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HvJNK, a Hydra member of the c-Jun NH2-terminal kinase gene family, is expressed during nematocyte differentiation
Gene Expression Patterns 5 (2005) 397–402 www.elsevier.com/locate/modgep
HvJNK, a Hydra member of the c-Jun NH2-terminal kinase gene family, is expressed during nematocyte differentiation Isabelle Philippa, Thomas W. Holsteinb, Bert Hobmayera,* a Division of Ultrastructural Research and Evolutionary Biology, Institute of Zoology and Limnology, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria b Department of Molecular Evolution and Genomics, Zoological Institute, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg, Germany
Received 21 July 2004; received in revised form 7 September 2004; accepted 14 September 2004
Abstract C-jun NH2-terminal kinases (JNKs) represent a subgroup of mitogen-activated protein kinases (MAPKs). MAPK pathways are important regulators of cell proliferation, apoptosis, and gene expression throughout higher metazoans. We report here the characterization of a highly conserved Hydra JNK orthologue (HvJNK) that exhibits amino acid sequence identity of 61% as compared with human JNK1a. Phylogenetic analysis places HvJNK in a cluster with other metazoan JNKs. HvJNK is expressed in the nematocyte differentiation pathway. Double in situ hybridizations demonstrate overlapping expression with two other genes specifically activated during nematocyte differentiation, HyZic and Nowa, and restrict the phase of HvJNK expression to late proliferating nematoblasts and early differentiating nematocytes. Our results indicate that JNKs might have acted in cell differentiation in simple, pre-bilaterian animals. q 2004 Elsevier B.V. All rights reserved. Keywords: JNK; SAPK; MAPK; Hydra; cnidaria; Evolution; Nematocyte differentiation; Cell signaling
1. Results and discussion Mitogen-activated protein kinase (MAPK) signaling pathways likely exist in all eukaryotic cells. They respond to a wide range of environmental stimuli like changes in available nutrients, heat shock, mechanical stress, ultraviolet radiation, and exposure to inflammatory cytokines, growth factors, and adhesion molecules (Kyriakis and Avruch, 2001; Weston and Davis, 2002). Three evolutionary conserved groups of MAPK signaling pathways are currently distinguished in mammals: extracellular signalregulated kinase (ERK) pathways, p38 pathways, and c-jun NH2-terminal kinase (JNK) pathways. JNK pathways have recently received particular attention, as they are not only involved in the response to environmental stimuli, but in * Corresponding author. Tel.: C43 512 507 6165; fax: C43 512 507 2930. E-mail address: [email protected] (B. Hobmayer). 1567-133X/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2004.09.007
addition have been demonstrated to act in metazoan embryonic development by controlling the cell cycle, inducing apoptosis, and organizing the cytoskeleton (Weston and Davis, 2002). At present, one of the currently most intensively studied aspects of signaling via JNK is its regulation of planar cell polarity and morphogenetic tissue movements in Drosophila and vertebrates (Boutros et al., 1998; Yamanaka et al., 2002). It is unclear, when in early metazoan phylogeny MAPK signaling pathways were coopted for their developmental functions (Caffrey et al., 1999). Recent identification of p38 and JNK ortholouges in the phylogenetically oldest extant metazoans, the sponges, suggested that at least p38 and JNK pathways evolved in the earliest multicellular animals (Mu¨ller et al., 2002). It is, however, unclear whether the sponge p38 and JNK genes act in embryogenesis. Furthermore, MAPKs have yet not been characterized in diploblastic cnidarians, the closest sister group to the triploblastic bilaterians. The ERK1-2 inhibitor apigenin is
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Fig. 1. HvJNK belongs to a metazoan JNK cluster. (A) Sequence alignment of the predicted amino acid sequences of HvJNK(Hydra vulgaris), SuJNK(Suberites domuncula), CaJNK1(Caenorhabditis elegans), DJNK (Drosophila melanogaster), and HoJNK1a (Homo sapiens). Alignment was done using CLUSTALw (www2.ebi.ac.uk/clustalw/) and visualized using GeneDoc v2.06.02 (http://www.psc.edu/biomed/genedoc/). The ser/thr-kinase domain is outlined by a black bar. A box indicates the JNK-specific activation peptide motive (MTPYVV). Conserved residues are shown with black background; semiconservative substitutions are shown in grey. (B) Phylogenetic tree of MAPKs including family members of the extracellular signal-regulated kinases (ERKs), p38 MAPKs,
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Fig. 2. HvJNK is expressed in the nematocyte differentiation pathway. (A) Whole mount in situ hybridization in a budding polyp. (B) Magnified view of the gastric region demonstrates HvJNK expression in nests of nematocyte precursors. Bar: 25 mm. (C) Quantitative analysis of the size of HvJNK expressing nests. Quantification was done in whole mount in situ hybridizations counterstained with the DNA-interacting fluorochrome DAPI to visualize cell nuclei. Each bar represents the meanCSD from three counts; 100 nests in various animals were analyzed in each count.
known to inhibit bud initiation in Hydra (Fabila et al., 2002), but the corresponding kinase has yet not been identified. Here, we describe the molecular cloning of the first cnidarian (Hydra) member of the JNK gene family, HvJNK, and demonstrate its specific expression during differentiation of interstitial stem cells into specialized sensory cells, the nematocytes (stinging cells). 1.1. Phylogeny of HvJNK A DNA fragment representing part of a JNK-specific ser/thr-kinase domain was cloned from a Hydra vulgaris cDNA template using degenerate PCR, and the translational open reading frame of the corresponding mRNA was then completed by RACE experiments (see experimental procedures). The domain structure of the predicted protein sequence closely resembles the structure of members of the JNK family from higher metazoans. It exhibits a N-terminal ser/thr-kinase domain and a phosphor-binding motive (MTPYVV), which is diagnostic for the JNK subgroup of MAPKs (Fig. 1A). Amino acid alignment including metazoan JNK members shows conservation throughout the predicted protein sequence with highest levels of identity in the ser/thr-kinase domain (Fig. 1A). Overall identity of the Hydra sequence with its human, Drosophila, Caenorhabditis, and sponge orthologs ranges between 54 to 61% (59–74% including conservative substitutions). Phylogenetic tree construction places the predicted Hydra JNK
protein with high statistical support into a cluster of metazoan JNKs (Fig. 1B). We therefore termed the corresponding gene HvJNK. 1.2. Expression of HvJNK during nematocyte differentiation Whole mount in situ hybridizations using intact polyps demonstrated that HvJNK was expressed in a large number of interstitial cell clusters in the ectoderm throughout the gastric region (Fig. 2A,B). Head (hypostome and tentacles) and foot (lower peduncle and basal disc) were free of HvJNK expressing cells (Fig. 2A). Furthermore, in situ hybridization did not detect HvJNK expression in ectodermal and endodermal epithelial cells, and HvJNK was neither upregulated during budding, nor during head or foot regeneration (data not shown). This expression pattern indicated that HvJNK is specifically activated in the nematocyte differentiation pathway. Nematocytes derive by differentiation from a population of multipotent, interstitial stem cells (David and Gierer, 1974; see Fig. 4). Those stem cells determined for the nematocyte pathway proliferate and form nests containing 2, 4, 8, or 16 nematoblasts. At any of these stages, nests can synchronously go through terminal mitosis. Thereafter, the resulting nests of 4, 8, 16, or 32 differentiating nematocytes start with nematocyst (capsule) formation. They finally break apart, when morphogenesis of the capsule is complete. The mature, individual nematocytes then
3 and JNKs. Metazoan sequences were selected from Hydra vulgaris (Hv), Homo sapiens (Ho), Drosophila melanogaster (D/Dr), Caenorhabditis elegans (Ca), and Suberites domuncula (Su). Further, the ancestral yeast MAPK precursors HOG from Emericella nidulans (Em) and Saccharomyces cerevisiae (Sa) were included. Alignment was performed using CLUSTALw (www2.ebi.ac.uk/clustalw/). The tree was then constructed by maximum-likelihood quartet puzzling with an E. nidulans HogA outgroup by using PUZZLE v4.0.2. (www.tree-puzzle.de) and visualized by using TreeView v.1.5.2. (http:// taxonomy.zoology.gla.ac.uk/rod/treeview.html). Branch supports over 60% are displayed, and the scale bar represents the number of amino acid substitutions per position.
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migrate towards their final point of action (primarily in the tentacles), where they are incorporated into ectodermal epithelial cells and mounted towards the outside of the body. A detailed inspection revealed that HvJNK was expressed in nests exhibiting no clearly visible capsule (Fig. 2B). This indicated that expression is restricted to proliferating nematoblasts and/or early differentiating nematocytes. Quantitative analysis showed that only few nests containing 4 or 8 cells expressed HvJNK, while the majority of HvJNK expressing nests consisted of 16 and 32 cells (Fig. 2C). The fact that only one out of four nematocyte sub-lineages, the desmonemes, forms nests of 32 cells (Rich and Tardent, 1969; David and Challoner, 1974) demontrates that HvJNK is involved in desmoneme differentiation. The large total number of HvJNK expressing nests in the body column (Fig. 2A) further suggests that upregulation also occurs in other sub-lineages, at least in holotrichous and atrichous
isorhizas, which differentiate in nests containing 8 and 16 cells (Rich and Tardent, 1969; David and Challoner, 1974). 1.3. Co-expression of HvJNK with other nematocyte-specific transcripts In order to determine the phase of HvJNK expression in this differentiation pathway in more detail, we performed double in situ hybridizations with two other nematocytespecific genes, HyZic and Nowa. HyZic, a member of the zic/odd-paired gene family of Zn-finger trancription factors, is expressed in the early phase of proliferating nematoblasts (Lindgens et al., 2004). Nowa, which encodes for a structural protein of the capsule wall, is expressed later in nests of differentiating nematocytes from terminal mitosis on to the early phase of capsule wall synthesis (Engel et al., 2002).
Fig. 3. HvJNK shows overlap in its expression with the nematocyte-specific transcripts HyZic and Nowa. (A–C) Double in situ hybridizations using HvJNK (blue) and HyZic (red) probes. (D–F) Double in situ hybridizations using HvJNK (blue) and Nowa (red) probes. (A,D) Whole polyp views. (B,E) Magnified views from the gastric region; arrow heads show co-expression of HvJNK/HyZic and HvJNK/Nowa in the same nests by a violett precipitate. Bars: 10 mm. (C,F) Quantitative analysis of overlapping expression. Each bar represents the meanCSD from four counts; 100 nests in various animals were analyzed in each count.
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In both HvJNK/HyZic (Fig. 3A,B) and HvJNK/Nowa (Fig. 3D,E) double in situ hybridizations, we found nests coexpressing the two genes. Hence, HvJNK is transcriptionally active in proliferating nematoblasts and in differentiating nematocytes. In HvJNK/HyZic experiments, the fraction of nests positive for both transcripts was small (16%), while 59% (HvJNK) and 24% (HyZic) of the stained nests expressed one transcript alone. As HyZic is expressed primarily in early nests containing 2–8 nematoblasts (Lindgens et al., 2004), and as the majority of HvJNK expressing nests contains 8 and 16 cells (Fig. 2C), the relatively small amount of co-expressing nests is most likely due to a situation that in the pathway HvJNK is upregulated, when HyZic expression is decreasing. In HvJNK/Nowa experiments (Fig. 3D–F), 25% of the stained nests expressed both genes, while 55% (HvJNK) and 19% (Nowa) exhibited upregulation of one gene alone (Fig. 3F). Assuming that transcriptional activation of HvJNK does not occur in two or more independent phases, the most parsimonious explanation for our results is that HvJNK transcription starts in a significant fraction of late proliferating nematoblasts, extends throughout terminal mitosis, and is finally downregulated in the early phase of capsule differentiation (Fig. 4).
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2. Experimental procedures 2.1. Cloning of HvJNK To identify DNA fragments of a putative Hydra JNK ser/thr-kinase domain, nested PCR was done using degenerated primers and single-stranded Hydra vulgaris cDNA. Primer combinations were as follows: HvJNKForw1/HvJNK-Rev2/HvJNK-Forw2/HvJNK-Rev3. Primer sequences (IUB-code 5 0 –3 0 ): HvJNK-Forw1: GGN WSN GGN GCN CAR GG, HvJNK-Forw2: GCN CAR GGN ATH GTN TG, HvJNK-Rev2: CCA YTG RTC DAT RTG RTC, HvJNK-Rev3: GGY TTN ARR TCN CKR TG. The PCR conditions for the first amplification round were: 1 cycle: 95 8C for 3 min; 30 cycles: 95 8C for 30 s, 42 8C for 30 s, 72 8C for 1 min 20 s; 1 cycle: 72 8C for 2 min. The conditions for the nested reaction were: 1 cycle: 95 8C for 3 min; 35 cycles: 95 8C for 45 s, 42 8C for 45 s, 72 8C for 1 min 20 s; 1 cycle: 72 8C for 3 min. Amplification products of expected size were characterized using standard methods. 5 0 and 3 0 RACE were carried out to clone the complete translational open reading frame of the HvJNK mRNA. 2.2. Whole mount in situ hybridization In situ hybridizations with one DIG-labeled RNA probe covering the whole HvJNK open reading frame were performed according to Grens et al. (1995) with the exception that NBT/BCIP substrates (Roche) were used. Double in situ hybridizations were done according to Hansen et al. (2000) using DIG- and fluorescein-labeled RNA probes simultaneously. Here, visualization of the DIG-labeled HvJNK probe was carried out with NBT/BCIP substrates and the reaction was stopped with 100% ethanol for 20 min. Then, the tissue was re-hydrated at room temperature by 5 min washes in 75%, 50% and 25% ethanol in MAB (100 mM maleinic acid, 150 mM NaCl; pH 7.5) and one 5 min wash in 100% MAB, followed by a one hour incubation in 1! blocking solution (Roche) at 4 8C. The blocking solution was replaced by antibody solution (antiFITC fab fragments conjugated to alkaline phosphatase; 1:2000 in 1! blocking solution) and incubated at 4 8C over night. Thereafter, antibody was removed, and the samples were equilibrated in NTMT (100 mM NaCl, 50 mM MgCl2, 0.1% Tween-20, 100 mM Tris–HCl; pH 9.5) two times for 5 min at room temperature. Finally, visualization of HyZic or Nowa hybridization was done with Fast Red substrate (Sigma). The reaction was stopped by washing the samples in 1! PBS.
Acknowledgements Fig. 4. Schematic view of nematocyte development from interstitial stem cells via proliferating nematoblasts into nests of differentiating nematocytes. M represents a terminal mitosis. Bars indicate the proposed periods of expression of HyZic, HvJNK, and Nowa genes.
We thank Dirk Lindgens and Ulrich Technau for providing data prior to publication and Thomas Posch for his help with microscopy. This work was supported by
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grants of the Austrian Fonds zur Fo¨rderung wissenschaftlicher Forschung (FWF P16685) and the Deutsche Forschungsgemeinschaft (DFG Ho1621/3-1, SFB 269 TPA5). The HvJNK sequence has been deposited in the GenBank database under the accession number AY651025.
References Boutros, M., Paricio, N., Strutt, D.I., Mlodzik, M., 1998. Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94, 109–118. Caffrey, D.R., O’Neill, L.A.J., Shields, D.C., 1999. The evolution of the MAP kinase pathways: coduplication of interacting proteins leads to new signaling cascades. J. Mol. Evol. 49, 567–582. David, C.N., Challoner, D., 1974. Distribution of interstitial cells and differentiating nematocysts in nests of Hydra attenuata. Am. Zool. 14, 537–542. David, C.N., Gierer, A., 1974. Cell cycle kinetics and development of Hydra attenuata. III. Nerve and nematocyte differentiation. J. Cell Sci. 16, 359–375. Engel, U., Oezbek, S., Engel, R., Petri, B., Lottspeich, F., Holstein, T.W., 2002. Nowa, a novel protein with minicollagen Cys-rich domains, is involved in nematocyst formation in Hydra. J. Cell Sci. 115, 3923– 3934.
Fabila, Y., Navarro, L., Fujisawa, T., Bode, H.R., Salgado, L.M., 2002. Selective inhibition of protein kinases blocks formation of a new axis, the beginning of budding, in Hydra. Mech. Dev. 119, 157–164. Grens, A., Mason, E., Marsh, J.L., Bode, H.R., 1995. Evolutionary conservation of a cell fate specification gene: the Hydra achaete scute homolog has proneural activity in Drosophila. Development 121, 4027–4035. Hansen, G.N., Williamson, M., Grimmelikhuijzen, C.J.P., 2000. Twocolour double-labeling in situ hybridization of whole mount Hydra using RNA probes for five different Hydra neuropeptide preprohormones: evidence for colocalization. Cell Tissue Res. 301, 245–253. Kyriakis, J.M., Avruch, J., 2001. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol. Rev. 81, 807–869. Lindgens, D., Holstein, T.W., Technau, U., 2004. HyZic, the Hydra homolog of the neural effector gene zic, is involved in the early specification of the sensory nematocytes. Development 2004; (in press). Mu¨ller, W.E.G., Bo¨hm, M., Grebenjuk, V.A., Skorokhod, A., Mu¨ller, I.M., Gamulin, V., 2002. Conservation of the positions of metazoan introns from sponges to humans. Gene 295, 299–309. Rich, F., Tardent, P., 1969. Untersuchungen zur Nematozyten-Differenzierung bei Hydra attenuata. Rev. Suisse Zool. 76, 779–789. Weston, C.R., Davis, R.J., 2002. The JNK signal transduction pathway. Curr. Opin. Genet. Dev. 12, 14–21. Yamanaka, H., Moriguchi, T., Masuyama, N., Kusakabe, M., Hanafusa, H., Takada, R., et al., 2002. JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. Eur. Mol. Biol. Org. Rep. 3, 69–75.
HvJNK, a Hydra member of the c-Jun NH2-terminal kinase gene family, is expressed during nematocyte differentiation Isabelle Philippa, Thomas W. Holsteinb, Bert Hobmayera,* a Division of Ultrastructural Research and Evolutionary Biology, Institute of Zoology and Limnology, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria b Department of Molecular Evolution and Genomics, Zoological Institute, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg, Germany
Received 21 July 2004; received in revised form 7 September 2004; accepted 14 September 2004
Abstract C-jun NH2-terminal kinases (JNKs) represent a subgroup of mitogen-activated protein kinases (MAPKs). MAPK pathways are important regulators of cell proliferation, apoptosis, and gene expression throughout higher metazoans. We report here the characterization of a highly conserved Hydra JNK orthologue (HvJNK) that exhibits amino acid sequence identity of 61% as compared with human JNK1a. Phylogenetic analysis places HvJNK in a cluster with other metazoan JNKs. HvJNK is expressed in the nematocyte differentiation pathway. Double in situ hybridizations demonstrate overlapping expression with two other genes specifically activated during nematocyte differentiation, HyZic and Nowa, and restrict the phase of HvJNK expression to late proliferating nematoblasts and early differentiating nematocytes. Our results indicate that JNKs might have acted in cell differentiation in simple, pre-bilaterian animals. q 2004 Elsevier B.V. All rights reserved. Keywords: JNK; SAPK; MAPK; Hydra; cnidaria; Evolution; Nematocyte differentiation; Cell signaling
1. Results and discussion Mitogen-activated protein kinase (MAPK) signaling pathways likely exist in all eukaryotic cells. They respond to a wide range of environmental stimuli like changes in available nutrients, heat shock, mechanical stress, ultraviolet radiation, and exposure to inflammatory cytokines, growth factors, and adhesion molecules (Kyriakis and Avruch, 2001; Weston and Davis, 2002). Three evolutionary conserved groups of MAPK signaling pathways are currently distinguished in mammals: extracellular signalregulated kinase (ERK) pathways, p38 pathways, and c-jun NH2-terminal kinase (JNK) pathways. JNK pathways have recently received particular attention, as they are not only involved in the response to environmental stimuli, but in * Corresponding author. Tel.: C43 512 507 6165; fax: C43 512 507 2930. E-mail address: [email protected] (B. Hobmayer). 1567-133X/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2004.09.007
addition have been demonstrated to act in metazoan embryonic development by controlling the cell cycle, inducing apoptosis, and organizing the cytoskeleton (Weston and Davis, 2002). At present, one of the currently most intensively studied aspects of signaling via JNK is its regulation of planar cell polarity and morphogenetic tissue movements in Drosophila and vertebrates (Boutros et al., 1998; Yamanaka et al., 2002). It is unclear, when in early metazoan phylogeny MAPK signaling pathways were coopted for their developmental functions (Caffrey et al., 1999). Recent identification of p38 and JNK ortholouges in the phylogenetically oldest extant metazoans, the sponges, suggested that at least p38 and JNK pathways evolved in the earliest multicellular animals (Mu¨ller et al., 2002). It is, however, unclear whether the sponge p38 and JNK genes act in embryogenesis. Furthermore, MAPKs have yet not been characterized in diploblastic cnidarians, the closest sister group to the triploblastic bilaterians. The ERK1-2 inhibitor apigenin is
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Fig. 1. HvJNK belongs to a metazoan JNK cluster. (A) Sequence alignment of the predicted amino acid sequences of HvJNK(Hydra vulgaris), SuJNK(Suberites domuncula), CaJNK1(Caenorhabditis elegans), DJNK (Drosophila melanogaster), and HoJNK1a (Homo sapiens). Alignment was done using CLUSTALw (www2.ebi.ac.uk/clustalw/) and visualized using GeneDoc v2.06.02 (http://www.psc.edu/biomed/genedoc/). The ser/thr-kinase domain is outlined by a black bar. A box indicates the JNK-specific activation peptide motive (MTPYVV). Conserved residues are shown with black background; semiconservative substitutions are shown in grey. (B) Phylogenetic tree of MAPKs including family members of the extracellular signal-regulated kinases (ERKs), p38 MAPKs,
I. Philipp et al. / Gene Expression Patterns 5 (2005) 397–402
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Fig. 2. HvJNK is expressed in the nematocyte differentiation pathway. (A) Whole mount in situ hybridization in a budding polyp. (B) Magnified view of the gastric region demonstrates HvJNK expression in nests of nematocyte precursors. Bar: 25 mm. (C) Quantitative analysis of the size of HvJNK expressing nests. Quantification was done in whole mount in situ hybridizations counterstained with the DNA-interacting fluorochrome DAPI to visualize cell nuclei. Each bar represents the meanCSD from three counts; 100 nests in various animals were analyzed in each count.
known to inhibit bud initiation in Hydra (Fabila et al., 2002), but the corresponding kinase has yet not been identified. Here, we describe the molecular cloning of the first cnidarian (Hydra) member of the JNK gene family, HvJNK, and demonstrate its specific expression during differentiation of interstitial stem cells into specialized sensory cells, the nematocytes (stinging cells). 1.1. Phylogeny of HvJNK A DNA fragment representing part of a JNK-specific ser/thr-kinase domain was cloned from a Hydra vulgaris cDNA template using degenerate PCR, and the translational open reading frame of the corresponding mRNA was then completed by RACE experiments (see experimental procedures). The domain structure of the predicted protein sequence closely resembles the structure of members of the JNK family from higher metazoans. It exhibits a N-terminal ser/thr-kinase domain and a phosphor-binding motive (MTPYVV), which is diagnostic for the JNK subgroup of MAPKs (Fig. 1A). Amino acid alignment including metazoan JNK members shows conservation throughout the predicted protein sequence with highest levels of identity in the ser/thr-kinase domain (Fig. 1A). Overall identity of the Hydra sequence with its human, Drosophila, Caenorhabditis, and sponge orthologs ranges between 54 to 61% (59–74% including conservative substitutions). Phylogenetic tree construction places the predicted Hydra JNK
protein with high statistical support into a cluster of metazoan JNKs (Fig. 1B). We therefore termed the corresponding gene HvJNK. 1.2. Expression of HvJNK during nematocyte differentiation Whole mount in situ hybridizations using intact polyps demonstrated that HvJNK was expressed in a large number of interstitial cell clusters in the ectoderm throughout the gastric region (Fig. 2A,B). Head (hypostome and tentacles) and foot (lower peduncle and basal disc) were free of HvJNK expressing cells (Fig. 2A). Furthermore, in situ hybridization did not detect HvJNK expression in ectodermal and endodermal epithelial cells, and HvJNK was neither upregulated during budding, nor during head or foot regeneration (data not shown). This expression pattern indicated that HvJNK is specifically activated in the nematocyte differentiation pathway. Nematocytes derive by differentiation from a population of multipotent, interstitial stem cells (David and Gierer, 1974; see Fig. 4). Those stem cells determined for the nematocyte pathway proliferate and form nests containing 2, 4, 8, or 16 nematoblasts. At any of these stages, nests can synchronously go through terminal mitosis. Thereafter, the resulting nests of 4, 8, 16, or 32 differentiating nematocytes start with nematocyst (capsule) formation. They finally break apart, when morphogenesis of the capsule is complete. The mature, individual nematocytes then
3 and JNKs. Metazoan sequences were selected from Hydra vulgaris (Hv), Homo sapiens (Ho), Drosophila melanogaster (D/Dr), Caenorhabditis elegans (Ca), and Suberites domuncula (Su). Further, the ancestral yeast MAPK precursors HOG from Emericella nidulans (Em) and Saccharomyces cerevisiae (Sa) were included. Alignment was performed using CLUSTALw (www2.ebi.ac.uk/clustalw/). The tree was then constructed by maximum-likelihood quartet puzzling with an E. nidulans HogA outgroup by using PUZZLE v4.0.2. (www.tree-puzzle.de) and visualized by using TreeView v.1.5.2. (http:// taxonomy.zoology.gla.ac.uk/rod/treeview.html). Branch supports over 60% are displayed, and the scale bar represents the number of amino acid substitutions per position.
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migrate towards their final point of action (primarily in the tentacles), where they are incorporated into ectodermal epithelial cells and mounted towards the outside of the body. A detailed inspection revealed that HvJNK was expressed in nests exhibiting no clearly visible capsule (Fig. 2B). This indicated that expression is restricted to proliferating nematoblasts and/or early differentiating nematocytes. Quantitative analysis showed that only few nests containing 4 or 8 cells expressed HvJNK, while the majority of HvJNK expressing nests consisted of 16 and 32 cells (Fig. 2C). The fact that only one out of four nematocyte sub-lineages, the desmonemes, forms nests of 32 cells (Rich and Tardent, 1969; David and Challoner, 1974) demontrates that HvJNK is involved in desmoneme differentiation. The large total number of HvJNK expressing nests in the body column (Fig. 2A) further suggests that upregulation also occurs in other sub-lineages, at least in holotrichous and atrichous
isorhizas, which differentiate in nests containing 8 and 16 cells (Rich and Tardent, 1969; David and Challoner, 1974). 1.3. Co-expression of HvJNK with other nematocyte-specific transcripts In order to determine the phase of HvJNK expression in this differentiation pathway in more detail, we performed double in situ hybridizations with two other nematocytespecific genes, HyZic and Nowa. HyZic, a member of the zic/odd-paired gene family of Zn-finger trancription factors, is expressed in the early phase of proliferating nematoblasts (Lindgens et al., 2004). Nowa, which encodes for a structural protein of the capsule wall, is expressed later in nests of differentiating nematocytes from terminal mitosis on to the early phase of capsule wall synthesis (Engel et al., 2002).
Fig. 3. HvJNK shows overlap in its expression with the nematocyte-specific transcripts HyZic and Nowa. (A–C) Double in situ hybridizations using HvJNK (blue) and HyZic (red) probes. (D–F) Double in situ hybridizations using HvJNK (blue) and Nowa (red) probes. (A,D) Whole polyp views. (B,E) Magnified views from the gastric region; arrow heads show co-expression of HvJNK/HyZic and HvJNK/Nowa in the same nests by a violett precipitate. Bars: 10 mm. (C,F) Quantitative analysis of overlapping expression. Each bar represents the meanCSD from four counts; 100 nests in various animals were analyzed in each count.
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In both HvJNK/HyZic (Fig. 3A,B) and HvJNK/Nowa (Fig. 3D,E) double in situ hybridizations, we found nests coexpressing the two genes. Hence, HvJNK is transcriptionally active in proliferating nematoblasts and in differentiating nematocytes. In HvJNK/HyZic experiments, the fraction of nests positive for both transcripts was small (16%), while 59% (HvJNK) and 24% (HyZic) of the stained nests expressed one transcript alone. As HyZic is expressed primarily in early nests containing 2–8 nematoblasts (Lindgens et al., 2004), and as the majority of HvJNK expressing nests contains 8 and 16 cells (Fig. 2C), the relatively small amount of co-expressing nests is most likely due to a situation that in the pathway HvJNK is upregulated, when HyZic expression is decreasing. In HvJNK/Nowa experiments (Fig. 3D–F), 25% of the stained nests expressed both genes, while 55% (HvJNK) and 19% (Nowa) exhibited upregulation of one gene alone (Fig. 3F). Assuming that transcriptional activation of HvJNK does not occur in two or more independent phases, the most parsimonious explanation for our results is that HvJNK transcription starts in a significant fraction of late proliferating nematoblasts, extends throughout terminal mitosis, and is finally downregulated in the early phase of capsule differentiation (Fig. 4).
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2. Experimental procedures 2.1. Cloning of HvJNK To identify DNA fragments of a putative Hydra JNK ser/thr-kinase domain, nested PCR was done using degenerated primers and single-stranded Hydra vulgaris cDNA. Primer combinations were as follows: HvJNKForw1/HvJNK-Rev2/HvJNK-Forw2/HvJNK-Rev3. Primer sequences (IUB-code 5 0 –3 0 ): HvJNK-Forw1: GGN WSN GGN GCN CAR GG, HvJNK-Forw2: GCN CAR GGN ATH GTN TG, HvJNK-Rev2: CCA YTG RTC DAT RTG RTC, HvJNK-Rev3: GGY TTN ARR TCN CKR TG. The PCR conditions for the first amplification round were: 1 cycle: 95 8C for 3 min; 30 cycles: 95 8C for 30 s, 42 8C for 30 s, 72 8C for 1 min 20 s; 1 cycle: 72 8C for 2 min. The conditions for the nested reaction were: 1 cycle: 95 8C for 3 min; 35 cycles: 95 8C for 45 s, 42 8C for 45 s, 72 8C for 1 min 20 s; 1 cycle: 72 8C for 3 min. Amplification products of expected size were characterized using standard methods. 5 0 and 3 0 RACE were carried out to clone the complete translational open reading frame of the HvJNK mRNA. 2.2. Whole mount in situ hybridization In situ hybridizations with one DIG-labeled RNA probe covering the whole HvJNK open reading frame were performed according to Grens et al. (1995) with the exception that NBT/BCIP substrates (Roche) were used. Double in situ hybridizations were done according to Hansen et al. (2000) using DIG- and fluorescein-labeled RNA probes simultaneously. Here, visualization of the DIG-labeled HvJNK probe was carried out with NBT/BCIP substrates and the reaction was stopped with 100% ethanol for 20 min. Then, the tissue was re-hydrated at room temperature by 5 min washes in 75%, 50% and 25% ethanol in MAB (100 mM maleinic acid, 150 mM NaCl; pH 7.5) and one 5 min wash in 100% MAB, followed by a one hour incubation in 1! blocking solution (Roche) at 4 8C. The blocking solution was replaced by antibody solution (antiFITC fab fragments conjugated to alkaline phosphatase; 1:2000 in 1! blocking solution) and incubated at 4 8C over night. Thereafter, antibody was removed, and the samples were equilibrated in NTMT (100 mM NaCl, 50 mM MgCl2, 0.1% Tween-20, 100 mM Tris–HCl; pH 9.5) two times for 5 min at room temperature. Finally, visualization of HyZic or Nowa hybridization was done with Fast Red substrate (Sigma). The reaction was stopped by washing the samples in 1! PBS.
Acknowledgements Fig. 4. Schematic view of nematocyte development from interstitial stem cells via proliferating nematoblasts into nests of differentiating nematocytes. M represents a terminal mitosis. Bars indicate the proposed periods of expression of HyZic, HvJNK, and Nowa genes.
We thank Dirk Lindgens and Ulrich Technau for providing data prior to publication and Thomas Posch for his help with microscopy. This work was supported by
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grants of the Austrian Fonds zur Fo¨rderung wissenschaftlicher Forschung (FWF P16685) and the Deutsche Forschungsgemeinschaft (DFG Ho1621/3-1, SFB 269 TPA5). The HvJNK sequence has been deposited in the GenBank database under the accession number AY651025.
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