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We review the anatomical and functional features of circumventricular organs in vertebrates and their homologous neurohemal organs in invertebrates. Focusing on cyclostomes (lamprey) and urochordates (ascidians), we discuss the evolutionary origin of these organs as a function of their cell type specification and morphogenesis.

Chantier qualité GA; International audience; Every model species requires its own developmental table. Astyanax mexicanus, a teleost fish comprising both sighted river and blind cave populations, is becoming more and more important in the field of developmental and evolutionary biology. As such, a developmental staging table is increasingly necessary, particularly since comparative analysis of early developmental events is widely employed by researchers. We collected freshly spawned embryos from surface fish and Pachón cavefish populations. Embryos were imaged every 10-12 min during the first day of development, and less frequently in the following days. The results provide an illustrated comparison of selected developmental stages from one cell to hatching of these two populations. The two morphs show an essentially synchronous development regarding major events such as epiboly, neurulation, somitogenesis, heart beating, or hatching. We also present data on particular morphological characters appearing during larval development, such as eye size, yolk regression, swim bladder, and fin development. Some details about the development of F1 Pachón cave×surface hybrids are also given. Comparisons are made with Danio rerio (zebrafish) development.

International audience; The highly conserved POU genes encode homeodomain transcription factors involved in various developmental events, with some, the Brn genes, playing key roles in neurogenesis. We investigated the evolutionary relationships between these genes, by studying the POU gene complement of a model teleost, the medaka (Oryzias latipes). We identified 17 POU genes and carried out a comprehensive in situ hybridization analysis focusing on the optic tectum, a cortical structure of the mesencephalon, in which cell positions and their differentiation states are spatially and temporally correlated. Six POU genes displayed patterned expression in the optic tectum: two genes were expressed in the center of the organ (a zone with differentiated neurons), two in an intermediate zone in which cells exit the cell cycle and two in the peripheral proliferation zone. These results suggest that POU genes may play key roles in both late neurogenesis and in multipotent neural progenitors.

International audience; A major prerequisite for the investigation of tissue-specific processes is the identification of cis-regulatory elements. No generally applicable technique is available to distinguish them from any other type of genomic non-coding sequence. Therefore, researchers often have to identify these elements by elaborate in vivo screens, testing individual regions until the right one is found. Here, based on many examples from the literature, we summarize how functional enhancers have been isolated from other elements in the genome and how they have been characterized in transgenic animals. Covering computational and experimental studies, we provide an overview of the global properties of cis-regulatory elements, like their specific interactions with promoters and target gene distances. We describe conserved non-coding elements (CNEs) and their internal structure, nucleotide composition, binding site clustering and overlap, with a special focus on developmental enhancers. Conflicting data and unresolved questions on the nature of these elements are highlighted. Our comprehensive overview of the experimental shortcuts that have been found in the different model organism communities and the new field of high-throughput assays should help during the preparation phase of a screen for enhancers. The review is accompanied by a list of general guidelines for such a project.

International audience; All subdivisions of the adult zebrafish brain maintain niches of constitutive neurogenesis, sustained by quiescent and multipotent progenitor populations. In the telencephalon, the latter potential neural stem cells take the shape of radial glia aligned along the ventricle, and are controlled by Notch signalling. With the aim of identifying new markers of this cell type, and of comparing the effectors of embryonic and adult neurogenesis, we focused on the family of hairy/enhancer of split E(spl) genes. We report the expression of seven hairy/E(spl) (her) and the new helt gene in three neurogenic areas of the zebrafish adult brain - the telencephalon, hypothalamus and midbrain - in relation to radial glia, proliferation and neurogenesis. We show that the expression of most her genes in the adult brain characterizes quiescent radial glia, while only few are expressed in progenitor domains engaged in active proliferation or neurogenesis. The low proliferation status of most her-positive progenitors contrasts with the embryonic nervous system, where her genes are expressed in actively dividing progenitors. Likewise, we demonstrate largely overlapping expression domains of a set of her genes in the adult brain, which is in striking contrast to their distinct embryonic expression profiles. Together our data provide a consolidated map of her expression, quiescent glia, proliferation and neurogenesis in these various subdivisions of the adult brain and suggest distinct regulation and function of Her factors in the embryonic and adult contexts. J. Comp. Neurol., 2011. © 2011 Wiley-Liss, Inc.

International audience; 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.

The identification of genes that regulate proliferation is of great importance to developmental biology, regenerative medicine and cancer research. Using an in situ screen on a cortical structure of the medaka fish brain, we identified the simplet gene (smp), which is homologous to the human FAM53B gene. smp was expressed in actively proliferating cells of the CNS throughout embryogenesis. It belongs to a family of vertebrate-specific genes with no characterized biochemical domains. We showed that FAM53B bound 14-3-3 chaperones, as well as SKIIP proteins, adaptor proteins connecting DNA-binding proteins to modulators of transcription. smp inactivation with morpholinos led to delayed epiboly and reduced embryonic size. Absence of Smp activity did not induce apoptosis, but resulted in a reduced cell proliferation rate and enlarged blastomeres. Moreover, smp was shown to control the expression of the pluripotency-associated oct4/pou5f1 gene. We propose that smp is a novel vertebrate-specific gene needed for cell proliferation and that it is probably associated with the maintenance of a pluripotent state.

International audience; Investigating neural stem cell (NSC) behaviour in vivo, which is a major area of research, requires NSC models to be developed. We carried out a multilevel characterisation of the zebrafish embryo peripheral midbrain layer (PML) and identified a unique vertebrate progenitor population. Located dorsally in the transparent embryo midbrain, these large slow-amplifying progenitors (SAPs) are accessible for long-term in vivo imaging. They form a neuroepithelial layer adjacent to the optic tectum, which has transitory fast-amplifying progenitors (FAPs) at its margin. The presence of these SAPs and FAPs in separate domains provided the opportunity to data mine the ZFIN expression pattern database for SAP markers, which are co-expressed in the retina. Most of them are involved in nucleotide synthesis, or encode nucleolar and ribosomal proteins. A mutant for the cad gene, which is strongly expressed in the PML, reveals severe midbrain defects with massive apoptosis and sustained proliferation. We discuss how fish midbrain and retina progenitors might derive from ancient sister cell types and have specific features that are not shared with other SAPs.

International audience; We aimed to identify cis-regulatory elements that control gene expression in progenitors of the cerebral cortex. A list of 975 putative enhancers were retrieved from a ChIP-Seq experiment performed in NS5 mouse stem cells with antibodies to Sox2, Brn2/Pou3f2, or Brn1/Pou3f3. Through a selection pipeline including gene ontology and expression pattern, we reduced the number of candidate enhancer sequences to 20. Ex vivo electroporation of green fluorescent pProtein (GFP) reporter constructs in the telencephalon of mouse embryos showed that 35% of the 20 selected candidate sequences displayed enhancer activity in the developing cortex at E13.5. In silico transcription factor binding site (TFBS) searches and mutagenesis experiments showed that enhancer activity is related to the presence of Sox/Pou TFBS pairs in the sequence. Comparative genomic analyses showed that enhancer activity is not related to the evolutionary conservation of the sequence. Finally, the combination of in utero electroporation of GFP reporter constructs with immunostaining for Tbr2 (basal progenitor marker) and phospho-histoneH3 (mitotic activity marker) demonstrated that each enhancer is specifically active in precise subpopulations of progenitors in the cortical germinal zone, highlighting the heterogeneity of these progenitors in terms of cis-regulation.

International audience; Peptide hormones and their receptors are widespread in metazoans, but the knowledge we have of their evolutionary relationships remains unclear. Recently, accumulating genome sequences from many different species have offered the opportunity to reassess the relationships between protostomian and deuterostomian peptidergic systems (PSs). Here we used sequences of all human rhodopsin and secretin-type G protein-coupled receptors as bait to retrieve potential homologs in the genomes of 15 bilaterian species, including nonchordate deuterostomian and lophotrochozoan species. Our phylogenetic analysis of these receptors revealed 29 well-supported subtrees containing mixed sets of protostomian and deuterostomian sequences. This indicated that many vertebrate and arthropod PSs that were previously thought to be phyla specific are in fact of bilaterian origin. By screening sequence databases for potential peptides, we then reconstructed entire bilaterian peptide families and showed that protostomian and deuterostomian peptides that are ligands of orthologous receptors displayed some similarity at the level of their primary sequence, suggesting an ancient coevolution between peptide and receptor genes. In addition to shedding light on the function of human G protein-coupled receptor PSs, this work presents orthology markers to study ancestral neuron types that were probably present in the last common bilaterian ancestor.

Background The retina of craniates/vertebrates has been proposed to derive from a photoreceptor prosencephalic territory in ancestral chordates, but the evolutionary origin of the different cell types making the retina is disputed. Except for photoreceptors, the existence of homologs of retinal cells remains uncertain outside vertebrates. Methods The expression of genes expressed in the sensory vesicle of the ascidian Ciona intestinalis including those encoding components of the monoaminergic neurotransmission systems, was analyzed by in situ hybridization or in vivo transfection of the corresponding regulatory elements driving fluorescent reporters. Modulation of photic responses by monoamines was studied by electrophysiology combined with pharmacological treatments. Results We show that many molecular characteristics of dopamine-synthesizing cells located in the vicinity of photoreceptors in the sensory vesicle of the ascidian Ciona intestinalis are similar to those of amacrine dopamine cells of the vertebrate retina. The ascidian dopamine cells share with vertebrate amacrine cells the expression of the key-transcription factor Ptf1a, as well as that of dopamine-synthesizing enzymes. Surprisingly, the ascidian dopamine cells accumulate serotonin via a functional serotonin transporter, as some amacrine cells also do. Moreover, dopamine cells located in the vicinity of the photoreceptors modulate the light-off induced swimming behavior of ascidian larvae by acting on alpha2-like receptors, instead of dopamine receptors, supporting a role in the modulation of the photic response. These cells are located in a territory of the ascidian sensory vesicle expressing genes found both in the retina and the hypothalamus of vertebrates (six3/6, Rx, meis, pax6, visual cycle proteins). Conclusion We propose that the dopamine cells of the ascidian larva derive from an ancestral multifunctional cell population located in the periventricular, photoreceptive field of the anterior neural tube of chordates, which also gives rise to both anterior hypothalamus and the retina in craniates/vertebrates. It also shows that the existence of multiple cell types associated with photic responses predates the formation of the vertebrate retina.

International audience; Metazoans begin their life as a single cell. Then, this cell enters a more or less protracted period of active cell proliferation, which can be considered as the default cellular state. A crucial event, the developmental cell cycle exit, occurs thereafter. This phenomenon allows for differentiation to happen and regulates the final size of organs and organisms. Its control is still poorly understood. Herein, we review some transcriptional mechanisms of cell cycle exit in animals, and propose to use cellular conveyor belts as model systems for its study. We finally point to evidence that suggests that the mechanisms of developmental cell cycle arrest may have to be maintained in adult tissues.

The gonadal soma-derived factor (GSDF) is a new member of the transforming growth factor beta (TGF-beta) superfamily that regulates the proliferation of the primordial germ cells (PGC) in developing embryos and spermatogonia in juvenile male trout. The gsdf transcripts are expressed in the somatic cells supporting germ cell development. In zebrafish, we show that GSDF is encoded by a single copy gene that generates polymorphic transcripts and proteins. We determined that gsdf gene expression occurs before gonadal differentiation and is restricted to the gonads. Gene expression is maintained in adult granulosa cells and Sertoli cells but decreases in the cells that are in contact with meiotic and postmeiotic germ cells. Using zebrafish transgenic lines, we demonstrate that the 2-kb proximal promoter region of the gsdf gene targets high levels of transgene expression in the Sertoli and granulosa cells, and is sufficient to mimic the temporal expression pattern of the endogenous gsdf gene from 16 days postfertilization onward. We identified within the first 500 bp evolutionarily conserved DNA motifs that may be involved in Sertoli and granulosa cell-specific expression. However, the 2-kb proximal promoter region failed to drive efficient expression of the transgene in the gonads in four transgenic medaka lines. We propose that the proximal promoter region can be used to target candidate gene deregulation in zebrafish granulosa and Sertoli cells. Furthermore, the green fluorescent protein-expressing zebrafish lines produced in the present study are new valuable models for cell lineage tracing during sex differentiation and gametogenesis.

Background Teeth and tooth-like structures, together named odontodes, are repeated organs thought to share a common evolutionary origin. These structures can be found in gnathostomes at different locations along the body: oral teeth in the jaws, teeth and denticles in the oral-pharyngeal cavity, and dermal denticles on elasmobranch skin. We, and other colleagues, had previously shown that teeth in any location were serially homologous because: i) pharyngeal and oral teeth develop through a common developmental module; and ii) the expression patterns of the Dlx genes during odontogenesis were highly divergent between species but almost identical between oral and pharyngeal dentitions within the same species. Here we examine Dlx gene expression in oral teeth and dermal denticles in order to test the hypothesis of serial homology between these odontodes. Results We present a detailed comparison of the first developing teeth and dermal denticles (caudal primary scales) of the dogfish (Scyliorhinus canicula) and show that both odontodes develop through identical stages that correspond to the common stages of oral and pharyngeal odontogenesis. We identified six Dlx paralogs in the dogfish and found that three showed strong transcription in teeth and dermal denticles (Dlx3, Dlx4 and Dlx5) whereas a weak expression was detected for Dlx1 in dermal denticles and teeth, and for Dlx2 in dermal denticles. Very few differences in Dlx expression patterns could be detected between tooth and dermal denticle development, except for the absence of Dlx2 expression in teeth. Conclusions Taken together, our histological and expression data strongly suggest that teeth and dermal denticles develop from the same developmental module and under the control of the same set of Dlx genes. Teeth and dermal denticles should therefore be considered as serial homologs developing through the initiation of a common gene regulatory network (GRN) at several body locations. This mechanism of heterotopy supports the 'inside and out' model that has been recently proposed for odontode evolution.

International audience; The developing vertebrate nervous system contains a remarkable array of neural cells organized into complex, evolutionarily conserved structures. The labeling of living cells in these structures is key for the understanding of brain development and function, yet the generation of stable lines expressing reporter genes in specific spatio-temporal patterns remains a limiting step. In this study we present a fast and reliable pipeline to efficiently generate a set of stable lines expressing a reporter gene in multiple neuronal structures in the developing nervous system in medaka. The pipeline combines both the accurate computational genome-wide prediction of neuronal specific cis-regulatory modules (CRMs) and a newly developed experimental setup to rapidly obtain transgenic lines in a cost-effective and highly reproducible manner. 95% of the CRMs tested in our experimental setup show enhancer activity in various and numerous neuronal structures belonging to all major brain subdivisions. This pipeline represents a significant step towards the dissection of embryonic neuronal development in vertebrates.

International audience; One of the goals of evolutionary developmental biology is to link specific adaptations to changes in developmental pathways. The dentition of cypriniform fishes, which in contrast to many other teleost fish species possess pharyngeal teeth but lack oral teeth, provides a suitable model to study the development of feeding adaptations. Here, we have examined the involvement of retinoic acid (RA) in tooth development and show that RA is specifically required to induce the pharyngeal tooth developmental program in zebrafish. Perturbation of RA signaling at this stage abolished tooth induction without affecting the development of tooth-associated ceratobranchial bones. We show that this inductive event is dependent on RA synthesis from aldh1a2 in the ventral posterior pharynx. Fibroblast growth factor (FGF) signaling has been shown to be critical for tooth induction in zebrafish, and its loss has been associated with oral tooth loss in cypriniform fishes. Pharmacological treatments targeting the RA and FGF pathways revealed that both pathways act independently during tooth induction. In contrast, we find that in Mexican tetra and medaka, species that also possess oral teeth, both oral and pharyngeal teeth are induced independently of RA. Our analyses suggest an evolutionary scenario in which the gene network controlling tooth development obtained RA dependency in the lineage leading to the cypriniforms. The loss of pharyngeal teeth in this group was cancelled out through a shift in aldh1a2 expression, while oral teeth might have been lost ultimately due to deficient RA signaling in the oral cavity.-Gibert, Y., Bernard, L., Debiais-Thibaud, M., Bourrat, F., Joly, J.-S., Pottin, K., Meyer, A., Retaux, S., Stock, D. W., Jackman, W. R., Seritrakul, P., Begemann, G., Laudet, V. Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling.

International audience; Few adult neural stem cells have been characterized in vertebrates. Although teleosts continually generate new neurons in many regions of the brain after embryogenesis, only two types of neural stem cells (NSCs) have been reported in zebrafish: glial cells in the forebrain resembling mammalian NSCs, and neuroepithelial cells in the cerebellum. Here, following our previous studies on dividing progenitors (Nguyen et al. [1999]: J Comp Neurol 413:385-404.), we further evidenced NSCs in the optic tectum (OT) of juvenile and adult in the medaka, Oryzias latipes. To detect very slowly cycling progenitors, we did not use the commonly used BrdU/PCNA protocol, in which PCNA may not be present during a transiently quiescent state. Instead, we report the optimizations of several protocols involving long subsequent incubations with two thymidine analogs (IdU and CldU) interspaced with long chase times between incubations. These protocols allowed us to discriminate and localize fast and slow cycling cells in OT of juvenile and adult in the medaka. Furthermore, we showed that adult OT progenitors are not glia, as they express neither brain lipid-binding protein (BLBP) nor glial fibrillary acidic protein (GFAP). We also showed that expression of pluripotency-associated markers (Sox2, Musashi1 and Bmi1) colocalized with OT progenitors. Finally, we described the spatio-temporally ordered population of NSCs and progenitors in the medaka OT. Hence, the medaka appears as an invaluable model for studying neural progenitors that will open the way to further exciting comparative studies of neural stem cells in vertebrates. (c) 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 693-713, 2010.

International audience; Ascidians have powerful capacities for regeneration but the underlying mechanisms are poorly understood. Here we examine oral siphon regeneration in the solitary ascidian Ciona intestinalis. Following amputation, the oral siphon rapidly reforms oral pigment organs (OPO) at its distal margin prior to slower regeneration of proximal siphon parts. The early stages of oral siphon reformation include cell proliferation and re-growth of the siphon nerves, although the neural complex (adult brain and associated organs) is not required for regeneration. Young animals reform OPO more rapidly after amputation than old animals indicating that regeneration is age dependent. UV irradiation, microcautery, and cultured siphon explant experiments indicate that OPOs are replaced as independent units based on local differentiation of progenitor cells within the siphon, rather than by cell migration from a distant source in the body. The typical pattern of eight OPOs and siphon lobes is restored with fidelity after distal amputation of the oral siphon, but as many as 16 OPOs and lobes can be reformed following proximal amputation near the siphon base. Thus, the pattern of OPO regeneration is determined by cues positioned along the proximal distal axis of the oral siphon. A model is presented in which columns of siphon tissue along the proximal-distal axis below pre-existing OPO are responsible for reproducing the normal OPO pattern during regeneration. This study reveals previously unknown principles of oral siphon and OPO regeneration that will be important for developing Ciona as a regeneration model in urochordates, which may be the closest living relatives of vertebrates.

One of the striking findings of comparative developmental genetics was that expression patterns of core transcription factors are extraordinarily conserved in bilaterians. However, it remains unclear whether cis-regulatory elements of their target genes also exhibit common signatures associated with conserved embryonic fields. To address this question, we focused on genes that are active in the anterior neuroectoderm and non-neural ectoderm of the ascidian Ciona intestinalis. Following the dissection of a prototypic anterior placodal enhancer, we searched all genomic conserved non-coding elements for duplicated motifs around genes showing anterior neuroectodermal expression. Strikingly, we identified an over-represented pentamer motif corresponding to the binding site of the homeodomain protein OTX, which plays a pivotal role in the anterior development of all bilaterian species. Using an in vivo reporter gene assay, we observed that 10 of 23 candidate cis-regulatory elements containing duplicated OTX motifs are active in the anterior neuroectoderm, thus showing that this cis-regulatory signature is predictive of neuroectodermal enhancers. These results show that a common cis-regulatory signature corresponding to K50-Paired homeodomain transcription factors is found in non-coding sequences flanking anterior neuroectodermal genes in chordate embryos. Thus, field-specific selector genes impose architectural constraints in the form of combinations of short tags on their target enhancers. This could account for the strong evolutionary conservation of the regulatory elements controlling field-specific selector genes responsible for body plan formation., Author Summary Regional identity in embryos is defined by a few specific transcription factors that activate a large number of target genes through binding to common tags in regulatory sequences. In chordates it is unclear if such tags can be identified in the cis-regulatory regions of regionally expressed genes. To address this question we focused on the anterior nervous system where Otx codes for a transcription factor that triggers expression of many other head-specific genes. We analyzed an element that is active in the region bordering the anterior nervous system in the marine invertebrate Ciona intestinalis. We found that the crucial binding sites have to be duplicated and close enough. One of the pairs is bound by OTX. We showed that anterior nervous system genes are often flanked by duplicated OTX binding sites. We confirmed by transgenic assays that about half of these genomic sequences are active and drive expression anteriorly. This study unravels a simple regulatory logic in the anterior enhancers. It indicates that although there are major changes in the organization of the binding sites at short evolutionary range, conserved expression patterns are partly generated by a duplicated organization of conserved binding sites for region-specific transcription factors.

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.

International audience; Anteroposterior patterning of the ectoderm in the invertebrate chordate Ciona intestinalis first relies on key zygotic activators, such as FoxA, and later on the coordinated responses to inducing signals from the underlying mesendoderm. Here, we focus on a mechanism of coordination of these responses by looking at the cis-regulatory logics of Ci-Rora and Ci-Rorb, which are coding for putative non-canonical transmembrane Wnt receptors, and are present in tandem along the C. intestinalis chromosome 08q. We showed that during cleavage stages, both Ci-Rora and Ci-Rorb genes are initially expressed in all blastomeres of the anterior ectoderm (a-line), as sFRP1/5 (Lamy, C., Rothb?er, U., Caillol, D., Lemaire, P., 2006. Ci-FoxA-a is the earliest zygotic determinant of the ascidian anterior ectoderm and directly activates Ci-sFRP1/5. Development 133, 2835-2844.). We then carried out a functional analysis of cis-regulatory regions and showed that both genes have elements enriched in Ci-FoxA-a binding sites. We dissected one of these early enhancers, and showed that it is directly activated by Ci-FoxA-a, as one sFRP1/5 cis-regulatory element. We also showed that although FoxA binding sites are abundant in genomes, dense clusters of these sites are found upstream from very few genes, and have a high predictive value of a-line expression. These data indicate an important role for FoxA in anterior specification, via the transcriptional regulation of target genes belonging to various signalling pathways.

The blind cavefish and its surface counterpart of the teleost species Astyanax mexicanus constitute an excellent model to study the evolution of morphological features. During adaptation to their lives in perpetual darkness, the cave population has lost eyes (and pigmentation), but has gained several constructive traits. Recently, the demonstration that an increase in Shh (Sonic Hedgehog) midline signalling was indirectly responsible for the loss of eyes in cavefish led to new ways to search for possible modifications in the forebrain of these cavefish, as this anterior-most region of the vertebrate central nervous system develops under close control of the powerful Shh morphogen. In this review, we summarize the recent progress in the understanding of forebrain and eye modifications in cavefish. These include major changes in cell death, cell proliferation and cell migration in various parts of the forebrain when compared with their surface counterparts with eyes. The outcome of these modifications, in terms of neuronal circuitry, morphological and behavioral adaptations are discussed.

Text-mining technologies can be integrated with genome annotation systems, increasing the availability of annotated cis-regulatory data., Background Decoding transcriptional regulatory networks and the genomic cis-regulatory logic implemented in their control nodes is a fundamental challenge in genome biology. High-throughput computational and experimental analyses of regulatory networks and sequences rely heavily on positive control data from prior small-scale experiments, but the vast majority of previously discovered regulatory data remains locked in the biomedical literature. Results We develop text-mining strategies to identify relevant publications and extract sequence information to assist the regulatory annotation process. Using a vector space model to identify Medline abstracts from papers likely to have high cis-regulatory content, we demonstrate that document relevance ranking can assist the curation of transcriptional regulatory networks and estimate that, minimally, 30,000 papers harbor unannotated cis-regulatory data. In addition, we show that DNA sequences can be extracted from primary text with high cis-regulatory content and mapped to genome sequences as a means of identifying the location, organism and target gene information that is critical to the cis-regulatory annotation process. Conclusion Our results demonstrate that text-mining technologies can be successfully integrated with genome annotation systems, thereby increasing the availability of annotated cis-regulatory data needed to catalyze advances in the field of gene regulation.

The sighted surface-dwelling (surface fish, SF) and the blind cave-living (cavefish, CF) forms of Astyanax mexicanus offer a unique opportunity to study the evolutionary changes in developmental mechanisms that lead to retinal degeneration. Previous data have shown the role of increased midline Sonic Hedgehog (Shh) signalling in cavefish eye degeneration (Yamamoto et al. [2004] Nature 431:844-847). Here, we have compared the major steps of eye development in SF and CF between 14 hours and 5 days of development. We have analyzed cell proliferation through PCNA and phospho-histone H3 staining and apoptosis through TUNEL and live LysoTracker analysis. We have assessed the expression of the major eye development signalling factors Shh and Fgf8, and the eye patterning genes Pax6, Lhx2, Lhx9, and Vax1, together with the differentiation marker GAD65. We show that eye development is retarded in CF and that cell proliferation in CF retina is proportionately similar to SF during early development, yet the retina degenerates after massive apoptosis in the lens and widespread cell death throughout the neuroretina. Moreover, and surprisingly, the signalling, patterning, and differentiation processes leading to the establishment of retinal layers and cell types happen almost normally in CF, although some signs of disorganization, slight heterochronies, and a lack of expression gradients are observable. Our data demonstrate that the evolutionary process of eye degeneration in the blind CF does not occur because of patterning defects of the retina and are consistent with the proposed scenario in which the trigger for eye degeneration in CF is lens apoptosis. J. Comp. Neurol. 505:221-233, 2007. (c) 2007 Wiley-Liss, Inc.

Through whole-mount in situ hybridisation screen on medaka (Oryzias latipes) brain, Ol-insm1b, a member of the Insm1/Mlt1 subfamily of SNAG-domain containing genes, has been isolated. It is strongly expressed during neurogenesis and pancreas organogenesis, with a pattern that suggests a role in cell cycle exit. Here, we describe Ol-insm1b expression pattern throughout development and in adult brain, and we report on its functional characterisation. Our data point to a previously unravelled role for Ol-insm1b as a down-regulator of cell proliferation during development, as it slows down the cycle without triggering apoptosis. Clonal analysis demonstrates that this effect is cell-autonomous, and, through molecular dissection studies, we demonstrate that it is likely to be non-transcriptional, albeit mediated by zinc-finger domains. Additionally, we report that Ol-insm1b mRNA, when injected in one cell of two-cell stage embryos, exhibits a surprising behaviour: it does not spread uniformly amongst daughter cells but remains cytoplasmically localised in the progeny of the injected blastomere. Our experiments suggest that Insm1 is a negative regulator of cell proliferation, possibly through mechanisms that do not involve modulation of transcription.

International audience; Gnathostome teeth are one of the most promising models for developmental evolutionary studies, they are the most abundant organ in the fossil record and an excellent example of organogenesis. Teeth have a complex morphology and are restricted to the mouth in mammals, whereas actinopterygian teeth have a simple morphology and are found in several locations, notably on pharyngeal bones. Morphological and developmental similarities support the hypothesis that oral and pharyngeal teeth are serially homologous. Gene expression data from the mouse and some teleosts have shown that the gene families involved in pharyngeal odontogenesis are also involved in oral tooth formation, with the notable exception of the evx gene family. Here, we present a complete description of early odontogenesis in the medaka (Oryzias latipes), which has both oral and pharyngeal dentition. We show that oral and pharyngeal teeth share deep developmental similarities. In the medaka, like in the zebrafish, eve1 is the only evx gene expressed during odontogenesis. In each forming tooth, regardless of its location, eve1 transcription is activated in the placode, then becomes restricted to the inner dental epithelium and is activated in the dental mesenchyme during early differentiation, and finally ceases at late differentiation. Thus eve1 expression is not specific to pharyngeal teeth development as was previously suggested. Because it permits direct comparisons between oral and pharyngeal teeth by molecular, development and functional studies, the medaka is an excellent model to develop further insights into the evolution of odontogenesis in gnathostomes.. 2007. Development of oral and pharyngeal teeth in the medaka (Oryzias latipes): comparison of morphology and expression of eve1 gene. J. Exp. Zool. (Mol. Dev. Evol.) 308B:693-708. Teeth are regarded by both palaeontologists and developmental geneticists as an excellent model for studying evolutionary mechanisms for several reasons. They are the most abundant organ in the vertebrate fossil record, chiefly because they are mainly composed of hard tissues and are partially hyper mineralised. They have undergone extensive morphological variation during the course of evolution, are therefore a very informative character, and have been used in a wide range of palaeontological studies. Finally, although the tooth is one of the simplest organs that form during gnathostomes development, it represents a model of organ ontogeny.

Improvements in closed-system culturing methods for marine invertebrates are important prerequisites for the generalized use of transgenic lines. We discuss here the effects of several closed-system conditions on the growth and survival of the solitary ascidian, Ciona intestinalis. In Shimoda, close to the sea, a small-tank system was used to ensure that tanks and systems were reasonably equipped, water exchange was rapid, and animals separated to minimize the risk of infection. In Gif-sur-Yvette, an inland site, we tried to determine the optimal conditions to limit handling operations, and to save artificial seawater by avoiding water pollution. A mixture of at least two types of live algae was better than any single-organism diet. With these maintenance protocols, we were able to obtain several generations of Ciona intestinalis, including several transgenic lines. Because these systems make it easier to rear Ciona intestinalis in laboratories, they increase the potentialities of this model organism for research. Developmental Dynamics 236:1832–1840, 2007. © 2007 Wiley-Liss, Inc.

With the availability of an increasing number of whole genome sequences in chordates, exhaustive comparisons of multigene families become feasible. Relationships of orthology/paralogy can not only be inferred from sequence similarity but also by comparing synteny conservation on chromosomes. More accurate scenarios for gene and expression domain gain or loss can now be proposed. Here, we take benefit from the recent release of the medaka (Oryzias latipes) genome to analyse the orthology relationships and expression patterns of the three different sub-families of the pitx homeobox genes belonging to the paired class. They are involved in a wide variety of developmental processes and have pleiotropic expression patterns, especially in the case of the pitx2 sub-family. The emerging picture is a strong conservation of expression domains, suggesting that most functions have been present in the common ancestor of actinopterygians and sarcopterygians. Almost all pitx genes are expressed in anterior placodes in all species studied so far, including medaka. It has previously been shown that in mammals, pitx1 and 2 are expressed in the pituitary. Interestingly we demonstrate here that only pitx3 is expressed in medaka pituitary. It will be interesting to analyze what are the corresponding changes in the regulatory elements of pitx genes.

The use of classic genetics is emerging in the ascidian Ciona intestinalis; recent advances in genomics and high-quality developmental and evolutionary studies have made this animal an attractive model for research purposes. Genetic mapping in Ciona will likely make a major contribution to ascidian genomics and developmental biology by providing support for genome assembly and annotation and for the isolation of genes with particular mutations, while construction of genetic maps advances classic genetics in this species. Two major issues must be overcome before fine genetic maps can be constructed: the choice of proper genetic backgrounds and the establishment of laboratory strains. A high degree of polymorphism is useful for genetic mapping if we consider particular combinations of genetic backgrounds and techniques, although it is necessary to pay attention to the confused classification of C. intestinalis. Thus, it is preferred to establish laboratory strains instead of using samples with various genetic backgrounds. As these issues are unresolved, only amplified fragment length polymorphism-based maps have been created, while bulk segregant analysis is expected to isolate markers flanking mutant loci. However, rich genomic resources should facilitate the next stage of genetic map construction based on type I markers using coding sequences. The meiotic events that occur in crossing experiments for mapping purposes should shed light on population genetics and speciation issues. The results of such investigations may provide feedback for comparative genomics and developmental genetics in the near future.

In chordates, the oral ectoderm is positioned at the anterior neural boundary and is characterized by pituitary homeobox (Pitx) and overlapping Dlx and Six3 expressions. Recent studies have shown that the ectoderm molecular map is also conserved in hemichordates and echinoderms. However, the mouth develops in a more posterior position in these animals, in a domain characterized by Nkx2.1 and Goosecoid expression, in a manner similar to that observed in protostomes. Furthermore, BMP signaling antagonizes mouth development in echinoderms and hemichordates, but seems to promote oral ectoderm specification in chordates. Conversely, Nodal signaling appears to be required for oral ectoderm specification in sea urchins but not in chordates. The Nodal/BMP antagonism at work during ectoderm patterning thus seems to constitute a conserved feature in deuterostomes, and mouth relocation may have been accompanied by a change in the influence of BMP/Nodal signals on oral ectoderm specification. We suggest that the mouth primordium was located at the anterior neural boundary, in early chordate evolution. In extant chordate embryos, subsequent mouth positioning differ between urochordates and vertebrates, presumably as a consequence of surrounding tissues remodelling. We illustrate these morphogenetic movements by means of morphological data obtained by the confocal imaging of ascidian tailbud embryos, and provide a table for determining the tailbud stages of this model organism.

International audience; Summary The central nervous system (cerebral ganglion) of adult ascidians is linked to the neural gland complex (NGC), which consists of a dorsal tubercle, a ciliated duct and a neural gland. The function of the NGC has been the subject of much debate. The recent publication of the complete genomic sequence of Ciona intestinalis provides new opportunities to examine the presence and distribution of protein families in this basal chordate. We focus here on the ascidian neuropeptide G-protein-coupled receptors (GPCRs), the vertebrate homologues of which are involved in homeostasis. In situ hybridization revealed that five Ciona GPCRs [vasopressin receptor, somatostatin receptor, CRH (corticotropin-releasing hormone) receptor, angiotensin receptor and tachykinin receptor] are expressed in the NGC of adult ascidians. These findings, together with histological and ultrastructural data, provide evidence to support a role for the ascidian NGC in maintaining ionic homeostasis. We further speculate about the potential similarities between the ascidian NGC and the vertebrate choroid plexus, a neural peri-ventricular organ.

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.

International audience; BACKGROUND: New, practical models of central nervous system regeneration are required and should provide molecular tools and resources. We focus here on the tunicate Ciona intestinalis, which has the capacity to regenerate nerves and a complete adult central nervous system, a capacity unusual in the chordate phylum. We investigated the timing and sequence of events during nervous system regeneration in this organism. METHODOLOGY/PRINCIPAL FINDINGS: We developed techniques for reproducible ablations and for imaging live cellular events in tissue explants. Based on live observations of more than 100 regenerating animals, we subdivided the regeneration process into four stages. Regeneration was functional, as shown by the sequential recovery of reflexes that established new criteria for defining regeneration rates. We used transgenic animals and labeled nucleotide analogs to describe in detail the early cellular events at the tip of the regenerating nerves and the first appearance of the new adult ganglion anlage. CONCLUSIONS/SIGNIFICANCE: The rate of regeneration was found to be negatively correlated with adult size. New neural structures were derived from the anterior and posterior nerve endings. A blastemal structure was implicated in the formation of new neural cells. This work demonstrates that Ciona intestinalis is as a useful system for studies on regeneration of the brain, brain-associated organs and nerves.