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
© Prexl et al; licensee BioMed Central Ltd. 2011
Received: 23 November 2010
Accepted: 7 September 2011
Published: 7 September 2011
The Notch signalling pathway is conserved in pre-bilaterian animals. In the Cnidarian Hydra it is involved in interstitial stem cell differentiation and in boundary formation during budding. Experimental evidence suggests that in Hydra Notch is activated by presenilin through proteolytic cleavage at the S3 site as in all animals. However, the endogenous ligand for HvNotch has not been described yet.
We have cloned a cDNA from Hydra, which encodes a bona-fide Notch ligand with a conserved domain structure similar to that of Jagged-like Notch ligands from other animals. Hyjagged mRNA is undetectable in adult Hydra by in situ hybridisation but is strongly upregulated and easily visible at the border between bud and parent shortly before bud detachment. In contrast, HyJagged protein is found in all cell types of an adult hydra, where it localises to membranes and endosomes. Co-localisation experiments showed that it is present in the same cells as HvNotch, however not always in the same membrane structures.
The putative Notch ligand HyJagged is conserved in Cnidarians. Together with HvNotch it may be involved in the formation of the parent-bud boundary in Hydra. Moreover, protein distribution of both, HvNotch receptor and HyJagged indicate a more widespread function for these two transmembrane proteins in the adult hydra, which may be regulated by additional factors, possibly involving endocytic pathways.
Early embryonic development is regulated in all animals by common signalling pathways such as the Wnt-, FGF-, Hedgehog-, TGFβ/BMP- and Notch pathways. They have been shown to be present in basal metazoans such as Cnidarians [1–5]. The fresh water polyp Hydra consists of a simple tube-like body made of two epithelial layers, the inner endoderm and the outer ectoderm separated by an acellular mesoglea. It further possesses a hypostome and a ring of tentacles at the apical end and a foot at its basal end. Interstitial cells are located in the spaces between the epithelial cells of both cell layers. They constitute a pluripotent stem cell lineage that differentiates nerve cells, gland cells, germ cells and nematocytes. The latter carry nematocyte capsules used for catching prey and are unique to Cnidarians [6–9].
The Notch signalling pathway has recently been shown to be involved in nematocyte and female germ cell differentiation, as well as in budding [5, 10]. Whilst an inductive function of Notch for terminal differentiation of nematoblasts and oocytes has been suggested the precise mechanisms are completely unclear. During budding Notch signalling was shown to be important for the formation of a sharp boundary between the bud and the parent animal. This seemed to be the preposition for the bud to make a constriction at its base and form a foot. As has been shown at many other developmental boundaries Notch signalling here appears to have the potential to create patterns where two adjacent cells adopt different fates. These are signal receiving cells, which have the Notch receptor, and signal sending cells carrying a Notch ligand. Notch ligands possess a characteristic domain, the DSL (D elta, S errate, L ag) domain, followed by several EGF repeats and a transmembrane domain. Serrate/Jagged-like ligands have an extra domain, the cysteine rich von Willebrand type C (VWC) domain, between the EGF repeats and the transmembrane domain. Interaction with Notch receptors takes place via the DSL domains of the ligand and the EGF repeats of the receptor .
We have now identified a potential Notch ligand in Hydra. Here we present its sequence and expression pattern. Comparison of its amino acid sequence with that of other Notch ligands of the DSL family shows similarity to Serrate/Jagged-like ligands. We thus named it HyJagged. Its mRNA expression levels in adult animals were not detectable by in situ hybridisation with the exception of a remarkable upregulation at final stages of budding in cells of the parent animal at the site where the bud detaches. However, immunofluorescence studies with anti-HyJagged antibody revealed the presence of HyJagged in all cells of adult animals where it is localised at membranes and in endosomes.
Isolation and characterization of the Hydra Jagged gene
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).
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 . This is in accordance with the fact that no ESTs encoding HyJagged could be identified in the libraries.
Protein localisation of HyJagged
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 ).
In this work we have identified a potential ligand for HvNotch in Hydra, HyJagged. It has the same modular structure as Notch ligands from higher animals and the closely related Cnidarian Nematostella, which is also reflected in the conserved exon-intron structure of its gene between Hydra and mouse. The protein includes a DSL domain, which is responsible for the interaction with the extracellular EGF repeats of the Notch receptors in flies and mammals . Moreover, HyJagged possesses a relatively small number of EGF repeats and a transmembrane domain. Comparison of the abundance of EGF repeats in Notch ligands and receptors shows some interesting similarities: Species with large extracellular domains in their Notch receptors (e.g. Nematostella, Drosophila and mouse) also have a high number of EGF repeats in their Serrate/Jagged-like proteins (however not in Delta) and vice versa (Figure 3 and ).
Thus, although we have not provided experimental evidence, we suggest that HyJagged constitutes the only canonical Notch ligand in Hydra. A second gene encoding a DSL domain is also present in Hydra. It has been identified previously and was shown to be expressed in gland cells . The resulting protein is 332 amino acids long, has a signal peptide and a DSL domain and thus shows some structural homology with DSL2, a soluble Notch ligand from C.elegans. It would be interesting to see whether this protein is involved in Notch signalling in Hydra.
In situ hybridisation showed that Hyjagged was not visibly expressed on the mRNA level in intact Hydra with the exception of a very strong upregulation at budding stage 9. We have previously described that Notch signalling is important for late budding stages in Hydra. Whilst as early as at budding stage 5-6 HvWnt8 and the Hydra FGFR-homolog kringelchen demarcate the boundary between the parent animal and the bud [20, 21] budding cannot be completed without Notch signalling. The gene expression zone of kringelchen remains diffuse and a crucial metalloprotease, MMP-A3 is not induced. Thus the bud cannot constrict and does not form a foot. Two stripes of gene expression are required for correct patterning of the bud, one leading to constriction involving MMP-A3 and the other leading to differentiation of the buds foot cells involving expression of foot specific genes such as ppod-1. For these to form we proposed a model including mutual inhibition and lateral activation on the basis of Notch signalling at this boundary .
Our model required that cells which express kringelchen at the boundary express a Notch ligand. The data shown here are partially in accordance with this assumption because Hyjagged is expressed in cells that also express kringelchen at the boundary between parent and bud. However, the timing of this upregulation is unexpected. While constriction begins in budding stage 8, Hyjagged mRNA could only be detected at the beginning of budding stage 9. Hyjagged expression during earlier budding stages probably occurs in too low amounts to be detectable by in situ hybridisation experiments. Antibody staining clearly shows the presence of HyJagged at the basis of buds, however, only a weak increase in its level at this position in comparison with the remaining tissue is detectable (Figure 9). At this point we clearly have not used techniques suited to uncover precise quantitative differences in the amounts of HyJagged mRNA and protein at the cellular level during the final stages of budding. Therefore we cannot exclude the possibility that the observed requirement for Notch signalling at the boundary between parent and bud involves a mechanism that does not include a direct HyJagged-HvNotch interaction.
In contrast to the generally undetectable levels of mRNA expression outside the boundary between parent and bud, HyJagged protein is clearly present in all cells of the animals. The same is true for HvNotch. Although this makes it difficult to conclude whether there is active Notch signalling going on, the presence of the proteins is not unexpected. Notch signalling is highly regulated by factors involved in post-translational modification of the receptors (e.g. Fringe) , by the activity of metalloproteases like ADAM  and of presenilin as catalytic subunit of the γ-secretase complex and also by endocytosis of both, the Notch receptors and the ligands [24, 25]. Endocytosis is part of a mechanism that leads to mutual inhibition of the signal sending and signal receiving cells. This means that signalling cells have higher levels of ligand but inhibit the propagation of Notch signals from adjacent cells and vice versa . That way signalling can be induced by small differences in the concentration of receptors and ligands in this system, which might be hard to detect in immunofluorescence experiments . We have shown that GFP/RFP-tagged HyJagged and HvNotch are indeed localised in endosomes (this work and ). However, the fluorescently tagged protein did not completely co-localise with each other within these structures. Differences in the localisation of the endogeneous proteins were also seen in immunofluorescence experiments. HyJagged appeared stronger localised at membranes whereas HvNotch showed a slightly stronger staining of punctae in the vicinity of the membranes. In interstitial cells membrane staining was completely lacking with the HvNotch antibody.
In Hydra, Notch signalling might also be regulated through post-translational modification of receptor and ligand as genes encoding metalloproteases of the ADAM family, the glycosyltransferase Fringe and the ubiquitin ligase Mindbomb, which regulates endocytosis of Notch ligands, are present in the genome . Their expression patterns might be conclusive in the future in order to understand the precise molecular mechanism by which Notch signalling exerts its profound impact on pattern formation and stem cell differentiation in Hydra.
We report here the identification of the Hyjagged gene, which encodes a protein with great similarity to canonical Notch ligands from worms, insects and vertebrates. Its mRNA is especially strongly expressed at the constriction site of budding animals, confirming the conserved role for Notch ligands during boundary formation. Furthermore, both HvNotch and HyJagged proteins are found in all cells of adult animals indicating a general role for Notch signalling in regulating cellular communication in Hydra. The differential distribution of both proteins in endosomes indicates that endosomal pathways may play an important role in modulating Notch signalling.
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
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 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, -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).
We thank Prof. Charles N. David for assistance during database searches of the Hydra genome. We also thank Prof. Charles N. David and Prof. Harry MacWilliams for helpful discussions. We furthermore thank Dr. Olga Alexandrova for assistance with immunofluorescence stainings and confocal microscopy. The study was supported by grants BO1748-2 and BO1748-5 from the Deutsche Forschungsgemeinschaft awarded to A.B.
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