ABSTRACT Defects in the evolutionarily conserved protein-glycosylation machinery during embryonic development are often fatal. Consequently, congenital disorders of glycosylation (CDG) in human are rare. We modelled a putative hypomorphic mutation described in an alpha-1,3/1,6-mannosyltransferase (ALG2) index patient (ALG2-CDG) to address the developmental consequences in the teleost medaka ( Oryzias latipes ). We observed specific, multisystemic, late-onset phenotypes, closely resembling the patient's syndrome, prominently in the facial skeleton and in neuronal tissue. Molecularly, we detected reduced levels of N -glycans in medaka and in the patient's fibroblasts. This hypo- N -glycosylation prominently affected protein abundance. Proteins of the basic glycosylation and glycoprotein-processing machinery were over-represented in a compensatory response, highlighting the regulatory topology of the network. Proteins of the retinal phototransduction machinery, conversely, were massively under-represented in the alg2 model. These deficiencies relate to a specific failure to maintain rod photoreceptors, resulting in retinitis pigmentosa characterized by the progressive loss of these photoreceptors. Our work has explored only the tip of the iceberg of N -glycosylation-sensitive proteins, the function of which specifically impacts on cells, tissues and organs. Taking advantage of the well-described human mutation has allowed the complex interplay of N -glycosylated proteins and their contribution to development and disease to be addressed.

ABSTRACT How the body and organs balance their relative growth is of key importance for coordinating size and function. This is of particular relevance in organisms, which continue to grow over their entire life span. We addressed this issue in the neuroretina of medaka fish ( Oryzias latipes ), a well-studied system with which to address vertebrate organ growth. We reveal that a central growth regulator, Igf1 receptor (Igf1r), is necessary and sufficient for proliferation control in the postembryonic retinal stem cell niche: the ciliary marginal zone (CMZ). Targeted activation of Igf1r signaling in the CMZ uncouples neuroretina growth from body size control, and we demonstrate that Igf1r operates on progenitor cells, stimulating their proliferation. Activation of Igf1r signaling increases retinal size while preserving its structural integrity, revealing a modular organization in which progenitor differentiation and neurogenesis are self-organized and highly regulated. Our findings position Igf signaling as a key module for controlling retinal size and composition, with important evolutionary implications.

ABSTRACT Yap1/Taz are well-known Hippo effectors triggering complex transcriptional programs controlling growth, survival and cancer progression. Here, we describe yap1b , a new Yap1 / Taz family member with a unique transcriptional activation domain that cannot be phosphorylated by Src/Yes kinases. We show that yap1b evolved specifically in euteleosts (i.e. including medaka but not zebrafish) by duplication and adaptation of yap1. Using DamID-seq, we generated maps of chromatin occupancy for Yap1, Taz (Wwtr1) and Yap1b in gastrulating zebrafish and medaka embryos. Our comparative analyses uncover the genetic programs controlled by Yap family proteins during early embryogenesis, and show largely overlapping targets for Yap1 and Yap1b. CRISPR/Cas9-induced mutation of yap1b in medaka does not result in an overt phenotype during embryogenesis or adulthood. However, yap1b mutation strongly enhances the embryonic malformations observed in yap1 mutants. Thus yap1 −/− ; yap1b −/− double mutants display more severe body flattening, eye misshaping and increased apoptosis than yap1 −/− single mutants, thus revealing overlapping gene functions. Our results indicate that, despite its divergent transactivation domain, Yap1b cooperates with Yap1 to regulate cell survival and tissue morphogenesis during early development.

ABSTRACT Patterning of a continuously growing naive field in the context of a life-long growing organ such as the teleost eye is of high functional relevance. Intrinsic and extrinsic signals have been proposed to regulate lineage specification in progenitors that exit the stem cell niche in the ciliary marginal zone (CMZ). The proper cell-type composition arising from those progenitors is a prerequisite for retinal function. Our findings in the teleost medaka ( Oryzias latipes ) uncover that the Notch-Atoh7 axis continuously patterns the CMZ. The complement of cell types originating from the two juxtaposed progenitors marked by Notch or Atoh7 activity contains all constituents of a retinal column. Modulation of Notch signalling specifically in Atoh7-expressing cells demonstrates the crucial role of this axis in generating the correct cell-type proportions. After transiently blocking Notch signalling, retinal patterning and differentiation is re-initiated de novo . Taken together, our data show that Notch activity in the CMZ continuously structures the growing retina by juxtaposing Notch and Atoh7 progenitors that give rise to distinct complementary lineages, revealing coupling of de novo patterning and cell-type specification in the respective lineages.

DNA adenine methyltransferase identification (DamID) has emerged as an alternative method to profile protein-DNA interactions; however, critical issues limit its widespread applicability. Here, we present iDamIDseq, a protocol that improves specificity and sensitivity by inverting the steps Dpn I- Dpn II and adding steps that involve a phosphatase and exonuclease. To determine genome-wide protein-DNA interactions efficiently, we present the analysis tool iDEAR (iDamIDseq Enrichment Analysis with R). The combination of DamID and iDEAR permits the establishment of consistent profiles for transcription factors, even in transient assays, as we exemplify using the small teleost medaka ( Oryzias latipes ). We report that the bacterial Dam-coding sequence induces aberrant splicing when it is used with different promoters to drive tissue-specific expression. Here, we present an optimization of the sequence to avoid this problem. This and our other improvements will allow researchers to use DamID effectively in any organism, in a general or targeted manner.

Recently, a stochastic model of symmetrical stem cell division followed by neutral drift has been proposed for intestinal stem cells (ISCs), which has been suggested to represent the predominant mode of stem cell progression in mammals. In contrast, stem cells in the retina of teleost fish show an asymmetric division mode. To address whether the mode of stem cell division follows phylogenetic or ontogenetic routes, we analysed the entire gastrointestinal tract of the teleost medaka ( Oryzias latipes ). X-ray microcomputed tomography shows a correlation of 3D topography with the functional domains. Analysis of ISCs in proliferation assays and via genetically encoded lineage tracing highlights a stem cell niche in the furrow between the long intestinal folds that is functionally equivalent to mammalian intestinal crypts. Stem cells in this compartment are characterized by the expression of homologs of mammalian ISC markers – sox9 , axin2 and lgr5 – emphasizing the evolutionary conservation of the Wnt pathway components in the stem cell niche of the intestine. The stochastic, sparse initial labelling of ISCs ultimately resulted in extended labelled or unlabelled domains originating from single stem cells in the furrow niche, contributing to both homeostasis and growth. Thus, different modes of stem cell division co-evolved within one organism, and in the absence of physical isolation in crypts, ISCs contribute to homeostatic growth.

Regenerative responses in the vertebrate CNS depend on quiescent radial glia stem cells, which re-enter the cell cycle and eventually differentiate into neurons. The entry into the cell cycle and the differentiation into neurons are events of opposite nature, and therefore efforts to force quiescent radial glia into neurons require different factors. Here, we use fish to show that a single neurogenic factor, Atoh7, directs retinal radial glia (Müller glia, MG) into proliferation. The resulting neurogenic clusters differentiate in vivo into various retinal neurons. We use signaling reporters to demonstrate that the Atoh7-induced regeneration-like response of MG cells is mimicked by Notch, resembling the behavior of early progenitors during retinogenesis. Activation of Notch signaling in MG cells is sufficient to trigger proliferation and differentiation. Our results uncover a new role for Atoh7 as a universal neurogenic factor, and illustrate how signaling modules are re-employed in diverse contexts to trigger different biological responses.

The troglomorphic phenotype shared by diverse cave-dwelling animals is regarded as a classical example of convergent evolution. One unresolved question is whether the characteristic eye loss in diverse cave species is based on interference with the same genetic program. Phreatichthys andruzzii , a Somalian cavefish, has evolved under constant conditions in complete darkness and shows severe troglomorphic characteristics, such as complete loss of eyes, pigments and scales. During early embryonic development, a complete eye is formed but is subsequently lost. In Astyanax mexicanus , another blind cavefish, eye loss has been attributed to interference during eye field patterning. To address whether similar pathways have been targeted by evolution independently, we investigated the retinal development of P. andruzzii , studying the expression of marker genes involved in eye patterning, morphogenesis, differentiation and maintenance. In contrast to Astyanax , patterning of the eye field and evagination of the optic vesicles proceeds without obvious deviation. However, the subsequent differentiation of retinal cell types is arrested during generation of the first-born cell type, retinal ganglion cells, which also fail to project correctly to the optic tectum. Eye degeneration in both species is driven by progressive apoptosis. However, it is retinal apoptosis in Phreatichthys that progresses in a wave-like manner and eliminates progenitor cells that fail to differentiate, in contrast to Astyanax , where lens apoptosis appears to serve as a driving force. Thus, evolution has targeted late retinal differentiation events, indicating that there are several ways to discontinue the development and maintenance of an eye.

The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic origin in the medaka retina. To analyze whether NSC potency is affected by developmental progression, as reported for the mammalian brain, we developed an inducible toolkit for clonal labeling and non-invasive fate tracking. We used this toolkit to address post-embryonic stem cells in different tissues and to functionally differentiate transient progenitor cells from permanent, bona fide stem cells in the retina. Using temporally controlled clonal induction, we showed that post-embryonic retinal NSCs are exclusively multipotent and give rise to the complete spectrum of cell types in the neural retina. Intriguingly, and in contrast to any other vertebrate stem cell system described so far, long-term analysis of clones indicates a preferential mode of asymmetric cell division. Moreover, following the behavior of clones before and after external stimuli, such as injuries, shows that NSCs in the retina maintained the preference for asymmetric cell division during regenerative responses. We present a comprehensive analysis of individual post-embryonic NSCs in their physiological environment and establish the teleost retina as an ideal model for studying adult stem cell biology at single cell resolution.

A unique feature of humans is the complexity of our central nervous system. A fully functional brain requires that billions of neurons make specific contacts in a highly coordinated way, an issue that is still not well understood. The neural retina constitutes an excellent system with which to analyze key aspects of neurogenesis and circuit formation in the central nervous system. Here, we provide an overview of retinal neurogenesis in vertebrates and discuss implications of the developmental mechanisms involved for regenerative therapy approaches.

Established transgenesis methods for fish model systems allow efficient genomic integration of transgenes. However, thus far a way of controlling copy number and integration sites has not been available, leading to variable transgene expression caused by position effects. The integration of transgenes at predefined genomic positions enables the direct comparison of different transgenes, thereby improving time and cost efficiency. Here, we report an efficient PhiC31-based site-specific transgenesis system for medaka. This system includes features that allow the pre-selection of successfully targeted integrations early on in the injected generation. Pre-selected embryos transmit the correctly integrated transgene through the germline with high efficiency. The landing site design enables a variety of applications, such as reporter and enhancer switch, in addition to the integration of any insert. Importantly, this allows assaying of enhancer activity in a site-specific manner without requiring germline transmission, thus speeding up large-scale analyses of regulatory elements.

The vertebrate central nervous system develops from an epithelium where cells are polarized along the apicobasal axis. Loss of this polarity results in abnormal organ architecture, morphology and proliferation. We found that mutations of the guanine nucleotide exchange factor ArhGEF18 affect apicobasal polarity of the retinal neuroepithelium in medaka fish. We show that ArhGEF18 -mediated activation of the small GTPase RhoA is required to maintain apicobasal polarity at the onset of retinal differentiation and to control the ratio of neurogenic to proliferative cell divisions. RhoA signals through Rock2 to regulate apicobasal polarity, tight junction localization and the cortical actin cytoskeleton. The human ArhGEF18 homologue can rescue the mutant phenotype, suggesting a conserved function in vertebrate neuroepithelia. Our analysis identifies ArhGEF18 as a key regulator of tissue architecture and function, controlling apicobasal polarity and proliferation through RhoA activation. We thus identify the control of neuroepithelial apicobasal polarity as a novel role for RhoA signaling in vertebrate development.

During embryogenesis, tissue specification is triggered by the expression of a unique combination of developmental genes and their expression in time and space is crucial for successful development. Synexpression groups are batteries of spatiotemporally co-expressed genes that act in shared biological processes through their coordinated expression. Although several synexpression groups have been described in numerous vertebrate species, the regulatory mechanisms that orchestrate their common complex expression pattern remain to be elucidated. Here we performed a pilot screen on 560 genes of the vertebrate model system medaka ( Oryzias latipes ) to systematically identify synexpression groups and investigate their regulatory properties by searching for common regulatory cues. We find that synexpression groups share DNA motifs that are arranged in various combinations into cis-regulatory modules that drive co-expression. In contrast to previous assumptions that these genes are located randomly in the genome, we discovered that genes belonging to the same synexpression group frequently occur in synexpression clusters in the genome. This work presents a first repertoire of synexpression group common signatures, a resource that will contribute to deciphering developmental gene regulatory networks.

Different levels and timing of Hedgehog (Hh) signalling activity have been proposed to specify three distinct cell types in the zebrafish myotome. Two of these, the medial fast-twitch fibres (MFFs) and the slow-twitch muscle pioneers (MPs) are characterised by expression of eng1a , - 1b and - 2a and require the highest levels of Hh for their specification. We have defined a minimal eng2a element sufficient to drive reporter expression specifically in MPs and MFFs. This element binds both Gli2a, a mediator of Hh signalling, and activated Smads (pSmads), mediators of bone morphogenic protein (BMP) signalling, in vivo. We found a strict negative correlation between nuclear accumulation of pSmad, and eng2a expression in myotomal cells and show that abrogation of pSmad accumulation results in activation of eng2a , even when Hh signalling is attenuated. Conversely, driving nuclear accumulation of pSmad suppresses the induction of eng expression even when Hh pathway activity is maximal. Nuclear accumulation of pSmads is depleted by maximal Hh pathway activation. We show that a synthetic form of the Gli2 repressor interacts with Smad1 specifically in the nuclei of myotomal cells in the developing embryo and that this interaction depends upon BMP signalling activity. Our results demonstrate that the eng2a promoter integrates repressive and activating signals from the BMP and Hh pathways, respectively, to limit its expression to MPs and MFFs. We suggest a novel basis for crosstalk between the Hh and BMP pathways, whereby BMP-mediated repression of Hh target genes is promoted by a direct interaction between Smads and truncated Glis, an interaction that is abrogated by Hh induced depletion of the latter.

Although the vertebrate retina is a well-studied paradigm for organogenesis, the morphogenetic mechanisms that carve the architecture of the vertebrate optic cup remain largely unknown. Understanding how the hemispheric shape of an eye is formed requires addressing the fundamental problem of how individual cell behaviour is coordinated to direct epithelial morphogenesis. Here, we analyze the role of ojoplano ( opo ), an uncharacterized gene whose human ortholog is associated with orofacial clefting syndrome, in the morphogenesis of epithelial tissues. Most notably,when opo is mutated in medaka fish, optic cup folding is impaired. We characterize optic cup morphogenesis in vivo and determine at the cellular level how opo affects this process. opo encodes a developmentally regulated transmembrane protein that localizes to compartments of the secretory pathway and to basal end-feet of the neuroepithelial precursors. We show that Opo regulates the polarized localization of focal adhesion components to the basal cell surface. Furthermore, tissue-specific interference with integrin-adhesive function impairs optic cup folding,resembling the ocular phenotype observed in opo mutants. We propose a model of retinal morphogenesis whereby opo -mediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup.

Recent studies on vertebrate eye development have focused on the molecular mechanisms of specification of different retinal cell types during development. Only a limited number of genes involved in this process has been identified. In Drosophila , BarH genes are necessary for the correct specification of R1/R6 eye photoreceptors. Vertebrate Bar homologues have been identified and are expressed in vertebrate retinal ganglion cells during differentiation; however, their retinal function has not yet been addressed. In this study, we report on the role of the Xenopus Bar homologue Xbh1 in retinal ganglion cell development and its interaction with the proneural genes Xath5 and Xath3 , whose ability to promote ganglion cell fate has been demonstrated. We show that XHB1 plays a crucial role in retinal cell determination, acting as a switch towards ganglion cell fate. Detailed expression analysis, animal cap assays and in vivo lipofection assays, indicate that Xbh1 acts as a late transcriptional repressor downstream of the atonal genes Xath3 and Xath5 . However, the action of Xbh1 on ganglion cell development is different and more specific than that of the Xath genes, and accounts for only a part of their activities during retinogenesis.

Six3 and Six6 are two genes required for the specification and proliferation of the eye field in vertebrate embryos,suggesting that they might be the functional counterparts of the Drosophila gene sine oculis (so) . Phylogenetic and functional analysis have however challenged this idea, raising the possibility that the molecular network in which Six3 and Six6 act may be different from that described for SO. To address this, we have performed yeast two-hybrid screens, using either Six3 or Six6 as a bait. In this paper,we report the results of the latter screen that led to the identification of TLE1 (a transcriptional repressor of the groucho family) and AES (a potential dominant negative form of TLE proteins) as cofactors for both SIX6 and SIX3. Biochemical and mutational analysis shows that the Six domain of both SIX3 and SIX6 strongly interact with the QD domain of TLE1 and AES, but that SIX3 also interacts with TLE proteins via the WDR domain. Tle1 and Aes are expressed in the developing eye of medaka fish( Oryzias latipes ) embryos, overlapping with the distribution of both Six3 and Six6 . Gain-of-function studies in medaka show a clear synergistic activity between SIX3/SIX6 and TLE1 ,which, on its own, can expand the eye field. Conversely, AES alone decreases the eye size and abrogates the phenotypic consequences of SIX3/6 over-expression. These data indicate that both Tle1 and Aes participate in the molecular network that control eye development and are consistent with the view that both Six3 and Six6 act in combination with either Tle1 and/or Aes.

The establishment of retinal identity and the subsequent patterning of the optic vesicle are the key steps in early vertebrate eye development. To date little is known about the nature and interaction of the genes controlling these steps. So far few genes have been identified that, when over-expressed, can initiate ectopic eye formation. Of note is Six3 , which is expressed exclusively in the anterior neural plate. However, ‘loss of function’ analysis has not been reported. Using medaka fish, we show that vertebrate Six3 is necessary for patterning of the anterior neuroectoderm including the retina anlage. Inactivation of Six3 function by morpholino knock-down results in the lack of forebrain and eyes. Corroborated by gain-of-function experiments, graded interference reveals an additional role of Six3 in the proximodistal patterning of the optic vesicle. During both processes of vertebrate eye formation, Six3 cooperates with Pax6 .

The role of Pax6 in eye development in insects and vertebrates supports the view that their eyes evolved from simple pigment-cup ocelli present in their last common ancestors (Urbilateria). The cerebral eyes in errant polychaetes represent prototype invertebrate pigment-cup ocelli and thus resemble the presumed ancestral eyes. We have analysed expression of conserved eye specification genes in the early development of larval and adult pigment-cup eyes in Platynereis dumerilii (Polychaeta, Annelida, Lophotrochozoa). Both larval and adult eyes form in close vicinity of the optic anlagen on both sides of the developing brain ganglia. While pax6 is expressed in the larval, but not in the developing, adult eyes, expression of six1/2 from trochophora stages onwards specifically outlines the optic anlagen and thus covers both the developing larval and adult eyes. Using Platynereis rhabdomeric opsin as differentiation marker, we show that the first pair of adult eye photoreceptor cells is detected within bilateral clusters that transitorily express ath , the Platynereis atonal orthologue, thus resembling proneural sensory clusters. Our data indicate that – similar to insects, but different from the vertebrates – polychaete six1/2 expression outlines the entire visual system from early developmental stages onwards and ath -positive clusters generate the first photoreceptor cells to appear. We propose that pax6 -, six1/2- and ath-positive larval eyes, as found in today’s trochophora, were present already in Urbilateria.