0000000000597928
AUTHOR
Uwe Warnken
A Wnt-specific astacin proteinase controls head formation inHydra
AbstractTheHydrahead 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 the identification of an astacin family proteinase as a Wnt processing factor.Hydraastacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone.HAS-7siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is re…
Additional file 11 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 11: Fig. S9. Uncropped Western blot and gel images
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 proteo…
Additional file 12 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 12: Table S3. LNA and RNA probe sequences used for WISH.
Additional file 13 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 13: Table S4. siRNA and qPCR primer sequences.
Additional file 2 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 2: Table S1. (a) Secretome of Hydra HL HyWnt3 (+) fraction. (b) Secretome of Hydra HL HyWnt3 (-) fraction.
Additional file 3 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 3: Table S2. Complete proteome data of HyWnt3(+) and HyWnt3(-) HL fractions.
Additional file 10 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 10: Fig. S8. Developmental balance between ectopic structures. (a) siHAS-7/siNdr electroporation blocks ectopic axis formation after subsequent AZK treatment. (b-c) siHAS-7/Wnt8 electroporation and AZK treatment reduces ectopic tentacle development in double axis animals (b) and leads to multiple secondary axis formation in a fraction of the treated animals (c). Red arrows denote secondary axes. The asterisk denotes the peduncle region. (d) Ectopic tentacle inhibition is clearly evident in animals electroporated with siWnt8 followed by AZK treatment. Note that few residual ectopic tentacles are detectable in c and d mostly on the side not directly hit by the electroporation …
Additional file 5 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 5: Fig. S3. Expression of HAS genes in the interstitial stem cell cluster. (a) t-SNE representation of interstitial cells with clusters labeled by cell state as presented in [25]. (b) Interstitial cell cluster annotation of HyDkk1/2/4 and ataxin genes identified in HyWnt3(+) head lysate fraction. The cells in the t-SNE plots were colored based on expression levels for the respective gene. The transcript IDs are as follows: HMP1: t1098aep, HAS-1: t20535aep, HAS-2: t18494aep, HAS-3: t22149aep, HAS-4: t11453aep, HAS-5: t596aep, HAS-6: t19593aep, HAS-7: t16296aep, HAS-8: t22154aep, HAS-9: t3416aep, HAS-10: t10258aep, HAS-11: t19316aep. HyDkk1/2/4: t8678aep. Cluster label abbrevi…
Additional file 6 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 6: Fig. S4. Detection of HAS-7 by Western blot. (a) Antigenic peptide competition demonstrates the specificity of the HAS-7 antibody. A Western blot for tissue lysates as in Fig. 3a was performed using primary antibody solution with (right panel) or without (left panel) 1 mg/ml of the antigenic peptide used for generating the HAS-7 antibody. The HAS-7 peptide effectively reduces the detection of specific bands at ~ 40 and 70 kDa. (b) Ni-NTA affinity purified recombinant HAS-7. Separation by 12% SDS-PAGE was followed by staining with Coomassie brilliant blue (left) or transfer to PVDF and immunodetection (right) using the Penta-His-antibody as described above. For each lane 1…
Additional file 8 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 8: Fig. S6. Representative images of knockdown and transgenic phenotypes. Representative images of HAS-7 siRNA treated (a-c), HAS-7 siRNA/AZK treated (d-g) or transgenic actin::HyWnt3 (h-i) animals. Scale bars: 200 μm. The inset in Fig. S6a shows an early stage of ectopic axis formation recorded 4 days after electroporation. Red arrows indicate the hypostome areas of the two heads.
Additional file 7 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 7: Fig. S5. Evidence for normal function and morphology of ectopic heads and tentacles. (a-b) Both heads in a HAS-7 siRNA treated animal with a double axis are able to capture and feed on artemia. The arrow denotes an ectopic foot induced by the secondary head. Scale bars = 500 μm. (c-e) Ectopic tentacles induced by ALP treatment show anatomic and molecular features of functional tentacles as demonstrated by immunocytochemistry using a nematocyst-specific antibody (anti-CPP-1) [53]. CPP-1 is a structural component of mature nematocysts in battery cells of tentacles. (c) Overview of CPP-1-stained hydra with ectopic tentacles. Scale bar = 200 μm. (d-e) Enlargement from boxed a…
Additional file 1 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 1: Fig. S1. Ion exchange chromatogram of hydra head lysate pool. (a) 7 fractions of 0.5 ml exceeding an absorption unit threshold of 0.175 were collected as indicated. The cut-off was chosen to provide a critical total protein concentration (> 80 μg) for the subsequent proteome analysis. (b) Peak fractions from (a) were re-screened for HyWnt3-His processing activity. A fragment of Hydra cadherin extracellular domain comprising the first two N-terminal cadherin repeats (HmCadherin1-2) was used as control substrate to monitor unspecific matrix metalloproteinase activity. Accordingly, fractions 4-5 were pooled and analyzed by mass spectrometry as HyWnt3-His(+) sample, fracti…
Additional file 14 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 14: The individual data values for Figs. 3f and 5f.
Additional file 9 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 9: Fig. S7. Function of HAS-7 in regeneration. Animals bisected after HAS-7 siRNA electroporation do not show axis duplication in head (a-b) or foot (c-d) regenerates. Animals were bisected at 50% of body length at day 6 after electroporation and documented at day 0 (a, c) and day 4 (b, d) after bisection. Representatives of 25 bisected hydras examined. Scale bars: 200 μm. (e) Heat map showing the dynamics of transcript levels for HyWnt3(+) astacin genes compared to HyWnt3 and beta-Catenin. Only components that were significantly differentially expressed (P
Additional file 4 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra
Additional file 4: Fig. S2. Phylogenetic tree of astacin metalloproteinases established by PhyLM 3.0 (SEAVIEW package) and based on an alignment of the catalytic domains only, omitting pro-sequences and multiple C-terminal domains. Numbers indicate probability values (in %) obtained from 100 bootstrap replications. Protein abbreviations from bottom: fAST, flavastacin (Flavobacterium meningosepticum, i.e. Chryseobacterium meningosepticum, i.e. Elisabethkingia meningoseptica, Q47899, used as outgroup); HEA-1, Hydractinia echinata astacin-1 (Q2MCX9); HEA-3, H. echinata astacin-3 (Q2MCX7); HEA-4, H. echinata astacin-4 (Q2MCX6); HMP1, Hydra vulgaris metalloproteinase-1 (NP_001296695.1), AST, ast…