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.

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.

Transcriptional mechanisms of developmental cell cycle arrest: problems and models.

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.

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.

The homology of odontodes in gnathostomes: insights from Dlx gene expression in the dogfish, Scyliorhinus canicula.

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.

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.

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.

Medaka as a model system for the characterisation of cell cycle regulators: a functional analysis of Ol-Gadd45gamma during early embryogenesis.

Numerous studies, mostly performed on mammalian cell cultures, have implicated the Gadd45 family of small acidic proteins in cell cycle control (arrest and/or engagement in the apoptotic pathway). We report here the cloning, detailled expression pattern and functional characterisation in embryonic development of Ol-Gadd45gamma, the Oryzias latipes ortholog of mammalian Gadd45gamma. Its expression pattern, notably in the developing brain (optic tectum) strongly suggests that it is involved in cell cycle exit. Gain-of-function experiments (through mRNA injection) slowed down early development, and produced embryos clearly reduced in size, while morpholino knockdowns resulted in small embryos over-sensitive to DNA damage (UV irradiation). We further demonstrated that, following Ol-Gadd45gamma overexpression, cells are proliferation-arrested before both G1/S and G2/M cell cycle checkpoints, while in the MO-Ol-Gadd45 loss-of-function experiments cells are engaged in apoptosis rather than prevented from proliferating. These results show that Ol-Gadd45gamma is likely to play an important role in coordinating cell fate decisions during neurogenesis; they also demonstrate that the medakafish is a promising model to analyse in vivo the developmental control of the cell cycle.

Development of the pancreas in medaka.

In the present work, pancreatic organogenesis has been studied in the medaka (Oryzias latipes), a teleost fish with several advantages as an experimental system in developmental biology. We demonstrated that the pancreas develops from three primordia budding from the dorsal and ventral faces of the gut epithelium. Such buds then fuse to form a single endocrine islet surrounded by exocrine tissue. Interestingly, the endocrine tissue forms only from the dorsal bud. We next analyzed a collection of medakas that had been hybridized with cDNAs derived from an anterior brain library. We found new clones expressed in the pancreatic region demonstrating that the medaka can be used to define new genes expressed in the pancreatic region that follow a specific spatial and temporal pattern of expression.

Cloning and developmental expression patterns of Dlx2, Lhx7 and Lhx9 in the medaka fish (Oryzias latipes).

We have isolated three homeodomain and LIM-homeodomain developmental transcription factors from the medaka fish (Oryzias latipes): OlDlx2, OlLhx7, and OlLhx9, and we have studied their expression patterns in the developing and adult brain. This analysis showed that OlDlx2 and OlLhx7 (together with OlNkx2.1b) delineate the subpallial divisions of the medaka telencephalon, and that OlLhx9 exhibits a typical and specific topology of expression in the pallium and diencephalic neuromeres. The expression patterns of these three genes, when compared in details with those of their tetrapod homologs, reveal both commonalities and differences in the basic organization of the developing teleost and vertebrate forebrain.

I-SceI meganuclease mediates highly efficient transgenesis in fish.

The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection.

Cell proliferation in the developing and adult hindbrain and midbrain of trout and medaka (teleosts): a segmental approach.

We report here the histogenesis of the brainstem of the trout (Salmo trutta fario) and the medaka (Oryzias latipes) chosen as examples of teleosts with slow and fast growth, respectively. Our results reveal that the sequence of formation of brain structures is rather similar in the teleosts species examined so far, but some interspecific differences do exist in terms of brainstem maturation at particular developmental stages, such as the end of the gastrulation and hatching periods. This sequence includes the subdivision of the brainstem in different transverse segments and longitudinal zones, where morphologically discernible boundaries are observed along the caudorostral and ventrodorsal axis. The boundary formation and subsequent subdivision of the trout and medaka brainstems, together with the proliferation pattern observed by immunohistochemistry with an antibody against the proliferating cell nuclear antigen (PCNA), support a segmental model throughout the brainstem. The spatiotemporal pattern of PCNA immunoreactivity is similar in the mesencephalon and rhombencephalon of the two teleosts species studied, although proliferation centers are less clearly defined in the medaka. Moreover, the segmental appearance of the brainstem, as revealed by PCNA immunohistochemistry, is blurred earlier in the medaka than in the trout. Thus, the trout brain appears a suitable model for morphogenetic studies because it allows more gradual survey of the changes throughout development.

Expression domains suggest cell-cycle independent roles of growth-arrest molecules in the adult brain of the medaka, Oryzias latipes.

Teleost fish are unique for their enormous potential to produce new neurons in the adult brain. Nevertheless, the regulation of this adult neurogenesis remains to be characterized. Does it resort to the same molecular mechanisms as those at play in embryonic development? Here, we analyse the expression of the neurogenic gene Ol-DeltaA in the brain of medaka (Oryzias latipes) embryos and adults. To determine the relationships between neurogenic and growth-arrest genes in the adult brain, we compare the expression domains of Ol-DeltaA with those of Ol-KIP and Ol-Gadd45gamma, two well-characterized genes involved in cell-cycle arrest and growth inhibition. While it is widely assumed that genes controlling cell-cycle exit show restricted expression domains next to proliferating cells (in the sites of prospective cell differentiation), we observe highly particular expression domains of Ol-KIP and Ol-Gadd45gamma not associated to proliferating areas of the adult brain, suggesting locally different and cell-cycle independent roles of these molecules in the adult brain.

Pitx genes in Tunicates provide new molecular insight into the evolutionary origin of pituitary.

We have initiated a project aimed at documenting molecular and cellular changes underlying the emergence of the hypothalamo-hypophyseal axis in Chordates. Considering the phylogenetic position of Tunicates and the 'pan-hypophyseal' expression pattern of Pitx genes in Vertebrate pituitary, we searched for a Pitx-related homeobox gene in the ascidian Ciona intestinalis, and identified Ci-Pitx (ona intestinalis uitary homeobo gene). We also isolated Cs-Pitx and Bs-Pitx, the Ci-Pitx respective counterparts of Ciona savignyi and Botryllus schlosseri, two other Tunicate species. Ci-Pitx mRNA encodes a putative protein exhibiting the diagnostic K50-Paired-class homeodomain and a conserved C-terminal Aristaless domain. Embryonic expression pattern of Ci-Pitx revealed a conserved expression domain in the anterior neural ridge and subsequently in the pharyngeal primordium, defined in Vertebrates as the stomodeal ectomere, which encompasses the presumptive pituitary territory. This shows that expression at early steps of pituitary development is a feature of Pitx-related genes that was already present in the last common ancestor of Chordates.

Effects of microcystin-LR on development of medaka fish embryos (Oryzias latipes).

Chronic and subchronic toxicity from exposure to microcystins, cyclic hepatotoxic heptapeptides from cyanobacteria, receives increasing attention as a public human health biohazard. So far, the effects of microcystin on fish have been studied mainly in adults, rather than during early life stages. Limitations of direct ambient exposure experiments to fish egg have resulted from the difficult access of microcystin through the egg chorion. Using a microinjection technology, we have introduced microcystin-LR (MC-LR) directly into one-cell stage embryos or into the vitellus of late neurula embryos (stage 19) or into the vitellus of stage 25 embryos of medaka (Oryzias latipes) at the onset of the liver anlage. Microinjection (100 pl; stage 1 or 2 nl; stage 19 or 25) of MC-LR resulted in a dose dependent mortality of embryos. Survival rates were reduced up to 90% with microcystin concentrations of 10 or 1 microg/ml (corresponding to 1-20 pg or 0.1-2 pg of toxin injected), injected either at stages 1, 19 or 25. Also, a dose dependent advanced embryonic hatching processing was observed; hatching being brought forward from 2 or 3 days compared to controls in most of the microcystin injected groups. In agreement with the known hepatotoxic effects of microcystin, injected embryos consistently displayed hepatobiliary abnormalities such as liver hypertrophy and hepatic hemorrhage, also evidenced in post-hatching juveniles. Thus, the methodology presented in this paper should be valuable tool to analyze the effects of toxins on the development of aquatic vertebrate embryos.

Combining computational prediction of cis-regulatory elements with a new enhancer assay to efficiently label neuronal structures in the medaka fish.

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.

Evidence for neural stem cells in the medaka optic tectum proliferation zones.

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.

Ol-insm1b, a SNAG family transcription factor involved in cell cycle arrest during medaka development.

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.

Morphological and gene expression similarities suggest that the ascidian neural gland may be osmoregulatory and homologous to vertebrate peri-ventricular organs.

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.