Comparative 3D genome analysis between neural retina and retinal pigment epithelium reveals differential cis-regulatory interactions at retinal disease loci.

BACKGROUND: Vision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space. RESULTS: To understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4, which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis-regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4. CONCLUSIONS: Through comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases.

Evolutionary conservation of embryonic DNA methylome remodelling in distantly related teleost species.

Methylation of cytosines in the CG context (mCG) is the most abundant DNA modification in vertebrates that plays crucial roles in cellular differentiation and identity. After fertilization, DNA methylation patterns inherited from parental gametes are remodelled into a state compatible with embryogenesis. In mammals, this is achieved through the global erasure and re-establishment of DNA methylation patterns. However, in non-mammalian vertebrates like zebrafish, no global erasure has been observed. To investigate the evolutionary conservation and divergence of DNA methylation remodelling in teleosts, we generated base resolution DNA methylome datasets of developing medaka and medaka-zebrafish hybrid embryos. In contrast to previous reports, we show that medaka display comparable DNA methylome dynamics to zebrafish with high gametic mCG levels (sperm: ∼90%; egg: ∼75%), and adoption of a paternal-like methylome during early embryogenesis, with no signs of prior DNA methylation erasure. We also demonstrate that non-canonical DNA methylation (mCH) reprogramming at TGCT tandem repeats is a conserved feature of teleost embryogenesis. Lastly, we find remarkable evolutionary conservation of DNA methylation remodelling patterns in medaka-zebrafish hybrids, indicative of compatible DNA methylation maintenance machinery in far-related teleost species. Overall, these results suggest strong evolutionary conservation of DNA methylation remodelling pathways in teleosts, which is distinct from the global DNA methylome erasure and reestablishment observed in mammals.

The Shh/Gli3 gene regulatory network precedes the origin of paired fins and reveals the deep homology between distal fins and digits.

One of the central problems of vertebrate evolution is understanding the relationship among the distal portions of fins and limbs. Lacking comparable morphological markers of these regions in fish and tetrapods, these relationships have remained uncertain for the past century and a half. Here we show that Gli3 functions in controlling the proliferative expansion of distal progenitors are shared among dorsal and paired fins as well as tetrapod limbs. Mutant knockout gli3 fins in medaka (Oryzias latipes) form multiple radials and rays, in a pattern reminiscent of the polydactyly observed in Gli3-null mutant mice. In limbs, Gli3 controls both anterior-posterior patterning and cell proliferation, two processes that can be genetically uncoupled. In situ hybridization, quantification of proliferation markers, and analysis of regulatory regions reveal that in paired and dorsal fins, gli3 plays a main role in controlling proliferation but not in patterning. Moreover, gli3 down-regulation in shh mutant fins rescues fin loss in a manner similar to how Gli3 deficiency restores digits in the limbs of Shh mutant mouse embryos. We hypothesize that the Gli3/Shh gene pathway preceded the origin of paired appendages and was originally involved in modulating cell proliferation. Accordingly, the distal regions of dorsal fins, paired fins, and limbs retain a deep regulatory and functional homology that predates the origin of paired appendages.

Analysis of gene network bifurcation during optic cup morphogenesis in zebrafish.

Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.

Genetic developmental timing revealed by inter-species transplantations in fish.

The path from a fertilised egg to an embryo involves the coordinated formation of cell types, tissues and organs. Developmental modules comprise discrete units specified by self-sufficient genetic programs that can interact with each other during embryogenesis. Here, we have taken advantage of the different span of embryonic development between two distantly related teleosts, zebrafish (Danio rerio) and medaka (Oryzias latipes) (3 and 9 days, respectively), to explore modularity principles. We report that inter-species blastula transplantations result in the ectopic formation of a retina formed by donor cells - a module. We show that the time taken for the retina to develop follows a genetic program: an ectopic zebrafish retina in medaka develops with zebrafish dynamics. Heterologous transplantation results in a temporal decoupling between the donor retina and host organism, illustrated by two paradigms that require retina-host interactions: lens recruitment and retino-tectal projections. Our results uncover a new experimental system for addressing temporal decoupling along embryonic development, and highlight the presence of largely autonomous but interconnected developmental modules that orchestrate organogenesis.

Retina Development in Vertebrates: Systems Biology Approaches to Understanding Genetic Programs: On the Contribution of Next-Generation Sequencing Methods to the Characterization of the Regulatory Networks Controlling Vertebrate Eye Development.

The ontogeny of the vertebrate retina has been a topic of interest to developmental biologists and human geneticists for many decades. Understanding the unfolding of the genetic program that transforms a field of progenitors cells into a functionally complex and multi-layered sensory organ is a formidable challenge. Although classical genetic studies succeeded in identifying the key regulators of retina specification, understanding the architecture of their gene network and predicting their behavior are still a distant hope. The emergence of next-generation sequencing platforms revolutionized the field unlocking the access to genome-wide datasets. Emerging techniques such as RNA-seq, ChIP-seq, ATAC-seq, or single cell RNA-seq are used to characterize eye developmental programs. These studies provide valuable information on the transcriptional and cis-regulatory profiles of precursors and differentiated cells, outlining the trajectories that connect each intermediate state. Here, recent systems biology efforts are reviewed to understand the genetic programs shaping the vertebrate retina.

yap1b, a divergent Yap/Taz family member, cooperates with yap1 in survival and morphogenesis via common transcriptional targets.

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.

Genetics of congenital eye malformations: insights from chick experimental embryology.

Embryological manipulations in chick embryos have been pivotal in our understanding of many aspects of vertebrate eye formation. This research was particularly important in uncovering the role of tissue interactions as drivers of eye morphogenesis and to dissect the function of critical genes. Here, we have highlighted a few of these past experiments to endorse their value in searching for hitherto unknown causes of rare congenital eye anomalies, such as microphthalmia, anophthalmia and coloboma. We have also highlighted a number of similarities between the chicken and human eye, which might be exploited to address other eye pathologies, including degenerative ocular diseases.

A conserved Shh cis-regulatory module highlights a common developmental origin of unpaired and paired fins.

Despite their evolutionary, developmental and functional importance, the origin of vertebrate paired appendages remains uncertain. In mice, a single enhancer termed ZRS is solely responsible for Shh expression in limbs. Here, zebrafish and mouse transgenic assays trace the functional equivalence of ZRS across the gnathostome phylogeny. CRISPR/Cas9-mediated deletion of the medaka (Oryzias latipes) ZRS and enhancer assays identify the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolishes shh expression and completely truncates pectoral fin formation. Strikingly, deletion of ZRS results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and median fins and that paired fins likely emerged by the co-option of developmental programs established in the median fins of stem gnathostomes. Shh function was later reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness.

Coordinated Morphogenetic Mechanisms Shape the Vertebrate Eye.

The molecular bases of vertebrate eye formation have been extensively investigated during the past 20 years. This has resulted in the definition of the backbone of the gene regulatory networks controlling the different steps of eye development and has further highlighted a substantial conservation of these networks among vertebrates. Yet, the precise morphogenetic events allowing the formation of the optic cup from a small group of cells within the anterior neural plate are still poorly understood. It is also unclear if the morphogenetic events leading to eyes of very similar shape are indeed comparable among all vertebrates or if there are any species-specific peculiarities. Improved imaging techniques have enabled to follow how the eye forms in living embryos of a few vertebrate models, whereas the development of organoid cultures has provided fascinating tools to recapitulate tissue morphogenesis of other less accessible species. Here, we will discuss what these advances have taught us about eye morphogenesis, underscoring possible similarities and differences among vertebrates. We will also discuss the contribution of cell shape changes to this process and how morphogenetic and patterning mechanisms integrate to assemble the final architecture of the eye.

Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo.

Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina.

Report of the European Zebrafish Principal Investigator Meeting in Padua, Italy, March 18–22, 2010.

Abstract The zebrafish community has been steadily growing in the last 20 years in Europe. Given the federal structure of Europe, this increase in zebrafish research generated a need for a strategic forum to identify and discuss exciting new areas of research and funding opportunities as well as to address infrastructural and legal issues of experimentation, transport, and husbandry of zebrafish. To foster this exchange, the European Union (EU)-funded network EuFishBioMed (Cost Action BM0804) organized an international scientific meeting of zebrafish principal investigators in Padova, Italy, in March this year. More than 120 researchers from all over the globe presented their latest work in talks and posters. A number of workshops addressed future directions of research and infrastructural issues.

Shaping the vertebrate eye.

For over a century, the vertebrate eye has served as a paradigm for organogenesis. It forms through a complex sequence of morphogenetic events, involving the lateral evagination of the optic vesicles and their subsequent folding into the optic cups. Through intensive studies by experimental embryologists, anatomical descriptions of the process were available since many decades. Recent genetic and molecular work has illuminated essential features of the stereotyped cellular behaviour driving eye morphogenesis. The first pieces of the molecular machinery operating in each individual progenitor cell have been identified. These studies now set the groundwork for a system-wide approach towards understanding the cellular and molecular mechanisms involved in shaping the vertebrate eye.

A global survey identifies novel upstream components of the Ath5 neurogenic network.

BACKGROUND: Investigating the architecture of gene regulatory networks (GRNs) is essential to decipher the logic of developmental programs during embryogenesis. In this study we present an upstream survey approach, termed trans-regulation screen, to comprehensively identify the regulatory input converging on endogenous regulatory sequences. RESULTS: Our dual luciferase-based screen queries transcriptome-scale collections of cDNAs. Using this approach we study the regulation of Ath5, the central node in the GRN controlling retinal ganglion cell (RGC) specification in vertebrates. The Ath5 promoter integrates the input of upstream regulators to enable the transient activation of the gene, which is an essential step for RGC differentiation. We efficiently identified potential Ath5 regulators that were further filtered for true positives by an in situ hybridization screen. Their regulatory activity was validated in vivo by functional assays in medakafish embryos. CONCLUSIONS: Our analysis establishes functional groups of genes controlling different regulatory phases, including the onset of Ath5 expression at cell-cycle exit and its down-regulation prior to terminal RGC differentiation. These results extent the current model of the GRN controlling retinal neurogenesis in vertebrates.

Cloning of mouse ojoplano, a reticular cytoplasmic protein expressed during embryonic development.

Ojoplano (Opo) is a morphogenetic gene playing an important role during embryogenesis in medaka. This report focuses on the identification and characterization of the mouse Opo gene. We examined Opo expression by whole-mount in situ hybridization and in situ hybridization on sagittal sections during mouse embryogenesis. First expression in whole-mounts was detected at Theiler stages 15-17 (E 9.5-10.5dpc) as a spotted specific staining in migrating neural crest cells and in placodal structures. A complex expression pattern was observed in Theiler stage 22-23 (E 14.5dpc) in sagittal sections, including expression in skeletal structures (skull, vertebrae, ribs, bones of the locomotor system), in the nasal region, the heart and the eye. Fusion proteins revealed the localization of OPO within the cytoplasm with a reticular distribution that largely overlapped with the endoplasmic reticulum. Opo shows homology to human transcripts linked to a hereditary craniofacial malformation, orofacial cleft 1 (OFC1). The expression of mouse Opo in neural crest derivatives and skull elements further supports this link.

ojoplano-mediated basal constriction is essential for optic cup morphogenesis.

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.

New genes in the evolution of the neural crest differentiation program.

BACKGROUND: Development of the vertebrate head depends on the multipotency and migratory behavior of neural crest derivatives. This cell population is considered a vertebrate innovation and, accordingly, chordate ancestors lacked neural crest counterparts. The identification of neural crest specification genes expressed in the neural plate of basal chordates, in addition to the discovery of pigmented migratory cells in ascidians, has challenged this hypothesis. These new findings revive the debate on what is new and what is ancient in the genetic program that controls neural crest formation. RESULTS: To determine the origin of neural crest genes, we analyzed Phenotype Ontology annotations to select genes that control the development of this tissue. Using a sequential blast pipeline, we phylogenetically classified these genes, as well as those associated with other tissues, in order to define tissue-specific profiles of gene emergence. Of neural crest genes, 9% are vertebrate innovations. Our comparative analyses show that, among different tissues, the neural crest exhibits a particularly high rate of gene emergence during vertebrate evolution. A remarkable proportion of the new neural crest genes encode soluble ligands that control neural crest precursor specification into each cell lineage, including pigmented, neural, glial, and skeletal derivatives. CONCLUSION: We propose that the evolution of the neural crest is linked not only to the recruitment of ancestral regulatory genes but also to the emergence of signaling peptides that control the increasingly complex lineage diversification of this plastic cell population.

Proper patterning of the optic fissure requires the sequential activity of BMP7 and SHH.

The optic disc develops at the interface between optic stalk and retina, and enables both the exit of visual fibres and the entrance of mesenchymal cells that will form the hyaloid artery. In spite of the importance of the optic disc for eye function, little is known about the mechanisms that control its development. Here, we show that in mouse embryos, retinal fissure precursors can be recognised by the expression of netrin 1 and the overlapping distribution of both optic stalk (Pax2, Vax1) and ventral neural retina markers (Vax2, Raldh3). We also show that in the absence of Bmp7, fissure formation is not initiated. This absence is associated with a reduced cell proliferation and apoptosis in the proximoventral quadrant of the optic cup, lack of the hyaloid artery, optic nerve aplasia, and intra-retinal misrouting of RGC axons. BMP7 addition to organotypic cultures of optic vesicles from Bmp7-/- embryos rescues Pax2 expression in the ventral region, while follistatin, a BMP7 antagonist, prevents it in early, but not in late, optic vesicle cultures from wild-type embryos. The presence of Pax2-positive cells in late optic cup is instead abolished by interfering with Shh signalling. Furthermore, SHH addition re-establishes Pax2 expression in late optic cups derived from ocular retardation (or) embryos, where optic disc development is impaired owing to the near absence of SHH-producing RGC. Collectively, these data indicate that BMP7 is required for retinal fissure formation and that its activity is needed, before SHH signalling, for the generation of PAX2-positive cells at the optic disc.