The Notch-signalling pathway plays an important role in pattern formation in Hydra. Using pharmacological Notch inhibitors (DAPT and SAHM1), it has been demonstrated that HvNotch is required for head regeneration and tentacle patterning in Hydra. HvNotch is also involved in establishing the parent-bud boundary and instructing buds to develop feet and detach from the parent. To further investigate the functions of HvNotch, we successfully constructed NICD (HvNotch intracellular domain)-overexpressing and HvNotch-knockdown transgenic Hydra strains. NICD-overexpressing transgenic Hydra showed a pronounced inhibition on the expression of predicted HvNotch-target genes, suggesting a dominant negative effect of ectopic NICD. This resulted in a “Y-shaped” phenotype, which arises from the parent-bud boundary defect seen in polyps treated with DAPT. Additionally, “multiple heads”, “two-headed” and “ectopic tentacles” phenotypes were observed. The HvNotch-knockdown transgenic Hydra with reduced expression of HvNotch exhibited similar, but not identical phenotypes, with the addition of a “two feet” phenotype. Furthermore, we observed regeneration defects in both, overexpression and knockdown strains. We integrated these findings into a mathematical model based on long-range gradients of signalling molecules underlying sharply defined positions of HvNotch-signalling cells at the Hydra tentacle and bud boundaries.

Hydra head regeneration consists of hypostome/organizer and tentacle development, and involves Notch and Wnt/β-catenin signaling. Notch inhibition blocks hypostome/organizer regeneration, but not the appearance of the tentacle tissue. β-Catenin inhibition blocks tentacle, but not hypostome/organizer regeneration. Gene expression analyses during head regeneration revealed the Notch-promoting expression of HyWnt3, HyBMP2/4, and the transcriptional repressor genes CnGsc, Sp5, and HyHes, while blocking HyBMP5/8b and the c-fos-related gene HyKayak β-Catenin promotes the expression of the tentacle specification factor HyAlx, but not of HyWnt3 This suggests HyWnt3 and HyBMP4 as parts of a hypostome/organizer gene module, and BMP5/8, HyAlx, and β-catenin as parts of a tentacle gene module. Notch then functions as an inhibitor of tentacle production to allow regeneration of a hypostome/head organizer. HyKayak is a candidate target gene for HvNotch-induced repressor genes. Inhibiting HyKayak attenuated the expression of HyWnt3 Polyps of Craspedacusta do not have tentacles and thus after head removal only regenerate a hypostome structure. Notch signaling was not needed for head regeneration in Craspedacusta, corroborating the idea of its requirement during Hydra head regeneration to harmonize two co-operating pattern-forming processes.

Wnt- and Notch signaling pathways are involved in regulating Hydra head regeneration. The Notch-inhibitor DAPT blocks expression of transcriptional repressor genes including CnGsc and HyHes and attenuates hypostomal HyWnt3 - and HyBMP2/4 expression in regenerating Hydra heads while increasing expression of HyBMP5/8b and the c-fos-related gene HyKayak . Inhibition of the transcriptional activity of β-catenin blocks expression of HyAlx during Hydra head regeneration and prevents formation of tentacles, however hypostome regeneration and Wnt3 expression patterns remain intact. This indicates competing pathways for hypostome and tentacle regeneration. We tested this hypothesis on fresh water polyps of Craspedacusta, which do not have tentacles and thus after head removal only regenerate a hypostome with a crescent of nematocytes around the mouth opening. We found that head regeneration in Craspedacusta was inhibited by the β-catenin-inhibitor iCRT14, but not by the Notch-inhibitor DAPT, indirectly confirming that in Hydra Notch-signaling maybe needed to balance separate signaling modules for hypostome and tentacle formation, involving Wnt3/BMP2/4 or HyAlx/HyBMP5/8b expression, respectively. We suggest that during head regeneration, Notch works by inhibiting HyBMP5/8b expression and HyKayak mediated repression of HyWnt3 , rather than directly activating transcription at the HyWnt3 promoter . We thus conclude that HvNotch mediates between two pattern forming systems in Hydra by coordinating a lateral inhibition process restricting the tentacle system.### Competing Interest StatementThe authors have declared no competing interest.

In Hydra, Notch inhibition causes defects in head patterning and prevents differentiation of proliferating nematocyte progenitor cells into mature nematocytes. To understand the molecular mechanisms by which the Notch pathway regulates these processes, we performed RNA-seq and identified genes that are differentially regulated in response to 48 h of treating the animals with the Notch inhibitor DAPT. To identify candidate direct regulators of Notch signalling, we profiled gene expression changes that occur during subsequent restoration of Notch activity and performed promoter analyses to identify RBPJ transcription factor-binding sites in the regulatory regions of Notch-responsive genes. Interrogating the available single-cell sequencing data set revealed the gene expression patterns of Notch-regulated Hydra genes. Through these analyses, a comprehensive picture of the molecular pathways regulated by Notch signalling in head patterning and in interstitial cell differentiation in Hydra emerged. As prime candidates for direct Notch target genes, in addition to Hydra (Hy)Hes, we suggest Sp5 and HyAlx. They rapidly recovered their expression levels after DAPT removal and possess Notch-responsive RBPJ transcription factor-binding sites in their regulatory regions.

Tumour necrosis factor receptors (TNF-Rs) and their ligands, tumour necrosis factors, are highly conserved proteins described in all metazoan phyla. They function as inducers of extrinsic apoptotic signalling and facilitate inflammation, differentiation and cell survival. TNF-Rs use distinct adaptor molecules to activate signalling cascades. Fas-associated protein with death domain (FADD) family adaptors often mediate apoptosis, and TNF-R-associated factor (TRAF) family adaptors mediate cell differentiation and inflammation. Most of these pathway components are conserved in cnidarians, and, here, we investigated the Hydra TNF-R. We report that it is related to the ectodysplasin receptor, which is involved in epithelial cell differentiation in mammals. In Hydra, it is localised in epithelial cells with incorporated nematocytes in tentacles and body column, indicating a similar function. Further experiments suggest that it interacts with the Hydra homologue of a TRAF adaptor, but not with FADD proteins. Hydra FADD proteins colocalised with Hydra caspases in death effector filaments and recruited caspases, suggesting that they are part of an apoptotic signalling pathway. Regulating epithelial cell differentiation via TRAF adaptors therefore seems to be an ancient function of TNF-Rs, whereas FADD-caspase interactions may be part of a separate apoptotic pathway.

The Notch pathway is highly conserved and essential for animal development. We investigated the function of Notch-signalling in Hydra by using the presenilin inhibitor DAPT, which efficiently blocks propagation of Notch-signals. In Hydra, DAPT treatment prevents differentiation of proliferating nematocyte progenitor cells into mature nematocytes. Moreover, it causes defects in the Hydra head by compromising the head organizer. In order to understand the molecular mechanisms by which the Notch pathway regulates these processes we performed RNAseq to identify genes that are differentially regulated in response to 48 hours of DAPT-exposure. This revealed downregulation of 624 genes and upregulation of 207 genes. To identify candidate direct regulators of Notch-signalling, we also profiled gene expression changes that occur during restoration of Notch-activity 3 and 6 hours after DAPT-removal. We then analysed gene expression patterns of these Notch-responsive genes in untreated animals by interrogating the available single cell sequencing data set for untreated animals and found that almost half of the Notch responsive genes were specifically expressed in nematocytes and nematocyte progenitors. This confirms the critical role for Notch-signalling in nematocyte development. Promoter analyses and gene expression profiling after DAPT-removal suggested an indirect role for Notch in regulating a POU-transcription factor, which is critical for nematogenesis. In support of a role for Notch-signalling in head organizer formation, we identified several head organizer genes in the Notch regulated gene data set, including Cngsc, a homologue of goosecoid, a gene associated with the Spemann organizer, and the Wnt pathway genes Sp5, Tcf and Wnt-7. Finally, the expression levels of the tentacle patterning genes HyAlx and Sp5 rapidly recovered after DAPT removal. Given that these genes possess Notch-responsive RBPJ transcription factor binding sites in their regulatory regions, these genes are likely directly targeted by Notch signalling. In summary, our data provide a comprehensive picture of the molecular pathways regulated by Notch signalling in interstitial cell differentiation and formation of the oral-aboral axis in Hydra.

Jumonji-domain-containing protein 6 (JMJD6) is a Fe(II) and 2-oxogluterate (2OG) dependent oxygenase involved in gene regulation through post-translationally modifying nuclear proteins. It is highly expressed in many cancer types and linked to tumor progression and metastasis. Four alternatively-spliced jmjd6 transcripts were annotated. Here, we focus on the two most abundantly expressed ones, which we call jmjd6-2 and jmjd6-Ex5. TCGA SpliceSeq data revealed a significant decrease of jmjd6-Ex5 transcripts in patients and postmortem tissue of several tumors. The two protein isoforms are distinguished by their C-terminal sequences, which include a serine-rich region (polyS-domain) in JMJD6-2 that is not present in JMJD6-Ex5. Immunoprecipitation followed by LC-MS/MS for JMJD6-Ex5 shows that different sets of proteins interact with JMJD6-2 and JMJD6-Ex5 with only a few overlaps. In particular, we found TFIIF-associating CTD phosphatase (FCP1), proteins of the survival of motor neurons (SMN) complex, heterogeneous nuclear ribonucleoproteins (hnRNPs) and upstream binding factor (UBF) to interact with JMJD6-Ex5. Like JMJD6-2, both UBF and FCP1 comprise a polyS-domain. The polyS domain of JMJD6-2 might block the interaction with polyS-domains of other proteins. In contrast, JMJD6-2 interacts with many SR-like proteins with arginine/serine-rich (RS)-domains, including several splicing factors. In an HIV-based splicing reporter assay, co-expression of JMJD6-2 inhibited exon inclusion, whereas JMJD6-Ex5 did not have any effect. Furthermore, the silencing of jmjd6 by siRNAs favored jmjd6-Ex5 transcripts, suggesting that JMJD6 controls splicing of its own pre-mRNA. The distinct molecular properties of JMJD6-2 and JMJD6-Ex5 open a lead into the functional implications of the variations of their relative abundance in tumors.

Mechanisms of programmed cell death differ between animals, plants and fungi. In animals, apoptotic cell death depends on caspases and Bcl-2 family proteins. These protein families are only found in multicellular animals, including cnidarians, insects and mammals. In contrast, members of the TMBIM-family of transmembrane proteins are conserved across all eukaryotes. Sequence comparisons of cell death related proteins between phyla indicate strong conservation of the genes involved. However, often it is not known whether this is paralleled by conservation of function. Here we present the first study to support an anti-apoptotic function of Bcl-2 like proteins in the cnidarian Hydra within a physiological context. We used transgenic Hydra expressing GFP-tagged HyBcl-2-like 4 protein in epithelial cells. The protein was localised to mitochondria and able to protect Hydra epithelial cells from apoptosis induced by either the PI(3) kinase inhibitor wortmannin or by starvation. Moreover, we identified members of the TMBIM-family in Hydra including HyBax-Inhibitor-1, HyLifeguard-1a and -1b and HyLifeguard 4. Expressing these TMBIM-family members in Hydra and human HEK cells, we found HyBax-inhibitor-1 protein localised to ER-membranes and HyLifeguard-family members localised to the plasma membrane and Golgi-vesicles. Moreover, HyBax-inhibitor-1 protected human cells from camptothecin induced apoptosis. This work illustrates that the investigated Bcl-2- and TMBIM-family members represent evolutionarily conserved mitochondrial, ER, Golgi and plasma membrane proteins with anti-apoptotic functions. The participation of ER and Golgi proteins in the regulation of programmed cell death might be a very ancient feature.

TNF-R, TNF, and FADD family members are conserved in the fresh water polyp Hydra. Moreover, Hydra expresses HyTNF-R adaptor proteins similar to the vertebrate TNF-receptor associated factors TRAF-4 and TRAF-6. HyTNF-R is closest related to the human ectodysplasin receptor EDAR, which is involved in epithelial cell differentiation, e.g. the formation of hair and tooth cells in mammals. Consistent with a similar function in Hydra , we show here that HyTNF-R protein is localised very specifically in battery cells and in such epithelial cells of the body column that incorporate nematocytes. Epithelial cell differentiation is therefore an evolutionary ancient function of TNF-R/TNF-protein superfamily members. We also show that two Hydra -FADD proteins co-localise with Hydra caspases possessing death (DD) or death effector (DED) domains in death effector filaments in human cells. Caspase recruitment by members of the FADD-protein family might therefore also be an ancient trait. Future research will have to discover the up-stream pathways, which govern this potential apoptotic pathway in Hydra and whether it is extrinsically or intrinsically induced.

Compared to GPCRs, the set of plant-derived compounds that target ion channels appears much more limited. For voltage-gated channels, most known toxins are derived from animals such as snails, spiders and snakes. Also, many ligand-gated channels are targeted by few to none known plant-derived drugs. Nevertheless, ion channels are the target of some of the most potent plant poisons and most commonly used plant-derived drugs.

The presence of GPRCs in plants is still a question of debate. While G-protein coupled signalling exists, the signalling cycle is typically not activated by seven transmembrane-spanning receptors. By contrast, many plant secondary metabolites are known to affect human GPRCs. Some are very specific for a single type of receptor; however, many others act on more than one type, albeit with often strongly different affinities.

Background Mechanisms of programmed cell death differ considerably between animals, plants and fungi. In animals they depend on caspases and Bcl-2 family proteins and this kind of cell death is called apoptosis. Most gene families encoding proteins involved in apoptosis are found in multicellular animals already in the eldest phyla but their functional conservation is still being studied. Much older protein families have cytoprotective functions across all kingdoms of life. This includes the TMBIMP-family, the presence and function of which in early metazoans has not been investigated yet. Methods We quantified apoptosis in transgenic Hydra overexpressing HyBcl-2-like 4. Moreover, we investigated putative TMBIMP-family members in Hydra by sequence comparison. By overexpression of TMBIMP-family members in Hydra and human HEK cells we analysed their subcellular localisation and in one case their capacity to protect cells from camptothecin induced apoptosis. Results HyBcl-2-like 4, as previously shown in a heterologous system, was localised to mitochondria and able to protect Hydra epithelial cells from apoptosis. The TMBIMP-family in Hydra includes HyBax-Inhibitor-1, HyLifeguard-1a and -1b and HyLifeguard 4 proteins. HyBax-inhibitor-1 protein was found localised to ER-membranes, HyLifeguard-family members were found at the plasma membrane and in Golgi-vesicles. Moreover, HyBax-inhibitor-1 protected human cells from apoptosis. Conclusion This work provides the first functional study to support an anti-apoptotic function of Bcl-2 like proteins in pre-bilaterians within a physiological context. Furthermore it illustrates that genes that were “inherited” from non-animal ancestors, like the TMBIMP-family, were recruited to carry out cell protective anti-apoptotic functions already in early metazoans.

Plants usually contain many different secondary metabolites, but some species contain very specific subsets of secondary metabolites. The amount of the compounds and the kind of compounds vary between different cells, tissues and developmental stages and can be influenced by external stressors. This means that the enzymatic pathways have to be tightly controlled. To synthetize secondary metabolites, plants use products from the primary metabolism as building blocks. The metabolites are further fused and modified by different processes to lead to the variations observed in nature.

While many of the secondary metabolites produced by plants make them unpalatable or toxic, the specific capacities of natural plant products have also been exploited by humans for a long time. In its simplest form, they have been used as spices and aroma compounds to give flavour to food. While it is rather easy to imagine this development, it is also obvious that so-called medicinal plants have a long history of use as pharmaceuticals, hallucinogens or painkillers, even though this often requires careful adjustment of the dose to avoid toxic effects. Last but not the least, since the onset of modern science and technical development, plant secondary metabolites and their derivatives have been exploited for technical applications.