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 [Thomsen, S., Till, A., Wittlieb, J., Beetz, C., Khalturin, K., Bosch, T. C., 2004. Control of foot differentiation in Hydra: in vitro evidence that the NK-2 homeobox factor CnNK-2 autoregulates its own expression and uses pedibin as target gene. Mech. Dev. 121, 195-204]. 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-3β, a vertebrate Forkhead transcription factor, in the CnNK-2 5′-flanking region. The Hydra HNF-3β 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 [Martinez, D.E., Dirksen, M.L., Bode, P. M., Jamrich, M., Steele, R.E., Bode, H.R., 1997. Budhead, a fork head/HNF-3β homolog, is expressed during axis formation and head specification in hydra. Dev. Biol. 192, 523-536]. By electrophoretic mobility shift assays we demonstrate an in vitro interaction between recombinant budhead protein and the interspecific conserved HNF-3β 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.