Elevated expression of human bHLH factor ATOH7 accelerates cell cycle progression of progenitors and enhances production of avian retinal ganglion cells.

The production of vertebrate retinal projection neurons, retinal ganglion cells (RGCs), is regulated by cell-intrinsic determinants and cell-to-cell signaling events. The basic-helix-loop-helix (bHLH) protein Atoh7 is a key neurogenic transcription factor required for RGC development. Here, we investigate whether manipulating human ATOH7 expression among uncommitted progenitors can promote RGC fate specification and thus be used as a strategy to enhance RGC genesis. Using the chicken retina as a model, we show that cell autonomous expression of ATOH7 is sufficient to induce precocious RGC formation and expansion of the neurogenic territory. ATOH7 overexpression among neurogenic progenitors significantly enhances RGC production at the expense of reducing the progenitor pool. Furthermore, forced expression of ATOH7 leads to a minor increase of cone photoreceptors. We provide evidence that elevating ATOH7 levels accelerates cell cycle progression from S to M phase and promotes cell cycle exit. We also show that ATOH7-induced ectopic RGCs often exhibit aberrant axonal projection patterns and are correlated with increased cell death during the period of retinotectal connections. These results demonstrate the high potency of human ATOH7 in promoting early retinogenesis and specifying the RGC differentiation program, thus providing insight for manipulating RGC production from stem cell-derived retinal organoids.

Müller glial cell-dependent regeneration of the neural retina: An overview across vertebrate model systems.

Retinal dystrophies are a major cause of blindness for which there are currently no curative treatments. Transplantation of stem cell-derived neuronal progenitors to replace lost cells has been widely investigated as a therapeutic option. Another promising strategy would be to trigger self-repair mechanisms in patients, through the recruitment of endogenous cells with stemness properties. Accumulating evidence in the past 15 year0s has revealed that several retinal cell types possess neurogenic potential, thus opening new avenues for regenerative medicine. Among them, Müller glial cells have been shown to be able to undergo a reprogramming process to re-acquire a stem/progenitor state, allowing them to proliferate and generate new neurons for repair following retinal damages. Although Müller cell-dependent spontaneous regeneration is remarkable in some species such as the fish, it is extremely limited and ineffective in mammals. Understanding the cellular events and molecular mechanisms underlying Müller cell activities in species endowed with regenerative capacities could provide knowledge to unlock the restricted potential of their mammalian counterparts. In this context, the present review provides an overview of Müller cell responses to injury across vertebrate model systems and summarizes recent advances in this rapidly evolving field. Developmental Dynamics 245:727-738, 2016. © 2015 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc.

Differentiation of retinal ganglion cells and photoreceptor precursors from mouse induced pluripotent stem cells carrying an Atoh7/Math5 lineage reporter.

The neural retina is a critical component of the visual system, which provides the majority of sensory input in humans. Various retinal degenerative diseases can result in the permanent loss of retinal neurons, especially the light-sensing photoreceptors and the centrally projecting retinal ganglion cells (RGCs). The replenishment of lost RGCs and the repair of optic nerve damage are particularly challenging, as both RGC specification and their subsequent axonal growth and projection involve complex and precise regulation. To explore the developmental potential of pluripotent stem cell-derived neural progenitors, we have established mouse iPS cells that allow cell lineage tracing of progenitors that have expressed Atoh7/Math5, a bHLH transcription factor required for RGC production. These Atoh7 lineage reporter iPS cells encode Cre to replace one copy of the endogenous Atoh7 gene and a Cre-dependent YFP reporter in the ROSA locus. In addition, they express pluripotent markers and are capable of generating teratomas in vivo. Under anterior neural induction and neurogenic conditions in vitro, the Atoh7-Cre/ROSA-YFP iPS cells differentiate into neurons that co-express various RGC markers and YFP, indicating that these neurons are derived from Atoh7-expressing progenitors. Consistent with previous in vivo cell lineage studies, the Atoh7-Cre/ROSA-YFP iPS cells also give rise to a subset of Crx-positive photoreceptor precursors. Furthermore, inhibition of Notch signaling in the iPSC cultures results in a significant increase of YFP-positive RGCs and photoreceptor precursors. Together, these results show that Atoh7-Cre/ROSA-YFP iPS cells can be used to monitor the development and survival of RGCs and photoreceptors from pluripotent stem cells.

PTEN regulates retinal interneuron morphogenesis and synaptic layer formation.

The lipid phosphatase PTEN is a critical negative regulator of extracellular signal-induced PI3K activities, yet the roles of PTEN in the neural retina remain poorly understood. Here, we investigate the function of PTEN during retinal development. Deletion of Pten at the onset of neurogenesis in retinal progenitors results in the reduction of retinal ganglion cells and rod photoreceptors, but increased Müller glial genesis. In addition, PTEN deficiency leads to elevated phosphorylation of Akt, especially in the developing inner plexiform layer, where high levels of PTEN are normally expressed. In Pten mutant retinas, various subtypes of amacrine cells show severe dendritic overgrowth, causing specific expansion of the inner plexiform layer. However, the outer plexiform layer remains relatively undisturbed in the Pten deficient retina. Physiological analysis detects reduced rod function and augmented oscillatory potentials originating from amacrine cells in Pten mutants. Furthermore, deleting Pten or elevating Akt activity in individual amacrine cells is sufficient to disrupt dendritic arborization, indicating that Pten activity is required cell autonomously to control neuronal morphology. Moreover, inhibiting endogenous Akt activity attenuates inner plexiform layer formation in vitro. Together, these findings demonstrate that suppression of PI3K/Akt signaling by PTEN is crucial for proper neuronal differentiation and normal retinal network formation.

Function and mechanism of CNTF/LIF signaling in retinogenesis.

Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) exhibit multiple biological effects in the developing vertebrate retina. CNTF/LIF inhibits rod photoreceptor, and promotes bipolar cells and Muller glia differentiation. In addition, CNTF/LIF has been shown to have proliferative and apoptotic effects. Moreover, LIF also inhibits retinal vascular development. CNTF/LIF signaling components CNTFRalpha, LIFRbeta, gp130, and a number of STAT proteins are expressed in the retina. CNTF/LIF activates Jak-STAT, ERK, and Notch pathways during retinal development. Perturbation of CNTF induced signal transduction reveals that different combinations of CNTF/LIF signaling pathways regulate differentiation of retinal neurons and glia. Gene expression studies show that CNTF/LIF affects retinogenesis by regulating various genes involved in transcription, signal transduction, protein modification, apoptosis, protein localization, and cell ion homeostasis. Most past studies have deployed ectopic expression or addition of exogenous CNTF/LIF, thus further ana-lysis of mice with conditional mutations in CNTF/LIF signaling components will allow better understanding of in-vivo functions of CNTF/LIF associated signaling events in retinogenesis.

Role of the immune modulator programmed cell death-1 during development and apoptosis of mouse retinal ganglion cells.

PURPOSE: Mammalian programmed cell death (PD)-1 is a membrane-associated receptor regulating the balance between T-cell activation, tolerance, and immunopathology; however, its role in neurons has not yet been defined. The hypothesis that PD-1 signaling actively promotes retinal ganglion cell (RGC) death within the developing mouse retina was investigated. METHODS: Mature retinal cell types expressing PD-1 were identified by immunofluorescence staining of vertical retina sections; developmental expression was localized by immunostaining and quantified by Western blot analysis. PD-1 involvement in developmental RGC survival was assessed in vitro using retinal explants and in vivo using PD-1 knockout mice. PD-1 ligand gene expression was detected by RT-PCR. RESULTS: PD-1 is expressed in most adult RGCs and undergoes dynamic upregulation during the early postnatal window of retinal cell maturation and physiological programmed cell death (PCD). In vitro blockade of PD-1 signaling during this time selectively increases the survival of RGCs. Furthermore, PD-1-deficient mice show a selective increase in RGC number in the neonatal retina at the peak of developmental RGC death. Lastly, gene expression of the immune PD-1 ligand genes Pdcd1lg1 and Pdcd1lg2 was found throughout postnatal retina maturation. CONCLUSIONS: These findings collectively support a novel role for a PD-1-mediated signaling pathway in developmental PCD during postnatal RGC maturation.

Molecular and cellular alterations induced by sustained expression of ciliary neurotrophic factor in a mouse model of retinitis pigmentosa.

PURPOSE: To characterize molecular and cellular changes induced by sustained expression of ciliary neurotrophic factor (CNTF) in the rds mutant mouse retina. METHODS: Recombinant adeno-associated virus (rAAV) expressing CNTF was injected subretinally, for transduction of peripherin/rds(+/)(-) transgenic mice that carry the P216L mutation found in human retinitis pigmentosa. Characterization of retinal neurons and glia was performed by immunocytochemistry with cell-type-specific markers. Activation of signaling molecules was examined by Western blot and immunostaining. Alterations of gene transcription profiles were studied by microarray analyses. RESULTS: CNTF viral transduction maintained rhodopsin expression in surviving rod photoreceptors, but greatly reduced both S- and M-opsin normally expressed in cones. In addition, CNTF treatment resulted in increased numbers and dispersion of Müller glia and Chx10-positive bipolar cells within the inner nuclear layer. Persistent CNTF signaling also caused enhanced phosphorylation of STAT1, STAT3, and p42/44 ERK, as well as their levels of expression. Moreover, altered transcription profiles were detected for a large number of genes. Among these, Crx and Nrl involved in photoreceptor differentiation and several genes involved in phototransduction were suppressed. CONCLUSIONS: Despite the rescue from cell death, continuous exposure to CNTF changed photoreceptor cell profiles, especially resulting in the loss of cone immunoreactivity. In addition, the Müller glia and bipolar cells became disorganized, and the number of cells expressing Müller and bipolar cell markers increased. Constitutive CNTF production resulted in sustained activation of cytokine signal transduction and altered the expression of a large number of genes. Therefore, stringent regulation of CNTF may be necessary for its therapeutic application in preventing retinal degeneration.

VEGF activates divergent intracellular signaling components to regulate retinal progenitor cell proliferation and neuronal differentiation.

During vertebrate neurogenesis, multiple extracellular signals influence progenitor cell fate choices. The process by which uncommitted progenitor cells interpret and integrate signals is not well understood. We demonstrate here that in the avascular chicken retina, vascular endothelial growth factor (VEGF) secreted by postmitotic neurons acts through the FLK1 receptor present on progenitor cells to influence cell proliferation and commitment. Augmenting VEGF signals increases progenitor cell proliferation and decreases retinal ganglion cell genesis. Conversely, absorbing endogenous VEGF ligand or disrupting FLK1 activity attenuates cell proliferation and enhances retinal ganglion cell production. In addition, we provide evidence that VEGF signals transmitted by the FLK1 receptor activate divergent intracellular signaling components, which regulate different responses of progenitor cells. VEGF-induced proliferation is influenced by the MEK-ERK pathway, as well as by the basic helix-loop-helix factor HES1. By contrast, VEGF-dependent ganglion cell suppression does not require MEK-ERK activation, but instead relies on VEGF-stimulated HES1 activity, which is independent of NOTCH signaling. Moreover, elevated HES1 expression promotes progenitor cell proliferation and prevents overproduction of retinal ganglion cells owing to the loss of VEGF or sonic hedgehog (SHH), another signal that suppresses ganglion cell development. Based on previous and current findings, we propose that HES1 serves as a convergent signaling node within early retinal progenitor cells to integrate various cell-extrinsic cues, including VEGF and SHH, in order to control cell proliferation and neuronal specification.

Roles of cell-extrinsic growth factors in vertebrate eye pattern formation and retinogenesis.

Formation of the vertebrate visual system involves complex interplays of cell-extrinsic cues and cell-intrinsic determinants. Studies in several vertebrate species demonstrate that multiple classes of signaling molecules participate in pattern formation of the eye and neurogenesis of the retina. Certain signals, such as hedgehog, BMP, and FGF molecules, are repeatedly deployed at varying concentration thresholds and in different cellular contexts. Accumulating evidence reveals a striking conservation of molecular mechanisms regulating the neurogenic process between Drosophila and vertebrate retinas. The remaining challenge is to understand how these well-characterized signaling pathways are activated and integrated to impact eye morphogenesis and retinal progenitor cell fate determination.

Expression of cytokine signal transduction components in the postnatal mouse retina.

PURPOSE: Members of the ciliary neurotrophic factor (CNTF) family of cytokines have been shown to influence neuronal differentiation during retinal development and enhance cell survival in various retinal degeneration models. However, the cellular mechanism of CNTF signaling and the target cell types for CNTF in the developing retina remain unidentified. The purpose of this study is to characterize expression patterns of proteins involved in cytokine signal transduction in the mouse retina, thus to assess the potential responsiveness of different retinal cell types to CNTF-like cytokine signals. METHODS: The expression profiles of various cytokine signal transduction components, including receptor subunits CNTF receptor alpha (CNTFRa) and gp130, intracellular protein kinases, Jak2 and Tyk2, as well as latent transcription factors, STAT1 and STAT3, were determined by immunohistochemical staining of mouse retinal sections derived from different postnatal stages. In addition, the distribution of ERK was studied by immunofluorescent staining. RESULTS: In the neonatal retina, intense staining signals for gp130, CNTFRalpha, Jak2, Tyk2, STAT1, and STAT3 were present in the differentiated ganglion cell layer and the developing inner plexiform layer of the mouse retina. Detectable staining signals were also observed in the ventricular zone of the early postnatal mouse retina. From P5 to P10, cytokine signaling molecules also accumulated in the developing outer plexiform layer. In the adult retina, cytokine signaling components examined were localized to the ganglion cell layer, the inner nuclear layer, and the two plexiform layers. In addition, regions corresponding to the inner and/or outer segments of the photoreceptor cells showed positive staining for cytokine signaling components. In contrast, the ERK2 protein kinase was found throughout the neonatal retina. In the mature retina, ERK2 was concentrated in the ganglion cells and the inner plexiform layer, while a lesser expression of ERK2 was detected in the inner nuclear layer, the outer plexiform layers, and the outer nuclear layer. CONCLUSIONS: In the neonatal mouse retina, signaling components of the Jak-STAT pathway and ERK2 are differentially expressed. All cytokine signaling components included in this study are expressed in the differentiated inner retina as well as in cells occupying the ventricular zone, suggesting that both postmitotic neurons and proliferative progenitors may directly respond to CNTF-like cytokines during postnatal development. The distribution of cytokine signaling pathway components in the adult mouse retina is consistent with previous findings that ganglion cells and Müller glia are the primary target cell types for CNTF.

Expression of the Flk1 receptor and its ligand VEGF in the developing chick central nervous system.

The receptor tyrosine kinase Flk1 is known to mediate signals of vascular endothelial growth factor (VEGF) during vasculogenesis and hematopoiesis. We demonstrate by in situ hybridization that in addition to endothelial cells, chick Flk1 mRNA is also expressed in the notochord and in the neural epithelial cells of the ventral diencephalon, hindbrain, and spinal cord. During the development of the avascular chick retina, Flk1 mRNA is detected in the proliferative zone of the neural epithelium, whereas the VEGF ligand is expressed by differentiated retinal ganglion cells. Moreover, expression patterns of Flk1 in the retina are conserved among chick, quail and mouse, thus suggesting a distinct role of Flk1 and VEGF in the development of the vertebrate central nervous system.

Retrovirus-mediated gene expression during chick visual system development.

The chick embryo is an excellent model for studying eye morphogenesis, retinal cell fate determination, and retinotectal projections due to its accessibility and the available molecular tools. Avian replication-competent retroviruses allow efficient infection of proliferating cells and stable integration of the viral genome, including up to 2.3kb of foreign cDNA, into the host chromosome. High-titer retroviruses are produced by transient transfection of avian DF-1 cells followed by centrifugation of the culture medium. Targeted infection of the optic vesicle, the lens vesicle, the retina and pigmented epithelium, the periocular mesenchyme, and the tectum can be performed at different developmental stages in ovo. In addition, retroviruses can be used to transduce genes of interest into various ocular tissue explants or cells in vitro. Virus-mediated gene expression can be detected within 12h of infection. Therefore, avian replication-competent retroviruses serve as powerful tools to misexpress wild-type and mutant gene products and to study molecular mechanisms underlying vertebrate visual system development.