Search

BackgroundVertebrate eye formation requires coordinated inductive interactions between different embryonic tissue layers, first described in amphibians. A network of transcription factors and signaling molecules controls these steps, with mutations causing severe ocular, neuronal, and craniofacial defects. In eyeless mutant axolotls, eye morphogenesis arrests at the optic vesicle stage, before lens induction, and development of ventral forebrain structures is disrupted.ResultsWe identified a 5-bp deletion in the rax (retina and anterior neural fold homeobox) gene, which was tightly linked to the recessive eyeless (e) axolotl locus in an F2 cross. This frameshift mutation, in exon 2, truncates RAX protein within the homeodomain (P154fs35X). Quantitative RNA analysis shows that mutant and wild-type rax transcripts are equally abundant in E/e embryos. Translation appears to initiate from dual start codons, via leaky ribosome scanning, a conserved feature among gnathostome RAX proteins. Previous data show rax is expressed in the optic vesicle and diencephalon, deeply conserved among metazoans, and required for eye formation in other species.ConclusionThe eyeless axolotl mutation is a null allele in the rax homeobox gene, with primary defects in neural ectoderm, including the retinal and hypothalamic primordia.

The retinal ganglion cell (RGC) competence factor ATOH7 is dynamically expressed during retinal histogenesis. ATOH7 transcription is controlled by a promoter-adjacent primary enhancer and a remote shadow enhancer (SE). Deletion of the ATOH7 human SE causes nonsyndromic congenital retinal nonattachment (NCRNA) disease, characterized by optic nerve aplasia and total blindness. We used genome editing to model NCRNA in mice. Deletion of the murine SE reduces Atoh7 messenger RNA (mRNA) fivefold but does not recapitulate optic nerve loss; however, SEdel/knockout (KO) trans heterozygotes have thin optic nerves. By analyzing Atoh7 mRNA and protein levels, RGC development and survival, and chromatin landscape effects, we show that the SE ensures robust Atoh7 transcriptional output. Combining SE deletion and KO and wild-type alleles in a genotypic series, we determined the amount of Atoh7 needed to produce a normal complement of adult RGCs, and the secondary consequences of graded reductions in Atoh7 dosage. Together, these data reveal the workings of an evolutionary fail-safe, a duplicate enhancer mechanism that is hard-wired in the machinery of vertebrate retinal ganglion cell genesis.

In vertebrate retinal progenitor cells, the proneural factor Atoh7 exhibits a dynamic tissue and cellular expression pattern. Although the resulting Atoh7 retinal lineage contains all seven major cell types, only retinal ganglion cells require Atoh7 for proper differentiation. Such specificity necessitates complex regulation of Atoh7 transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous in vivo mouse genetic studies established the cell autonomous suppression of Atoh7 transcription by Notch1, Rbpj and Hes1. Here we identify four CSL binding sites within the Atoh7 proximal regulatory region and demonstrate Rbpj protein interaction at these sequences by in vitro electromobility shift, calorimetry and luciferase assays and, in vivo via colocalization and chromatin immunoprecipitation. We found that Rbpj simultaneously represses Atoh7 transcription using both Notch-dependent and -independent pathways.

The Atoh7 transcription factor catalyzes the rate-limiting step in the specification of retinal ganglion cells (RGCs). As a tool to study vertebrate retinal development, we validate an antibody that recognizes human and mouse Atoh7 polypeptide, using informative knockout and transgenic mouse tissues and overexpression experiments. The transient features of Atoh7 protein expression during retinal neurogenesis match the expected pattern at the tissue and cellular level. Further, we compare endogenous Atoh7 to established RGC markers, reporter mouse lines and cell cycle markers, demonstrating the utility of the antibody to investigate molecular mechanisms of retinal histogenesis.

The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Teadresponsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yapdependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosagedependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson's chorioretinal atrophy and congenital retinal coloboma. [ABSTRACT FROM AUTHOR]

This chapter provides an overview of the early developmental origins of six ocular tissues: the cornea, lens, ciliary body, iris, neural retina, and retina pigment epithelium. Many of these tissue types are concurrently specified and undergo a complex set of morphogenetic movements that facilitate their structural interconnection. Within the context of vertebrate eye organogenesis, we also discuss the genetic hierarchies of transcription factors and signaling pathways that regulate growth, patterning, cell type specification and differentiation., (© 2019 Elsevier Inc. All rights reserved.)