Hydra vulgaris (Basel) was grown at a temperature of 18°C in hydra medium (0.4 mM CaCl2, 0.6 mM MgSO4, 0.5 mM NaHCO3, 0.08 mM K2CO3). The animals were fed regularly with freshly hatched Artemia nauplii.
PCR amplification of Hyjagged sequences
Hydra cDNA for RACE experiments was obtained with the GeneRacer Kit with Superscript III (Invitrogen). 5' and 3'RACE experiments were performed using the gene specific primers 5'-CCCATTTGTT GTGAGGCGTA AAGCTGATGT AAGTGC-3' and 5'-GGAGTTGCAA AATGTACAGA TGCATGGTGT GG-3'. Full-length Hyjagged was then amplified from Hydra cDNA by PCR.
Whole mount in situ hybridisation experiments
Single whole mount in situ hybridisation experiments were carried out using Digoxigenin-labeled RNA probes as described before [17, 28].
Construction of GFP and RFP fusion proteins
Full-length Hvnotch was cloned into the NheI and SmaI sites of the HoTRed expression vector. Full-length Hyjagged as well as Hyzic were cloned into the modified EGFP expression vector HoTG(reen)  into the NheI and SmaI sites for overexpression from the Hydra actin promoter in hydra cells.
For expression in mammalian cells HvNotch and Hyjagged genes were cloned into the EcoRI and SmaI sites of the expression vector pEGFP (Clontech), leading to expression of C-terminally GFP-tagged proteins.
Transfection of Hydra cells
GFP- and RFP-constructs were introduced into hydra cells using a particle gun (Biorad) as described previously . After 1-2 days cells expressing the GFP and RFP fusion proteins were clearly visible.
Production of recombinant proteins
To allow expression in E.coli the coding sequences for HvN
(HvNotch without its extracellular domain), HvNICD , Hyjagged-ICD, Hyzic, HyHes and Cnash were cloned into the vector pRSET (Invitrogen) using the BamHI and EcoRI sites for HvN
, HvNICD, Hyjagged-IC and Hyzic, and the EcoRI and XhoI sites for Cnash and HyHes.
Denatured His6-tagged-HyJagged-ICD, -HvNΔE , -HvNICD, -HyZic, -HyHES and -CnASH were produced in E.coli and affinity-purified using nickel sepharose (Amersham Biosciences). Recombinant HvNΔE and HyZic were used for antibody production, recombinant HyJagged-ICD, HvNICD, HyZic, HyHES and CnASH were analysed by SDS-PAGE and Western Blot using anti-Notch (dilution 1:100), anti-JAG-IC (dilution 1:500), anti-ZIC7A12 (dilution 1:1), anti-HES (dilution 1:500) and anti-CNA7B10 (dilution 1:1) antibodies.
Anti-Notch antibody was produced in chicken by Davids Biotechnologie GmbH by immunisation with recombinant HvNΔE. A portion of the resulting polyclonal antibody was then affinity purified.
For the anti-JAG-IC antibody a peptide corresponding to a stretch of the intracellular domain of HyJagged (VNKDNLKKGIFKTISRKS) was produced by Davids Biotechnologie GmbH and used for immunisation of rabbit. The resulting antibody was then affinity purified.
For the anti-HES antibody a peptide corresponding to a stretch of the region shortly before and the begin of the basic domain of HyHES (RHPMKEKRRANKPLLER) was produced by Davids Biotechnologie GmbH and used for immunisation of rabbit. The resulting antibody was then affinity purified.
Monoclonal anti-ZIC7A12- and anti-CNA7B10-antibodies were produced in rat as described in . Recombinant HyZic and CnASH were used for immunisation.
Cultivation and transfection of HEK293T
Human embryonic kidney cells (293T) cells were cultured in DMEM (Sigma) supplemented with 10% FCS and 5% Pen/Strep at 37°C and 5% CO2. 293T cells were transfected using PEI pH 7,0.
Antibody staining of HEK293T
HEK293T cells at 70-80% confluence were seeded on glass coverslips in 6-well culture plates and incubated for 24 h at 37°C. After that the cells were transfected with the specific DNA. After 24 h the coverslips were washed twice with PBS, and the cells were fixed in 4% PFA in PBS for 15 minutes at room temperature. After washing the cells again, they were permeabilized for 15 minutes with 1% Triton X-100 in PBS, washed twice with PBS and incubated in blocking solution (10% FCS, 0,2% Tween 20, PBS) for one hour at room temperature. Cells were then incubated with primary antibody (anti-Notch antibody (dilution 1:100) or anti-JAG-IC antibody (dilution 1:50)) for further 60 minutes at room temperature. After that, they were washed three times for 10 minutes in washing solution (1% BSA, 0,2% Tween 20, PBS) before they were incubated in secondary antibody for one hour at room temperature. Finally, cells were washed three times for 10 minutes with washing solution, incubated with TO-PRO 3 (Molecular probes, 1 μg/ml in PBS) for 5 minutes and mounted on slides with Vectashield (Alexis Biochemicals).
500 hydra were dissociated into single cells in dissociation medium (3.6 mM KCl; 6 mM CaCl2; 1.2 mM MgSO4; 6 mM sodium citrate; 6 mM sodium pyruvate; 6 mM glucose; 12.5 mM TES; and 50 mg/ml rifampicin, pH6.9) by pipetting. After centrifugation the resulting cell pellet was resuspended and incubated for 4 hours at 18°C on a rotator in 20 μM of the proteasome inhibitor MG-132 (Sigma). The cells were then incubated on ice in 500 mM Saccharose, 10 mM Tris, 2 mM EGTA pH7,4, 10 ng/ml Pepstatin A, 10 ng/ml Aprotinin, 10 ng/ml Leupeptin, 0,5 mg/ml Pefablock for 20 min before homogenization with a Potter-Dounce homogenizer. The homogenate was differentially centrifuged. The 1000 g and 100.000 g pellets were separated by SDS-PAGE and Western Blots were stained with the anti-JAG-IC (dilution 1:500), rabbit preimmune serum (1:1000), anti-HES (dilution 1:500), anti-ZIC7A12 (dilution1:1) or anti-CNA7B10 (dilution1:1) respectively.
Antibody staining of whole Hydra
For membrane staining of endogenous HvNotch and endogenous HyJagged whole animals were relaxed with 2% urethane and fixed with PFA/EtOH (2% PFA, 70% EtOH, PBS) for 30 minutes. They were then washed twice for 5 minutes with PBS followed by an ethanol treatment. Therefore hydra were treated with 20%, 30%, 40%, 50%, 75% EtOH in PBS and 100% EtOH, followed by 75%, 50%, 40%, 30% and 20% EtOH in PBS, each for 5 minutes. Further staining was performed as below, starting with permeabilization.
Other immunofluorescence staining experiments were performed by relaxing animals with 2% urethane and fixation with 4% PFA in hydra medium for 1 hour. Hydra were then washed three times for 10 minutes with PBS before they were permeabilized for 15 minutes with 0,5% Triton X-100 in PBS. Animals were blocked for 15 minutes with 0,1% Triton X-100, 1% BSA in PBS and incubated in anti-Notch antibody (dilution 1:100), anti-JAG-IC antibody (dilution 1:50), anti-Nv1 (dilution 1:2) or anti-ZIC7A12 (dilution 1:1) for 1 h at room temperature. Hydra were washed three times 10 minutes in PBS followed by incubation in secondary antibody for 2 h at room temperature. Before the animals were stained with TO-PRO3 (Molecular probes, 1 μg/ml in PBS) they were washed in PBS three times 10 minutes. Finally they were mounted on slides with Vectashield (Alexis Biochemicals).
Living animals with GFP-expressing cells were stained with FM4-64 (Molecular Probes) for detection of plasma membranes and endosomes. 1 μl of 500 μM FM4-64 in hydra medium was injected into the gastric cavity of Hydra. The animals were then incubated in 50 μM FM4-64 in hydra medium for 20 minutes to 20 hours.
Living animals with GFP- and RFP-expressing cells were relaxed in 2% urethane and scanned immediately.
Light optical serial sections were acquired with a Leica (Leica Microsystems, Wetzlar, Germany) TCS SP confocal laser-scanning microscope equipped with an oil immersion Plan-Apochromat 100/1.4 NA objective lens. Fluorochromes were visualized with an argon laser with an excitation wavelength of 488 nm and emission filters of 520-540 nm for EGFP and Alexa488 and 640-760 nm for FM4-64, and with a helium-neon laser with an excitation wavelength of 633 nm and emission filter of 660-760 nm for TO-PRO3. For Cy3 and RFP the excitation wavelength was 561 nm and an emission filter of 570-580 nm was used. Image resolution was 512 × 512 pixel. The 8-bit grey scale single-channel images were overlaid to an RGB image assigning false color to each channel, and then assembled into tables using Adobe Photoshop 10.0 and ImageJ 1.37 k software.
GenBank accession numbers for genes and proteins used in alignments and domain structures are as follows:
HyJagged (JN036823); Mouse Jagged1 (NP_038850); Human Jagged1 (AAC51731); Human Delta-like1 (NP_005609); Drosophila Serrate (CAA40148); Drosophila DeltaD1 (AAR21456); C.elegans Apx-1 (NP_503882); C.elegans Lag-2 (NP_503877); Hydra HvNotch (ABV68547).