Control of foot differentiation in Hydra: Phylogenetic footprinting indicates interaction of head, bud and foot patterning systems

Homeodomain transcription factor CnNK-2 seems to play a major role in foot formation in Hydra. Recently, we reported in vitro evidence indicating that CnNK-2 has autoregulatory features and regulates expression of the morphogenetic peptide pedibin. We proposed that CnNK-2 and pedibin synergistically orchestrate foot differentiation processes. Here, we further analyzed the regulatory network controlling foot formation in Hydra. By phylogenetic footprinting we compared the CnNK-2 5'-flanking sequence from two closely related species, Hydra vulgaris and Hydra oligactis. Unexpectedly, we detected a highly conserved binding site for HNF-3beta, a vertebrate Forkhead transcription factor, in the CnNK-2 5'-flanking region. The Hydra HNF-3beta homolog budhead is predominantly expressed in the apical region of the body column and early during budding. Budhead is absent from tissue expressing CnNK-2 and thought to be involved in determining tissue for head differentiation. By electrophoretic mobility shift assays we demonstrate an in vitro interaction between recombinant budhead protein and the interspecific conserved HNF-3beta binding motif in the CnNK-2 5'-flanking region. Our results strengthen the view of CnNK-2 as an important regulator during foot patterning processes. Furtheron, they point to budhead as a candidate for a transcriptional regulator of CnNK-2 and to an interaction of foot and head patterning processes in Hydra on the molecular level.