Retinogenesis of the Human Fetal Retina: An Apical Polarity Perspective

Abstract

The Crumbs complex has prominent roles in the control of apical cell polarity, in the coupling of cell density sensing to downstream cell signaling pathways, and in regulating junctional structures and cell adhesion. The Crumbs complex acts as a conductor orchestrating multiple downstream signaling pathways in epithelial and neuronal tissue development. These pathways lead to the regulation of cell size, cell fate, cell self-renewal, proliferation, differentiation, migration, mitosis, and apoptosis. In retinogenesis, these are all pivotal processes with important roles for the Crumbs complex to maintain proper spatiotemporal cell processes. Loss of Crumbs function in the retina results in loss of the stratified appearance resulting in retinal degeneration and loss of visual function. In this review, we begin by discussing the physiology of vision. We continue by outlining the processes of retinogenesis and how well this is recapitulated between the human fetal retina and human embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC)-derived retinal organoids. Additionally, we discuss the functionality of in utero and preterm human fetal retina and the current level of functionality as detected in human stem cell-derived organoids. We discuss the roles of apical-basal cell polarity in retinogenesis with a focus on Leber congenital amaurosis which leads to blindness shortly after birth. Finally, we discuss Crumbs homolog (CRB)-based gene augmentation.

Keywords: Leber congenital amaurosis; PAR complex; adeno-associated virus (AAV); apical polarity; crumbs complex; fetal retina; gene augmentation; retinal organoids; retinogenesis.

Figure 1

Transmission of light. (A) Light is electromagnetic radiation that travels as waves consisting of perpendicular oscillating electric and magnetic fields. (B) Visible light is a narrow group of wavelengths between approximately 400 nm (short wavelength) and 760 nm (long wavelength) which we interpret as a spectrum of different colors. Wavelengths outside this range are not visible to humans. (C) Light can be reflected, absorbed and refracted.

Figure 2

Processing of light. (A) Schematic picture of the eye. The eye is comprised of the aqueous humor, ciliary body, cornea, iris, lens, optic nerve, pupil, retina, retinal pigment epithelium, retinal vasculature, sclera, vitreous body, and zonal fibers. When light first enters the eye, it is refracted by the cornea through the pupil, whose size is controlled by the iris. The iris, the colored part of the eye, controls the amount of light entering the eye while the lens focuses the light through the vitreous humor and on to the proximal surface of the retina. (B) Schematic picture of the retina. The retina is composed of seven cell types: amacrine cells (red), bipolar cells (blue), cones (orange), ganglion cells (green), horizontal cells (purple), Müller glial cells (yellow), and rods (pink). When light first enters the retina, it goes through the ganglion cell layer (GCL), then the inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), and outer nuclear layer (ONL). As light is passing through the retina it is absorbed by its light responsive cells: rod and cone photoreceptors and the intrinsically-photosensitive retinal ganglion cells (ipRGCs). This creates electrophysiological signals that then are further propagated to the inner retina and can be propagated through many different cell to cell pathways to the ganglion cells. (C) Schematic picture of the visual pathway. The axons of the retinal ganglion cells exit the eyes as bundles, the optic nerve, and extend to the optic chiasm were the nasal axons of each eye crossover and combine with the contralateral eyes temporal axons and subsequently via the optic tract travel to the lateral geniculate nucleus (LGN) and superior colliculus (SC). The LGN, SC, and pulvinar nucleus are all involved in the process of relaying and refining visual information to the visual cortex. Visual information is relayed to the visual cortex via optic radiations which extend from the LGN.

Figure 3

The organization of the developing retina. Schematic picture of early retinal development. From the blastocyst which contains the pluripotent cell mass gastrulation and neurulation occur forming the neural plate. The eye field specifies at the medial region of the anterior neural plate and contains all the progenitors which go on to form all the neural-derived cell types and structures of the eye. Bilateral optic sulci develop from the eye field forming the optic vesicles which extend towards the surface ectoderm. The optic vesicles invaginate forming the two-layered optic cups and the lens vesicle forms and sits behind the surface ectoderm. The outer layer of the optic cup remains as a single cuboidal layer becoming the retinal pigment epithelium. The single inner layer of the optic cup proliferates and differentiates forming the multilayered neural retina. EF: eye field; CMZ: ciliary marginal zone.

Figure 4

Retinogenesis. (A) Radial progenitor cells undergoing interkinetic nuclear migration during cell cycle phases G1, S, G2, and M. The mitosis (M) phase takes place at the apical side, whereas the DNA synthesis (S) phase takes place more basally. (B) Symmetric versus asymmetric cell division. (C) Genesis of retinal cells born during the development of the human eye can be divided into an early phase (ganglion cells, cone photoreceptors, horizontal cells, and amacrine cells) and an overlapping late phase (rod photoreceptors, Müller glial cells, and bipolar cells; see Aldiri et al. 2017 [40]). FWK—fetal week.

Figure 5

Tangential and Radial migration. (A) Tangential migration can be described in three steps: (1) Early born cell type progenitors localize to their correct laminar position (Cones: orange, Bipolar cells: purple, amacrine cells: red, ganglion cells: green), (2) they undergo morphological differentiation, (3) tangential migration coincides with morphological differentiation allowing subsets of early born cell types to move a short distance within their laminar position (see Reese et al. 1999 [43]). (B) Glial cell-guided, apically born neurons become initially detached and subsequently attach to radial glial progenitor cells. They then migrate along the radial glial progenitor cells to the target laminar location where they fully integrate. (C) Somal translocation, apically born nuclei can move along there inherited basally attached process from. Once they move to their final laminar location they fully integrate (This process can also occur with only apically inherited processes). (D) Multipolar migratory mode, in rare case apically born neurons can loses both apical and basal attachments but can move to their final laminar position and integrate due to a multipolar mode. (E) No translocation, inefficient migration due to retention of the apical or basal process and slow release of opposing process. For further details see Icha et al. 2016 and Amini et al. 2018 [45,47].

Figure 6

Schematic picture of Epithelial polarity. (A) Epithelial cell polarity consist of apical-cell polarity and orthogonal to this axis planar cell polarity (PCP). PCP is the collective alignment of cell polarity across the tissue plane and involves asymmetric segregation of proximal (purple) and distal (blue) PCP components. Apical-basal polarity involves the antagonistic, functional, and spatial segregation of apical (red) and basal (black) components. (B) In the retina the CRB and PAR complexes are located at the sub-apical region (green), below the apical surface (red) and adjacent to the adherens junctions (yellow). Scribbled (SCRIB), discs large (DLG), and lethal giant larvae (LGL) form a basolateral domain (black) extending below the adherens junctions. (C) Schematic overview of the prototypic CRB1, CRB2, and CRB3 proteins. Mutations from the three iPSC CRB1 RP patients lines recently derived to retinal organoids are mapped to their protein location in CRB1. Adapted from Quinn et al. 2017 [144].

Figure 7

Schematic picture of the localization of the CRB proteins in mammalian adult retinal Müller glial and photoreceptor cells. (A–E) CRB1 (red) and CRB2 (blue) proteins localize at the subapical region in mouse (A) and human (B) retinal progenitors cells and adult Müller glial cells of mice (C), non-human primates (D) and humans (E). However, the expression of CRB1 in the early developing human retina is low and sporadic. Additionally, the expression patterns of CRB1 and CRB2 at the subapical region of adult photoreceptors differs between the three species. CRB2 is present at the subapical region of mouse photoreceptors, whereas CRB1 is not (C). In non-human primates both CRB1 and CRB2 are present in photoreceptors (D). In cadaveric human retinas collected 2-days after death CRB1 is present at the subapical region whereas CRB2 is present in the photoreceptor inner segments but at some distance from the subapical region (E). PRC: Photoreceptor; MGC: Müller glial cells.

Figure 8

Disruptions at the outer limiting membrane in retinal organoids from CRB1 retinitis pigmentosa (RP) patients. Schematic depiction of healthy (A) and CRB1 patient (B,C) derived retinal organoids. (A) In healthy organoids CRB1 is located at the subapical region adjacent to the adherens junctions. (B,D) In retinal organoids from CRB1 retinitis pigmentosa patient (LUMC0116iCRB) disruptions of the outer limiting membrane (OLM) and ectopic photoreceptors have been found. (C,E) Additionally, budding of the apical membrane and mislocalization of the CRB1 variant in the NBL also occurs. Scale bars (D,E), 20 µm. Panels (C,D) are modified from [178] under a Creative Commons license.