Sexual and asexual reproduction in the colonial ascidian Botryllus schlosseri.

The colonial tunicate Botryllus schlosseri is a widespread filter-feeding ascidian that lives in shallow waters and is easily reared in aquaria. Its peculiar blastogenetic cycle, characterized by the presence of three blastogenetic generations (filtering adults, buds, and budlets) and by recurrent generation changes, has resulted in over 60 years of studies aimed at understanding how sexual and asexual reproduction are coordinated and regulated in the colony. The possibility of using different methodological approaches, from classical genetics to cell transplantation, contributed to the development of this species as a valuable model organism for the study of a variety of biological processes. Here, we review the main studies detailing rearing, staging methods, reproduction and colony growth of this species, emphasizing the asymmetry in sexual and asexual reproduction potential, sexual reproduction in the field and the laboratory, and self- and cross-fertilization. These data, opportunely matched with recent tanscriptomic and genomic outcomes, can give a valuable help to the elucidation of some important steps in chordate evolution.

Evolutionary diversification of secondary mechanoreceptor cells in tunicata.

BACKGROUND: Hair cells are vertebrate secondary sensory cells located in the ear and in the lateral line organ. Until recently, these cells were considered to be mechanoreceptors exclusively found in vertebrates that evolved within this group. Evidence of secondary mechanoreceptors in some tunicates, the proposed sister group of vertebrates, has recently led to the hypothesis that vertebrate and tunicate secondary sensory cells share a common origin. Secondary sensory cells were described in detail in two tunicate groups, ascidians and thaliaceans, in which they constitute an oral sensory structure called the coronal organ. Among thaliaceans, the organ is absent in salps and it has been hypothesised that this condition is due to a different feeding system adopted by this group of animals. No information is available as to whether a comparable structure exists in the third group of tunicates, the appendicularians, although different sensory structures are known to be present in these animals. RESULTS: We studied the detailed morphology of appendicularian oral mechanoreceptors. Using light and electron microscopy we could demonstrate that the mechanosensory organ called the circumoral ring is composed of secondary sensory cells. We described the ultrastructure of the circumoral organ in two appendicularian species, Oikopleura dioica and Oikopleura albicans, and thus taxonomically completed the data collection of tunicate secondary sensory cells. To understand the evolution of secondary sensory cells in tunicates, we performed a cladistic analysis using morphological data. We constructed a matrix consisting of 19 characters derived from detailed ultrastructural studies in 16 tunicate species and used a cephalochordate and three vertebrate species as outgroups. CONCLUSIONS: Our study clearly shows that the circumoral ring is the appendicularian homologue of the coronal organ of other tunicate taxa. The cladistic analysis enabled us to reconstruct the features of the putative ancestral hair cell in tunicates, represented by a simple monociliated cell. This cell successively differentiated into the current variety of oral mechanoreceptors in the various tunicate lineages. Finally, we demonstrated that the inferred evolutionary changes coincide with major transitions in the feeding strategies in each respective lineage.

Developmental signature, synaptic connectivity and neurotransmission are conserved between vertebrate hair cells and tunicate coronal cells.

In tunicates, the coronal organ represents a sentinel checking particle entrance into the pharynx. The organ differentiates from an anterior embryonic area considered a proto-placode. For their embryonic origin, morphological features and function, coronal sensory cells have been hypothesized to be homologues to vertebrate hair cells. However, vertebrate hair cells derive from a posterior placode. This contradicts one of the principle historical criteria for homology, similarity of position, which could be taken as evidence against coronal cells/hair cells homology. In the tunicates Ciona intestinalis and C. robusta, we found that the coronal organ expresses genes (Atoh, Notch, Delta-like, Hairy-b, and Musashi) characterizing vertebrate neural and hair cell development. Moreover, coronal cells exhibit a complex synaptic connectivity pattern, and express neurotransmitters (Glu, ACh, GABA, 5-HT, and catecholamines), or enzymes for their synthetic machinery, involved in hair cell activity. Lastly, coronal cells express the Trpa gene, which encodes an ion channel expressed in hair cells. These data lead us to hypothesize a model in which competence to make secondary mechanoreceptors was initially broadly distributed through placode territories, but has become confined to different placodes during the evolution of the vertebrate and tunicate lineages.

Thalassemia minor, the Gilbert mutation, and the risk of gallstones.

BACKGROUND AND OBJECTIVES: Gallstones are a frequent complication of hemolytic anemias. The association with the mutation of the A(TA)nTAA motif of the promoter of the bilirubin UDP-glucuronosyltransferase gene has also been reported to increase the risk of gallstones. We studied the prevalence of cholelithiasis in thalassemia minor and the role of the Gilbert mutation. DESIGN AND METHODS: A group of 143 women obligate carriers of beta-thalassemia, and a control group of 170 hematologically normal women were compared. In both groups serum bilirubin, total cholesterol, and alanine-aminotransferase were measured and analysis of the mutation of the UGT-1A gene was performed. On the same occasion the women underwent ultrasonography. RESULTS: Total and unconjugated bilirubin were significantly higher in beta-thalassemia heterozygotes. Carriers of thalassemia had a higher prevalence of gallstones (20.3% vs 10.6% OR=2.15). Among the control group, the prevalence of gallstones did not differ significantly in relation to UGT1-A1 genotype, while in women carriers of beta-thalassemia it increased in an allele dose-dependent fashion. As compared to the controls, the odds ratios for the development of gallstones in thalassemic women were 1.68 (95% C.I.: 0.70-4.03) for those who had the normal UGT1-A1 genotype [(TA)6/(TA)6], 2.31 (95% C.I.: 1.06-5.02) for heterozygote carriers of the mutated genotype [(TA)7/(TA)6] and 3.88 (95% C.I.: 1.31-11.55) for those homozygous for the mutated genotype [(TA)7/(TA)7]. INTERPRETATION AND CONCLUSIONS: Thalassemia minor represents a risk factor for cholelithiasis and the Gilbert mutation further increases this risk. This is an additional example of how two genotypes can interact and modify a phenotype.

Testing an unusual in vivo vessel network model: a method to study angiogenesis in the colonial tunicate Botryllus schlosseri.

Tunicates are the closest relatives to vertebrates and include the only chordate species able to reproduce both sexually and asexually. The colonial tunicate Botryllus schlosseri is embedded in a transparent extracellular matrix (the tunic) containing the colonial circulatory system (CCS). The latter is a network of vessels external to zooids, limited by a simple, flat epithelium that originated from the epidermis. The CCS propagates and regenerates by remodelling and extending the vessel network through the mechanism of sprouting, which typically characterises vertebrate angiogenesis. In exploiting the characteristics of B. schlosseri as a laboratory model, we present a new experimental and analysis method based on the ability to obtain genetically identical subclones representing paired samples for the appropriate quantitative outcome statistical analysis. The method, tested using human VEGF and EGF to induce angiogenesis, shows that the CCS provides a useful in vivo vessel network model for testing the effects of specific injected solutes on vessel dynamics. These results show the potentiality of B. schlosseri CCS as an effective complementary model for in vivo studies on angiogenesis and anticancer therapy. We discuss this potentiality, taking into consideration the origin, nature, and roles of the cellular and molecular agents involved in CCS growth.

The oral sensory structures of Thaliacea (Tunicata) and consideration of the evolution of hair cells in Chordata.

We analyzed the mouth of three species, representative of the three orders of the class Thaliacea (Tunicata)--Pyrosoma atlanticum (Pyrosomatida), Doliolum nationalis (Doliolida), and Thalia democratica (Salpida)--to verify the presence of mechanoreceptors, particularly hair cells. In vertebrates, hair cells are well-known mechanoreceptors of the inner ear and lateral line, typically exhibiting an apical hair bundle composed of a cilium and stereovilli but lacking an axon. For a long time, hair cells were thought to be exclusive to vertebrates. However, evidence of a mechanosensory organ (the coronal organ) employing hair cells in the mouth of tunicates, considered the sister group of vertebrates, suggests that tunicate and vertebrate hair cells may share a common origin. This study on thaliaceans, a tunicate group not yet investigated, shows that both P. atlanticum and D. nationalis possess a coronal organ, in addition to sensory structures containing peripheral neurons (i.e., cupular organs and triads of sensory cells). In contrast, in T. democratica, we did not recognize any oral multicellular sensory organ. We hypothesize that in T. democratica, hair cells were secondarily lost, concomitantly with the loss of branchial fissures, the acquisition of a feeding mechanism based on muscle activity, and a mechanosensory apparatus based on excitable epithelia. Our data are consistent with the hypothesis that hair cells were present in the common ancestor of tunicates and vertebrates, from which hair cells progressively evolved.

Cytodifferentiation of hair cells during the development of a basal chordate.

Tunicates are unique animals for studying the origin and evolution of vertebrates because they are considered vertebrates' closest living relatives and share the vertebrate body plan and many specific features. Both possess neural placodes, transient thickenings of the cranial ectoderm that give rise to various types of sensory cells, including axonless secondary mechanoreceptors. In vertebrates, these are represented by the hair cells of the inner ear and the lateral line, which have an apical apparatus typically bearing cilia and stereovilli. In tunicates, they are found in the coronal organ, which is a mechanoreceptor located at the base of the oral siphon along the border of the velum and tentacles and is formed of cells bearing a row of cilia and short microvilli. The coronal organ represents the best candidate homolog for the vertebrate lateral line. To further understand the evolution of secondary sensory cells, we analysed the development and cytodifferentiation of coronal cells in the tunicate ascidian Ciona intestinalis for the first time. Here, coronal sensory cells can be identified as early as larval metamorphosis, before tentacles form, as cells with short cilia and microvilli. Sensory cells gradually differentiate, acquiring hair cell features with microvilli containing actin and myosin VIIa; in the meantime, the associated supporting cells develop. The coronal organ grows throughout the animal's lifespan, accompanying the growth of the tentacle crown. Anti-phospho Histone H3 immunostaining indicates that both hair cells and supporting cells can proliferate. This finding contributes to the understanding of the evolution of secondary sensory cells, suggesting that both ancestral cell types were able to proliferate and that this property was progressively restricted to supporting cells in vertebrates and definitively lost in mammals.