Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus ( Branchiostoma lanceolatum ) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis -regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that—in vertebrates—over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

The retina pigment epithelium (RPE) is a highly specialised epithelium that serves as a multifunctional and indispensable component of the vertebrate eye. Although a great deal of attention has been paid to its transdifferentiation capabilities and its ancillary functions in neural retina development, little is known about the molecular mechanisms that specify the RPE itself. Recent advances in our understanding of the genetic network that controls the progressive specification of the eye anlage in vertebrates have provided some of the initial cues to the mechanisms responsible for RPE patterning. Here, we have outlined many recent findings that suggest that a limited number of transcription factors, including Otx2, Mitf and Pax6 and a few signalling cascades, are the elements required for the onset of RPE specification in vertebrates. Furthermore, using this information and the data available on the specification of the pigmented cells of primitive chordates, we have ventured some hypotheses on the origin of RPE cells during evolution.

In vertebrates, midline-derived sonic hedgehog and nodal are crucial for the initial proximal-distal patterning of the eye. The establishment of the distal optic stalk is in turn a prerequisite to initiate retinogenesis. However, the signal that activates this process is unknown. Here, we demonstrate that in both chick and fish, the initiation of retinal differentiation is triggered by a species-specific localized Fgf signaling center that acts as mediator of the midline signals. The concerted activity of Fgf8 and Fgf3 is both necessary and sufficient to coordinate retinal differentiation independent of the connecting optic stalk.

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.

Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to induce RNA degradation in yeast, plants and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-Cas13d is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that both zygotically-expressed and maternally-provided transcripts are efficiently targeted, resulting in an 80% average decrease in transcript level and the recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish and mouse embryos. Altogether our results demonstrate that CRISPR-Cas13d is an efficient knock-down platform to interrogate gene function in animal embryos..

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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 opomediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup.

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.