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The transcription factor Pax6 controls multiple aspects of forebrain development. Conditional deletion of Pax6 in embryonic mouse cortex causes increased proliferation of cortical progenitor cells and a concomitant decrease in neural differentiation. Notch signaling regulates the balance between proliferation and differentiation of cortical progenitor cells, suggesting a possible connection between Pax6 and Notch signaling. We investigated how expression of the Notch ligand delta-like 1 ( Dll1 ) is altered by loss of Pax6. Acute cortex-specific deletion of Pax6 resulted in a widespread decrease in the density of Dll1 + cells at embryonic days 12.5 and 13.5 (E12.5 and E13.5). In constitutive loss-of-function mutants, decreases in the densities of Dll1 + cells were more limited both spatially and temporally. Controlled over-expression of Pax6 had no detectable effect on Dll1 expression. The proneural transcription factor Neurog2 is a target of Pax6 that can activate Dll1 expression and we found clear co-expression of Neurog2 and Dll1 in radial glial progenitors, suggesting that Pax6's effect on Dll1 could be mediated through Neurog2. However, we found no change in Dll1 + cells in Neurog2 -/- cortex suggesting either that Neurog2 is not directly involved, or that its loss of function in embryonic cortex can be compensated for.

Heparan sulfate (HS) is a cell surface and extracellular matrix carbohydrate extensively modified by differential sulfation. HS interacts physically with canonical fibroblast growth factor (FGF) proteins that signal through the extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) pathway. At the embryonic mouse telencephalic midline, FGF/ERK signaling drives astroglial precursor somal translocation from the ventricular zone of the corticoseptal boundary (CSB) to the induseum griseum (IG), producing a focus of Slit2 -expressing astroglial guidepost cells essential for interhemispheric corpus callosum (CC) axon navigation. Here, we investigated the cell and molecular function of a specific form of HS sulfation, 2-O HS sulfation catalyzed by the enzyme Hs2st, in midline astroglial development and in regulating FGF protein levels and interaction with HS. Hs2st -/- embryos of either sex exhibit a grossly enlarged IG due to precocious astroglial translocation and conditional Hs2st mutagenesis and ex vivo culture experiments show that Hs2st is not required cell autonomously by CC axons or by the IG astroglial cell lineage, but rather acts non-cell autonomously to suppress the transmission of translocation signals to astroglial precursors. Rescue of the Hs2st -/- astroglial translocation phenotype by pharmacologically inhibiting FGF signaling shows that the normal role of Hs2st is to suppress FGF-mediated astroglial translocation. We demonstrate a selective action of Hs2st on FGF protein by showing that Hs2st (but not Hs6st1 ) normally suppresses the levels of Fgf17 protein in the CSB region in vivo and use a biochemical assay to show that Hs2st (but not Hs6st1 ) facilitates a physical interaction between the Fgf17 protein and HS. SIGNIFICANCE STATEMENT We report a novel non-cell-autonomous mechanism regulating cell signaling in developing brain. Using the developing mouse telencephalic midline as an exemplar, we show that the specific sulfation modification of the cell surface and extracellular carbohydrate heparan sulfate (HS) performed by Hs2st suppresses the supply of translocation signals to astroglial precursors by a non-cell-autonomous mechanism. We further show that Hs2st modification selectively facilitates a physical interaction between Fgf17 and HS and suppresses Fgf17 protein levels in vivo , strongly suggesting that Hs2st acts selectively on Fgf17 signaling. HS interacts with many signaling proteins potentially encoding numerous selective interactions important in development and disease, so this class of mechanism may apply more broadly to other biological systems., (Copyright © 2019 Clegg et al.)

Differences in the growth and maturation of diverse forebrain tissues depend on region-specific transcriptional regulation. Individual transcription factors act simultaneously in multiple regions that develop very differently, raising questions about the extent to which their actions vary regionally. We found that the transcription factor Pax6 affects the transcriptomes and the balance between proliferation and differentiation in opposite directions in the diencephalon versus cerebral cortex. We tested several possible mechanisms to explain Pax6's tissue-specific actions and found that the presence of the transcription factor Foxg1 in the cortex but not in the diencephalon was most influential. We found that Foxg1 is responsible for many of the differences in cell cycle gene expression between the diencephalon and cortex and, in cortex lacking Foxg1, Pax6's action on the balance of proliferation versus differentiation becomes diencephalon like. Our findings reveal a mechanism for generating regional forebrain diversity in which one transcription factor completely reverses the actions of another., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pax6 is a key regulator of the rates of progenitor cell division in cerebral corticogenesis. Previous work has suggested that this action is mediated at least in part by regulation of the cell cycle gene Cdk6 , which acts largely on the transition from G1 to S phase. We began the present study by investigating whether, in addition to Cdk6 , other Pax6-regulated cell cycle genes are likely to be primary mediators of Pax6's actions on cortical progenitor cell cycles. Following acute cortex-specific deletion of Pax6, Cdk6 showed changes in expression a day earlier than any other Pax6-regulated cell cycle gene suggesting that it is the primary mediator of Pax6's actions. We then used flow cytometry to show that progenitors lacking Pax6 have a shortened G1 phase and that their Cdk6 levels are increased in all phases. We found that Cdk6 levels oscillate during the cell cycle, increasing from G1 to M phase. We propose a model in which loss of Pax6 shortens G1 phase by raising overall Cdk6 levels, thereby shortening the time taken for Cdk6 levels to cross a threshold triggering transition to S phase.

The cerebral cortex contains an enormous number of neurons, allowing it to perform highly complex neural tasks. Understanding how these neurons develop at the correct time and place and in accurate numbers constitutes a major challenge. Here, we demonstrate a novel role for Gli3, a key regulator of cortical development, in cortical neurogenesis. We show that the onset of neuron formation is delayed in Gli3 conditional mouse mutants. Gene expression profiling and cell cycle measurements indicate that shortening of the G1 and S phases in radial glial cells precedes this delay. Reduced G1 length correlates with an upregulation of the cyclin-dependent kinase gene Cdk6 , which is directly regulated by Gli3. Moreover, pharmacological interference with Cdk6 function rescues the delayed neurogenesis in Gli3 mutant embryos. Overall, our data indicate that Gli3 controls the onset of cortical neurogenesis by determining the levels of Cdk6 expression, thereby regulating neuronal output and cortical size., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Introduction: To review important studies examining focal laser for diabetic macular edema (DME), to examine real-world data regarding actual treatments patients are receiving, to present long-term visual outcomes in real-world practice, and to suggest an evidence-based approach for the use of focal laser., Methods: This study is a review of landmark studies evaluating focal laser and pharmacologic therapy for DME. In addition, the authors include a retrospective review of 102 consecutive eyes of 53 patients in our practice setting in rural Alabama. A chart review was performed, and patients were included if they were diagnosed with DME and were treated with both focal laser and bevacizumab. Bevacizumab and focal laser were given on a "as needed basis" at the discretion of one treating physician (J.O.M.). Worse visual acuity or worsening macular edema were indications for additional treatment. Statistical analysis was performed using frequencies and percentages. Best-corrected visual acuity (BCVA) was recorded at baseline and at the end of treatment (mean of 5 years) in the medical record. Primary outcome measures were BCVA, patients with better than 20/40 BCVA, patients with worse than 20/200 BCVA, and patients with stable BCVA., Results: Anti-vascular endothelial growth factor (VEGF) therapies are the first-line treatment for DME, but real-world claims data suggest that diabetic patients cannot come in for monthly injections as in large clinical trials. In our series, after a mean of 5 lasers and 5.5 injections, 90% of eyes had stable or better BCVA, 65% were ≥20/40, and only 13% were ≤20/200., Conclusions: Laser treatment for DME remains an important adjunctive therapy., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: To evaluate the outcomes of choroidal melanoma 25-gauge vitrectomy needle biopsy and iodine 125 ( 125 I) brachytherapy performed by supervised fellows compared with experienced faculty., Design: Retrospective, observational case series., Participants: Sixty-three consecutive eyes of 63 patients with treatment-naïve medium choroidal melanomas without extraocular extension., Methods: Sixty-three consecutive eyes of 63 patients undergoing simultaneous 125 I brachytherapy and 25-gauge vitrectomy melanoma biopsy for choroidal melanoma. One attending surgeon performed 31 surgeries and 2 fellows performed 32 surgeries. All cases treated by fellows were under direct supervision of the attending physician., Main Outcome Measures: Percentage of adequate biopsy sample, tumor regression, and postoperative complications., Results: There were no baseline differences between the groups. The biopsy yield for gene expression profile and the melanoma regression rate were equal in both groups (63/63 [100%]; P = 1.0). Complications occurred in 1 of 31 faculty surgery patients (transient vitreous hemorrhage) and in 0 of 32 fellow surgery patients (P = 1.0)., Conclusions: With proper supervision, fellows can achieve equally high biopsy yield and melanoma regression rate with few complications compared with experienced faculty., (Copyright © 2017 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: Transvitreal and transscleral needle biopsy can result in complications including vitreous hemorrhage and retinal detachment. This study evaluated a technique using 25-gauge vitrectomy as an adjunct to needle biopsy immediately before brachytherapy to minimize these complications and preserve good visual acuity., Design: Retrospective, observational case series., Participants: Fifty-seven consecutive eyes of 57 patients with treatment-naïve medium choroidal melanomas without extraocular extension from July 2012 through September 2015., Methods: Fifty-seven consecutive eyes of 57 patients with a clinically diagnosed choroidal melanoma underwent complete 25-gauge posterior vitrectomy followed by transvitrector port fine-needle aspiration biopsy of the tumor immediately before implantation of a radioactive iodine 125 plaque as treatment for the tumor. Cytopathologic analysis was not performed on the tumor aspirates in this study., Main Outcome Measures: Best-corrected postoperative visual acuity, postoperative complications of the reported technique, implantation tumor development, local tumor recurrence, presence of metastatic disease after surgery, and sufficiency of the tumor aspirates obtained by the reported technique for successful gene expression profile testing and prognostic classification., Results: Mean preoperative and postoperative visual acuities were similar (20/60 vs. 20/80, respectively). Mean tumor thickness was 5.0 mm (range, 2.5-10 mm) and mean tumor basal diameter was 13.1 mm (range, 7-22 mm). Only 1 of 57 eyes (1.8%) showed a transient vitreous hemorrhage, biopsy yield was 100% for genetic analysis, and no patients showed recurrence or implantation tumor at the vitrector site., Conclusions: Combined 25-gauge posterior vitrectomy and 25-gauge trans-vitrector port needle aspiration biopsy immediately before brachytherapy is excellent for obtaining tumor aspirate for gene expression profiling while controlling for hemostasis, resulting in few complications., (Copyright © 2017 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Background: The tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/- hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3., Results: Although TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/- hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/- hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex., Conclusions: Mosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing.

Background: We studied whether regulation of Cdca7 (Cell division cycle associated 7) expression by transcription factor Pax6 contributes to Pax6's cellular actions during corticogenesis. The function of Cdca7 in mediating Pax6's effects during corticogenesis has not been explored. Pax6 is expressed by radial glial progenitors in the ventricular zone of the embryonic cortical neuroepithelium, where it is required for the development of a normal complement of Tbr2-expressing intermediate progenitor cells in the subventricular zone. Pax6's expression levels are graded across the ventricular zone, with highest levels laterally where Tbr2-expressing progenitors are generated in greatest numbers at early stages of corticogenesis., Methods: We used in situ hybridization and immunohistochemistry to analyse patterns of Cdca7 and Pax6 expression in cortical tissue from wild-type and Pax6 -/- embryos. In each genotype we compared the graded expression of the two genes quantitatively at several ages. To test whether defects in Cdca7 expression in lateral cortical cells might contribute to the cellular defects in this region caused by Pax6 loss, we electroporated a Cdca7 expression vector into wild-type lateral cortex and examined the effect on the production of Tbr2-expressing cells., Results: We found that Cdca7 is co-expressed with Pax6 in cortical progenitors, at levels opposite to those of Pax6. Lowest levels of Cdca7 are found in the radial glial progenitors of lateral cortex, where Pax6 levels are highest. Higher levels of Cdca7 are found in ventral telencephalon, where Pax6 levels are low. Loss of Pax6 causes Cdca7 expression to increase in the lateral cortex. Elevating Cdca7 in normal lateral cortical progenitors to levels close to those normally found in ventral telencephalon reduces their production of Tbr2-expressing cells early in lateral cortical formation., Conclusion: Our results suggest that Pax6 normally represses Cdca7 expression in the lateral cortex and that repression of Cdca7 in cells of this region is required for their production of a normal complement of Tbr2-expressing intermediate progenitors.

Purpose . To identify treatment preferences of patients with diabetic macular edema (DME) having undergone laser and intravitreal injections. Methods . Patients with DME who received lasers and injections were surveyed, measuring preferences toward specific treatments. 66/210 diabetic patients met the criteria for our survey assessing preference for lasers and/or injections, incorporating demographics and treatment preference questions. Outcome measures included treatment preference (laser or injections), how often patients are willing to be treated, and how much vision they will sacrifice to avoid being treated every month. Results . 66 patients completed the survey. The mean diabetes duration was 20.7 years, the mean retina follow-up was 4.4 years, and patients received a mean of 4.82 lasers and 4.86 injections. 56% preferred injections, 33% preferred laser, and 11% had no preference. Regarding treatment effectiveness, 38% found no difference, 36% chose laser, and 25% chose injections. Regarding anxiety, 56% reported injection anxiety. While 50% versus 38% reported that laser was easier than injections. 91% would give up zero lines on the eye chart, and 76% would come in 12 times yearly for treatment to maintain vision. Conclusion . Patients with DME have no profound preference regarding laser versus intravitreal injections but prefer aggressive treatment and are unwilling to sacrifice vision for less visits.

Background: The transcription factor Barhl2 is an antiproneural transcription factor with roles in neuronal differentiation. The functions of its homologue in Drosophila development are better understood than its functions in mammalian brain development. Existing evidence suggests that its expression in the embryonic forebrain of the mouse is regional and may complement that of another transcription factor that is important for forebrain development, Pax6. The aim of this study is to provide a more detailed description of the Barhl2 expression pattern in the embryonic forebrain than is currently available, to relate its expression domains to those of Pax6 and to examine the effects of Pax6 loss on Barhl2 expression., Results: We found that Barhl2 is expressed in the developing diencephalon from the time of anterior neural tube closure. Its expression initially overlaps that of Pax6 in a central region of the alar diencephalon but over the following days their domains of expression become complementary in most forebrain regions. The exceptions are the thalamus and pretectum, where countergradients of Pax6 and Barhl2 expression are established by embryonic day 12.5, before overall Pax6 levels in these regions decline greatly while Barhl2 levels remain relatively high. We found that Barhl2 expression becomes upregulated in specifically the thalamus and pretectum in Pax6-null mice., Conclusions: The region-specific expression pattern of Barhl2 makes it likely to be an important player in the development of region-specific differences in embryonic mouse forebrain. Repression of its expression in the thalamus and pretectum by Pax6 may be crucial for allowing proneural factors to promote normal neuronal differentiation in this region.

Background: Acute Multifocal Hemorrhagic Retinal Vasculitis (AMHRV) is a rare disease with unknown incidence that presents with abrupt onset of visual loss associated with retinal vasculitis, retinal hemorrhage, non-confluent posterior retinal infiltrates, vitreous cellular inflammation and papillitis in, otherwise, healthy adult individuals. The reported treatment options for Acute Multifocal Hemorrhagic Retinal Vasculitis are oral corticosteroids, intravitreal ganciclovir and laser photocoagulation or vitrectomy. We report a child with Acute Multifocal Hemorrhagic Retinal Vasculitis who was treated with aggressive immunosuppressive therapy resulting in a favorable visual outcome., Case Presentation: A retrospective case report of a 10-year-old African American girl who developed unilateral Acute Multifocal Hemorrhagic Retinal Vasculitis, which later on progressed bilaterally. We conducted a review of the clinical, laboratory and photographic records to evaluate her functional and anatomic outcome after aggressive immunosuppressive treatment. During the first 4 months of treatment of OD with intravitreal ganciclovir, intravitreal dexamethasone and systemic prednisone, the change in vision in OD improved from light perception (LP) to counting fingers (CF). During the next 18 months of aggressive systemic treatment of OD and the newly affected left eye (OS), the change in vision improved from CF in OD and CF in OS to 20/200 in OD and 20/80 in OS. Management during the 18-month interval included rituximab infusions, cyclophosphamide/methylprednisolone infusions, prednisone and mycophenolate., Conclusions: This is the first reported case of Acute Multifocal Hemorrhagic Retinal Vasculitis occurring in a child. Ophthalmologists should be aware of the need to treat severe Acute Multifocal Hemorrhagic Retinal Vasculitis with aggressive immunosuppressive agents in collaboration with rheumatologists to obtain the best possible visual outcome.

The recent development of organoid techniques, in which embryonic brain-like tissue can be grown from human or mouse stem cells in vitro offers the potential to transform the way in which brain development is studied. In this review, we summarize key aspects of the embryonic development of mammalian forebrains, focussing in particular on the cerebral cortex and highlight significant differences between mouse and primates, including human. We discuss recent work using cerebral organoids that has revealed key similarities and differences between their development and that of the brain in vivo. Finally, we outline the ways in which cerebral organoids can be used in combination with CRISPR/Cas9 genome editing to unravel genetic mechanisms that control embryonic development of the cerebral cortex, how this can help us understand the causes of neurodevelopmental disorders and some of the key challenges which will have to be resolved before organoids can become a mainstream tool to study brain development., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6 (tm1Ued) (Pax6 (fl) ) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6 (fl/fl) and heterozygous Pax6 (fl/+) mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6 (fl/fl) corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6 (Sey-Neu) (Pax6 (-)) null allele. Pax6 (fl/-) compound heterozygotes had more severe eye abnormalities than Pax6 (+/-) heterozygotes, implying that Pax6 (fl) differs from the wild-type Pax6 (+) allele. Immunohistochemistry showed that the Pax6 (fl/-) corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6 (fl) allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles.

Purpose: To evaluate common vitreoretinal surgeries performed by retinal fellows under direct faculty supervision, compared with experienced faculty members., Methods: Retrospective study analyzing 592 consecutive eyes undergoing retinal surgery from 2009 to 2011 at Retina Consultants of Alabama/University of Alabama at Birmingham, Department of Ophthalmology. Vitreoretinal surgeries included macular hole, macular pucker, retinal detachment, diabetic vitreous hemorrhage, and diabetic tractional retinal detachment. Three fellows performed 390 cases (divided into first or second year fellows), while 4 faculty members performed 202 cases. All 390 fellow-performed cases were under direct supervision. Chi-square analysis was used to compare outcomes., Results: There were no baseline differences between the groups. The mean postoperative visual improvement was statistically significant and equal in all groups, as well as between each physician (P ≤ 0.0001). Complications occurred in 29/592 cases (4.8%), whereas reoperations occurred in 21/592 cases (3.5%) and were equally distributed across groups. There were no differences in complications and reoperations when comparing first-year with second-year fellows., Conclusion: With proper supervision, vitreoretinal fellows can achieve an equally high visual improvement with low complication and reoperation rates compared with experienced faculty. The year of fellowship does not significantly influence outcomes or complications. Quality outcomes after vitreoretinal surgery can be obtained throughout fellowship training.

Purpose: To report a very rare case of bilateral isolated choroidal melanocystosis., Methods: Clinical case report and literature review., Results: A 24-year-old asymptomatic African American woman presented with bilateral diffuse choroidal pigmentation. The diagnosis of bilateral isolated choroidal melanocytosis was made, and optical coherence tomography was remarkable for increased choroidal thickness with a normal inner and outer retina., Conclusion: Although extremely rare, bilateral isolated choroidal melanocytosis may occur in young patients, as well as in those who are African American. Longer follow-up of this case and those in the literature will elucidate whether these choroidal lesions enlarge or have a risk of developing uveal melanoma.

Understanding brain development remains a major challenge at the heart of understanding what makes us human. The neocortex, in evolutionary terms the newest part of the cerebral cortex, is the seat of higher cognitive functions. Its normal development requires the production, positioning, and appropriate interconnection of very large numbers of both excitatory and inhibitory neurons. Pax6 is one of a relatively small group of transcription factors that exert high-level control of cortical development, and whose mutation or deletion from developing embryos causes major brain defects and a wide range of neurodevelopmental disorders. Pax6 is very highly conserved between primate and non-primate species, is expressed in a gradient throughout the developing cortex and is essential for normal corticogenesis. Our understanding of Pax6's functions and the cellular processes that it regulates during mammalian cortical development has significantly advanced in the last decade, owing to the combined application of genetic and biochemical analyses. Here, we review the functional importance of Pax6 in regulating cortical progenitor proliferation, neurogenesis, and formation of cortical layers and highlight important differences between rodents and primates. We also review the pathological effects of PAX6 mutations in human neurodevelopmental disorders. We discuss some aspects of Pax6's molecular actions including its own complex transcriptional regulation, the distinct molecular functions of its splice variants and some of Pax6's known direct targets which mediate its actions during cortical development.

The corticothalamic and thalamocortical tracts play essential roles in the communication between the cortex and thalamus. During development, axons forming these tracts have to follow a complex path to reach their target areas. While much attention has been paid to the mechanisms regulating their passage through the ventral telencephalon, very little is known about how the developing cortex contributes to corticothalamic/thalamocortical tract formation. Gli3 encodes a zinc finger transcription factor widely expressed in telencephalic progenitors which has important roles in corticothalamic and thalamocortical pathfinding. Here, we conditionally inactivated Gli3 in dorsal telencephalic progenitors to determine its role in corticothalamic tract formation. In Emx1Cre;Gli3(fl/fl) mutants, only a few corticothalamic axons enter the striatum in a restricted dorsal domain. This restricted entry correlates with a medial expansion of the piriform cortex. Transplantation experiments showed that the expanded piriform cortex repels corticofugal axons. Moreover, expression of Sema5B, a chemorepellent for corticofugal axons produced by the piriform cortex, is similarly expanded. Finally, time course analysis revealed an expansion of the ventral pallial progenitor domain which gives rise to the piriform cortex. Hence, control of lateral cortical development by Gli3 at the progenitor level is crucial for corticothalamic pathfinding., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: The aim of this work is to evaluate the abundance, origins, and phenotypes of actively proliferating cells in proliferative diabetic retinopathy (PDR)., Methods: Eleven epiretinal membranes from patients undergoing surgery for PDR were evaluated by indirect immunofluorescence for evidence of cell proliferation using the nuclear cell proliferation marker Ki67 and for cell identities using glial fibrillary acidic protein (GFAP), glutamine synthetase, and α-smooth muscle actin (αSMA)., Results: Ki67 positivity was consistently rare in PDR epiretinal membranes at 3.02 ± 1.42 % of the total cell population. The majority of the Ki67-positive cells were also positive for GFAP (74.0 %) with lower proportions positive for αSMA (30.7 %) and glutamine synthetase (1.5 %). Co-localization studies using glial and myoid markers revealed that virtually all (92 %) of the αSMA-positive cells are also GFAP positive and thus derive from glia., Conclusions: Entry into cell cycle and thus cell proliferation appears to be a rare phenomenon in PDR involving only a small percentage of the total cell population. Glia and/or glial-derived myofibroblasts appear to be the predominate cell types in epiretinal scar tissues and also account for the majority of the actively proliferating cells.

Background: Little is known about motor vehicle collision (MVC) risk in older drivers with age-related macular degeneration (AMD). The purpose of this study is to examine associations between MVC involvement and AMD presence and severity., Methods: In a retrospective cohort study pooling the samples from four previous studies, we examined associations between MVC rate and older drivers with early, intermediate or advanced AMD as compared with those in normal eye health. MVC data were based on accident reports obtained from the state agency that compiles this information., Results: MVC rate was highest among those in normal eye health and progressively declined among those with early and intermediate disease, and then increased for those with advanced AMD. However, only for drivers with intermediate AMD was the MVC rate significantly different (lower) as compared with those in normal eye health, regardless of whether the rate was defined in terms of person-years (RR 0.34, 95% CI 0.13 to 0.89) or person-miles (RR 0.35, 95% CI 0.13 to 0.91) of driving., Conclusions: These results suggest that older drivers with intermediate AMD have a reduced risk of collision involvement. Further research should investigate whether self-regulatory driving practices by these drivers (avoiding challenging driving situations) underlies this reduced risk.

During embryonic development of the visual system, retinal ganglion cells (RGCs) project their axons towards the brain, passing through the optic chiasm. Axons are guided on this journey by molecular cues in the environment. The heparan sulphate sulphotransferase (Hst) enzymes Hs2st and Hs6st1 are each known to be required for specific aspects of axon guidance in the developing visual system, as revealed by studies of Hs2st(-/-) and Hs6st1(-/-) mutant embryos. However, it remained possible that these two enzymes have additional, overlapping, functions in RGC axon guidance; but that no effect is manifest in single mutant embryos, because the other enzyme is sufficient to fulfil the shared function. To investigate this possibility, we generated a set of Hs2st;Hs6st1 double mutant embryos that had reduced gene dosage of each of these Hsts, reasoning that any additional phenotypes in these animals would indicate the presence of functional overlap. We first characterised the structure of the mutant Hs6st1 locus, identifying the insertion site of the gene trap vector, to allow us to genotype compound mutants reliably. We found that Hs2st(-/-) ;Hs6st1(-/-) mutants that lack both enzymes died prior to E15.5. As the optic chiasm has not formed by this stage, we were unable to determine the effect of complete loss of Hs2st and Hs6st1 on chiasm formation. However, compound mutant embryos lacking one Hst and heterozygous for the other were viable. We found that RGC axon guidance defects in such compound mutants were no more severe than those found in the single mutant embryos. We also found that expression of the Hs6st1 isoform Hs6st3 overlaps with that of Hs6st1 in the developing visual system, suggesting that some Hs6st activity remains present in this region of Hs6st1(-/-) mutant embryos., (© 2011 The Authors. Journal of Anatomy © 2011 Anatomical Society of Great Britain and Ireland.)

Aim: To examine associations between retinal thickness and rod-mediated dark adaptation in older adults with non-exudative age-related maculopathy (ARM) or normal macular health., Methods: A cross-sectional study was conducted with 74 adults ≥ 50 years old from the comprehensive ophthalmology and retina services of an academic eye centre. ARM presence and disease severity in the enrolment eye was defined by the masked grading of stereofundus photos using the Clinical Age-Related Maculopathy grading system. High-definition, spectral-domain optical coherence tomography was used to estimate retinal thickness in a grid of regions in the macula. Rod-mediated dark adaptation, recovery of light sensitivity after a photo-bleach, was measured over a 20-min period for a 500 nm target presented at 5° on the inferior vertical meridian. Main outcomes of interest were retinal thickness in the macula (μm) and parameters of rod-mediated dark adaptation (second slope, third slope, average sensitivity, final sensitivity)., Results: In non-exudative disease retinal thickness was decreased in greater disease severity; thinner retina was associated with reductions in average and final rod-mediated sensitivity even after adjustment for age and visual acuity., Conclusions: Impairment in rod-mediated dark adaptation in non-exudative ARM is associated with macular thinning.

The zinc finger transcription factor Gli3 is essential for normal development of the forebrain. Mutant mice with no functional Gli3 (extra-toes, Gli3(Xt/Xt) mutants) display a massive reduction in the size of the telencephalic lobes and absence of dorsomedial telencephalic structures, including the cortical hem, which normally expresses a number of Wnt molecules essential for patterning the hippocampus. Dorsomedial telencephalic Wnt activity, transduced through the Wnt/β-catenin signaling pathway, is also required for hippocampal specification and dorsoventral telencephalic patterning. Wnts whose normal expression is restricted to the cortical hem are completely absent in Gli3(Xt/Xt) embryos, but some expression of those Wnts with a broader expression domain persists, raising the possibility that Wnt/β-catenin signaling may still be active in this mutant. We examined whether the Wnt expression that persists in the Gli3(Xt/Xt) mutant neocortex activates Wnt/β-catenin signaling, using the BAT-gal transgenic reporter. We found Wnt/β-catenin signaling consistently decreased in the forebrains of Gli3(Xt/Xt) mutants, even prior to the formation of the cortical hem. This is accompanied by a severe reduction in expression of Wnt7b and Wnt8b at the lateral edges of the anterior neural plate that will give rise to the pallium. In addition, we found a significant increase in the expression of rostroventral markers of the anterior neural plate that will give rise to the basal forebrain. Our data reveal that Gli3 is required at the neural plate stage to regulate Wnt expression and Wnt/β-catenin signaling in the presumptive forebrain and confirm its previously proposed role in patterning the anterior neural plate., (Copyright © 2011 Wiley-Liss, Inc.)

The Gli3 zinc finger transcription factor is expressed in developing forebrain, with the highest levels of expression in dorsal telencephalon. In Gli3(-/-) embryos the dorsal telencephalon is abnormally small and fails to develop dorsomedial telencephalic structures, including hippocampus and cortical hem, while the ventral telencephalon appears to expand. A hurdle to understanding the underlying mechanisms is that abnormalities of developing Gli3(-/-) telencephalic cells in Gli3(-/-) mutants result from a combination of their own cell autonomous defects and defects in the Gli3(-/-) cells that surround them. Here we used chimeras to identify some of the defects of Gli3(-/-) telencephalic cells that are likely to be autonomous by studying how Gli3(-/-) cells develop when surrounded by a majority of wild-type cells. We found that Gli3(-/-) cells are present in all components of the Gli3(-/-)<-->Gli3(+/+) chimeric forebrain, including dorsomedial structures, in proportions that either equal or exceed proportions found elsewhere in the embryo. Gli3(-/-) cells segregate from Gli3(+/+) cells to form many abnormal structures particularly in dorsal telencephalon. Gli3(-/-) cells in some locations are misspecified: in those parts of the dorsal telencephalon near to its boundaries with the diencephalon and the ventral telencephalon, mutant cells express sets of transcription factors expressed by wild-type cells on the other side of the boundary. Elsewhere in the dorsal telencephalon, in the diencephalon and in the ventral telencephalon, mutant cells express sets of transcription factors similar to those expressed by their immediately surrounding wild-type cells. We propose that an important cell autonomous action of Gli3 is to regulate the competence of dorsal telencephalic cells, preventing cells near to its boundaries expressing regulatory factors normally restricted to adjacent tissues.

The transcription factor Gli3 is expressed throughout developing telencephalon. Previous studies have focused on Gli3's role in dorsal telencephalon, which is greatly reduced in size in Gli3(Xt/Xt) mutants. We examined the effects of loss of Gli3 on early development of ventral telencephalon. Ventral telencephalon was defined in both wildtypes and Gli3(Xt/Xt) mutants on the basis of its expression of Olig2, Nkx2.1, Mash1, and Foxg1 and its lack of expression of Pax6. We found that at embryonic day (E)10.5 the volume of the ventral telencephalon is about 50% greater in Gli3(Xt/Xt) mutants than in wildtypes. By E12.5, however, the volume of the ventral telencephalon is about 20% lower in Gli3(Xt/Xt) mutants than in wildtypes. We observed a significant increase in the number of both apoptotic cells and newly differentiated neurons in the E10.5 Gli3(Xt/Xt) ventral telencephalon, suggesting that increased cell death and withdrawal of cells from the cell cycle might account for the failure of the Gli3(Xt/Xt) ventral telencephalon to grow normally by E12.5. We found no changes in the lengths of the cell cycles of proliferating ventral telencephalic cells at E10.5. We used marker analysis and optical projection tomography to assess the Gli3(Xt/Xt) forebrain in three dimensions and found that the Gli3(Xt/Xt) diencephalon is shifted relatively rostrally. We conclude that in the absence of Gli3 an abnormally large portion of the newly formed telencephalon is specified to a ventral fate but this then suffers impaired growth, due to defects of cell differentiation and death, contributing to severe distortion of the forebrain.

This interventional case report retrospectively reviews the outcome of a 56-year-old woman who suffered an intraoperative choroidal hemorrhage at commencement of macular hole surgery. Due to the early intraoperative choroidal hemorrhage, vitrectomy was not performed. One month postoperatively, the macular hole was noted to be closed and remained closed 2 years later, leaving the patient with a good visual outcome. The authors postulate that the temporal location of the choroidal hemorrhage may have exerted mechanical displacement of the retina toward the macular hole margins, resulting in closure. Macular hole closure directly following intraoperative choroidal hemorrhage is possible.

Purpose: To evaluate the efficacy of intravitreal injection of bevacizumab (Avastin) (IVA) in preventing panretinal photocoagulation (PRP)-induced macular thickening and visual dysfunction in eyes with severe proliferative diabetic retinopathy., Methods: A retrospective review of 60 consecutive eyes (30 patients) with severe proliferative diabetic retinopathy whose visual acuity was 20/30 or better (<0.18 in logarithm of the minimum angle of resolution acuity) and average foveal thickness (FT) was 280 microm or less, and whose retinopathy was bilateral and symmetrical, was performed. In all eyes, PRP was performed in two sessions. In the interventional group, 1.25 mg of IVA was injected to each eye 1 week before initiation of PRP. Foveal thickness was measured by optical coherence tomography before treatment, and the clinical course was monitored by best corrected visual acuity (BCVA) and FT for 24 weeks after beginning PRP., Results: Before treatment, mean BCVA and FT was 0.073 +/- 0.071 microm and 278.8 +/- 29.5 microm in the IVA-injected group and 0.069 +/- 0.076 microm and 273.5 +/- 27.7 microm in the control group, respectively. After the IVA injection and PRP completion, FT in the IVA-injected eyes was significantly decreased, with a mean FT of 257.2 microm at 12 weeks and 264.3 microm at 24 weeks. In the control group, FT increased dramatically and reached 307.3 microm at 12 weeks and 298.2 microm at 24 weeks. The difference in final FT between groups was significant (P = 0.001). Best corrected visual acuity in the control group decreased with time to 0.149 +/- 0.113 at 24 weeks; in contrast, BCVA in the IVA-injected eyes improved over time to 0.039 +/- 0.054 at 24 weeks. This difference in BCVA was statistically significant (P< or =0.0001). Seven eyes (23.3%) in the control group had worse vision by > or =2 lines and increased FT by > or =50 microm at 24 weeks, whereas none of the eyes in the IVA group had either worse vision or a significant increase in FT (P = 0.011)., Conclusions: A single IVA injection given before standard PRP may be beneficial in preventing PRP-induced visual dysfunction and foveal thickening in eyes with severe proliferative diabetic retinopathy and good vision.

Purpose: To evaluate the visual prognosis in eyes with branch retinal artery occlusion (BRAO)., Design: Retrospective, observational, consecutive case series., Methods: Retrospective medical record review of 52 consecutive eyes of 52 patients with BRAO seen at two vitreoretinal practices in Birmingham, Alabama. Only eyes with decreased central macular perfusion on fluorescein angiography (FA) were included. Eyes with anterior segment or optic nerve disease, lack of retinal whitening or a delay in arterial filling on FA, central retinal artery occlusion, and cilioretinal artery occlusion were excluded. The main outcome measure was presenting best-corrected visual acuity (BCVA) and its relationship to final BCVA., Results: On presentation, 54% of eyes with BRAO had BCVA of 20/40 or better. At the mean 14-month visit, 60% of all eyes had visual acuity (VA) of 20/40 or better. VAs of 20/40 or better were retained by 89% of eyes with baseline BCVA of 20/40 or better. Only 14% of eyes with 20/100 or worse BCVA improved to 20/40 or better. Neither visible emboli (P > or = .244) nor the region of macular involvement (P = .142) were significant with respect to visual improvement., Conclusions: Visual prognosis after BRAO seems to be correlated to presenting VA. Eyes with initial VA of 20/40 or better usually remained at 20/40 or better. Individuals with poor VA of 20/100 or worse generally did not show the significant improvement reported in previous studies.

Background and Objective: Evaluation of surgical treatment of full-thickness macular holes secondary to diabetic tractional retinal detachments was conducted., Patients and Methods: A retrospective review of medical records, fluorescein angiograms, fundus photographs, optical coherence tomography images, and operative findings of six consecutive patients with full-thickness macular holes and diabetic tractional retinal detachments was completed. Each eye was treated with pars plana vitrectomy, tractional retinal detachment repair, membranectomy, indocyanine green-assisted internal limiting membrane peeling, and intraocular gas tamponade., Results: Surgical intervention resulted in the closure of all full-thickness macular holes. Mean best-corrected visual acuity was 20/250 preoperatively and 20/100 postoperatively, with all patients having improvement after a mean follow-up of 10 months., Conclusion: Closure of tractional retinal detachments related to full-thickness macular holes can be achieved via pars plana vitrectomy, complete membranectomy, and intraocular gas. Vitrectomy with dissection of proliferative membranes helps to relieve the tractional forces responsible for full-thickness macular hole formation, enabling successful closure of the diabetic full-thickness macular holes and resulting in visual acuity improvement.

The Wnt family of secreted signaling proteins regulates many aspects of animal development and the behavior of several types of stem cells, including embryonic stem (ES) cells. Activation of canonical Wnt signaling has been shown to either inhibit or promote the differentiation of ES cells into neurons, depending on the stage of differentiation. Here, we describe the expression of all 19 mouse Wnt genes during this process. Using the well-established retinoic acid induction protocol we found that all Wnt genes except Wnt8b are expressed as ES cells differentiate into neurons, many of them in dynamic patterns. The expression pattern of 12 Wnt genes was analyzed quantitatively at 2-day intervals throughout neural differentiation, showing that multiple Wnt genes are expressed at each stage. A large proportion of these, including both canonical and noncanonical Wnts, are expressed at highest levels during later stages of differentiation. The complexity of the patterns observed indicates that disentangling specific roles for individual Wnt genes in the differentiation process will be a significant challenge.

The development of the neural crest is orchestrated by a complex interplay between intercellular signalling molecules and transcription factors. Here, we demonstrate a direct interaction between two such factors, the paired-type transcription factor Pax3 and the secretory glycoprotein Wnt1. We found that the Wnt1 promoter can be regulated by Pax3 in a dose-dependent manner. Sequence analysis predicted a conserved binding site for Pax3 within the Wnt1 promoter region. Deletion or mutation of this sequence abolished the promoter response to Pax3. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated that Pax3 interacts with the Wnt1 promoter in vivo. These data indicate that Pax3 directly regulates the expression of Wnt1 in the developing embryo.

Background and Objectives: To determine whether irrigation of the sub-Tenon's space with anesthetic agents during pars plana vitrectomy (PPV) involving general anesthesia decreases postoperative pain, analgesic use, or nausea., Patients and Methods: A prospective, controlled trial of 46 consecutive patients requesting general anesthesia for PPV who were randomized to receive or not receive a sub-Tenon's space injection prior to surgery. A mixture of 3 mL of 2% lidocaine with hyaluronidase and 3 mL of 0.5% bupivacaine was used to induce local blockade. Pain, postoperative nausea, and analgesia use were evaluated., Results: Local blockade did not significantly alter the proportion of reported pain at 30 minutes and 2, 4, and 24 hours after the operation. The local blockade had no effect on reducing postoperative nausea or the number of patients requiring pain medication., Conclusions: Local blockade prior to surgery in patients undergoing PPV under general anesthesia does not significantly decrease postoperative pain, analgesic use, or nausea.

Pax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus. In this 420 kb YAC a tauGFP-IRES-Neomycin reporter cassette has been inserted into the PAX6 translational start site in exon 4. The YAC has been further engineered to insert LoxP sites flanking a 35 kb long, distant downstream regulatory region (DRR) containing previously described DNaseI hypersensitive sites, to allow direct comparison between the presence or absence of this region in the same genomic context. Five independent transgenic lines were obtained that vary in the extent of downstream PAX6 locus that has integrated. Analysis of transgenic embryos carrying full-length and truncated versions of the YAC indicates the location and putative function of several novel tissue-specific enhancers. Absence of these distal regulatory elements abolishes expression in specific tissues despite the presence of more proximal enhancers with overlapping specificity, strongly suggesting interaction between these control elements. Using plasmid-based reporter transgenic analysis we provide detailed characterization of one of these enhancers in isolation. Furthermore, we show that overexpression of a short PAX6 isoform derived from an internal promoter in a multicopy YAC transgenic line results in a microphthalmia phenotype. Finally, direct comparison of a single-copy line with the floxed DRR before and after Cre-mediated deletion demonstrates unequivocally the essential role of these long-range control elements for PAX6 expression.

Purpose: To report the use of intravitreal bevacizumab (Avastin) as an adjunctive treatment for proliferative diabetic retinopathy (PDR)., Design: Retrospective case review., Methods: Institutional review board approval to review patient data was obtained for this retrospective study. Three patients underwent intravitreal injection of bevacizumab as part of their treatment for PDR after informed consent was signed. Each patient also underwent fundus photographs before the bevacizumab injection and then one to three weeks after., Results: All three patients showed complete regression of their neovascularization elsewhere (NVE) and neovascularization of the disk (NVD) between one and three weeks after injection., Conclusions: The speed and degree of neovascular regression after the injection of intravitreal bevacizumab may make this procedure an important adjunctive treatment in the management of selected cases with severe PDR.

Purpose: To assess the visual outcome following vitrectomy for diabetic retinopathy compared with previous studies; to evaluate risk factors for light perception (LP) and no light perception (NLP) vision after diabetic vitrectomy., Design: Retrospective medical record review., Methods: The charts of 100 consecutive patients who underwent vitrectomy for proliferative diabetic retinopathy between November 1, 1997, and November 30, 1998, were reviewed. Surgical technique included standard pars plana vitrectomy with combination of delamination and segmentation of gliotic tractional membranes using bimanual techniques. All patients had post-operative follow-up of at least 12 months. Several factors were analyzed for their effect on poor visual outcome (LP and NLP) using Fisher's exact test., Results: Post-vitrectomy, 73% of diabetic patients had stable or improved vision; 16% had worsened but functional vision, defined as worse but still >or= 20/400; 4% had worsened but ambulatory vision, defined as worse but still count fingers (CF) or hand motion (HM); and 7% had poor visual outcome, LP or NLP. Resultant visual acuity was >or=20/40 in 38% of patients, 20/50 to 20/100 in 34%, 20/120 to CF in 18%, HM in 3%, LP in 4%, and NLP in 3%. Risk factors for eyes with LP and NLP vision included pre-operative iris neovascularization (INV), P = .05, post-operative INV, P = .02, post-operative macular ischemia, P = .0001, and post-operative vitreous hemorrhage (VH), P = .02., Conclusions: Pre-operative and post-operative INV, post-operative macular ischemia, and post-operative VH appear to be risk factors for LP and NLP vision following diabetic vitrectomy, whereas overall improvements in surgical technique and visual outcome continue to be reported.

Purpose: To explore a clinically observed association between central serous chorioretinopathy (CSC) and gastroesophageal reflux disease (GERD) DESIGN: A retrospective case-control study., Participants and Controls: Sixty-nine consecutive patients diagnosed with CSC were compared with a control group of 55 non-CSC patients., Methods: The records of 69 patients with CSC were retrospectively reviewed and compared with the records of 55 controls. All patients and controls were examined in a referral setting., Results: Patients with CSC were significantly more likely to have GERD compared with controls (odds ratio 6.05; 95% confidence interval 2.14-17.11; P =.0003). Central serous chorioretinopathy patients were also more likely than controls to have used oral corticosteroid medications (odds ratio 16.30; 95% confidence interval 2.09-127.33; P =.0006) and antacid/antireflux medications (odds ratio 15.00; 95% confidence interval 1.91-117.58; P =.001)., Conclusions: This study identifies an association between CSC and GERD. There are biochemical factors common to both diseases that support this association, giving further insight into the pathogenesis of CSC.

Objective: To test the hypothesis that retinochoroidal collateral veins (RCVs), or alternatively, retinociliary or optociliary shunts/collaterals/veins or opticociliary anastomoses, act protectively against the development of anterior segment neovascularization (ASN) following central retinal vein occlusion (CRVO)., Design: Case-control retrospective medical record review of patients with CRVO., Patients: We identified 107 patients with CRVO, of whom 34 had developed ASN, by reviewing their medical records. After applying exclusion criteria, a case group and an age-, sex-, and visual acuity-matched control group were selected. We analyzed these groups for the presence or absence of RCVs and noted the time course involved in their development., Main Outcome Measures: Anterior segment neovascularization (including neovascularization of the iris and/or anterior chamber angle), neovascular glaucoma, and RCV development., Results: Only 1 (5.4%) of 19 individuals who developed ASN did so in the presence of RCVs. In contrast, 11 (57.9%) of 19 individuals in the control group developed RCVs. Statistical analysis revealed that patients who developed ASN were roughly 25 times less likely to have had RCVs than individuals who never developed ASN (odds ratio = 24.74; P =.001)., Conclusion: Retinochoroidal collateral veins are negatively associated with ASN post-CRVO and may function in a protective manner against such an outcome.