ß-Catenin acts in a position-independent regeneration response in the simple eumetazoan Hydra.

Wnt/ß-Catenin signaling plays crucial roles in regenerative processes in eumetazoans. It also acts in regeneration and axial patterning in the simple freshwater polyp Hydra, whose morphallactic regenerative capacity is unparalleled in the animal kingdom. Previous studies have identified ß-catenin as an early response gene activated within the first 30min in Hydra head regeneration. Here, we have studied the role of ß-Catenin in more detail. First, we show that nuclear ß-Catenin signaling is required for head and foot regeneration. Loss of nuclear ß-Catenin function blocks head and foot regeneration. Transgenic Hydra tissue, in which ß-Catenin is over-expressed, regenerates more heads and feet. In addition, we have identified a set of putative ß-Catenin target genes by transcriptional profiling, and these genes exhibit distinct expression patterns in the hypostome, in the tentacles, or in an apical gradient in the body column. All of them are transcriptionally up-regulated in the tips of early head and foot regenerates. In foot regenerates, this is a transient response, and expression starts to disappear after 12-36h. ChIP experiments using an anti-HydraTcf antibody show Tcf binding at promoters of these targets. We propose that gene regulatory ß-Catenin activity in the pre-patterning phase is generally required as an early regeneration response. When regenerates are blocked with iCRT14, initial local transcriptional activation of ß-catenin and the target genes occurs, and all these genes remain upregulated at the site of both head and foot regeneration for the following 2-3 days. This indicates that the initial regulatory network is followed by position-specific programs that inactivate fractions of this network in order to proceed to differentiation of head or foot structures. brachyury1 (hybra1) has previously been described as early response gene in head and foot regeneration. The HyBra1 protein, however, appears in head regenerating tips not earlier than about twelve hours after decapitation, and HyBra1 translation does not occur in iCRT14-treated regenerates. Foot regenerates never show detectable levels of HyBra1 protein at all. These results suggest that translational control mechanisms may play a decisive role in the head- and foot-specific differentiation phase, and HyBra1 is an excellent candidate for such a key regulator of head specification.

Minimal ProtoHox cluster inferred from bilaterian and cnidarian Hox complements.

Bilaterian animals have a Hox gene cluster essential for patterning the main body axis, and a ParaHox gene cluster. Comparison of Hox and ParaHox genes has led workers to postulate that both clusters originated from the duplication of an ancient cluster named ProtoHox, which contained up to four genes with at least the precursors of anterior and posterior Hox/ParaHox genes. However, the way in which genes diversified within the ProtoHox, Hox and ParaHox clusters remains unclear because no systematic study of non-bilaterian animals exists. Here we characterize the full Hox/ParaHox gene complements and genomic organization in two cnidarian species (Nematostella vectensis and Hydra magnipapillata), and suggest a ProtoHox cluster simpler than originally thought on the basis of three arguments. First, both species possess bilaterian-like anterior Hox genes, but their non-anterior genes do not appear as counterparts of either bilaterian central or posterior genes; second, two clustered ParaHox genes, Gsx and a gene related to Xlox and Cdx, are found in Nematostella vectensis; and third, we do not find clear phylogenetic support for a common origin of bilaterian Cdx and posterior genes, which might therefore have appeared after the ProtoHox cluster duplication. Consequently, the ProtoHox cluster might have consisted of only two anterior genes. Non-anterior genes could have appeared independently in the Hox and ParaHox clusters, possibly after the separation of bilaterians and cnidarians.

Cnidarians: an evolutionarily conserved model system for regeneration?

Cnidarians are among the simplest metazoan animals and are well known for their remarkable regeneration capacity. They can regenerate any amputated head or foot, and when dissociated into single cells, even intact animals will regenerate from reaggregates. This extensive regeneration capacity is mediated by epithelial stem cells, and it is based on the restoration of a signaling center, i.e., an organizer. Organizers secrete growth factors that act as long-range regulators in axis formation and cell differentiation. In Hydra, Wnt and TGF-beta/Bmp signaling pathways are transcriptionally up-regulated early during head regeneration and also define the Hydra head organizer created by de novo pattern formation in aggregates. The signaling molecules identified in Cnidarian regeneration also act in early embryogenesis of higher animals. We suppose that they represent a core network of molecular interactions, which could explain at least some of the mechanisms underlying regeneration in vertebrates.

Brachyury, the blastopore and the evolution of the mesoderm.

The role of Brachyury and other T-box genes in the differentiation of mesoderm and endoderm of vertebrates is well established. Recently, homologues of Brachyury have been isolated from an increasing number of diverse organisms ranging from Cnidaria to vertebrates and insects. Comparative expression and function analysis allows the origin of the mesoderm and the evolution of the developmental role of Brachyury gene family in metazoans to be traced. The data suggest that an ancestral function of Brachyury was to designate a blastoporal region that had distinct properties in induction and axis elongation. A subset of blastoporal cells expressing Brachyury and other genes that convey specific mesodermal functions may have segregated as a distinct cell population from this region in the course of mesoderm evolution.

Evolution of the bilaterian larval foregut.

Bilateria are subdivided into Protostomia and Deuterostomia. Indirect development through primary, ciliary larvae occurs in both of these branches; however, the closing blastopore develops into mouth and anus in Protostomia and into anus only in Deuterostomia. Because of this important difference in larval gut ontogeny, the tube-shaped guts in protostome and deuterostome primary larvae are thought to have evolved independently. To test this hypothesis, we have analysed the expression of brachyury, otx and goosecoid homologues in the polychaete Platynereis dumerilii, which develops by means of a trochophora larva-the primary, ciliary larva prototypic for Protostomia. Here we show that brachyury expression in the ventral portion of the developing foregut in Platynereis and also otx expression along ciliated bands in the mouth region of the trochophora larva parallels expression in primary larvae in Deuterostomia. In addition, goosecoid expression in the foregut of Platynereis mirrors the function in higher Deuterostomia. We present molecular evidence for the evolutionary conservation of larval foreguts and mouth regions of Protostomia and Deuterostomia. Our data indicate that Urbilateria, the common bilaterian ancestors, developed through a primary, ciliary larva that already possessed a tripartite tube-shaped gut.

Parameters of self-organization in Hydra aggregates.

Self-organization has been demonstrated in a variety of systems ranging from chemical-molecular to ecosystem levels, and evidence is accumulating that it is also fundamental for animal development. Yet, self-organization can be approached experimentally in only a few animal systems. Cells isolated from the simple metazoan Hydra can aggregate and form a complete animal by self-organization. By using this experimental system, we found that clusters of 5-15 epithelial cells are necessary and sufficient to form de novo head-organizing centers in an aggregate. Such organizers presumably arise by a community effect from a small number of cells that express the conserved HyBra1 and HyWnt genes. These local sources then act to pattern and instruct the surrounding cells as well as generate a field of lateral inhibition that ranges up to 1,000 microm. We propose that conserved patterning systems in higher animals originate from extremely robust and flexible molecular self-organizing systems that were selected for during early metazoan evolution.

Oocyte development in Hydra involves selection from competent precursor cells.

We have investigated oocyte development in Hydra vulgaris, a member of one of the oldest metazoan phyla. We show that oocyte determination involves a mechanism that establishes a subset of precursor interstitial cells competent to differentiate into oocytes. The oocyte is singled out from this subset and the competence of the remaining cells to become oocytes dramatically decreases as they adopt the alternative nurse cell fate. Progression through the nurse cell differentiation program requires the presence of the oocyte. When the oocyte is removed from the egg field, nurse cells abort their differentiation program, undergo apoptosis, and are phagocytosed and degraded by somatic epithelial cells. However, in the presence of the oocyte, nurse cells differentiate and enter an unusual apoptosis program where they are phagocytosed by the oocyte, but are not degraded. We show that the oocyte is able to induce this unusual apoptosis program in immature nurse cells that have not completed differentiation. A new model for oocyte development in Hydra is discussed.

HyBra1, a Brachyury homologue, acts during head formation in Hydra.

A homologue of the T-box gene, Brachyury, has been isolated from hydra. The gene, termed HyBra1, is expressed in the endoderm and is associated with the formation of the hypostome, the apical part of the head in four different developmental situations. In adults, which are continuously undergoing patterning, HyBra1 is continuously expressed in the hypostome. During budding, hydra's asexual form of reproduction, the gene is expressed in a small area that will eventually form the hypostome of the developing bud before any morphological sign of budding is apparent. The gene is also expressed very early during head regeneration and is confined to the region that will form the hypostome. During embryogenesis, HyBra1 is expressed shortly before hatching in the region that will form the apical end of the animal, the hypostome. The absence of expression at the apical end of decapitated animals of reg-16, a head formation-deficient mutant, provides additional evidence for a role of HyBra1 during head formation. Further, treatments that alter the head activation gradient have no effect on HyBra1 expression indicating the role of the gene is confined to head formation. Transplantation experiments indicate that the expression occurs before head determination has occurred, but expression does not irreversibly commit tissue to forming a head. A comparison of the function of the Brachyury homologues suggests an evolutionary conservation of a molecular mechanism that has been co-opted for a number of developmental processes throughout evolution.

Phenotypic maturation of neurons and continuous precursor migration in the formation of the peduncle nerve net in Hydra.

Mechanisms of nerve net formation in Hydra were analyzed using a monoclonal antibody (L96) directed against neurons of the peduncle, the basal end of the polyp's body axis. L96+ neurons express RFamide neuropeptides and constitute 70-80% of all ectodermal neurons in the lower peduncle. L96+ neurons arise from neuronal precursors which immigrate from the gastric region into the upper peduncle and first differentiate into neurons lacking the L96 antigen. By tissue movement, these L96- neurons become displaced to the lower peduncle where L96 antigen expression is initiated. The entire L96 neuron differentiation pathway requires about 4 days, but regeneration stimuli shorten it to only 36 hr. Our experiments indicate that local extrinsic signals released by epithelial cells in the peduncle control the L96+ neuron differentiation pathway. Ectopic L96+ neuron differentiation can be induced by LiCl treatment, which also stimulates ectopic feet in the gastric region. Further experiments show that intrinsic signals are also involved in the L96+ neuron differentiation pathway. Neurons of the gastric region become continuously displaced to the peduncle by tissue movement, but these "old" neurons fail to express the L96 antigen in response to the altered epithelial environment. Gastric neurons also fail to express the L96 antigen after LiCl treatment or regeneration in stem cell-depleted polyps. Thus, the competence of neurons to respond to environmental cues with L96 antigen expression is strongly age-dependent. We define this age-dependent acquisition of the neuronal phenotype as phenotypic maturation controlled by the target tissue.

Cell sorting during the regeneration of Hydra from reaggregated cells.

The role of cell sorting in the reorganization of Hydra cell reaggregates was studied. We quantitatively labeled ectodermal and endodermal cells by incubating whole animals in fluorescent beads or by injecting the beads into the gastric cavity. Beads were stably incorporated into the cells by phagocytosis. Our data show that dramatic cell sorting processes drive the formation of ectoderm and endoderm within the first 12 hr of reaggregation. After the ectoderm is established, no further rearrangement could be observed. We also tested the ability of cells to sort out with respect to their original position in Hydra by dissociating labeled apical and basal pieces of Hydra and measuring the clumping of labeled cells during reorganization. There was no increase in the clumping of cells during reorganization indicating that cell sorting is not involved in the formation of early activation centers. There was also no preferential incorporation of apically derived (presumptive head) tissue into tentacles that subsequently formed, indicating that after dissociation into single cells there is no predisposition of erstwhile presumptive head tissue to form heads.