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1.  Distinct neurogenic potential in the retinal margin and the pars plana of mammalian eye 
Unlike many other vertebrates, a healthy mammalian retina does not grow throughout life and lacks a ciliary margin zone capable of actively generating new neurons. The isolation of stem-like cells from the ciliary epithelium has led to speculation that the mammalian retina and/or surrounding tissues may retain neurogenic potential capable of responding to retinal damage. Using genetically altered mouse lines with varying degrees of retinal ganglion cell loss, we show that the retinal margin responds to ganglion cell loss by prolonging specific neurogenic activity, as characterized by increased numbers of Atoh7LacZ expressing cells. The extent of neurogenic activity correlated with the degree of ganglion cell deficiency. In the pars plana, but not the retinal margin, cells remain proliferative into adulthood, marking the junction of pars plana and retinal margin as a niche capable of producing proliferative cells in the mammalian retina and a potential cellular source for retinal regeneration.
PMCID: PMC3462447  PMID: 22973003
Math5; Atoh7; ciliary body; pars plana; CMZ; retinal stem cell
2.  Birth of Cone Bipolar Cells, but Not Rod Bipolar Cells, Is Associated with Existing RGCs 
PLoS ONE  2014;9(1):e83686.
Retinal ganglion cells (RGCs) play important roles in retinogenesis. They are required for normal retinal histogenesis and retinal cell number balance. Developmental RGC loss is typically characterized by initial retinal neuronal number imbalance and subsequent loss of retinal neurons. However, it is not clear whether loss of a specific non-RGC cell type in the RGC-depleted retina is due to reduced cell production or subsequent degeneration. Taking advantage of three knockout mice with varying degrees of RGC depletion, we re-examined bipolar cell production in these retinas from various aspects. Results show that generation of the cone bipolar cells is correlated with the existing number of RGCs. However, generation of the rod bipolar cells is unaffected by RGC shortage. Results report the first observation that RGCs selectively influence the genesis of subsequent retinal cell types.
PMCID: PMC3879276  PMID: 24392091
3.  Adenosine and dopamine receptors co-regulate photoreceptor coupling via gap junction phosphorylation in mouse retina 
Gap junctions in retinal photoreceptors suppress voltage noise and facilitate input of rod signals into the cone pathway during mesopic vision. These synapses are highly plastic and regulated by light and circadian clocks. Recent studies have revealed an important role for connexin36 (Cx36) phosphorylation by protein kinase A (PKA) in regulating cell-cell coupling. Dopamine is a light-adaptive signal in the retina, causing uncoupling of photoreceptors via D4 receptors (D4R), which inhibits adenylyl cyclase (AC) and reduces PKA activity. We hypothesized that adenosine, with its extracellular levels increasing in darkness, may serve as a dark signal to co-regulate photoreceptor coupling through modulation of gap junction phosphorylation. Both D4R and A2a receptor (A2aR) mRNAs were present in photoreceptors, inner nuclear layer neurons, and ganglion cells in C57BL/6 mouse retina, and showed cyclic expression with partially overlapping rhythms. Pharmacologically activating A2aR or inhibiting D4R in light-adapted daytime retina increased photoreceptor coupling. Cx36 among photoreceptor terminals, representing predominantly rod-cone gap junctions but possibly including some rod-rod and cone-cone gap junctions, was phosphorylated in a PKA-dependent manner by the same treatments. Conversely, inhibiting A2aR or activating D4R in daytime dark-adapted retina decreased Cx36 phosphorylation with similar PKA dependence. A2a-deficient mouse retina showed defective regulation of photoreceptor gap junction phosphorylation, fairly regular dopamine release, and moderately down-regulated expression of D4R and AC type I mRNA. We conclude that adenosine and dopamine co-regulate photoreceptor coupling through opposite action on the PKA pathway and Cx36 phosphorylation. In addition, loss of the A2aR hampered D4R gene expression and function.
PMCID: PMC3711184  PMID: 23407968
4.  Near complete loss of retinal ganglion cells in the math5/brn3b double knockout elicits severe reductions of other cell types during retinal development 
Developmental biology  2008;316(2):214-227.
Retinal ganglion cells (RGCs) are the first cell type to differentiate during retinal histogenesis. It has been postulated that specified RGCs subsequently influence the number and fate of the remaining progenitors to produce the rest of the retinal cell types. However, several genetic knockout models have argued against this developmental role for RGCs. Although it is known that RGCs secrete cellular factors implicated in cell proliferation, survival, and differentiation, until now, limited publications have shown that reductions in the RGC number cause significant changes in these processes. In this study, we observed that Math5 and Brn3b double null mice exhibited over a 99% reduction in the number of RGCs during development. This severe reduction of RGCs is accompanied by a drastic loss in the number of all other retinal cell types that was never seen before. Unlike Brn3b null or Math5 null animals, mice null for both alleles lack an optic nerve and have severe retinal dysfunction. Results of this study support the hypothesis that RGCs play a pivotal role in the late phase of mammalian retina development.
PMCID: PMC2483850  PMID: 18321480
math5; ath5; atoh7; brn3b; POU4f2; RGC; progenitor; retina; development; mouse
5.  Retinal Ganglion Cell Death and Optic Nerve Degeneration by Genetic Ablation in Adult Mice 
Experimental Eye Research  2008;88(3):542-552.
Despite the magnitude of the problem, no effective treatments exist to prevent retinal ganglion cell (RGC) death and optic nerve degeneration from occurring in diseases affecting the human eye. Animal models currently available for developing treatment strategies suffer from cumbersome procedures required to induce RGC death or rely on mutations that induce defects in developing retinas rather than in mature retinas of adults. Our objective was to develop a robust genetically engineered adult mouse model for RGC loss and optic nerve degeneration based on genetic ablation. To achieve this, we took advantage of Pou4f2 (Brn3b), a gene activated immediately as RGCs begin to differentiate and expressed throughout life. We generated adult mice whose genomes harbored a conditional Pou4f2 allele containing a floxed-lacZ-stop-diphtheria toxin A cassette and a CAGG-Cre-ER™ transgene. In this bigenic model, Cre recombinase is fused to a modified estrogen nuclear receptor in which the estrogen-binding domain binds preferentially to the estrogen agonist tamoxifen rather than to endogenous estradiol. Upon binding to the estrogen-binding domain, tamoxifen derepresses Cre recombinase, leading to the efficient genomic deletion of the floxed lacZ-stop DNA sequence and expression of diphtheria toxin A. Tamoxifen administered to adult mice at different ages by intraperitoneal injection led to rapid RGC loss, reactive gliosis, progressive degradation of the optic nerve over a period of several months, and visual impairment. Perhaps more reflective of human disease, partial loss of RGCs was achieved by modulating the tamoxifen treatment. Especially relevant for RGC death and optic nerve degeneration in human retinal pathologies, RGC-ablated retinas maintained their structural integrity, and other retinal neurons and their connections in the inner and outer plexiform layers appeared unaffected by RGC ablation. These events are hallmarks of progressive optic nerve degeneration observed in human retinal pathologies and demonstrate the validity of this model for use in developing stem cell therapies for replacing dead RGCs with healthy ones.
PMCID: PMC3398624  PMID: 19109949
Retinal ganglion cells; optic nerve degeneration; Pou4f2/Brn3b; diphtheria toxin A; genetic ablation; retinal disease mouse model
6.  Gene expression in the developing mouse retina by EST sequencing and microarray analysis 
Nucleic Acids Research  2001;29(24):4983-4993.
Retinal development occurs in mice between embryonic day E11.5 and post-natal day P8 as uncommitted neuroblasts assume retinal cell fates. The genetic pathways regulating retinal development are being identified but little is understood about the global networks that link these pathways together or the complexity of the expressed gene set required to form the retina. At E14.5, the retina contains mostly uncommitted neuroblasts and newly differentiated neurons. Here we report a sequence analysis of an E14.5 retinal cDNA library. To date, we have archived 15 268 ESTs and have annotated 9035, which represent 5288 genes. The fraction of singly occurring ESTs as a function of total EST accrual suggests that the total number of expressed genes in the library could approach 27 000. The 9035 ESTs were categorized by their known or putative functions. Representation of the genes involved in eye development was significantly higher in the retinal clone set compared with the NIA mouse 15K cDNA clone set. Screening with a microarray containing 864 cDNA clones using wild-type and brn-3b (–/–) retinal cDNA probes revealed a potential regulatory linkage between the transcription factor Brn-3b and expression of GAP-43, a protein associated with axon growth. The retinal EST database will be a valuable platform for gene expression profiling and a new source for gene discovery.
PMCID: PMC97568  PMID: 11812828

Results 1-6 (6)