Generation and Staging of Human Retinal Organoids Based on Self-Formed Ectodermal Autonomous Multi-Zone System

Methods for stem cell-derived, three-dimensional retinal organoids induction have been established and shown great potential for retinal development modeling and drug screening. Herein, we reported an exogenous-factors-free and robust method to generate retinal organoids based on “self-formed ectodermal autonomous multi-zone” (SEAM) system, a two-dimensional induction scheme that can synchronously generate multiple ocular cell lineages. Characterized by distinct morphological changes, the differentiation of the obtained retinal organoids could be staged into the early and late differentiation phases. During the early differentiation stage, retinal ganglion cells, cone photoreceptor cells (PRs), amacrine cells, and horizontal cells developed; whereas rod PRs, bipolar cells, and Müller glial cells were generated in the late differentiation phase, resembling early-phase and late-phase retinogenesis in vivo. Additionally, we modified the maintenance strategy for the retinal organoids and successfully promoted their long-term survival. Using 3D immunofluorescence image reconstruction and transmission electron microscopy, the substantial mature PRs with outer segment, inner segment and ribbon synapse were demonstrated. Besides, the retinal pigment epithelium (RPE) was induced with distinct boundary and the formation of ciliary margin was observed by co-suspending retina organoids with the zone containing RPE. The obtained RPE could be expanded and displayed similar marker expression, ultrastructural feature and functional phagocytosis to native RPE. Thus, this research described a simple and robust system which enabled generation of retina organoids with substantial mature PRs, RPE and the ciliary margin without the need of exogenous factors, providing a new platform for research of retinogenesis and retinal translational application.

Phase transition specified by a binary code patterns the vertebrate eye cup

The developing vertebrate eye cup is partitioned into the neural retina (NR), the retinal pigmented epithelium (RPE) and the ciliary margin (CM). By single cell analysis, we showed that a gradient of FGF signaling regulates demarcation and subdivision of the CM and controls its stem cell-like property of self-renewal, differentiation and survival. This regulation by FGF is balanced by an evolutionarily conserved Wnt signaling gradient induced by the lens ectoderm and the periocular mesenchyme, which specifies the CM and the distal RPE. These two morphogen gradients converge in the CM where FGF signaling promotes Wnt signaling by stabilizing β-catenin in a GSK3β-independent manner. We further showed that activation of Wnt signaling converts the NR to either the CM or the RPE depending on the level of FGF signaling. Conversely, activation of FGF transforms the RPE to the NR or CM dependent on Wnt activity. We demonstrated that the default fate of the eye cup is the NR, but synergistic FGF and Wnt signaling promotes CM formation both in vivo and in retinal organoid culture of human iPS cells. Our study reveals that the vertebrate eye develops through phase transition determined by a combinatorial code of FGF and Wnt signaling.

The transcription factor Maz is essential for normal eye development

ABSTRACTWnt/β-catenin signaling has an essential role in eye development. Faulty regulation of this pathway results in ocular malformations, owing to defects in cell-fate determination and differentiation. Herein, we show that disruption of Maz, the gene encoding Myc-associated zinc-finger transcription factor, produces developmental eye defects in mice and humans. Expression of key genes involved in the Wnt cascade, Sfrp2, Wnt2b and Fzd4, was significantly increased in mice with targeted inactivation of Maz, resulting in abnormal peripheral eye formation with reduced proliferation of the progenitor cells in the region. Paradoxically, the Wnt reporter TCF-Lef1 displayed a significant downregulation in Maz-deficient eyes. Molecular analysis indicates that Maz is necessary for the activation of the Wnt/β-catenin pathway and participates in the network controlling ciliary margin patterning. Copy-number variations and single-nucleotide variants of MAZ were identified in humans that result in abnormal ocular development. The data support MAZ as a key contributor to the eye comorbidities associated with chromosome 16p11.2 copy-number variants and as a transcriptional regulator of ocular development.

Deficient FGF signaling in the developing peripheral retina disrupts ciliary margin development and causes aniridia

ABSTRACTThe mammalian ciliary margin is a part of the developing peripheral neural retina that differentiates into the ciliary body and the iris. Canonical WNT signaling plays a critical role in the specification of the ciliary margin at the peripheral retina in the presence of strong FGF signaling in the central retina. The mechanism of how the boundary between the central retina and the ciliary margin is created has not been previously elucidated. Using genetic ablation and epistasis experiments, we show that loss of FGF signaling gradient in the peripheral retina causes expansion of WNT signaling towards the central retina thereby disrupting the neurogenic boundary and compartmentalization of the ciliary margin. Loss of WNT signaling displays a complimentary effect with expansion of FGF signaling into the ciliary marginal space. Using in vivo experiments, we elucidate the FGF signaling cascade involved in development of the ciliary margin. We also identify the surface ectoderm as the source of WNT ligands in eliciting WNT response at the ciliary margin. We show that an interaction between FGF and WNT signaling is required for generation of the ciliary marginal cells. Taken together, our results reveal that a gradient intersection of FGF and WNT signaling is required for specification of the ciliary margin.