFP is also maximally expressed apically but absent from the oral region, and remains high along the upper body column. During apical regeneration, gastrodermal HySp5:GFP appears early and diffusely, epidermal HySp5:GFP later. Upon alsterpaullone treatment, apical HySp5:GFP expression is shifted to the body column where epidermal HySp5:GFP transiently forms ectopic circular figures. After β-catenin(RNAi), only epidermal HySp5:GFP is down-regulated, while pseudo-bud structures expressing gastrodermal HySp5:GFP develop. Sp5(RNAi) highlights the negative autoregulation of Sp5 in epidermis, involving direct binding of Sp5 to its own promoter as observed in human HEK293T cells. In these cells, HyZic4, which can interact with huTCF1, regulates Wnt3 negatively and Sp5 positively. This differential regulation of the Wnt/β-catenin/TCF/Sp5/Zic4 network in epidermis and gastrodermis highlights distinct architectures and patterning roles in the hypostome, tentacle and body column, as well as distinct regulations in homeostatic and developmental organizers.

The Wnt/β-catenin/TCF/Sp5/Zic4 gene network that regulates head organizer activity in Hydra is differentially regulated in epidermis and gastrodermis