The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Abstract

Background The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. Results Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. Conclusions We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

How Dickkopf molecules and Wnt/β-catenin interplay to self-organise the Hydra body axis

“…Furthermore, they showed that this negative influence was mutual, given that removal of HyDkk1/2/4-A-expressing gland cells led to ectopic HyWnt3-expressing cell clusters throughout the body column. This inhibitory influence was additionally confirmed by analysis of the impact of Hydra HyDkk1/2/4-A on canonical Wnt signalling in Xenopus [14], and in another recent study showing that knockdown of HyDkk1/2/4-A in combination with ALP treatment led to the formation of ectopic axes in Hydra [40].…”

“…However, we want to point out that in late stages of these simulations, Dickkopf patterns re-establish, which might reflect the fact that terminal cell differentiation is not incorporated in our model and ALP-driven effects are only initially given and thus reversible. Finally, we also repeated recent HyDkk1/2/4-A-knockdown experiments [40] with and without ALP treatment (Fig. 4).…”

“…Indeed, mutual repression of β-catenin/HyWnt3 vs. HyDkk1/2/4-A effectively led to local self-activation (via the inhibition of an inhibition), as a mechanism proposed to drive local self-activation in other patterning systems [41]. In contrast, HyDkk1/2/4-C (and similarly HAS-7 [40]) appeared to realise long-range inhibition, being additionally activated by the SD respectively the secreted HyWnt3 molecules (possibly including additional fast-diffusing factors [40]) to achieve the long-range effect. Also here, the additional local inhibition from β-catenin/HyWnt3 to HyDkk1/2/4-C (respectively HAS-7) leads to a lack of Dkk in the head -in contrast to the maximal expression of the inhibitor proposed by the activator-inhibitor model [25].…”

“…Recently, the astacin protease HAS-7 has been identified and analysed in Hydra [40]. Similar to Dkks, canonical Wnt signalling is antagonised on the protein level, but here, based on degradation rather than receptor isolation.…”

“…Interestingly, the expression patterns of HyDkk1/2/4-A resemble that of a "substrate molecule" in the activator-depleted substrate model, a second type of model proposed by Gierer and Meinhardt [25]. However, this model requires that HyDkk1/2/4-A activates head-related molecules (such as canonical Wnt signalling), but rather the contrary is suggested from molecular knowledge [6] and functional studies in Hydra [40]. Additionally, expression of both Dkks appears to be downregulated after pharmacological GSK3 inhibition [14,15].…”