Sensory Vesicle Research Articles

Genomics and Developmental Approaches to an Ascidian Adenohypophysis Primordium

Ascidians, which are the closest phylogenetic relatives to vertebrates, lack a distinct pituitary gland, which is the major endocrine gland in vertebrates. Nevertheless, for the past 130 years, it has been debated that the ascidian neural complex (NC) is homologous to the pituitary. Of the three major components of the NC, the neural gland (NG) has mainly been thought to be the ascidian counterpart of the pituitary. Recently, however, the ciliated funnel, and not the NG, was postulated to be the adenohypophysis (AH) primordium because it is likely derived from oral ectoderm, and because the expression of several placodal genes is comparable to their expression in vertebrates. An extensive in silico survey of the Ciona intestinalis genome sequence revealed that genes encoding pituitary hormones are absent in ascidians. Under the circumstances, this thesis attempts to find a path that shows that the AH primordium is recognizable in the ascidian by revisiting molecular and developmental data from recent public resources on C. intestinalis, and through the use of advanced bio-imaging techniques. A putative Ciona genetic pathway, which was constructed by referring to data from mammals, shows that only a patchwork of the genetic network exists to achieve terminal differentiation of the AH endocrine cells in the Ciona genome. Re-annotation on glycoprotein hormone related proteins, a GPA2/ARP and two GPB5/BRP ones previously reported, reveals that the GPA2 locus contains two splicing variants, and one variant likely formed a three-dimensional conformation similar to that of human GPA2. No clone of the GPB5/BRP1 locus has been isolated, and another candidate, BRP2, is unlikely to be a GPB5. Next, I argued a possibility that endocrine activities of Ciona species could be specialized in association with its short generation time, and I suggest that not only Ciona species but also other ascidians should be studied in order to understand ascidian endocrinology. Confocal images of the stages of tailbud development reconfirmed the presence of an oral ectoderm placode, and I propose to update the stomodeum development by adding descriptions of a folded structure of the stomodeum and deeply positioned opening of the sensory vesicle. Finally, YFP expression driven by Ci-Six3 promoter demonstrated a boundary between the pharyngeal endoderm and other ectodermal and neuroectodermal tissues around the ciliated funnel. These updates on the ascidian model, which complement other lower chordates and vertebrates, shed light on the evolutionary origin of the pituitary primordium.

Neural Map of the Larval Central Nervous System in the AscidianCiona intestinalis

We examined the distribution patterns and axonal pathways of cholinergic, GABAergic, and dopaminergic neurons in the central nervous system of Ciona intestinalis larvae, based on the expression patterns of two reporter genes (GFP and LacZ) driven by the promoters of several neuron-specific genes (vesicular acetylcholine transporter, glutamic acid decarboxylase, tyrosine 3-hydroxylase and dopa decarboxylase). Putative cholinergic and GABAergic cells were found in the sensory vesicle (SV) and visceral ganglion (VG), while putative dopaminergic cells were found only in the SV. The axons of almost all putative cholinergic and GABAergic cells in the SV extend posteriorly towards the VG and seem to connect with motor neurons. Some cells extend axons to the proximal region of the tail beyond the trunk-tail boundary. As this tail region contains several neurons, these cells may modulate larval behavior through the latter neurons. We also found that some putative cholinergic and GABAergic cells in the dorsal VG form a complex and extend axons anteriorly to the SV, posteriorly to the tail, and possibly ventrally to some motor neurons. Finally, we observed that one pair of the anterior most putative cholinergic cells in the ventral VG extends axons contralaterally to the right and left caudal axon tracts. We discuss the similarity of these cells to the Mauthner cells in vertebrates.

Spatial and temporal expression of two transcriptional isoforms of Lhx3, a LIM class homeobox gene, during embryogenesis of two phylogenetically remote ascidians, Halocynthia roretzi and Ciona intestinalis

Lhx3 genes are members of one of the six subfamilies of the LIM class homeobox genes. In ascidians, Lhx3 is known to play a critical role in endoderm differentiation, while in vertebrates Lhx3 is involved in the development of pituitary and subsets of motor neurons. It has been shown recently, using RT-PCR analysis, that two transcriptional isoforms a and b are differentially expressed during the larval development of Ciona intestinalis (Christiaen et al., 2009). The present study provides an in-depth description of Lhx3 gene expression during the development of the two remote ascidian species, C. intestinalis and Halocynthia roretzi; for this, 5'RACE and whole-mount in situ hybridization (WISH) were employed. In both species, maternal expression of Lhx3a, but not Lhx3b, is evident. In H. roretzi, the maternal Lhx3a transcripts have been detected by WISH in the animal half of early cleavage stage embryos. In both species, transcriptional isoform a is also zygotically expressed in the sensory vesicle and the visceral ganglion lineages from the neurula stage onward. By contrast, Lhx3b transcripts are expressed only zygotically and localized in the endoderm, notochord and mesenchyme lineages during cleavage stage. Lhx3a, but not Lhx3b, transcripts are subjected to trans-splicing. Additionally, in C. intestinalis, other variations in the 5' region have been identified among Lhx3a transcripts. Although some differences are present, over-all developmental expression of Lhx3 is rather well conserved between the two ascidian species, which is quite different from that of vertebrate counterparts.

Distribution and structural diversity of cilia in tadpole larvae of the ascidian Ciona intestinalis

Accumulating evidence demonstrates that cilia play important roles in a variety of processes in embryogenesis. For functional survey of larval cilia at the cellular level, we exploited the simple cell organization of tadpole larvae in the ascidian Ciona intestinalis. Immunofluorescent microscopy showed distribution of cilia not only in previously described tissues but also in a subpopulation of ependymal cells in the sensory vesicle, gut primordium, papillae, apical trunk epidermal neurons, and the endodermal strand. Transmission electron microscopy revealed a variety of axonemal structures, including a 9+0 structure similar to vertebrate primary cilia, a 9+0 structure with electron-dense materials in the center, a 9+2 structure with no dynein arms, and an axoneme with a disorganized structure at the distal end. Extensive description of cilia in the present study gives important insights into the evolution of the ciliary structure and provides a basis for analysis of ciliary functions in establishment of chordate body plan.

Gene regulatory networks underlying the compartmentalization of theCionacentral nervous system

The tripartite organization of the central nervous system (CNS) may be an ancient character of the bilaterians. However, the elaboration of the more complex vertebrate brain depends on the midbrain-hindbrain boundary (MHB) organizer, which is absent in invertebrates such as Drosophila. The Fgf8 signaling molecule expressed in the MHB organizer plays a key role in delineating separate mesencephalon and metencephalon compartments in the vertebrate CNS. Here, we present evidence that an Fgf8 ortholog establishes sequential patterns of regulatory gene expression in the developing posterior sensory vesicle, and the interleaved ;neck' region located between the sensory vesicle and visceral ganglion of the simple chordate Ciona intestinalis. The detailed characterization of gene networks in the developing CNS led to new insights into the mechanisms by which Fgf8/17/18 patterns the chordate brain. The precise positioning of this Fgf signaling activity depends on an unusual AND/OR network motif that regulates Snail, which encodes a threshold repressor of Fgf8 expression. Nodal is sufficient to activate low levels of the Snail repressor within the neural plate, while the combination of Nodal and Neurogenin produces high levels of Snail in neighboring domains of the CNS. The loss of Fgf8 patterning activity results in the transformation of hindbrain structures into an expanded mesencephalon in both ascidians and vertebrates, suggesting that the primitive MHB-like activity predates the vertebrate CNS.

Effects of the azole fungicide Imazalil on the development of the ascidian Ciona intestinalis (Chordata, Tunicata): Morphological and molecular characterization of the induced phenotype

Imazalil (IMA) is a fungicide that is used extensively in fruit plantations and post-harvest treatments, but has teratogenic effects on vertebrate development, possibly due to the perturbation of retinoic acid (RA) levels in the embryo. Ascidians are sessile marine invertebrate chordates that develop through a tadpole larva, with a body plan that shares basic homologies with vertebrates. In this work, we tested the effects of IMA on the development of the solitary ascidian Ciona intestinalis by treating two-cell stage embryos with a range of concentrations (0.1, 0.5, 1, 2.5, 5, 10, 20 and 50 μM). The fungicide significantly altered ascidian development even at low concentrations and its effects were dose-dependent. Probit analysis revealed that the median lethal concentration, LC 50, was 4.87 μM and the median teratogenic concentration, TC 50, was 0.73 μM. Larvae developing from embryos exposed to IMA showed malformations of the anterior structures, which became more severe as IMA concentration increased. In particular, the anterior nervous system and the sensory vesicle were reduced, and the pigmented organs (the ocellus and the otolith) progressively lost their pigmentation. The larval phenotype induced by 5 μM IMA exposure was further characterized by means of molecular analysis, through whole mount in situ hybridization with probes for genes related to the nervous system: Ci-Otp, Ci-GAD, Ci-POU IV, which are markers of the anterior neuro-ectoderm, the central nervous system and the peripheral nervous system respectively, and Ci-Hox-1, a gene specifically activated by RA, and Ci-Aldh2, a gene for aldehyde dehydrogenase, which is involved in RA synthesis. The altered expression of Ci-Otp, Ci-GAD, Ci-POU IV in 5 μM IMA-exposed larvae compared to control larvae showed that this fungicide could affect the differentiation of the anterior nervous system, particularly of the sensory vesicle neurons. Recent studies suggest a similarity between IMA- and RA-induced phenotypes in tunicates, indicating that triazoles may also alter RA metabolism in ascidians. The observed Ci-Hox-1 and Ci-Aldh2 expression in control and treated larvae did not allow a direct link between IMA teratogenic potential and RA-dependent morphogenesis to be identified. It is likely that the fungicidal teratogenic mechanism involved RA signalling but that its effects on ascidian development depend on a more complex mechanism.

Trunk lateral cells are neural crest-like cells in the ascidian Ciona intestinalis: Insights into the ancestry and evolution of the neural crest

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.

Cis-regulatory organization of the Pax6 gene in the ascidian Ciona intestinalis

The Pax6 gene has attracted intense research interest due to its apparently important role in the development of eyes and the central nervous system (CNS) in many animal groups. Pax6 is also of interest for comparative genomics since it has not been duplicated in tetrapods, making for a direct orthology between the Ciona intestinalis gene CiPax6 and Pax6 in mammals. CiPax6 has been shown to be expressed in the anterior brain, caudal nerve cord, and in parts of the brain associated with the photoreceptive ocellus. This information was extended here using in-situ hybridization, and shows that CiPax6 transcripts mark the lateral regions of the nerve cord, remarkably similar to Pax6 expression in the mouse. As a means of dissecting the cis-regulation of CiPax6 we tested 8 kb of sequence using transient reporter transgene assays. Three separate regions were found that work together to drive the overall CiPax6 expression pattern. A 211 bp sequence 2 kb upstream of the first exon was found to be a major enhancer driving expression in the sensory vesicle (the anterior portion of the ascidian brain). Other upstream sequences were shown to work with the sensory vesicle enhancer to drive expression in the remainder of the CNS. An “eye enhancer” was localized to the first intron, which controls specific expression in the central portion of the sensory vesicle, including photoreceptor cells. The fourth intron was found to repress ectopic expression of the reporter gene in middle portions of the embryonic brain. Aspects of this overall regulatory organization are similar to the organization of the Pax6 homologs in mice and Drosophila, particularly the presence of intronic elements driving expression in the eye, brain and nerve cord.

Developmental expression of glutamic acid decarboxylase and of γ‐aminobutyric acid type B receptors in the ascidian Ciona intestinalis

We describe Ciona intestinalis gamma-aminobutyric acid (GABA)-ergic neurons during development, studying the expression pattern of Ci-GAD (glutamic acid decarboxylase: GABA synthesizing enzyme) by in situ hybridization. Moreover, we cloned two GABA(B) receptor subunits (Ci-GABA(B)Rs), and a phylogenetic analysis (neighbor-joining method) suggested that they clustered with their vertebrate counterparts. We compared Ci-GAD and Ci-GABA(B)Rs expression patterns in C. intestinalis embryos and larvae. At the tailbud stage, Ci-GAD expression was widely detected in central and peripheral nervous system (CNS/PNS) precursors, whereas Ci-GABA(B)Rs expression was evident at the level of the precursors of the visceral ganglion. GABA was localized by immunohistochemistry at the same developmental stage. In the larva, Ci-GAD transcripts and GABA immunofluorescence were also detected throughout the CNS and in some neurons of the PNS, whereas transcripts of both GABA(B) receptor subunits were found mainly in the CNS. The expression pattern of Ci-GABA(B)Rs appeared restricted to Ci-GAD-positive territories in the sensory vesicle, whereas, in the visceral ganglion, Ci-GABA(B)Rs transcripts were found in ventral motoneurons that did not express Ci-GAD. Insofar as GABAergic neurons are widely distributed also in the CNS and PNS of vertebrates and other invertebrate chordates, it seems likely that GABA signaling was extensively present in the protochordate nervous system. Results from this work show that GABA is the most widespread inhibitory neurotransmitter in C. intestinalis nervous system and that it can signal through GABA(B) receptors both pre- and postsynaptically to modulate different sensory inputs and subsequent swimming activity.

Neurons of the ascidian larval nervous system in Ciona intestinalis: I. Central nervous system

The tadpole larva of ascidians, basal living relatives of vertebrates, has a chordate body plan. The CNS has many homologies with that of vertebrates yet only about 100 neurons. These few, possibly fixed in number and composition, nevertheless govern a diverse repertoire of behaviors. To elucidate the circuits of the CNS first requires that we recognize each neuron type, for which we used electroporation to transfect precleavage embryos with a plasmid containing green fluorescent protein (GFP) driven by the promoter of the synaptotagmin gene. Hatched larvae were fixed and GFP 3-D reconstructions of confocal image stacks compiled into images of 31 whole or partial larvae, either with many GFP-labelled neurons or with few, each clearly visible. Neuron counts in the sensory vesicle (SV) and visceral ganglion (VG) indicated that between 75% (SV) and 69% (VG) of previously reported numbers of neurons were transfected. Based on their position, shape, and projections, the following neurons were identified in the SV: a prominent eminens neuron, possibly with direct input from papillar neurons, a large ventroposterior interneuron, photoreceptors of the ocellus, and putative antenna cells of the otolith. In the VG, we identified at least four subtypes of motor neuron, including an ovoid cell that may innervate distal tail muscle cells and contrapelo cells with ascending projections, unique among VG neurons. The caudal nerve cord contained the first reported neurons, the somata of planate neurons. These neurons are the first identified types, and will be used to construct a map of the nervous system for this model basal chordate.

Neurons of the ascidian larval nervous system in Ciona intestinalis: II. Peripheral nervous system

The peripheral nervous system of the ascidian tadpole larva comprises a distributed population of isolated receptor neurons, most of unproved function, organized along the trunk or tail epithelium. Previous reports using immunocytochemical methods failed to resolve the detailed morphology of the neurons and their axon pathways. Precleavage embryos of Ciona intestinalis transfected with the promoter of the neuron-specific synaptotagmin gene fused to a green fluorescent protein (GFP) gene yielded clearly labelled GFP profiles. These we examined in confocal image stacks of 31 larvae. Anchor cells, at least eight in each adhesive apical papilla, contribute axons to the papillar nerves that terminate in the sensory vesicle of the central nervous system. Two nerve bundles projected from each papilla, suggesting that at least two subpopulations of papillar neurons exist. Each bundle fasciculated with axons of the rostral trunk epidermal neurons (RTEN) in a stereotyped pattern. The RTEN had a hitherto unreported elaborate arbor of sensory dendrites within the tunic, suggesting that each has an extended sensorial field. Two subpopulations of apical trunk epidermal neurons (ATEN), anterior and posterior, were distinguished. As with the RTEN, these neurons extended dendritic arbors into the tunic. Two additional types of tail neuron, the caudal epidermal neurons (dorsal and ventral) as well as a novel bipolar interneuron, were identified. These identified neuron types are the substrate for the ascidian larva's entire peripheral sensory input, important during larval swimming and settlement.

Fluconazole induces teratogenic effects in the tunicate Phallusia mammillata

Fluconazole (FLUCO) is an azole derivative used to treat fungal and yeast infections. Embryotoxicity tests on the ascidian Phallusia mammillata were performed to evaluate the effects of this drug. FLUCO proved to have strong consequences on P. mammillata development. Incidence of malformations and of lethality increased in a dose dependent way. Probit analysis showed that FLUCO had a high TI value (Teratogenic Index, LC 50/TC 50), thus this substance could be classified as a teratogenic compound for ascidians. Larvae exposed to FLUCO showed a typical phenotype characterized by malformations restricted to the trunk region: the trunk appeared round in shape with flat palps, the sensory vesicle cavity was absent or reduced and the anterior central nervous system failed to correctly differentiate. These anomalies resulted similar to those induced by retinoic acid (RA) treatment. Thus, it could be hypothesized that FLUCO and RA may act with a similar pathogenic mechanism in ascidian larvae, as it has been proposed for mammals.

Precraniate origin of cranial motoneurons.

The craniate head is innervated by cranial sensory and motor neurons. Cranial sensory neurons stem from the neurogenic placodes and neural crest and are seen as evolutionary innovations crucial in fulfilling the feeding and respiratory needs of the craniate "new head." In contrast, cranial motoneurons that are located in the hindbrain and motorize the head have an unclear phylogenetic status. Here we show that these motoneurons are in fact homologous to the motoneurons of the sessile postmetamorphic form of ascidians. The motoneurons of adult Ciona intestinalis, located in the cerebral ganglion and innervating muscles associated with the huge "branchial basket," express the transcription factors CiPhox2 and CiTbx20, whose vertebrate orthologues collectively define cranial motoneurons of the branchiovisceral class. Moreover, Ciona's postmetamorphic motoneurons arise from a hindbrain set aside during larval life and defined as such by its position (caudal to the prosensephalic sensory vesicle) and coexpression of CiPhox2 and CiHox1, whose orthologues collectively mark the vertebrate hindbrain. These data unveil that the postmetamorphic ascidian brain, assumed to be a derived feature, in fact corresponds to the vertebrate hindbrain and push back the evolutionary origin of cranial nerves to before the origin of craniates.

The ascidian homolog of the vertebrate homeobox gene Rx is essential for ocellus development and function

The tadpole larvae prosencephalon of the ascidian Ciona intestinalis contains a single large ventricle, along the inner walls of which lie two sensory organs: the otolith (a gravity-sensing organ) and the ocellus (a photo-sensing organ composed of a single cup-shaped pigment cell, about 20 photoreceptor cells, and three lens cells). Comparison has been drawn between the morphology and physiology of photoreceptor cells in the ascidian ocellus and the vertebrate eye. The development of vertebrate and invertebrate eyes requires the activity of several conserved genes and it is regulated by precise expression patterns and cell fate decisions common to several species. We have isolated a Ciona homeobox gene ( Ci-Rx) that belongs to the paired-like class of homeobox genes. Rx genes have been identified from a variety of organisms and have been demonstrated to have a role in vertebrate eye formation. Ci-Rx is expressed in the anterior neural plate in the middle tailbud stage and subsequently in the larval stage in the sensory vesicle around the ocellus. Loss of Ci-Rx function leads to an ocellus-less phenotype that shows a loss of photosensitive swimming behavior, suggesting the important role played by Ci-Rx in basal chordate photoreceptor cell differentiation and ocellus formation. Furthermore, studies on Ci-Rx regulatory elements electroporated into Ciona embryos using LacZ or GFP as reporter genes indicate the presence of Ci-Rx in pigment cells, photoreceptors, and neurons surrounding the sensory vesicle. In Ci-Rx knocked-down larvae, neither basal swimming activity nor shadow responses develop. Thus, Rx has a role not only in pigment cells and photoreceptor formation but also in the correct development of the neuronal circuit that controls larval photosensitivity and swimming behavior. The results suggest that a Ci-Rx “retinal” territory exists, which consists of pigment cells, photoreceptors, and neurons involved in transducing the photoreceptor signals.

Prospective protochordate homologs of vertebrate midbrain and MHB, with some thoughts on MHB origins

The MHB (midbrain-hindbrain boundary) is a key organizing center in the vertebrate brain characterized by highly conserved patterns of gene expression. The evidence for an MHB homolog in protochordates is equivocal, the "neck" region immediately caudal to the sensory vesicle in ascidian larvae being the best accepted candidate. It is argued here that similarities in expression patterns between the MHB and the ascidian neck region are more likely due to the latter being the principal source of neurons in the adult brain, and hence where all the genes involved in patterning the latter will necessarily be expressed. The contrast with amphioxus is exemplified by pax2/5/8, expressed in the neck region in ascidian larvae, but more caudally, along much of the nerve cord in amphioxus. The zone of expression in each case corresponds with that part of the nerve cord ultimately responsible for innervating the adult body, which suggests the spatially restricted MHB-like expression pattern in ascidians is secondarily reduced from a condition more like that in amphioxus. Patterns resembling those of the vertebrate MHB are nevertheless found elsewhere among metazoans. This suggests that, irrespective of its modern function, the MHB marks the site of an organizing center of considerable antiquity. Any explanation for how such a center became incorporated into the chordate brain must take account of the dorsoventral inversion chordates have experienced relative to other metazoans. Especially relevant here is a concept developed by Claus Nielsen, in which the brain is derived from a neural center located behind the ancestral mouth. While this is somewhat counterintuitive, it accords well with emerging molecular data.

GABAergic synaptic transmission modulates swimming in the ascidian larva

To examine the role of the amino acid GABA in the locomotion of basal chordates, we investigated the pharmacology of swimming and the morphology of GABA-immunopositive neurones in tadpole larvae of the ascidians Ciona intestinalis and Ciona savignyi. We verified that electrical recording from the tail reflects alternating muscle activity during swimming by correlating electrical signals with tail beats using high-speed video recording. GABA reversibly reduced swimming periods to single tail twitches, while picrotoxin increased the frequency and duration of electrical activity associated with spontaneous swimming periods. Immunocytochemistry for GABA revealed extensive labelling throughout the larval central nervous system. Two strongly labelled regions on either side of the sensory vesicle were connected by an arc of labelled fibres, from which fibre tracts extended caudally into the visceral ganglion. Fibre tracts extended ventrally from a third, more medial region in the posterior sensory vesicle. Two rows of immunoreactive cell bodies in the visceral ganglion extended neurites into the nerve cord, where varicosities were seen. Thus, presumed GABAergic neurones form a network that could release GABA during swimming that is involved in modulating the time course and frequency of periods of spontaneous swimming. GABAergic and motor neurones in the visceral ganglion could interact at the level of their cell bodies and/or through the presumed GABAergic fibres that enter the nerve cord. The larval swimming network appears to possess some of the properties of spinal networks in vertebrates, while at the same time possibly showing a type of peripheral innervation resembling that in some protostomes.

Expression of a novel Ski-like gene in Xenopus development

Members of the Ski/Sno family of gene products contain a characteristic peptide domain involved in protein–protein or protein–DNA interaction. Here, we characterize the developmental expression of xDawg, in Xenopus laevis, of a new gene, related to the Ski/Sno family of transcription regulators. The Ski/Sno domain of xDawg is predicted to present an electropositive surface, consistent with a role in DNA binding. This gene is expressed in the marginal zone of early gastrulae, and in the brain, sensory vesicles, and cranial neural crest of neurula and tailbud embryos.

Urochordate βγ-Crystallin and the Evolutionary Origin of the Vertebrate Eye Lens

A refracting lens is a key component of our image-forming camera eye; however, its evolutionary origin is unknown because precursor structures appear absent in nonvertebrates. The vertebrate betagamma-crystallin genes encode abundant structural proteins critical for the function of the lens. We show that the urochordate Ciona intestinalis, which split from the vertebrate lineage before the evolution of the lens, has a single gene coding for a single domain monomeric betagamma-crystallin. The crystal structure of Ciona betagamma-crystallin is very similar to that of a vertebrate betagamma-crystallin domain, except for paired, occupied calcium binding sites. The Ciona betagamma-crystallin is only expressed in the palps and in the otolith, the pigmented sister cell of the light-sensing ocellus. The Ciona betagamma-crystallin promoter region targeted expression to the visual system, including lens, in transgenic Xenopus tadpoles. We conclude that the vertebrate betagamma-crystallins evolved from a single domain protein already expressed in the neuroectoderm of the prevertebrate ancestor. The conservation of the regulatory hierarchy controlling betagamma-crystallin expression between organisms with and without a lens shows that the evolutionary origin of the lens was based on co-option of pre-existing regulatory circuits controlling the expression of a key structural gene in a primitive light-sensing system.

Structure of ocellus photoreceptors in the ascidian Ciona intestinalis larva as revealed by an anti‐arrestin antibody

Although there have been several studies on the structure of the ocellus photoreceptors in ascidian tadpole larvae using electron microscopy, the overall structure of these photoreceptor cells, especially the projection sites of the axons, has not been revealed completely. The number of photoreceptor cells is also controversial. Here, the whole structure of the ocellus photoreceptors in the larvae of the ascidian Ciona intestinalis was revealed by using an anti-arrestin (anti-Ci-Arr) antibody. The cell bodies of 30 photoreceptor cells covered the right side of the ocellus pigment cell and their outer segments extended through the pigment cell into the pigment cup. The axons of the photoreceptor cells were bundled together ventro-posteriorly in a single tract extending towards the midline. The nerve terminals diverged antero-posteriorly at the midline of the posterior sensory vesicle (SV). The Ci-arr gene was expressed throughout the SV at the embryonic mid-tailbud stage and it became restricted to the neighborhood of the ocellus pigment when ocellus pigmentation occurred. At the same time, the Ci-Arr protein was first detected, suggesting that the photoreceptor cells began to differentiate. The development of photoreceptor cells after hatching was also investigated using the anti-Ci-Arr antibody. Three hours after hatching, the photoreceptor terminals began to ramify and then expanded. Previous behavioral analysis showed that the larvae did not respond to the step-down of light until 2 h after hatching and then the photoresponse became robust. Accordingly, our results suggest that growth of the photoreceptor terminal is critical for the larvae to become photoresponsive.

The dopamine‐synthesizing cells in the swimming larva of the tunicate Ciona intestinalis are located only in the hypothalamus‐related domain of the sensory vesicle

Dopamine is a major neuromodulator synthesized by numerous cell populations in the vertebrate forebrain and midbrain. Owing to the simple organization of its larval nervous system, ascidian tunicates provide a useful model to investigate the anatomy, neurogenesis and differentiation of the dopaminergic neural network underlying the stereotypical swimming behaviour of its chordate-type larva. This study provides a high-resolution cellular analysis of tyrosine hydroxylase (TH)-positive and dopamine-positive cells in Ciona intestinalis embryos and larvae. Dopamine cells are present only in the sensory vesicle of the Ciona larval brain, which may be an ancestral chordate feature. The dopamine-positive cells of the ascidian sensory vesicle are located in the expression domain of homologues of vertebrate hypothalamic markers. We show here that the larval coronet cells also arise from this domain. As a similar association between coronet cells and the hypothalamus was reported in bony and cartilaginous fishes, we propose that part of the ascidian ventral sensory vesicle is the remnant of a proto-hypothalamus that may have been present in the chordate ancestor. As dopaminergic cells are specified in the hypothalamus in all vertebrates, we suggest that the mechanisms of dopamine cell specification are conserved in the hypothalamus of Ciona and vertebrates. To test this hypothesis, we have identified new candidate regulators of dopaminergic specification in Ciona based on their expression patterns, which can now be compared with those in vertebrates.