An evolutionary conserved Sp5-Wnt3 regulatory module contributes to restrict head organizer activity in Hydra

Head regeneration in Hydra requires the transformation of gastric tissue into a head organizer that produces a head activator and a head inhibitor. Here we report the decipherment of a long-standing question in developmental biology, the identification of the transcription factor Sp5 as a key head inhibitory component. We show that Sp5 has an apical to basal graded expression pattern in intact Hydra, its expression is induced upon Wnt/β-catenin signaling activation and when knocked-down triggers the formation of multiple heads and axes in homeostatic and regenerative conditions. Our data indicate that Sp5 acts in a feed-forward loop to robustly regulate its own expression and that Sp5 prevents head formation by repressing the Wnt3 promoter. This Sp5 mediated Wnt antagonism is conserved and was invented early in metazoan evolution to set up axial patterning. ChIP seq analysis of Sp5 occupancies in human HEK293 cells transfected with expression vectors coding either for Hydra Sp5 or Zebrafish Sp5 proteins.The genomic occupancies mutant versions of these proteins lacking the Sp5 DNA binding domain were also tested.