ZeBraInspector, a platform for the automated segmentation and analysis of body and brain volumes in whole 5 days post-fertilization zebrafish following simultaneous visualization with identical orientations.

In recent years, the zebrafish has become a well-established laboratory model. We describe here the ZeBraInspector (ZBI) platform for high-content 3D imaging (HCI) of 5 days post-fertilization zebrafish eleuthero-embryos (EEs). This platform includes a mounting method based on 3D-printed stamps to create a grid of wells in an agarose cast, facilitating batch acquisitions with a fast-confocal laser scanning microscope. We describe reference labeling in cleared fish with a fluorescent lipophilic dye. Based on this labeling, the ZBI software registers. EE 3D images, making it possible to visualize numerous identically oriented EEs on a single screen, and to compare their morphologies and any fluorescent patterns at a glance. High-resolution 2D snapshots can be extracted. ZBI software is therefore useful for diverse high-content analyses (HCAs). Following automated segmentation of the lipophilic dye signal, the ZBI software performs volumetric analyses on whole EEs and their nervous system white matter. Through two examples, we illustrate the power of these analyses for obtaining statistically significant results from a small number of samples: the characterization of a phenotype associated with a neurodevelopmental mutation, and of the defects caused by treatments with a toxic anti-cancer compound.

Fibrillarin is essential for S-phase progression and neuronal differentiation in zebrafish dorsal midbrain and retina.

Fibrillarin (Fbl) is a highly conserved protein that plays an essential role in ribosome biogenesis and more particularly in the methylation of ribosomal RNAs and rDNA histones. In cellular models, FBL was shown to play an important role in tumorigenesis and stem cell differentiation. We used the zebrafish as an in vivo model to study Fbl function during embryonic development. We show here that the optic tectum and the eye are severely affected by Fbl depletion whereas ventral regions of the brain are less impacted. The morphogenesis defects are associated with impaired neural differentiation and massive apoptosis. Polysome gradient experiments show that fbl mutant larvae display defects in ribosome biogenesis and activity. Strikingly, flow cytometry analyses revealed different S-phase profiles between wild-type and mutant cells, suggesting a defect in S-phase progression.

Postembryonic Fish Brain Proliferation Zones Exhibit Neuroepithelial-Type Gene Expression Profile.

In mammals, neuroepithelial cells play an essential role in embryonic neurogenesis, whereas glial stem cells are the principal source of neurons at postembryonic stages. By contrast, neuroepithelial-like stem/progenitor (NE) cells have been shown to be present throughout life in teleosts. We used three-dimensional (3D) reconstructions of cleared transgenic wdr12:GFP medaka brains to demonstrate that this cell type is widespread in juvenile and to identify new regions containing NE cells. We established the gene expression profile of optic tectum (OT) NE cells by cell sorting followed by RNA-seq. Our results demonstrate that most OT NE cells are indeed active stem cells and that some of them exhibit long G2 phases. We identified several novel pathways (e.g., DNA repair pathways) potentially involved in NE cell homeostasis. In situ hybridization studies showed that all NE populations in the postembryonic medaka brain have a similar molecular signature. Our findings highlight the importance of NE progenitors in medaka and improve our understanding of NE-cell biology. These cells are potentially useful not only for neural stem cell studies but also for improving the characterization of neurodevelopmental diseases, such as microcephaly. Stem Cells 2017;35:1505-1518.

The trouble with collective nouns for genome editing.

We should start as we mean to go on and try to avoid the confusion most of us experience when bombarded with acronyms with overstated significations. You will be familiar with the situation, you are in a seminar or a meeting and someone who has been using a set of acronyms for years, includes them in sentence after sentence that has you lost because you don't know what some or most of them stand for. Even worse when scientists start making verbs out of them, CRISPR seems to have fallen into this category; how many of us have heard someone asking if a mutation can be CRISPRed! Does it matter though? We are all familiar with informal language in scientific talks and discussions which is replaced by more formal dialect when research is published or presented to the general public. However, when an ill-defined acronym slips outside of laboratory chatter and is widely recognised by the general public, we need to proceed with caution to avoid misinterpretation and misunderstandings.

Zebrafish midbrain slow-amplifying progenitors exhibit high levels of transcripts for nucleotide and ribosome biogenesis.

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.

Molecular evolution of peptidergic signaling systems in bilaterians.

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.

Characterization of enhancers active in the mouse embryonic cerebral cortex suggests Sox/Pou cis-regulatory logics and heterogeneity of cortical progenitors.

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.

The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program.

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.

A cis-regulatory signature for chordate anterior neuroectodermal genes.

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.

Monoaminergic modulation of photoreception in ascidian: evidence for a proto-hypothalamo-retinal territory.

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.

When needles look like hay: how to find tissue-specific enhancers in model organism genomes.

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.

Genome-wide analysis of the POU genes in medaka, focusing on expression in the optic tectum.

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.

Molecular and Cellular Analysis of the Repair of Zebrafish Optic Tectum Meninges Following Laser Injury.

We studied cell recruitment following optic tectum (OT) injury in zebrafish (Danio rerio), which has a remarkable ability to regenerate many of its organs, including the brain. The OT is the largest dorsal layered structure in the zebrafish brain. In juveniles, it is an ideal structure for imaging and dissection. We investigated the recruited cells within the juvenile OT during regeneration in a Pdgfrß-Gal4:UAS-EGFP line in which pericytes, vascular, circulating, and meningeal cells are labeled, together with neurons and progenitors. We first performed high-resolution confocal microscopy and single-cell RNA-sequencing (scRNAseq) on EGFP-positive cells. We then tested three types of injury with very different outcomes (needle (mean depth in the OT of 200 µm); deep-laser (depth: 100 to 200 µm depth); surface-laser (depth: 0 to 100 µm)). Laser had the additional advantage of better mimicking of ischemic cerebral accidents. No massive recruitment of EGFP-positive cells was observed following laser injury deep in the OT. This type of injury does not perturb the meninx/brain-blood barrier (BBB). We also performed laser injuries at the surface of the OT, which in contrast create a breach in the meninges. Surprisingly, one day after such injury, we observed the migration to the injury site of various EGFP-positive cell types at the surface of the OT. The migrating cells included midline roof cells, which activated the PI3K-AKT pathway; fibroblast-like cells expressing numerous collagen genes and most prominently in 3D imaging; and a large number of arachnoid cells that probably migrate to the injury site through the activation of cilia motility genes, most likely being direct targets of the FOXJ1a gene. This study, combining high-content imaging and scRNAseq in physiological and pathological conditions, sheds light on meninges repair mechanisms in zebrafish that probably also operate in mammalian meninges.

Regeneration of oral siphon pigment organs in the ascidian Ciona intestinalis.

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.

The proximal promoter region of the zebrafish gsdf gene is sufficient to mimic the spatio-temporal expression pattern of the endogenous gene in Sertoli and granulosa cells.

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.

Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling.

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.

Similar regulatory logic in Ciona intestinalis for two Wnt pathway modulators, ROR and SFRP-1/5.

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., Rothbächer, 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.

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.

The role of neuro-epithelial-like and radial-glial stem and progenitor cells in development, plasticity, and repair.

Neural stem and progenitor cells (NSPCs) are the primary source of new neurons in the brain and serve critical roles in tissue homeostasis and plasticity throughout life. Within the vertebrate brain, NSPCs are located within distinct neurogenic niches differing in their location, cellular composition, and proliferative behaviour. Heterogeneity in the NSPC population is hypothesized to reflect varying capacities for neurogenesis, plasticity and repair between different neurogenic zones. Since the discovery of adult neurogenesis, studies have predominantly focused on the behaviour and biological significance of adult NSPCs (aNSPCs) in rodents. However, compared to rodents, who show lifelong neurogenesis in only two restricted neurogenic niches, zebrafish exhibit constitutive neurogenesis across multiple stem cell niches that provide new neurons to every major brain division. Accordingly, zebrafish are a powerful model to probe the unique cellular and molecular profiles of NSPCs and investigate how these profiles govern tissue homeostasis and regenerative plasticity within distinct stem cell populations over time. Amongst the NSPC populations residing in the zebrafish central nervous system (CNS), proliferating radial-glia, quiescent radial-glia and neuro-epithelial-like cells comprise the majority. Here, we provide insight into the extent to which these distinct NSPC populations function and mature during development, respond to experience, and contribute to successful CNS regeneration in teleost fish. Together, our review brings to light the dynamic biological roles of these individual NSPC populations and showcases their diverse regenerative modes to achieve vertebrate brain repair later in life.

Comparison of the expression of medaka (Oryzias latipes) pitx genes with other vertebrates shows high conservation and a case of functional shuffling in the pituitary.

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.