Edward M. Levine

Retinal remodeling triggered by photoreceptor degenerations

Many photoreceptor degenerations initially affect rods, secondarily leading to cone death. It has long been assumed that the surviving neural retina is largely resistant to this sensory deafferentation. New evidence from fast retinal degenerations reveals that subtle plasticities in neuronal form and connectivity emerge early in disease. By screening mature natural, transgenic, and knockout retinal degeneration models with computational molecular phenotyping, we have found an extended late phase of negative remodeling that radically changes retinal structure. Three major transformations emerge: 1) Müller cell hypertrophy and elaboration of a distal glial seal between retina and the choroid/retinal pigmented epithelium; 2) apparent neuronal migration along glial surfaces to ectopic sites; and 3) rewiring through evolution of complex neurite fascicles, new synaptic foci in the remnant inner nuclear layer, and new connections throughout the retina. Although some neurons die, survivors express molecular signatures characteristic of normal bipolar, amacrine, and ganglion cells. Remodeling in human and rodent retinas is independent of the initial molecular targets of retinal degenerations, including defects in the retinal pigmented epithelium, rhodopsin, or downstream phototransduction elements. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, it suggests that the neural retina may be more plastic than previously believed. J. Comp. Neurol. 464:1–16, 2003.

Multipotential stem cells and progenitors in the vertebrate retina

The vertebrate retina is derived from paired evaginations from the neural tube in embryonic development and is initially produced by progenitor cells similar to those that generate the neurons and glia of other areas of the central nervous system. In some amphibians and fish, the retina continues to grow along with the eye throughout the life of the animal. The new retinal cells are added at the ciliary margin of the eye from the mitotic activity of neural/glial stem cells in a region known as the germinal zone and are seamlessly incorporated into the existing retinal circuitry. Little is known about the cell or molecular biology of these stem cells; however, studies of retinal progenitor cells in chick and mammalian embryos have led to the identification of several factors that control their proliferation. Moreover, studies of retinal regeneration have shown that retinal stem cells can also be derived from two or perhaps three additional sources after retinal damage: (a) the retinal pigmented epithelium (RPE) in amphibians and embryonic chicks and mammals; (b) a specialized rod progenitor in fish; and (c) the Müller glial cells. While there is currently no evidence for a neural/glial stem cell in the adult mammalian retina, and the retina of the mature mammal does not show regenerative capacity after damage, there is a possibility for the reinitiation of stem cell potential at the peripheral retinal margin, from the RPE or from the Müller glial cells. The application of information derived from the studies of retinal progenitor cells in developing organisms should soon provide a test of these possibilities.

Genetic rescue of cell number in a mouse model of microphthalmia: interactions between Chx10 and G1-phase cell cycle regulators

Insufficient cell number is a primary cause of failed retinal development in the Chx10 mutant mouse. To determine if Chx10 regulates cell number by antagonizing p27Kip1 activity, we generated Chx10, p27Kip1 double null mice. The severe hypocellular defect in Chx10 single null mice is alleviated in the double null, and while Chx10-null retinas lack lamination, double null retinas have near normal lamination. Bipolar cells are absent in the double null retina, a defect that is attributable to a requirement for Chx10 that is independent of p27Kip1. We find that p27Kip1 is abnormally present in progenitors of Chx10-null retinas, and that its ectopic localization is responsible for a significant amount of the proliferation defect in this microphthalmia model system. mRNA and protein expression patterns in these mice and in cyclin D1-null mice suggest that Chx10 influences p27Kip1 at a post-transcriptional level, through a mechanism that is largely dependent on cyclin D1. This is the first report of rescue of retinal proliferation in a microphthalmia model by deletion of a cell cycle regulatory gene.

Soluble factors and the development of rod photoreceptors

Abstract. Photoreceptors are the most abundant cell type in the vertebrate neural retina. Like the other retinal neurons and the Müller glia, they arise from a population of precursor cells that are multipotent and intrinsic to the retina. Approximately 10 years ago, several studies demonstrated that retinal precursor cells (RPCs) are competent to respond to environmental factors that promote cell type determination and differentiation. Since those studies, significant effort has been directed at identifying the molecular nature of these environmental signals and understanding the precise mechanisms they employ to drive RPCs towards the different retinal fates. In this review, we describe the recent progress toward understanding how environmental factors influence the development of vertebrate rod photoreceptors.

Hes1 but not Hes5 regulates an astrocyte versus oligodendrocyte fate choice in glial restricted precursors

To determine the role of Hes genes in the differentiation process of neuroepithelial (NEP) cells to glial restricted precursor cells (GRPs) and subsequently GRPs to oligodendrocytes and astrocytes, we have examined the effects of Hes1 and Hes5 on glial differentiation. We find that both Hes1 and Hes5 are expressed by GRPs and that Hes1 can drive GRPs to an astrocyte cell fate at the expense of oligodendrocyte differentiation. Overexpression of Hes1 in GRPs results in the up‐regulation of the astrocyte markers glial fibrillary acidic protein and CD44 and the down‐regulation of oligodendrocyte markers myelin proteolipid protein/DM20, GalC, and CNPase. Transcription factors involved in oligodendrocyte differentiation, such as Nkx2.2, Olig1, and Mash1, are also down‐regulated in Hes1‐overexpressing cells. The effect of Hes1 on gliogenesis is stage‐specific as Hes1 does not direct NEP cells to an astrocytic fate. In contrast to Hes1, Hes5 does not promote astrocyte differentiation. Instead, it inhibits both astrocyte and oligodendrocyte differentiation. Overexpression of Notch1 has an effect on gliogenesis similar to that of Hes1 and the mRNA levels of Hes1 are up‐regulated in cells overexpressing Notch1, suggesting that Notch1 could be an upstream activator of Hes1. Developmental Dynamics 675–689, 2003.

ASCL1 reprograms mouse Müller glia into neurogenic retinal progenitors

Non-mammalian vertebrates have a robust ability to regenerate injured retinal neurons from Müller glia (MG) that activate the gene encoding the proneural factor Achaete-scute homolog 1 (Ascl1; also known as Mash1 in mammals) and de-differentiate into progenitor cells. By contrast, mammalian MG have a limited regenerative response and fail to upregulate Ascl1 after injury. To test whether ASCL1 could restore neurogenic potential to mammalian MG, we overexpressed ASCL1 in dissociated mouse MG cultures and intact retinal explants. ASCL1-infected MG upregulated retinal progenitor-specific genes and downregulated glial genes. Furthermore, ASCL1 remodeled the chromatin at its targets from a repressive to an active configuration. MG-derived progenitors differentiated into cells that exhibited neuronal morphologies, expressed retinal subtype-specific neuronal markers and displayed neuron-like physiological responses. These results indicate that a single transcription factor, ASCL1, can induce a neurogenic state in mature MG.

Vsx‐1 and Vsx‐2: Differential expression of two Paired‐like homeobox genes during zebrafish and goldfish retinogenesis

Vsx‐1 and Vsx‐2 are two homeobox genes that were cloned originally from an adult goldfish retinal library. They are members of the paired‐like:CVC gene family, which is characterized by the presence of a paired homeodomain and an additional conserved region, termed the CVC domain. To analyze the possible roles for Vsx‐1 and Vsx‐2 in eye development, we used in situ hybridization to examine their expression patterns in zebrafish and goldfish embryos. Vsx‐2 is initially expressed by proliferating neuroepithelial cells of the presumptive neural retina, then it is down‐regulated as differentiation begins, and it is finally reexpressed at later stages of differentiation in a subset of cells, presumed to be bipolar cells, in the inner nuclear layer. In contrast, Vsx‐1 is expressed only weakly in undifferentiated, presumptive neural retina and is then up‐regulated selectively in presumptive bipolar cells at early stages of differentiation (when Vsx‐2 is turned off), before decreasing to an intermediate level, which is maintained in the differentiated (adult) retina. The restricted expression patterns of Vsx‐2 correspond to the observed phenotypes in mice with the ocular retardation mutation (orJ), further supporting the notion that Vsx‐2 and Chx10 are homologues. The sequential complimentary and then corresponding expression patterns of Vsx‐1 and Vsx‐2 suggest that these similar transcription factors may be recruited for partially overlapping, but distinct, functions during the development of the retina. J. Comp. Neurol. 388:495–505, 1997.

The cyclin-dependent kinase inhibitors p19(Ink4d) and p27(Kip1) are coexpressed in select retinal cells and act cooperatively to control cell cycle exit.

Cyclin-dependent kinase inhibitors (cdkis), including p19(Ink4d) and p27(Kip1), mediate exit from the cell cycle. To determine the function of these cdkis in regulating neurogenesis, we examined retina from wild-type, Ink4d-null, and Ink4d/Kip1-double null animals. Ink4d was expressed in progenitors and select neurons in the mature retina. Ink4d-null retina showed an extended period of proliferation, followed by apoptosis. Colabeling for p19(Ink4d) and p27(Kip1) revealed that a subpopulation of cells expressed both inhibitors. Deletion of Ink4d and Kip1 resulted in continued proliferation that was synergistic. This hyperproliferation led to an increase in number of horizontal cells and differentiated neurons reentering the cell cycle. Deletion of Ink4d and Kip1 also exacerbated the retinal dysplasia observed in Kip1-null mice, which was shown to be partly dependent on p53. These data indicate that select retinal cells express both p19(Ink4d) and p27(Kip1) and that they act cooperatively to ensure cell cycle exit.

Retinal pigment epithelium development, plasticity, and tissue homeostasis.

The retinal pigment epithelium (RPE) is a simple epithelium interposed between the neural retina and the choroid. Although only 1 cell-layer in thickness, the RPE is a virtual workhorse, acting in several capacities that are essential for visual function and preserving the structural and physiological integrities of neighboring tissues. Defects in RPE function, whether through chronic dysfunction or age-related decline, are associated with retinal degenerative diseases including age-related macular degeneration. As such, investigations are focused on developing techniques to replace RPE through stem cell-based methods, motivated primarily because of the seemingly limited regeneration or self-repair properties of mature RPE. Despite this, RPE cells have an unusual capacity to transdifferentiate into various cell types, with the particular fate choices being highly context-dependent. In this review, we describe recent findings elucidating the mechanisms and steps of RPE development and propose a developmental framework for understanding the apparent contradiction in the capacity for low self-repair versus high transdifferentiation.

Lhx2 links the intrinsic and extrinsic factors that control optic cup formation

A crucial step in eye organogenesis is the transition of the optic vesicle into the optic cup. Several transcription factors and extracellular signals mediate this transition, but whether a single factor links them into a common genetic network is unclear. Here, we provide evidence that the LIM homeobox gene Lhx2, which is expressed in the optic neuroepithelium, fulfils such a role. In Lhx2-/- mouse embryos, eye field specification and optic vesicle morphogenesis occur, but development arrests prior to optic cup formation in both the optic neuroepithelium and lens ectoderm. This is accompanied by failure to maintain or initiate the expression patterns of optic-vesicle-patterning and lens-inducing determinants. Of the signaling pathways examined, only BMP signaling is noticeably altered and Bmp4 and Bmp7 mRNAs are undetectable. Lhx2-/- optic vesicles and lens ectoderm upregulate Pax2, Fgf15 and Sox2 in response to BMP treatments, and Lhx2 genetic mosaics reveal that transcription factors, including Vsx2 and Mitf, require Lhx2 cell-autonomously for their expression. Our data indicate that Lhx2 is required for optic vesicle patterning and lens formation in part by regulating BMP signaling in an autocrine manner in the optic neuroepithelium and in a paracrine manner in the lens ectoderm. We propose a model in which Lhx2 is a central link in a genetic network that coordinates the multiple pathways leading to optic cup formation.