The putative Notch ligand HyJagged is a transmembrane protein present in all cell types of adult Hydra and upregulated at the boundary between bud and parent

Isolation and characterization of the Hydra Jagged gene

Database searches of the Hydra genome [12] led to a core sequence prediction for a DSL protein with similarity to Jagged/Serrate-like Notch ligands. After performing 5' and 3' RACE experiments the full-length Hyjagged sequence was amplified from Hydra cDNA [GenBank:JN036823]. The resulting HyJagged protein sequence is shown in Figure 1 in an alignment with mouse Jagged1. It contains a signal peptide (1-22aa), a DSL (D elta, S errate, L ag) domain (181-252aa), which is characteristic for Notch ligands (Figure 2) [13], and five EGF repeats (255-540aa). A cysteine rich domain, the von Willebrand type C (VWC) domain (549-616aa), is located N-terminally of the transmembrane domain (763-785aa), followed by a short non-conserved intracellular region. Comparison with Notch ligands from worms, insects and vertebrates further shows that the modular structure is conserved in HyJagged (Figure 3). The presence of the von Willebrand type C domain, which is characteristic for Serrate/Jagged-like ligands, led us to name the Hydra protein HyJagged.

Interestingly, comparison of HyJagged with the predicted sequence of a Notch ligand from the sea anemone Nematostella vectensis, another Cnidarian, shows a generally similar domain structure between the two proteins but also an important difference (Figure 3). The Nematostella gene encodes a protein with 17 EGF repeats, whereas HyJagged has only six, which in both cases nearly matches the number of EGF repeats in their respective Notch receptors. The DSL domains between the Notch ligands from Nematostella and Hydra are also highly conserved (Figure 2).

HyJagged consists of 835 amino acids that are encoded by 16 exons on a stretch of 11.452 nucleotides. Comparison of the gene organisation of mouse jagged1 with Hyjagged is shown in Figure 4. All exon-intron boundaries are flanked by the conserved GT-AG sequences, 14 of these boundaries appear to be conserved between mouse and Hydra (Figure 4, indicated by arrows). These reflect the modular organisation of Jagged proteins in all animals. Most EGF repeats and the transmembrane domain are encoded by single exons. However the Hydra DSL domain and the mouse VWC domain, as well as the signal peptides of both proteins are encoded within two exons, respectively.

mRNA expression of Hyjagged

To look for expression of the Hyjagged gene in adult hydra in situ hybridisation experiments were performed. These showed no signal, indicating that Hyjagged is expressed at very low levels in contrast to the expression of HvNotch, which shows very high levels of mRNA all over the animal [5]. This is in accordance with the fact that no ESTs encoding HyJagged could be identified in the libraries.

However, when we looked for Hyjagged mRNA during budding we found a very strong expression of the gene during budding stage 9. This is when the bud begins to form foot cells. The signal was present in cells of the ectoderm of the parent animal and coincided with the expression zone of the Hydra FGF-receptor homolog kringelchen (Figure 5).

Protein localisation of HyJagged

In order to study the subcellular distribution of HyJagged in hydra cells the protein was overexpressed as a GFP fusion protein in single cells of intact animals and observed in vivo (Figure 6). For detection of plasma membranes and endosomes the membrane-selective dye FM4-64 was injected into the gastric cavity and the animals were imaged at different time points afterwards. FM4-64 is usually detected at membranes first but is then increasingly enriched in endosomes, which become larger over time [14]. In Figure 6 we show the distribution of FM4-64 in cells of live imaged animals over a period of 20 hours. HyJagged-GFP hardly co-localised with FM4-64 at membranes at the time points indicated. However, it was seen in punctuate and ring-like structures in the cytoplasm. Some of these clearly corresponded to FM4-64 positive structures and thus most likely constituted early and late endosomes. We then compared this HyJagged-GFP distribution with the localisation of HvNotch-RFP in epithelial cells. Figure 7 shows that both proteins are localised in punctuate and ring-like structures, however they only partially co-localised.

In the absence of detectable mRNA for the putative Notch ligand Hyjagged we thought to look for the endogenous HyJagged protein. We therefore raised an antibody in rabbit against a synthetic peptide corresponding to 18 amino acids of the intracellular domain of HyJagged. The antibody recognised the intracellular domain of HyJagged when it was expressed in E.coli after SDS-PAGE and Western blotting (Additional file 1A). In addition, on a Western blot with a vesicle fraction from Hydra the antibody recognised a band at an apparent molecular weight of 130 kDa. This is larger than the HyJagged primary sequence suggests (96 kDa), most probably due to post-translational modification of mature HyJagged. Heavy glycosylation of EGF repeats is well documented for Notch ligands (recently reviewed in [15]).

The anti-JAG-IC antibody also detected HyJagged in immunofluorescence experiments, when we expressed it as GFP fusion protein in human HEK293T cells. This is shown in Additional file 1 D-G. In a transient transfection experiment in HEK293T cells HyJagged-GFP did not reach the membrane but was instead trapped in the ER as can be seen by strong staining of the nuclear envelope. This incorrect distribution of HyJagged is most probably due to incomplete post-translational processing of the hydra protein in human cells [16]. Nevertheless this experiment clearly shows that the antibody recognises HyJagged specifically in immunofluorescence. It did not react with any other human protein in HEK293T cells. We therefore used this antibody for immunofluorescence on Hydra whole mounts. Here it stained membranes and punctuate structures in the cytoplasm of epithelial cells all over the animal (Figure 8). A weak increase of the staining in comparison with the surrounding tissue was detected in budding animals at stage 9 at the base of the bud in the ectoderm of the parental animal (Figure 9). This is in accordance with the in situ hybridisation data, which indicated strong mRNA expression in this region.

In addition to epithelial cells, cells of the interstitial cell lineage were also stained with the antibody. This was seen especially in nerve cells of the basal disc (Figure 10). Co-immunofluorescence experiments with the anti-JAG-IC- and anti-Nv1-antibodies were performed in order to relate HyJagged-positive cells to a specific cell type. The anti-Nv1-antibody is specific for sensory neurons in the tentacles and ganglion cells in the peduncle [17]. Co-staining with anti-JAG-IC showed that these ganglion cells also contained HyJagged, but in addition, HyJagged was also present in Nv-1 negative neurons (Figure 11).

In order to identify HyJagged proteins in cells of the nematocyte line we used a rat monoclonal antibody against HyZic protein since Hyzic mRNA is known to be expressed in proliferating nematocyte precursors [18]. Rats were immunized with HyZic protein that we produced in E.coli. The resulting monoclonal antibody anti-ZIC7A12 detected the recombinant protein on Western blots. Moreoever, it stained a band of 46 kDa, which corresponds to the predicted size of HyZic in Western blots from hydra lysates (Additional file 2). In addition, in immunofluorescence experiments it recognised a HyZic-GFP fusion protein when expressed in hydra cells (see Additional file 2). On Hydra whole mounts the anti-ZIC7A12 antibody strongly stained single and double interstitial cells and nests of nematoblasts before their exit from mitosis. Weaker staining was observed in post-mitotic nematoblasts, which already had formed a small vacuole. In further developed nematocytes HyZic was not detected anymore. This distribution of HyZic protein in Hydra is in good agreement with published data about the localisation of its mRNA in proliferating nematoblasts [18]. Figure 12 shows that single and pairs of interstitial cells highlighted with the anti-ZIC7A12 antibody also contained HyJagged in a punctuate pattern. This was also the case in proliferating nematoblasts. However, in finally differentiated nematocytes negative for HyZic very strong HyJagged staining was observed (Figure 12 Q-T, nc). In conclusion, while all cells of the nematocyte line seem to have HyJagged protein, its abundance increases in terminally differentiated cells.

Finally, we wanted to compare the cellular localisation of HyJagged with that of HvNotch. To this end we raised an antibody in chicken against HvN^ΔE (the Hydra Notch receptor HvNotch without its extracellular domain) after its expression in E.coli. It detected HvN^ΔE after expression in E.coli by SDS-PAGE and Western blotting down to a concentration of 8, 9 ng (see Additional file 3A). Furthermore it recognised overexpressed HvNotch-GFP fusion protein in human HEK293T cells in immunofluorescence experiments (see Additional file 3). We thus used it for co-immunofluorescence staining on Hydra whole mounts together with the anti-JAG-IC antibody. Both antibodies stained membranes of epithelial cells and cells in the interstitial spaces, however, the patterns did not completely overlap. Moreover, both antibodies stained punctuate structures in the cytoplasm. From co-localisation experiments of live images where HvNotch and HyJagged clearly co-localised with the membrane selective dye FM4-64 we concluded that these punctae most likely correspond to endosomes. However, HyJagged- and HvNotch antibody signals in endosomes do not generally co-localise. Moreover, HyJagged signals appear stronger localised on the membranes in comparison with Notch, this is especially obvious in interstitial cells (Figure 13).

Hydra culture

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) [29] 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 [29]. 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 ^ΔE (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 ^ΔE , HvNICD, Hyjagged-IC and Hyzic, and the EcoRI and XhoI sites for Cnash and HyHes.

Denatured His₆-tagged-HyJagged-ICD, -HvN^ΔE , -HvNICD, -HyZic, -HyHES and -CnASH were produced in E.coli and affinity-purified using nickel sepharose (Amersham Biosciences). Recombinant HvN^ΔE 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.

Antibody production

Anti-Notch antibody was produced in chicken by Davids Biotechnologie GmbH by immunisation with recombinant HvN^ΔE. 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 [30]. 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).

Subcellular fractionation

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).

Confocal imaging

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.

Accession numbers

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).