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1.  Cyclin D1 inactivation extends proliferation and alters histogenesis in the postnatal mouse retina 
Developmental Dynamics  2012;241(5):941-952.
The cell cycle regulator Cyclin D1 is expressed in embryonic retinal progenitor cells (RPCs) and regulates their cell cycle rate and neurogenic output. We report here that Cyclin D1 also has important functions in postnatal retinal histogenesis.
The initial production of Müller glia and bipolar cells was enhanced in Cyclin D1 knockout (Ccnd1−/−) retinas. Despite a steeper than normal rate of depletion of the RPC population at embryonic ages, postnatal Ccnd1−/− retinas exhibited an extended window of proliferation, neurogenesis and gliogenesis. Cyclin D3, normally confined to Müller glia, was prematurely expressed in Ccnd1−/− RPCs. However, Cyclin D3 did not compensate for Cyclin D1 in regulating cell cycle kinetics or neurogenic output.
The data presented in this study along with our previous finding that Cyclin D2 was unable to completely compensate for the absence of Cyclin D1 indicate that Cyclin D1 regulates retinal histogenesis in ways not shared by the other D-cyclins.
PMCID: PMC3361900  PMID: 22434780
2.  Lhx2 Balances Progenitor Maintenance with Neurogenic Output and Promotes Competence State Progression in the Developing Retina 
The Journal of Neuroscience  2013;33(30):12197-12207.
The LIM-Homeodomain transcription factor Lhx2 is an essential organizer of early eye development and is subsequently expressed in retinal progenitor cells (RPCs). To determine its requirement in RPCs, we performed a temporal series of conditional inactivations in mice with the early RPC driver Pax6 α-Cre and the tamoxifen-inducible Hes1CreERT2 driver. Deletion of Lhx2 caused a significant reduction of the progenitor population and a corresponding increase in neurogenesis. Precursor fate choice correlated with the time of inactivation; early and late inactivation led to the overproduction of retinal ganglion cells (RGCs) and rod photoreceptors, respectively. In each case, however, the overproduction was selective, occurring at the expense of other cell types and indicating a role for Lhx2 in generating cell type diversity. RPCs that persisted in the absence of Lhx2 continued to generate RGC precursors beyond their normal production window, suggesting that Lhx2 facilitates a transition in competence state. These results identify Lhx2 as a key regulator of RPC properties that contribute to the ordered production of multiple cell types during retinal tissue formation.
PMCID: PMC3721834  PMID: 23884928
3.  Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding 
PLoS Genetics  2012;8(9):e1002924.
The homeodomain and adjacent CVC domain in the visual system homeobox (VSX) proteins are conserved from nematodes to humans. Humans with missense mutations in these regions of VSX2 have microphthalmia, suggesting both regions are critical for function. To assess this, we generated the corresponding mutations in mouse Vsx2. The homeodomain mutant protein lacked DNA binding activity and the knock-in mutant phenocopied the null mutant, ocular retardation J. The CVC mutant protein exhibited weakened DNA binding; and, although the corresponding knock-in allele was recessive, it unexpectedly caused the strongest phenotype, as indicated by severe microphthalmia and hyperpigmentation of the neural retina. This occurred through a cryptic transcriptional feedback loop involving the transcription factors Mitf and Otx1 and the Cdk inhibitor p27Kip1. Our data suggest that the phenotypic severity of the CVC mutant depends on the weakened DNA binding activity elicited by the CVC mutation and a previously unknown protein interaction between Vsx2 and its regulatory target Mitf. Our data also suggest that an essential function of the CVC domain is to assist the homeodomain in high-affinity DNA binding, which is required for eye organogenesis and unhindered execution of the retinal progenitor program in mammals. Finally, the genetic and phenotypic behaviors of the CVC mutation suggest it has the characteristics of a recessive neomorph, a rare type of genetic allele.
Author Summary
Problems with the early development of the mammalian retina can cause congenital eye defects such as microphthalmia, in which the eye is dramatically smaller and functionally compromised. Severe microphthalmia is associated with mutations in the retinal-expressed visual system homeobox 2 (Vsx2) gene, but how Vsx2 controls retinal development, and ultimately eye formation, has remained unclear. We assessed the impact of two missense mutations, discovered in humans, on Vsx2 function and eye development in mice. One mutation altered a highly conserved residue of the homeodomain, and the other altered a highly conserved residue in the CVC domain, a region of unresolved function. Both mutations impacted the DNA binding properties of the protein, although to differing extents. Likewise, both mutations caused microphthalmia and disruptions in retinal development, also to differing extents and by distinct mechanisms. Our data suggest that Vsx2 acts as a gatekeeper of the retinal gene expression program by preventing the activation of interfering or competing gene expression programs. We propose that the evolutionary stable association between the VSX-class homeodomain and CVC domain set the stage for Vsx2 or its archetype to assume a gatekeeper function for retinal development and ultimately eye organogenesis.
PMCID: PMC3447932  PMID: 23028343
4.  Negative Regulation of Vsx1 by its Paralog Chx10/Vsx2 is Conserved in the Vertebrate Retina 
Brain Research  2007;1192:99-113.
and Vsx1 are the only Paired-like CVC (Prd-L:CVC) homeobox genes in the mouse genome. Both are expressed in the retina and have important but distinct roles in retinal development. Mutations in Chx10 cause reduced retinal progenitor cell (RPC) proliferation and an absence of bipolar cells, while mutations in Vsx1 impair differentiation of cone bipolar cells. Given their structural similarities and importance in retinal development, we sought to determine if a regulatory interaction exists between these genes and whether inactivation of both genes blocks initiation of retinal development. We found that Chx10 binds to a specific sequence in the Vsx1 5′-intergenic region and represses the activity of a luciferase reporter under the control of the Vsx1 promoter. This is consistent with our observation that there is an inverse relationship between the levels of Chx10 and Vsx1 immunostaining within the bipolar cell class. Furthermore, Vsx1 mRNA is upregulated in the RPCs of Chx10 deficient mice and zebrafish embryos injected with a chx10 morpholino. In mice deficient for both Chx10 and Vsx1 and zebrafish embryos co-injected with chx10 and vsx1 morpholinos, the changes in embryonic retinal development and marker expression are similar in magnitude to embryos with Chx10 loss of function only. From these studies, we propose that Vsx1 is a direct target of Chx10-mediated transcriptional repression. Although Vsx1 mRNA is upregulated in Chx10 deficient RPCs, Vsx1 does not genetically compensate for loss of Chx10, demonstrating that Prd-L:CVC genes, although important, are not absolutely required to initiate retinal development.
PMCID: PMC3315787  PMID: 17919464
homeobox; homeodomain; development; eye; microphthalmia; bipolar cell
5.  Vsx2 / Chx10 Ensures the Correct Timing and Magnitude of Hedgehog Signaling in the Mouse Retina 
Developmental biology  2008;317(2):560-575.
Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.
PMCID: PMC2671289  PMID: 18417110
retina; eye; ocular retardation J; histogenesis; stem cell; homeobox; homeodomain; Sonic Hedgehog; Hhip; Gli1
6.  Cyclin D1 fine-tunes the neurogenic output of embryonic retinal progenitor cells 
Neural Development  2009;4:15.
Maintaining the correct balance of proliferation versus differentiation in retinal progenitor cells (RPCs) is essential for proper development of the retina. The cell cycle regulator cyclin D1 is expressed in RPCs, and mice with a targeted null allele at the cyclin D1 locus (Ccnd1-/-) have microphthalmia and hypocellular retinas, the latter phenotype attributed to reduced RPC proliferation and increased photoreceptor cell death during the postnatal period. How cyclin D1 influences RPC behavior, especially during the embryonic period, is unclear.
In this study, we show that embryonic RPCs lacking cyclin D1 progress through the cell cycle at a slower rate and exit the cell cycle at a faster rate. Consistent with enhanced cell cycle exit, the relative proportions of cell types born in the embryonic period, such as retinal ganglion cells and photoreceptor cells, are increased. Unexpectedly, cyclin D1 deficiency decreases the proportions of other early born retinal neurons, namely horizontal cells and specific amacrine cell types. We also found that the laminar positioning of horizontal cells and other cell types is altered in the absence of cyclin D1. Genetically replacing cyclin D1 with cyclin D2 is not efficient at correcting the phenotypes due to the cyclin D1 deficiency, which suggests the D-cyclins are not fully redundant. Replacement with cyclin E or inactivation of cyclin-dependent kinase inhibitor p27Kip1 restores the balance of RPCs and retinal cell types to more normal distributions, which suggests that regulation of the retinoblastoma pathway is an important function for cyclin D1 during embryonic retinal development.
Our findings show that cyclin D1 has important roles in RPC cell cycle regulation and retinal histogenesis. The reduction in the RPC population due to a longer cell cycle time and to an enhanced rate of cell cycle exit are likely to be the primary factors driving retinal hypocellularity and altered output of precursor populations in the embryonic Ccnd1-/- retina.
PMCID: PMC2694796  PMID: 19416500
7.  Absence of Chx10 Causes Neural Progenitors to Persist in the Adult Retina 
Mutation of the Chx10 homeobox gene in mice and humans causes congenital blindness and microphthalmia (small eyes). This study used Chx10−/− (ocular retardation) mice to investigate how lack of Chx10 affects progenitor/stem cell behavior in the retina and ciliary epithelium (CE).
The distribution of mitotic retinal progenitor cells (RPCs) during embryonic development was analyzed using phosphohistone 3 (H3)-labeling. DNA flow cytometry was used to measure DNA content. The distribution and phenotype of dividing cells in the postnatal retina and CE was analyzed by incorporation of the thymidine analogue BrdU and immunohistochemistry.
The Chx10−/− embryonic retina maintained a constantly sized population of mitotic RPCs during development, causing the mitotic index to increase markedly over time compared with the wild type. Also, the proportion of cells in the G1 phase of the cell cycle was increased compared with the wild type. Of interest, division of RPC-like cells with neurogenic properties persisted in the adult Chx10−/− retina. Colabeling for BrdU and the neural progenitor marker nestin or the neuronal markers β3-tubulin, syntaxin, and VC1.1 showed that new amacrine-like neurons developed in the adult central retina. By contrast, cells with these characteristics were not observed in the mature wild-type retina. In the mature CE, BrdU-positive cells were observed in both wild-type and Chx10−/− mice. However, neurogenesis from this cell population was not evident.
Without Chx10, proliferative expansion of the embryonic RPC pool is markedly reduced. In the adult retina, lack of Chx10 results in a population of dividing neural progenitor cells that persist and produce new neurons in the central retina.
PMCID: PMC2423807  PMID: 16384989
8.  Defects in retinal pigment epithelium cell proliferation and retinal attachment in mutant mice with p27Kip1 gene ablation 
Molecular Vision  2007;13:273-286.
Little is known about the mechanisms that regulate cell cycle withdrawal of the retinal pigment epithelium (RPE) during development, or about the mechanisms maintaining epithelial cell quiescence in adult retinas. The present study examines the potential role of the negative cell cycle regulator p27Kip1 in controlling RPE proliferation, using mice with targeted ablation of the p27Kip1 gene.
Ocular tissues were obtained from wild-type and p27Kip1-null mice at several postnatal ages. Following aldehyde fixation, eyes were processed intact for JB-4 histology and electron microscopy. Alternatively, tissues were removed by manual or enzymatic dissection in order to obtain flat-mounts of the RPE attached to either the choroid-sclera or neural retina, respectively. Epithelial flat-mounts were either left unlabeled, in which case melanin pigment provided internal contrast, or labeled with Alexa Fluor 488-phalloidin and propidium iodide to visualize cell boundaries and nuclei, respectively.
Morphometric analysis using transverse plastic sections revealed a 96% increase in nuclear density and a 12% increase in thickness (apical to basal) for mutant vs. normal epithelia at postnatal day 35 (P35). These changes were not restricted to central or peripheral regions, and were uncorrelated with focal areas of dysplasia seen in the mutant neural retina. When similar tissues were viewed as flat-mounts, an observed 100% increase in nuclear density was accompanied by only a 46% enhancement in cellular density. This resulted in a larger proportion of multinucleated cells in the nullizygous RPE as compared with the wild-type epithelium (91 versus 47%). Such a pattern was achieved relatively early in development since, at P7 when the increase in RPE nuclear density was essentially complete, cellular density was augmented by only 39%. In addition to these proliferative changes, individual epithelial cells sometimes exhibited structural abnormalities, including an altered cortical actin cytoskeleton and displacement of nuclei from their normal central position. Surprisingly, while the RPE cells of null animals were similar ultrastructurally to those of the wild-type, interdigitation of their microvillous processes with outer segments was incomplete. Quantitative analysis revealed that such areas of detachment characterize, on average, 42% of the nullizygous retina, and that there is little correlation between detachment and neural retina dysplasia from one eye to another. Together with parallel evidence demonstrating a substantial decline in the apparent adhesiveness of mutant retinas relative to the normal tissue, the data is strongly indicative of an altered epithelium-photoreceptor interaction following gene ablation.
The absence of a functional p27Kip1 gene results in enhanced RPE nuclear division, without a commensurate increase in cell division. Although the mutant epithelium as a whole appears structurally normal, individual cells exhibit cytoskeletal changes and their interaction with the neural retina is compromised.
PMCID: PMC2633469  PMID: 17356514
9.  CDC42 Is Required for Tissue Lamination and Cell Survival in the Mouse Retina 
PLoS ONE  2013;8(1):e53806.
The small GTPase CDC42 has pleiotropic functions during development and in the adult. These functions include intra- as well as intercellular tasks such as organization of the cytoskeleton and, at least in epithelial cells, formation of adherens junctions. To investigate CDC42 in the neuronal retina, we generated retina-specific Cdc42-knockdown mice (Cdc42-KD) and analyzed the ensuing consequences for the developing and postnatal retina. Lack of CDC42 affected organization of the developing retina as early as E17.5, prevented correct tissue lamination, and resulted in progressive retinal degeneration and severely reduced retinal function of the postnatal retina. Despite the disorganization of the retina, formation of the primary vascular plexus was not strongly affected. However, both deeper vascular plexi developed abnormally with no clear layering of the vessels. Retinas of Cdc42-KD mice showed increased expression of pro-survival, but also of pro-apoptotic and pro-inflammatory genes and exhibited prolonged Müller glia hypertrophy. Thus, functional CDC42 is important for correct tissue organization already during retinal development. Its absence leads to severe destabilization of the postnatal retina with strong degeneration and loss of retinal function.
PMCID: PMC3553133  PMID: 23372671
10.  Proliferative reactive gliosis is compatible with glial metabolic support and neuronal function 
BMC Neuroscience  2011;12:98.
The response of mammalian glial cells to chronic degeneration and trauma is hypothesized to be incompatible with support of neuronal function in the central nervous system (CNS) and retina. To test this hypothesis, we developed an inducible model of proliferative reactive gliosis in the absence of degenerative stimuli by genetically inactivating the cyclin-dependent kinase inhibitor p27Kip1 (p27 or Cdkn1b) in the adult mouse and determined the outcome on retinal structure and function.
p27-deficient Müller glia reentered the cell cycle, underwent aberrant migration, and enhanced their expression of intermediate filament proteins, all of which are characteristics of Müller glia in a reactive state. Surprisingly, neuroglial interactions, retinal electrophysiology, and visual acuity were normal.
The benign outcome of proliferative reactive Müller gliosis suggests that reactive glia display context-dependent, graded and dynamic phenotypes and that reactivity in itself is not necessarily detrimental to neuronal function.
PMCID: PMC3203081  PMID: 21985191
reactive gliosis; retinal degeneration; neuronal degeneration; GFAP; Müller glia; p27Kip1; CDKN1B; intermediate filaments

Results 1-10 (10)