PURPOSE: Interphotoreceptor retinoid-binding protein's (IRBP) role in eye growth and its involvement in cell homeostasis remain poorly understood. One hypothesis proposes early conditional deletion of the IRBP gene could lead to a myopic response with retinal degeneration, whereas late conditional deletion (after eye size is determined) could cause retinal degeneration without myopia. Here, we sought to understand if prior myopia was required for subsequent retinal degeneration in the absence of IRBP. This study investigates if any cell type or developmental stage is more important in myopia or retinal degeneration. METHODS: IBRPfl/fl mice were bred with 5 Cre-driver lines: HRGP-Cre, Chx10-Cre, Rho-iCre75, HRGP-Cre Rho-iCre75, and Rx-Cre. Mice were analyzed for IRBP gene expression through digital droplet PCR (ddPCR). Young adult (P30) mice were tested for retinal degeneration and morphology using spectral-domain optical coherence tomography (SD-OCT) and hematoxylin and eosin (H&E) staining. Function was analyzed using electroretinograms (ERGs). Eye sizes and axial lengths were compared through external eye measurements and whole eye biometry. RESULTS: Across all outcome measures, when bred to IRBPfl/fl, HRGP-Cre and Chx10-Cre lines showed no differences from IRBPfl/fl alone. With the Rho-iCre75 line, small but significant reductions were seen in retinal thickness with SD-OCT imaging and postmortem H&E staining without increased axial length. Both the HRGP-Cre+Rho-iCre75 and the Rx-Cre lines showed significant decreases in retinal thickness and outer nuclear layer cell counts. Using external eye measurements and SD-OCT imaging, both lines showed an increase in eye size. Finally, function in both lines was roughly halved across scotopic, photopic, and flicker ERGs. CONCLUSIONS: Our studies support hypotheses that for both eye size determination and retinal homeostasis, there are two critical timing windows when IRBP must be expressed in rods or cones to prevent myopia (P7-P12) and degeneration (P21 and later). The rod-specific IRBP knockout (Rho-iCre75) showed significant retinal functional losses without myopia, indicating that the two phenotypes are independent. IRBP is needed for early development of photoreceptors and eye size, whereas Rho-iCre75 IRBPfl/fl knockout results in retinal degeneration without myopia.

• MeSH
• Retinal Degeneration * genetics   metabolism   physiopathology   MeSH
• Electroretinography * MeSH
• Disease Models, Animal * MeSH
• Myopia * genetics   metabolism   physiopathology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout * MeSH
• Mice MeSH
• Eye Proteins * genetics   metabolism   MeSH
• Tomography, Optical Coherence * MeSH
• Retinol-Binding Proteins * genetics   MeSH
• Retina metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• interstitial retinol-binding protein MeSH Browser
• Eye Proteins * MeSH
• Retinol-Binding Proteins * MeSH

Chromatin remodeling complexes are required for many distinct nuclear processes such as transcription, DNA replication, and DNA repair. However, the contribution of these complexes to the development of complex tissues within an organism is poorly characterized. Imitation switch (ISWI) proteins are among the most evolutionarily conserved ATP-dependent chromatin remodeling factors and are represented by yeast Isw1/Isw2, and their vertebrate counterparts Snf2h (Smarca5) and Snf2l (Smarca1). In this study, we focused on the role of the Snf2h gene during the development of the mammalian retina. We show that Snf2h is expressed in both retinal progenitors and post-mitotic retinal cells. Using Snf2h conditional knockout mice (Snf2h cKO), we found that when Snf2h is deleted, the laminar structure of the adult retina is not retained, the overall thickness of the retina is significantly reduced compared with controls, and the outer nuclear layer (ONL) is completely missing. The depletion of Snf2h did not influence the ability of retinal progenitors to generate all the differentiated retinal cell types. Instead, the Snf2h function is critical for the proliferation of retinal progenitor cells. Cells lacking Snf2h have a defective S-phase, leading to the entire cell division process impairments. Although all retinal cell types appear to be specified in the absence of the Snf2h function, cell-cycle defects and concomitantly increased apoptosis in Snf2h cKO result in abnormal retina lamination, complete destruction of the photoreceptor layer, and consequently, a physiologically non-functional retina.

• Keywords
• Smarca5, Snf2h, apoptosis, cell cycle, photoreceptors, retina,
• MeSH
• Adenosine Triphosphatases * metabolism   MeSH
• Cell Nucleus metabolism   MeSH
• Chromatin * metabolism   MeSH
• Chromosomal Proteins, Non-Histone * metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Cell Proliferation MeSH
• Chromatin Assembly and Disassembly * MeSH
• Retina MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Adenosine Triphosphatases * MeSH
• Chromatin * MeSH
• Chromosomal Proteins, Non-Histone * MeSH
• Smarca5 protein, mouse MeSH Browser

Iron accumulation has been implicated in degenerative retinal diseases. It can catalyze the production of damaging reactive oxygen species. Previous work has demonstrated iron accumulation in multiple retinal diseases, including age-related macular degeneration and diabetic retinopathy. In mice, systemic knockout of the ferroxidases ceruloplasmin (Cp) and hephaestin (Heph), which oxidize iron, results in retinal iron accumulation and iron-induced degeneration. To determine the role of Heph in the retina, we generated a neural retina-specific Heph knockout on a background of systemic Cp knockout. This resulted in elevated neural retina iron. Conversely, retinal ganglion cells had elevated transferrin receptor and decreased ferritin, suggesting diminished iron levels. The retinal degeneration observed in systemic Cp-/-, Heph-/- mice did not occur. These findings indicate that Heph has a local role in regulating neural retina iron homeostasis, but also suggest that preserved Heph function in either the RPE or systemically mitigates the degeneration phenotype observed in the systemic Cp-/-, Heph-/- mice.

• Keywords
• Age-related macular degeneration, Ceruloplasmin, Ferrous, Hephaestin, Iron, Retina,
• MeSH
• Ceruloplasmin genetics   metabolism   MeSH
• Homeostasis MeSH
• Macular Degeneration * genetics   MeSH
• Membrane Proteins * genetics   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Retina metabolism   MeSH
• Iron metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Ceruloplasmin MeSH
• Membrane Proteins * MeSH
• Iron MeSH

Iron-induced oxidative stress can cause or exacerbate retinal degenerative diseases. Retinal iron overload has been reported in several mouse disease models with systemic or neural retina-specific knockout (KO) of homologous ferroxidases ceruloplasmin (Cp) and hephaestin (Heph). Cp and Heph can potentiate ferroportin (Fpn) mediated cellular iron export. Here, we used retina-specific Fpn KO mice to test the hypothesis that retinal iron overload in Cp/Heph DKO mice is caused by impaired iron export from neurons and glia. Surprisingly, there was no indication of retinal iron overload in retina-specific Fpn KO mice: the mRNA levels of transferrin receptor in the retina were not altered at 7-10-months age. Consistent with this, levels and localization of ferritin light chain were unchanged. To "stress the system", we injected iron intraperitoneally into Fpn KO mice with or without Cp KO. Only mice with both retina-specific Fpn KO and Cp KO had modestly elevated retinal iron levels. These results suggest that impaired iron export through Fpn is not sufficient to explain the retinal iron overload in Cp/Heph DKO mice. An increase in the levels of retinal ferrous iron caused by the absence of these ferroxidases, followed by uptake into cells by ferrous iron importers, is most likely necessary.

• Keywords
• Age-related macular degeneration, Ceruloplasmin, Ferroportin, Ferrous, Iron, Retina,
• MeSH
• Ceruloplasmin genetics   metabolism   MeSH
• Ferroportin MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Iron Overload * MeSH
• Cation Transport Proteins * genetics   MeSH
• Retina metabolism   MeSH
• Iron metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Ceruloplasmin MeSH
• Ferroportin MeSH
• Cation Transport Proteins * MeSH
• Iron MeSH

Retinal progenitor cells (RPCs) divide in limited numbers to generate the cells comprising vertebrate retina. The molecular mechanism that leads RPC to the division limit, however, remains elusive. Here, we find that the hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) in an RPC subset by deletion of tuberous sclerosis complex 1 (Tsc1) makes the RPCs arrive at the division limit precociously and produce Müller glia (MG) that degenerate from senescence-associated cell death. We further show the hyperproliferation of Tsc1-deficient RPCs and the degeneration of MG in the mouse retina disappear by concomitant deletion of hypoxia-induced factor 1-alpha (Hif1a), which induces glycolytic gene expression to support mTORC1-induced RPC proliferation. Collectively, our results suggest that, by having mTORC1 constitutively active, an RPC divides and exhausts mitotic capacity faster than neighboring RPCs, and thus produces retinal cells that degenerate with aging-related changes.

• Keywords
• mTORC1, Hif1a, clonal expansion, developmental biology, glycolysis, hypoxia-induced factor 1-alpha, mechanistic target of rapamycin complex 1, mitotic division limit, mouse, retinal progenitor cell,
• MeSH
• Ependymoglial Cells pathology   MeSH
• Hypoxia-Inducible Factor 1, alpha Subunit genetics   metabolism   MeSH
• Tuberous Sclerosis Complex 1 Protein genetics   metabolism   MeSH
• Stem Cells pathology   MeSH
• Mitosis MeSH
• Mechanistic Target of Rapamycin Complex 1 genetics   metabolism   MeSH
• Mice MeSH
• Retina pathology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hypoxia-Inducible Factor 1, alpha Subunit MeSH
• Tuberous Sclerosis Complex 1 Protein MeSH
• Hif1a protein, mouse MeSH Browser
• Mechanistic Target of Rapamycin Complex 1 MeSH
• Tsc1 protein, mouse MeSH Browser

Members of the POU4F/Brn3 transcription factor family have an established role in the development of retinal ganglion cell (RGCs) types, the main transducers of visual information from the mammalian eye to the brain. Our previous work using sparse random recombination of a conditional knock-in reporter allele expressing alkaline phosphatase (AP) and intersectional genetics had identified three types of Brn3c positive (Brn3c+ ) RGCs. Here, we describe a novel Brn3cCre mouse allele generated by serial Dre to Cre recombination and use it to explore the expression overlap of Brn3c with Brn3a and Brn3b and the dendritic arbor morphologies and visual stimulus response properties of Brn3c+ RGC types. Furthermore, we explore brain nuclei that express Brn3c or receive input from Brn3c+ neurons. Our analysis reveals a much larger number of Brn3c+ RGCs and more diverse set of RGC types than previously reported. Most RGCs expressing Brn3c during development are still Brn3c positive in the adult, and all express Brn3a while only about half express Brn3b. Genetic Brn3c-Brn3b intersection reveals an area of increased RGC density, extending from dorsotemporal to ventrolateral across the retina and overlapping with the mouse binocular field of view. In addition, we report a Brn3c+ RGC projection to the thalamic reticular nucleus, a visual nucleus that was not previously shown to receive retinal input. Furthermore, Brn3c+ neurons highlight a previously unknown subdivision of the deep mesencephalic nucleus. Thus, our newly generated allele provides novel biological insights into RGC type classification, brain connectivity, and cytoarchitectonic.

• Keywords
• Brn3c, Cre recombinase, Pou4f3, deep mesencephalic nucleus, periaqueductal gray, retinal ganglion cells, superior colliculus, thalamic reticular nucleus, transcription factor,
• MeSH
• Alleles MeSH
• Gene Knock-In Techniques methods   MeSH
• Homeodomain Proteins genetics   metabolism   MeSH
• Integrases MeSH
• Brain cytology   metabolism   MeSH
• Mice MeSH
• Retinal Ganglion Cells cytology   metabolism   MeSH
• Transcription Factor Brn-3C genetics   metabolism   MeSH
• Visual Pathways cytology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cre recombinase MeSH Browser
• Homeodomain Proteins MeSH
• Integrases MeSH
• Pou4f3 protein, mouse MeSH Browser
• Transcription Factor Brn-3C MeSH

The vertebrate eye is derived from the neuroepithelium, surface ectoderm, and extracellular mesenchyme. The neuroepithelium forms an optic cup in which the spatial separation of three domains is established, namely, the region of multipotent retinal progenitor cells (RPCs), the ciliary margin zone (CMZ)-which possesses both a neurogenic and nonneurogenic potential-and the optic disk (OD), the interface between the optic stalk and the neuroretina. Here, we show by genetic ablation in the developing optic cup that Meis1 and Meis2 homeobox genes function redundantly to maintain the retinal progenitor pool while they simultaneously suppress the expression of genes characteristic of CMZ and OD fates. Furthermore, we demonstrate that Meis transcription factors bind regulatory regions of RPC-, CMZ-, and OD-specific genes, thus providing a mechanistic insight into the Meis-dependent gene regulatory network. Our work uncovers the essential role of Meis1 and Meis2 as regulators of cell fate competence, which organize spatial territories in the vertebrate eye.

• Keywords
• Meis, development, retina,
• MeSH
• Cell Differentiation genetics   MeSH
• Gene Knockdown Techniques MeSH
• Homeodomain Proteins genetics   metabolism   MeSH
• Stem Cells cytology   metabolism   MeSH
• Vertebrates MeSH
• Retina cytology   metabolism   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Gene Expression Regulation, Developmental MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Homeodomain Proteins MeSH
• Transcription Factors MeSH

Genome duplication leads to an emergence of gene paralogs that are essentially free to undergo the process of neofunctionalization, subfunctionalization or degeneration (gene loss). Onecut1 (Oc1) and Onecut2 (Oc2) transcription factors, encoded by paralogous genes in mammals, are expressed in precursors of horizontal cells (HCs), retinal ganglion cells and cone photoreceptors. Previous studies have shown that ablation of either Oc1 or Oc2 gene in the mouse retina results in a decreased number of HCs, while simultaneous deletion of Oc1 and Oc2 leads to a complete loss of HCs. Here we study the genetic redundancy between Oc1 and Oc2 paralogs and focus on how the dose of Onecut transcription factors influences abundance of individual retinal cell types and overall retina physiology. Our data show that reducing the number of functional Oc alleles in the developing retina leads to a gradual decrease in the number of HCs, progressive thinning of the outer plexiform layer and diminished electrophysiology responses. Taken together, these observations indicate that in the context of HC population, the alleles of Oc1/Oc2 paralogous genes are mutually interchangeable, function additively to support proper retinal function and their molecular evolution does not follow one of the typical routes after gene duplication.

• MeSH
• Alleles MeSH
• Amacrine Cells metabolism   pathology   MeSH
• Retinal Bipolar Cells metabolism   pathology   MeSH
• Retinal Cone Photoreceptor Cells metabolism   pathology   MeSH
• Ependymoglial Cells metabolism   pathology   MeSH
• Genetic Loci MeSH
• Genotype MeSH
• Hepatocyte Nuclear Factor 6 genetics   metabolism   MeSH
• Homeodomain Proteins genetics   metabolism   MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Eye growth & development   pathology   MeSH
• Retina cytology   pathology   physiology   MeSH
• Retinal Ganglion Cells cytology   metabolism   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hepatocyte Nuclear Factor 6 MeSH
• Homeodomain Proteins MeSH
• Onecut1 protein, mouse MeSH Browser
• ONECUT2 protein, mouse MeSH Browser
• Transcription Factors MeSH

The liver secretes hepcidin (Hepc) into the bloodstream to reduce blood iron levels. Hepc accomplishes this by triggering degradation of the only known cellular iron exporter ferroportin in the gut, macrophages, and liver. We previously demonstrated that systemic Hepc knockout (HepcKO) mice, which have high serum iron, develop retinal iron overload and degeneration. However, it was unclear whether this is caused by high blood iron levels or, alternatively, retinal iron influx that would normally be regulated by retina-produced Hepc. To address this question, retinas of liver-specific and retina-specific HepcKO mice were studied. Liver-specific HepcKO mice had elevated blood and retinal pigment epithelium (RPE) iron levels and increased free (labile) iron levels in the retina, despite an intact blood-retinal barrier. This led to RPE hypertrophy associated with lipofuscin-laden lysosome accumulation. Photoreceptors also degenerated focally. In contrast, there was no change in retinal or RPE iron levels or degeneration in the retina-specific HepcKO mice. These data indicate that high blood iron levels can lead to retinal iron accumulation and degeneration. High blood iron levels can occur in patients with hereditary hemochromatosis or result from use of iron supplements or multiple blood transfusions. Our results suggest that high blood iron levels may cause or exacerbate retinal disease.

• MeSH
• Retinal Degeneration etiology   metabolism   pathology   MeSH
• Blood-Retinal Barrier MeSH
• Hepcidins physiology   MeSH
• Liver metabolism   pathology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Iron Overload etiology   metabolism   pathology   MeSH
• Retina metabolism   pathology   MeSH
• Iron metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Hamp protein, mouse MeSH Browser
• Hepcidins MeSH
• Iron MeSH

During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.

• MeSH
• Endothelium, Vascular metabolism   MeSH
• Dopamine metabolism   MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Eye blood supply   enzymology   growth & development   metabolism   MeSH
• Opsins genetics   metabolism   MeSH
• Dopamine Plasma Membrane Transport Proteins antagonists & inhibitors   chemistry   metabolism   MeSH
• Retinal Ganglion Cells metabolism   radiation effects   MeSH
• Vitreous Body metabolism   MeSH
• Light * MeSH
• Threonine metabolism   MeSH
• Vesicular Inhibitory Amino Acid Transport Proteins physiology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Dopamine MeSH
• Membrane Proteins MeSH
• OPN5 protein, mouse MeSH Browser
• Opsins MeSH
• Dopamine Plasma Membrane Transport Proteins MeSH
• Slc6a3 protein, mouse MeSH Browser
• Threonine MeSH
• Vesicular Inhibitory Amino Acid Transport Proteins MeSH
• Viaat protein, mouse MeSH Browser