Ziegler B et al. (2021), The Wnt-specific astacin proteinase HAS-7 restr...

XB-ART-59010

BMC Biol 2021 Jun 09;191:120. doi: 10.1186/s12915-021-01046-9.

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

Ziegler B , Yiallouros I , Trageser B , Kumar S , Mercker M , Kling S , Fath M , Warnken U , Schnölzer M , Holstein TW , Hartl M , Marciniak-Czochra A , Stetefeld J , Stöcker W , Özbek S .

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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.

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Species referenced: Xenopus laevis
Genes referenced: pdlim3 wnt3 wnt8a
GO keywords: axis specification [+]

Phenotypes: Xla Wt + Hvu.wnt3 (Fig.4.C) [+]

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	Fig.4 HAS-7 inhibits HyWnt3 induced ectopic axis in Xenopus laevis axial duplication assay. a, b Xenopus Wnt8 (XWnt8) mRNA injection induces axis duplication in Xenopus embryos compared to uninjected controls. c HyWnt3 mRNA induces axis duplication comparable to XWnt8 mRNA. d Double injection of HyWnt and HAS-7 mRNAs reverts the axis duplication phenotype. e Quantification of embryos showing a double axis phenotype. Injected mRNA doses were as indicated. n indicates the number of embryos analyzed for each experimental condition. Arrows indicate cement glands. Scale bars = 100 μm.
	Fig. 1 Screen for HyWnt3 proteolytic activity in Hydra tissue lysates. a Schematic representation of the Hydra body plan. Body parts used for lysates in b–d are indicated. The hypostomal organizer, which harbors HyWnt3 expressing cells is marked in red. b Recombinant HyWnt3-His levels, monitored by Western blotting with anti-His antibody, were reduced after ~ 4 h incubation in the head lysate and after ~ 6 h in the upper body lysate. No cleavage was observed during incubation in tentacle and lower body lysates, while incubation in the PBS control showed unspecific cleavage at 24 h. No unspecific proteolysis of 1 μg BSA was detectable in HL over the time period of 24 h as detected by SDS-PAGE and Coomassie staining. HyWnt3-His cleavage activity in HL was completely blocked by the addition of broad zinc metalloproteinase inhibitors EDTA and Phenanthroline or the matrix metalloproteinase inhibitor Batimastat. c No cleavage was observed for the recombinant Wnt antagonist HyDkk1/2/4-His in the respective body tissue lysates during a 24-h incubation time. Mark that the double band appearance is an SDS-PAGE artifact. d Tissue lysates from different body parts of adult hydra polyps as indicated in the scheme were adjusted in total protein concentrations by tubulin Western blotting. e Distribution of protein classes in the Hydra HL secretome identified in HyWnt3(+) and HyWnt3(−) fractions as indicated. The full dataset is given in Additional file 2: Table S1a-b. f HyWnt3-His processing is inhibited by recombinant mouse Fetuin-B protein in a dose-dependent manner as indicated
	Fig. 2 Sequence features and expression patterns of HyWnt3(+) astacin genes. a Multiple sequence alignment of pro-domain and catalytic domain sequences of astacins identified in this study. For comparison, astacin sequences from diverse species outside the cnidarian phylum were included. Gene ID numbers are as follows: Astacin A. astacus (P07584), NAS-35 C. elegans (P98060), ZHE-1 Danio rerio (Q1LW01), HCE-1 O. latipes (P31580), Meprin α H. sapiens (Q16819), Meprin β H. sapiens (Q16820), SPAN S. purpuratus (P98068), HMP1 (NP_001296695.1), HEA-1 Hydractinia echinata astacin 1 (Q2MCX9), HEA-3 Hydractinia echinata astacin 3 (Q2MCX7), HEA-4 Hydractinia echinate astacin 4 (Q2MCX6), HEA-2 Hydractinia echinata astacin 2 (Q2MCX8), HAS-3 (XP_002166229.3), HAS-6 (XP_002157397.2), HAS-5 (XP_002164800.1), HAS-4 (XP_002162738.1), HAS-2 (XP_002162822.1), HAS-8 (XP_002153855.1), HAS-9 (XP_002161766.1), HAS-1 (XP_012565441.1), HAS-11 (XP_012561076.1), HAS-7 (XP_012560086.1), HAS-10 (XP_002159980.2). In red: the aspartate switch residue in the pro-peptide, the zinc-binding motif, and the Met-turn. Orange background: residues forming the S1 ′ sub-site. A red bullet denotes the activation site. b Domain structures of astacins detected in HyWnt3(+) head lysate fractions. S, signal peptide; Pro, pro-domain; ShKT, Stichodactyla toxin domain. c–k WISH experiments using antisense and sense oligonucleotide probes (c–e) or LNAs (f–k) show a collar-like expression pattern marking a transition zone between head and body column for the majority of astacin genes. Representative of 10 hydras examined. Scale bars: HAS-7, 200 μm; HMP-1, 200 μm; HAS-1, 200 μm; HAS-2, 100 μm; HAS-11, 50 μm; HAS-5, 100 μm; HAS-9, 100 μm; HAS-10, 200 μm, control LNA, 200 μm
	Fig. 3 Functional analysis of HAS-7 knockdown. a Detection of HAS-7 protein in body lysate samples using a polyclonal HAS-7 antibody. M = protein marker. Tubulin was used as loading control of the respective hydra lysates. The arrow indicates the position of the HAS-7 protein band, also in b and c. b Recombinant HAS-7 protein expressed in High Five cells compared to native protein in HL as detected by a HAS-7-specific antibody. Note that the slightly higher apparent molecular mass is due to the introduced histidine tag of recombinant HAS-7. c Knockdown effect of different siRNA combinations on HAS-7 protein levels was assayed by anti-HAS-7 Western blot analysis of complete hydras treated as indicated. Tubulin was used as loading control of the respective hydra lysates. The distinct band at 70 k Da in a-c likely represents a dimer of processed HAS-7. d HyWnt3-His proteolysis is impaired in HL of animals electroporated with siHAS-7 (2 + 3) as compared to siGFP control animals. Head lysates were generated 6 days after electroporation. Tubulin was used as loading control of the respective HL applied for each time point. e Relative intensities of the Western blot bands in d. f Quantitative real-time PCR analysis of HAS-7 expression in head tissues confirms the decreased expression in siHAS-7 treated animals compared to siGFP treated and untreated (steady-state AEP animals) controls. Relative expression levels are given in 2(-ΔΔCt). Results represent mean +/− S.D. from 3 independent experiments, analyzed by t tests. p < 0.05. The individual data values are shown in Additional file 14. g siGFP control electroporation without AZK treatment shows normal morphology. Scale bar = 500 μm. h siHAS-7 electroporation without AZK treatment shows animals with double axis lacking ectopic tentacles. Scale bar = 200 μm. i siGFP control animal showing ectopic tentacle formation after AZK treatment. Scale bar = 200 μm. j Double axis phenotype with ectopic tentacles near the head region in hydras treated with AZK after HAS-7 (2 + 3)/GFP siRNA electroporation. The asterisk denotes the peduncle region. Scale bar = 500 μm. k Both heads in HyWnt3P::HyWnt3 transgenic animal treated as in j exhibit hypostomal HyWnt3 expression (arrow). Smaller spots along the body column represent ectopic organizers that usually give rise to ectopic tentacles as in i. Scale bar = 200 μm. l Ectopic axis phenotype in actin::HyWnt3 transgenic hydra. Red arrows indicate multiple secondary heads. m No double axis was observed in hydras after HyDkk1/2/4/GFP siRNA electroporation. Scale bar = 500 μm. n Double axis phenotype in hydras after HyDkk1/2/4/GFP siRNA electroporation and AZK treatment. Scale bar = 500 μm. o Rescue of double axis phenotype in animals treated with AZK after electroporation with a combination of HAS-7 (2 + 3) and HyWnt3 siRNAs. Scale bar = 200 μm. p No double axes were observed in hydras treated with AZK after electroporation with HAS-1/GFP siRNAs. q Ratios of double axis phenotypes in hydras after electroporation with siGFP or combinations of siRNAs as indicated. In animals without subsequent AZK treatment double axes were counted 6 days after electroporation. In animals treated additionally with AZK, incubation was started 6 days after electroporation and the numbers of double axes in each group were counted 5 days after AZK removal. Total numbers of animals with double axis phenotype in each group were: siGFP/DMSO = 0/192 (n=5), siGFP/AZK = 10/230 (n=5), siHAS-7/siGFP = 90/186 (n=4), siHAS-7/siGFP/AZK = 203/248 (n=6), siHAS-7/siHyWnt3/AZK = 11/203 (n=3), siHyDkk1/2/4/siGFP/AZK = 65/290 (n=3), siHMP1/siGFP/AZK = 93/204 (n=3), siHAS-1/siGFP/AZK = 1/150 (n=3). Results from at least three independent experiments are shown. Each column represents the total percentage of one group, bars indicate the mean ± S.E.M. *P value < 0.0001, P value < 0.0005, *P value < 0.001. ns = not significant. The data were analyzed using an unpaired parametric T test with Welch’s correction followed by pairwise multiple comparisons of each group with the other groups. The individual data values are shown in Additional file 14
	Fig. 5 beta-Catenin dependent expression of HAS-7. a–c ISH analysis of HAS-7 expression after ALP treatment shows a global upregulation after 24 h and a shift towards the developing ectopic organizers along the body column after 48 h as compared to DMSO-treated controls (d). At 0 h after ALP wash (a) no change of the HAS-7 expression pattern compared to untreated controls (compare d and Fig. Fig.2c)2c) was evident. Scale bars = 150 μm. e HAS-7 expression is globally upregulated in the gastric region of a transgenic actin::HyWnt3 animal with ectopic axis. Scale bar = 250 μm. Arrows indicate head structures. Representatives of 10 hydras examined. f Quantitative real-time PCR analysis of HAS-7 expression confirms the upregulated expression in actin::HyWnt3 animals compared to steady-state AEP animals (lower panel). Inhibition of beta-Catenin by siRNA knockdown reduces HAS-7 expression levels compared to siGFP treated controls (lower panel). Relative expression level is given in 2(-ΔΔCt). Results represent mean +/− S.D. from 3 independent experiments, analyzed by t tests. *p < 0.05. The individual data values are shown in Additional file 14. g, h No detectable binding of Hydra TCF to the HAS-7 promoter. g ChIP analysis of the Hydra magnipapillata HAS-7 promoter. Upper site: Topography of the HAS-7 5′-untranslated region (nt 1 to 4529). The ATG indicates the translation start site. The position of a canonical TCF binding motif (5′-CTTTGTT-3′) is indicated by a blue bar. The localization of the 165-bp DNA segment flanked by the specific ChIP primer pair is visualized with a grey bar. Lower site: ChIP analysis of the Hydra HAS-7 promoter region using chromatin from untreated whole hydra animals (ctrl), and from animals treated with ALP. A polyclonal antibody directed against Hydra TCF was used for precipitation, followed by PCR amplification of the indicated fragment from the HAS-7 regulatory region. Reactions with normal rabbit serum (NRS) or total chromatin (Input) were used as controls (n = 2). PCR products were resolved by agarose gel electrophoresis, and visualized by ethidium bromide staining. h ChIP analysis of the HmTSP promoter performed under the same conditions as in g and used as a positive control. Upper site: Topography of the 3,000-bp HmTSP promoter (nt − 2191 to + 809). Black boxes depict the first two exons of the HmTSP gene. The arrow indicates the transcription start site of the HmTSP mRNA, and ATG the translation start site. The position of the tested canonical TCF binding motif (5′-AACAAAG-3′) is indicated by a red bar. The localization and size of a 164-bp DNA segment flanked by the specific ChIP primer pair is visualized with a grey bar. Lower site: ChIP analysis as described under (g) (n = 2).
	Fig. 6 Structural homology model of the putative HyWnt3:HAS-7 complex. a Overview of HAS-7 (surface potential) complexed with HyWnt3 (yellow cartoon presentation). The active site Zn-ion of the astacin is shown as pink sphere. b Detailed view inside the active site pocket, shown in standard orientation with the Zn-ion located at the bottom of the cleft. c Alignment of amino acid sequences in different Hydra Wnt proteins encompassing the putative DP cleavage motif in HyWnt3. NvWnt3, Nematostella vectensis Wnt3; mWnt3, mouse Wnt3; XWnt8, Xenopus Wnt8
	Fig. 7 Mathematical model of HAS-7 function. a Schematic representation of the model. Yellow blocks depict self-containing pattern formation systems consisting of an activator and an inhibitor. b–f Numerical simulations of different experimental scenarios. The blue color corresponds to high levels of HAS-7 expression, the red color to HyWnt3 expression, and the green color indicates the formation of tentacles. The scale of these expression intensities is similar across all simulation plots. g Schematic diagram summarizing the role of HAS-7 in restricting the organizer in steady-state hydras and double axis induction after experimental HAS-7 gene silencing

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