Metabolic co-dependence drives the evolutionarily ancient Hydra-Chlorella symbiosis.

Many multicellular organisms rely on symbiotic associations for support of metabolic activity, protection, or energy. Understanding the mechanisms involved in controlling such interactions remains a major challenge. In an unbiased approach we identified key players that control the symbiosis between Hydra viridissima and its photosynthetic symbiont Chlorella sp. A99. We discovered significant up-regulation of Hydra genes encoding a phosphate transporter and glutamine synthetase suggesting regulated nutrition supply between host and symbionts. Interestingly, supplementing the medium with glutamine temporarily supports in vitro growth of the otherwise obligate symbiotic Chlorella, indicating loss of autonomy and dependence on the host. Genome sequencing of Chlorella sp. A99 revealed a large number of amino acid transporters and a degenerated nitrate assimilation pathway, presumably as consequence of the adaptation to the host environment. Our observations portray ancient symbiotic interactions as a codependent partnership in which exchange of nutrients appears to be the primary driving force.

Blood system formation in the urochordate Ciona intestinalis requires the variable receptor vCRL1.

Adaptive immune systems are present only in vertebrates. How do all the remaining animals withstand continuous attacks of permanently evolving pathogens? Even in the absence of adaptive immunity, every organism must be able to unambiguously distinguish "self" cells from any imaginable "nonself." Here, we analyzed the function of highly polymorphic gene vCRL1, which is expressed in follicle and blood cells of Ciona intestinalis, pointing to possible recognition roles either during fertilization or in immune reactions. By using segregation analysis, we demonstrate that vCRL1 locus is not involved in the control of self-sterility. Interestingly, genetic knockdown of vCRL1 in all tissues or specifically in hemocytes results in a drastic developmental arrest during metamorphosis exactly when blood system formation in Ciona normally occurs. Our data demonstrate that vCRL1 gene might be essential for the establishment of a functional blood system in Ciona. Presumably, presence of the vCRL1 receptor on the surface of blood cells renders them as self, whereas any cell lacking it is referred to as nonself and will be consequently destroyed. We propose that individual-specific receptor vCRL1 might be utilized to facilitate somatic self/nonself discrimination.

Asexual propagation and regeneration in colonial ascidians.

Regeneration is widely distributed among the metazoans. However, clear differences exist as to the degree of regenerative capacity: some phyla can only replace missing body parts, whereas others can generate entirely new individuals. Ascidians are animals that possess a remarkable regenerative plasticity and exhibit a great diversity of mechanisms for asexual propagation and survival. They are marine invertebrate members of the subphylum Tunicata and represent modern-day descendants of the chordate ancestor; in their tadpole stage they exhibit a chordate body plan that is resorbed during metamorphosis. Solitary species grow into an adult that can reach several centimeters in length, whereas colonial species grow by asexual propagation, creating a colony of genetically identical individuals. In this review, we present an overview of the biology of colonial ascidians as a paradigm for study in stem cell and regenerative biology. Focusing on botryllid ascidians, we introduce the potential roles played by multipotent epithelia and multipotent/pluripotent stem cells as source of asexual propagation and regenerative plasticity in the different budding mechanisms, and consider the putative mechanism of body repatterning in a non-embryonic scenario. We also discuss the involvement of intra-colony homeostatic processes in regulating budding potential, and the functional link between allorecognition, chimerism, and regenerative potential.

In the urochordate Ciona intestinalis zona pellucida domain proteins vary among individuals.

Fertilization in hermaphroditic urochordates requires efficient mechanisms to prevent self-mating. By comparing gonadal transcripts of three genetically unrelated Ciona intestinalis individuals by suppression subtractive hybridization (SSH), we have previously identified a highly polymorphic gene coding for a transmembrane receptor which is expressed in follicle cells surrounding the mature oocyte [Kürn U, Sommer F, Hemmrich G, Bosch TCG, Khalturin K. Allorecogmition in urochordates: identification of a highly variable complement receptor-like protein expressed in follicle cells of Ciona. Dev Comp Immunol 2007; 31(4): 360-71]. Here, we show that in addition to follicle cells, oocytes also express variable genes. We have identified four novel Ciona genes coding for zona pellucida (ZP) domain proteins which are expressed in developing oocytes and which significantly vary among individuals. ZP containing proteins are thought to play a crucial and conserved role in controlling fertilization. Our results provide strong evidence that in Ciona components of both follicle cells and oocytes show a high degree of interindividual variability and, therefore, may take part in the prevention of self-mating as well as in the maintenance of species-specific fertilization.

Allorecognition in urochordates: identification of a highly variable complement receptor-like protein expressed in follicle cells of Ciona.

The evolutionary origin of allorecognition in vertebrates is unknown. Urochordates, being the closest living relatives of vertebrates [Delsuc F, Brinkmann H, Chourrout D, Philippe H]. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 2006; 439: 965-8], have efficient mechanisms to prevent both allogeneic fusion and self fertilization. To shed light on allorecognition in urochordates and on the molecules involved in preventing self fertilization, we compared gonadal cDNAs of three genetically unrelated Ciona intestinalis individuals by suppression subtractive hybridisation (SSH). Here, we report the discovery and characterization of a highly polymorphic gene coding for a transmembrane protein with several short consensus repeat domains (SCR/CCP). The protein, termed variable complement receptor-like 1 (vCRL1), is structurally similar to vertebrate complement receptors. However, in contrast to vertebrate complement receptors, vCRL1 shows an unprecedented high degree of amino acid variations among Ciona individuals and is expressed in follicle cells as well as in hemocytes. Based on our data we propose that in the absence of MHC Ciona uses variable components of the complement system as individuality markers.

Discovery of genes expressed in Hydra embryogenesis.

Hydra's remarkable capacity to regenerate, to proliferate asexually by budding, and to form a pattern de novo from aggregates allows studying complex cellular and molecular processes typical for embryonic development. The underlying assumption is that patterning in adult hydra tissue relies on factors and genes which are active also during early embryogenesis. Previously, we reported that in Hydra the timing of expression of conserved regulatory genes, known to be involved in adult patterning, differs greatly in adults and embryos (Fröbius, A.C., Genikhovich, G., Kürn, U., Anton-Erxleben, F. and Bosch, T.C.G., 2003. Expression of developmental genes during early embryogenesis of Hydra. Dev. Genes Evol. 213, 445-455). Here, we describe an unbiased screening strategy to identify genes that are relevant to Hydra vulgaris embryogenesis. The approach yielded two sets of differentially expressed genes: one set was expressed exclusively or nearly exclusively in the embryos, while the second set was upregulated in embryos in comparison to adult polyps. Many of the genes identified in hydra embryos had no matches in the database. Among the conserved genes upregulated in embryos is the Hydra orthologue of Embryonic Ectoderm Development (HyEED). The expression pattern of HyEED in developing embryos suggests that interstitial stem cells in Hydra originate in the endoderm. Importantly, the observations uncover previously unknown differences in genes expressed by embryos and polyps and indicate that not only the timing of expression of developmental genes but also the genetic context is different in Hydra embryos compared to adults.

Towards a molecular code for individuality in the absence of MHC: screening for individually variable genes in the urochordate Ciona intestinalis.

Urochordates possess several well described allorecognitions systems, the molecular nature of those is not yet understood. A prerequisite for any self-/nonself discimination system is the presence of a group of highly variable molecules, which should vary between individuals. Using suppression subtractive hybridisation (SSH) we surveyed Ciona intestinalis for individually variable genes. Our search so far identified two genes, ciS7 and ciMETA2, which both display an unexpected high degree of intra- and interindividual variability, code for secreted proteins, and contain multiple domains suitable for protein-protein interactions. The possible role of these molecules in allorecognition is discussed.

Expression of developmental genes during early embryogenesis of Hydra.

Hydra is a classical model to study key features of embryogenesis such as axial patterning and stem cell differentiation. In contrast to other organisms where these mechanisms are active only during embryonic development, in Hydra they can be studied in adults. The underlying assumption is that the machinery governing adult patterning mimics regulatory mechanisms which are also active during early embryogenesis. Whether, however, Hydra embryogenesis is governed by the same mechanisms which are controlling adult patterning, remains to be shown. In this paper, in precisely staged Hydra embryos, we examined the expression pattern of 15 regulatory genes shown previously to play a role in adult patterning and cell differentiation. RT-PCR revealed that most of the genes examined were expressed in rather late embryonic stages. In situ hybridization, nuclear run-on experiments, and staining of nucleolar organizer region-associated proteins indicated that genes expressed in early embryos are transcribed in the engulfed "nurse cells" (endocytes). This is the first direct evidence that endocytes in Hydra not only provide nutrients to the developing oocyte but also produce maternal factors critical for embryogenesis. Our findings are an initial step towards understanding the molecular machinery controlling embryogenesis of a key group of basal metazoans and raise the possibility that in Hydra there are differences in the mechanisms controlling embryogenesis and adult patterning.