Die Rolle des Notch-Signaltransduktionsweges bei Muster- und Grenzbildungsprozessen in Hydra vulgaris

Im Rahmen dieser Arbeit konnte gezeigt werden, dass der Notch-Signalweg auch in Hydra für die Ausbildung und Aufrechterhaltung zweier entwicklungsrelevanter Grenzen benötigt wird. Sowohl während der späten Knospung als auch während der Musterbildung eines Kopfes ist seine Aktivität für die Etablierung zweier benachbarter, scharf zueinander abgegrenzter Signalzentren verantwortlich. Es konnte gezeigt werden, dass dies Voraussetzung für die normale Entwicklung von Knospen aber auch für die Strukturierung eines Kopfes in seine drei Teilbereiche ist. Im Fall der Knospung war nach Inhibition des Notch-Signalweges die Knospenfußbildung inhibiert, weshalb sich die Knospen nicht an ihrer Basis einschnüren und vom Elterntier abspalten konnten. Dies resultierte in der Entwicklung stabiler Y-Tiere. Die Expression von HyHes und der Matrix-Metalloprotease MMP-A3 konnte in DAPT-behandelten Tieren nicht mehr detektiert werden. Dadurch konnte gezeigt werden, dass HyHes ein primäres Zielgen des Notch-Signalweges darstellt. Dies konnte auch in einem in vitro-Reportergenversuch bestätigt werden, indem HvNICD die Expression von EGFP ausgehend vom HyHes-Promotor über zwei Su(H)-Bindestellen induzieren konnte. Die Expression des FGF-Rezeptors kringelchen und des Gerüstproteins Hydsh war nach DAPT-Behandlung nicht inhibiert, sondern diffus und unbegrenzt. Eine Expression in klaren, streifenförmigen Domänen an der Knospenbasis wie in unbehandelten Tieren konnte nie beobachtet werden. Demnach wird die Aktivität des Notch-Signalweges im Verlauf der Knospung vermutlich für die Schärfung einer bestehenden Grenze zwischen Muttertier und Knospe benötigt. Dies ermöglicht die definierte, voneinander abgegrenzte Expression von Genen, die einerseits für die voranschreitende Einschnürung der Knospenbasis und andererseits für die Fußdifferenzierung der Knospe verantwortlich sind. Neben der Knospung wird der Notch-Signalweg auch für den Erhalt adulter Kopfstrukturen und für die de novo-Musterbildung eines Kopfes während der Kopfregeneration benötigt. Hvnotch wird in adulten an der Tentakelbasis (an der Grenze zwischen Tentakeln und Tentakelzone) und auch während der Kopfregeneration erhöht exprimiert. Die Inhibition des Notch-Signalweges führte auf morphologischer Ebene zu abnormen Kopfstrukturen und übermäßiger Produktion von Tentakelgewebe. Daraus konnte gefolgert werden, dass der Notch-Signalweg in unbehandelten Tieren die Ausbildung von Tentakelgewebe unterdrückt bzw. verhindert. Dies konnte auch auf molekularer Ebene durch Untersuchung der Expression von Hywnt3, HMMP und Hyalx gezeigt werden. Diese werden in unbehandelten Tieren je spezifisch in einem Teilbereich des Kopfes, nämlich dem Hypostom, den Tentakeln oder der Grenzregion zwischen Tentakeln und Tentakelzone exprimiert. In DAPT-behandelten adulten Tieren und Kopfregeneraten war ihre Expression inhibiert oder verändert und spiegelte die beobachteten Kopf-Fehlbildungen wider. Anhand von Transplantationsversuchen und der Untersuchung der Expression von Hywnt3 konnte überdies ein Einfluss des Notch-Signalweges auf die de novo-Ausbildung eines Kopforganisators während der Regeneration beobachtet werden

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

<p>Abstract</p> <p>Background</p> <p>The Notch signalling pathway is conserved in pre-bilaterian animals. In the Cnidarian <it>Hydra </it>it is involved in interstitial stem cell differentiation and in boundary formation during budding. Experimental evidence suggests that in <it>Hydra </it>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.</p> <p>Results</p> <p>We have cloned a cDNA from <it>Hydra</it>, which encodes a bona-fide Notch ligand with a conserved domain structure similar to that of Jagged-like Notch ligands from other animals. <it>Hyjagged </it>mRNA is undetectable in adult <it>Hydra </it>by <it>in situ </it>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.</p> <p>Conclusions</p> <p>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 <it>Hydra</it>. 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.</p

Horizontal gene transfer contributed to the evolution of extracellular surface structures

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment

Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth) families.

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment

Internal repeats and three-dimensional structure of <i>Hydra</i> PPOD.

<p>A) Alignment of six internal repeats detected within the PPOD4 sequence using the RADAR algorithm. B) Structural model of a single β-trefoil domain in PPOD4 as inferred by Phyre. Three internal sequence repeats (coloured ribbon models) correspond to three repeated supersecondary structures that form a single β-trefoil fold.</p

Immuno-EM localisation of PPOD.

<p>A: Immunogold labeling of PPOD (visualised by anti-chicken 10 nm colloidal gold) in the cuticle layer c5 and in subapical secretory granules (s) of ectodermal epithelial cells. Freeze-substitution with pure acetone followed by LR-white embedding. Scale bar: 200 nm. B: PPOD-positive secretory granule (s) in contact (arrow-head) with the plasma membrane (pm). Scale bar: 200 nm. C: Subapical secretory granules (s) are all PPOD-positive. A mitochondrium is lettered with (m), the plasma membrane with (pm). Scale bar: 200 nm. D: Scarce PPOD-immunogold-labelling can also be seen throughout cuticle layers c2–4 (marked by arrows), in addition to PPOD-labelling of layer c5 (double arrows). Scale bar: 500 nm.</p

PAS cytochemistry of <i>Hydra</i> cuticle.

<p>Periodic acid-thiocarbohydrazide-silver proteinate staining was performed on Epon sections from <i>H. vulgaris</i>. A: Cuticle layers c1–5 react positively as do apical secretory granules (s) and glycogen particles (arrow-heads) in a neighbouring nematocyst; the asterisk marks a vacuole. Scale bar: 500 nm. B: Negative control for the PAS-reaction (omission of periodic acid oxidation); faint unspecific staining results from binding of thiocarbohydrazide to the osmium tetroxide used for freeze-substitution. Scale bar: 500 nm.</p

Identification of PPOD proteins in the SDS-PAGE gel by mass spectrometry.

*<p>Numbers in parentheses refer to numbers of peptides identified by MS-spectra and MS/MS spectra respectively.</p>**<p>Numbers give the position of the peptides in the protein sequence.</p>***<p>There are two slightly different PPOD4 gene models (XP_002161930 and XP_002159894) based on NCBI annotation of the <i>Hydra</i> genome. Unique peptides corresponding to both gene models were found in the 27 kDa band. By comparison, only one PPOD4 gene model (Hma2.211683) corresponding to XP_002161930 is found in the <i>Hydra</i> genome browser (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052278#pone.0052278-Chapman1" target="_blank">[11]</a> for details of two genome assemblies).</p