Hydra as a model system for investigating the genetic and cellular regulation of developmental plasticity
In short
The past twenty years have represented a historical turn in our knowledge of the genetics of bilaterian development, namely that of vertebrates, arthropods and nematodes. In particular, genes have been cloned which control and execute important morphogenetic steps. Surprizingly, we realize now that these genes are present in all animals and that, consequently, most of the involved mechanisms are heavily conserved during evolution. Hydra belongs to Cnidaria, a phylum that is considered as a sister group to bilaterians and where ancestral molecular mechanisms driving basic developmental processes can be investigated. Moreover, upon bisection, hydra is capable of regenerating its missing part in few days, a property that made it a popular model system amongst cellular and developmental biologists. For these reasons, we propose to identify the genes and the cellular mechanisms involved in the amazing developmental plasticity of this animal.In the recent years, we established functional tools in hydra, i.e. expression of GFP-reporter constructs in live animals and silencing of gene expression through RNA interference, in order to investigate the function and the regulation of those genes. More specifically, we focus on the genetic mechanisms that underly the cross talk between the different cell lineages at the time the head developmental program is reactivated in order to dissect the respective functions of highly differentiated cells towards that of stem cells. We anticipate that these investigations will highlight the molecular and cellular bases of fundamental biological processes, like cellular plasticity, neurogenesis and apical patterning.
The hydra model system
Hydra is a freshwater cnidarian, which represents one of the most ancestral form of all living animals. It displays a rather simple cellular organization, with two cell layers, the ectoderm and the endoderm separated by an extracellular collagenous matrix, the mesogolea. Hydra shows an oral-aboral polarity, with a head region surrounded by a ring of tentacles that actively catches the food, thanks to its neuro-muscular system. Moreover, hydra regenerate all along their life: upon amputation, the endodermal gastric cells of the head-regenerating tip will acquire in several hours the properties of a ́head organizer centerî. Therefore, hydra offers a unique experimental model system to understand the ancestral mechanisms of neurogenesis (Gauchat et al., Dev. Biol. 2004; Miljkovic-Licina et al., Biosystems, 2004) as well as those that lead to the reactivation of a developmental program whatever the age of animal.We want to make use of this system to address the following questions :
1) What are the cellular and molecular mechanisms underlying the budding and regenerative processes in hydra ?2) How different are the budding and regeneration processes in hydra ?
3) What is the function of stem cells in these processes ?
4) How the highly differentiated cells from the gastroepidermis can transiently take over an organizer function in the regenerating tip after amputation or in the budding zone in intact animal?
5) What are the molecular mechanisms that keep the developmental program open all along the hydra life ?
6) Which of those mechanisms have been conserved along evolution and recruited for epimorphic regeneration in bilaterian species ?
7) What is the genetic circuitry that leads to neurogenesis ?
8) What are the developmental molecular mechanisms involved in the establishment of the apical - oral pole in cnidarians ?
9) What are the intrinsic relationships between neurogenesis and apical patterning in hydra ?
10) Which of those mechanisms have been conserved along evolution and recruited for anterior patterning in bilaterian species ?
Scheme showing the general body organization of hydra, which is basically a tube with a single opening, the mouth-anus, surrounded by a ring of tentacles. At the basal pole, the animal attaches to the substrate thanks the mucous cells. The different cell types from three distinct stem cell populations : myoepithelial cells located in the ectoderm, myoepithelial cells located in the endoderm and interstitial cells that are multipotent stem cells.Genes regulating cell differentiation and developmental processes are highly conserved from cnidarians to mammals
(for review Galliot and Schmid, Int. J. Dev. Biol., 2002)The RSK -> CREB -> CBP pathway during early head regeneration
In order to identify the regulatory genes, i.e. transcription factors, whose activity is required to set up soon after bisection the organizer activity that induces head regeneration, we used DNA-binding assays to screen for transcription factors that would exhibit modulations in their DNA-binding patterns upon regeneration. That way, we characterize the cAMP Response Element Binding (CREB) protein (Galliot et al., Development, 1995). Whereas the hydra CREB gene is itself an early marker for apical formation during regeneration (Chera et al., Biosystems, in press), we also identified a RSK-dependent phosphorylation event that takes place in endodermal cells of head-regenerating tips immediately after bisection. This immediate post-translational regulation of the CREB transcription factor is necessary to carry out the initiation of the regeneration process (Kaloulis et al., PNAS, 2004). In vertebrates, the phosphorylated form of CREB binds to the chromatin modifyer CBP in order to modulate gene expression. We isolated the cnidarian CBP cognate gene and found that silencing of either RSK or CREB or CBP indeed prevents the cellular reorganization normally observed in head-regenerating tips (Chera et al., unpublished). Hence, the functional dissection of this pathway will help us to trace the cellular remodelling at work in head-regenerating tips.Homeobox-containing genes
The recent discovery that all animals essentially share the same complement of genes convinced us of the paradigmatic value of this little animal and of its potential to understand some fundamental processes involved in the development of higher metazoans, such as e.g. morphogenetic induction through gradients of diffusible molecules. These processes had been previously investigated only at the cellular level. Fifteen years ago, in the laboratory of Chica Schaller, Heidelberg University, we therefore started to settle a molecular biology approach, which led in the cloning of several homeobox-containing genes, unexpectedly similar to their higher vertebrate counterparts (Schummer et al., EMBO J., 1992). In addition, their variations of expression during regeneration confirmed our expectation that important pathways had been conserved throughout evolution.Among evolutionary conserved head-specific regulatory genes, the paired-class homeobox gene prdl-a, is expressed in apical nerve cells in adult polyps but after bisection, transiently in endodermal cells of the head-regenerating tip. This result suggested that prdl-a is involved in hydra head organizer activity through inductive interactions from endodermal cells to the overlying ectodermal layer (Gauchat et al., Development, 1998). This result was puzzling as structurally-related genes perform similar tasks at the time head organizer activity is established in chordates, indicating thus some functional conservation of basic molecular mechanisms leading to head differentiation (Galliot and Miller, Trends Genet., 2000). Beside a direct demonstration of the head organizer activity of PRDL-A in hydra through gene silencing, our next interest will focus on deciphering the transcriptional regulatory elements required for this dual temporo-spatial regulation of prdl-a expression.
Considering Hox-related genes, phylogenetic analyses and expression data suggest that at least 5 different Hox/paraHox-related families are expressed in cnidarians, more or less significantly related to anterior and posterior genes. None of them show relatedness with bilaterian Hox/paraHox genes involved in trunk development. Such Hox-like genes were not found in poriferan, therefore, cnidarian Hox-like genes can be considered as representative of the proto-Hox genes (Galliot et al., Dev. Genes Evol., 1999; Gauchat et al., PNAS, 2000). Out of them, three show distinct temporal regulations during head differentiation, suggesting that these genes likely participate in apical patterning (Schummer et al., EMBO J., 1992; Gauchat et al., PNAS, 2000). Recently, we showed that in intact hydra the expression of the paraHox cnox-2/Gsx homolog gene is restricted to the neuronal and nematocyte cell lineages as well as their common proliferating precursor cell, while in budding or regenerating hydra, an early-late up-regulation of cnox-2 was observed in apical neurons of forming heads. Interestingly, cnox-2 silencing leads to alterations of the apical patterning process. These data support an evolutionarily-conserved function for cnox-2/Gsx in neurogenesis and a possible functional link between neurogenesis and head patterning at the early-late phase of head formation (Miljkovic-Licina et al., submitted).
Functional analysis of the genetic circuitry in hydra
Expression of GFP-reporter constructs in live animals
The promoter complexity in developmental genes is likely one of the key elements for the understanding of the apparent paradox between the surprisingly high level of conservation of developmental cascades and the large diversity in observed morphogenesis. As complete sexual development is not easily amenable to experimentation in hydra, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We established a procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to efficiently transfect hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals, and in epithelial as well as interstitial cell types of dissociated hydra. This GFP expression in hydra cells was directed by various promoters, either homologous like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken ß-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra, more specifically the genetic regulations that take place in endodermal cells at the time budding or regeneration are initiated (Miljkovic et al. Dev. Biol., 2002).Live recording of GFP-expressing cells showing the cell movements
in a head-regenerating tip during the first 24 hours after bisection
Gene silencing through RNA interference
The delivery of dsRNAs by feeding the animal on dsRNA-producing bacteria was first described in C. elegans (Timmons and Fire, Nature, 1998), more recently adapted to planarians (Newmark et al., PNAS, 2003). Within the past years, we established gene silencing in hydra through RNA interference by feeding hydra with dsRNAs produced in bacteria : we proved that this method is harmless, stepwise and efficient as for 6 different genes, we could induce gene-specific phenotypes (Chera, de Rosa et al., in revision). This strategy offers the possibility of systematic RNAi screens in hydra.
The feeding dsRNAi strategy in hydra adapted from Newmark et al. PNAS (2003).
Double-stranded RNAs are produced in bacteria and hydra are repeatedly fed with the bacteria-agarose mix.
In case of the protease inhibitor Kazal1, which is specifically expressed in gland cells of the gastrodermis, we observed a massive cell death of those cells together with an autophagy of their neighboring cells, the digestive cells, a phenotype that mimics the human SPINK1/mouse SPINK3 pancreatic phenotype (Ohmuraya et al., Gastroenterology, 2005). This provides a first example where a specific genetic defect affecting an evolutionarily-conserved gene in a cnidarian species, leads to cellular alterations that are similar to those observed in a human disease (Chera et al., in revision).
Perspectives
During the past 15 years, the cnidarian community, including our group, produced a large panel of molecular markers for developmental processes. Recently an EST sequencing project was carried out that provided about 13'000 gene sequences (www.hydrabase.org). Simultaneously, much efforts were carried out for establishing efficient functional strategies that will now help to decipher the molecular and cellular mechanisms that support the initiation of the regeneration and budding processes as well as the morphogenetic processes like head patterning.Beside hydra, several marine cnidarian species also provide useful experimental model systems that offer complementary advantages to that of hydra. Some marine cnidarian species readily follow a full life cycle where they alternate between the polyp and the medusa stages, like the hydrozoan jellyfish Podocoryne carnea, Clytia hemispherica, Cladonema californicum or the sycophozoan Aurelia aurita, those two last species differentiating sophisticated sensory organs including eyes. Finally, the anthozoan sea anemone Nematostella vectensis, which as all anthozoans is restricted to the polyp stage, also provides a model system easily amenable to experimentation to follow developmental and cell differentiation processes during sexual development. Comparisons between the cellular and molecular mechanisms at work in these different species will help us to identify the major common themes and the variations in the ancestral developmental mechanisms.
Schema depicting the position of cnidarians in the metazoan tree. Among cnidarians, anthozoans live exclusively as polyps, cubozoans and scyphozoans most often as jellyfish, while hydrozoans usually alternate between the polyp and the jellyfish stages during their life cycle. Hydra is a freshwater hydrozoan, which lost the medusa stage and displays the polyp stage exclusively.
