An evolutionarily-conserved Wnt3/β-catenin/Sp5 feedback loop restricts head organizer activity in Hydra [RNA-Seq]

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. To gain insights on the evolution of the transcriptional properties of the Sp5 gene, we analyzed the capacity of the hydra Sp5 and zebrafish Sp5a paralogs to bind to cis-regulatory elements of the contitutively active human genome and to regulate the expression of the corresponding target genes. To this end, we transfected HEK293 cells with plasmids encoding for either hySp5 or zfSp5a proteins and we performed ChIPseq and RNAseq experiments to analyze their genomic occupancies and the changes in the transcriptional profiles induced upon transfection.