LARGE SILICONE DROPLETS AFTER INTRAVITREAL BEVACIZUMAB (AVASTIN).

PURPOSE: Despite its off-label status, intravitreal bevacizumab is the most commonly used intraocular anti-vascular endothelial growth factor agent. Regulation of compounding pharmacies has recently increased to make compounded pharmaceuticals safer. Despite these changes, a marked increase in symptomatic, large silicone oil droplets following intravitreal bevacizumab injections was noticed. METHODS: Retrospective chart review was performed. Within a single private practice, patients who were noted to have large or symptomatic silicone oil bubbles after an intravitreal injection were reviewed. RESULTS: A recent, dramatic increase in the incidence of large or symptomatic silicone oil droplets was noted, with 23 cases noted in the past 5 months, compared with 1 in the previous decade. Patients frequently noted a circular floater consisting of a dark ring surrounding a bright central area immediately following an injection of intravitreal bevacizumab. All bevacizumab injections were from single-piece insulin syringes. B-scan ultrasonography produced a very characteristic reverberation pattern. No inflammation or visual acuity loss was noted because of the droplets; however, some patients were annoyed enough to consider vitrectomy. CONCLUSION: Patients should be carefully evaluated for this possibility, and the characteristic symptom of a round floater consisting of a dark ring surrounding a bright center, and the prominent reverberation pattern on B-scan ultrasonography may help increase detection. Changes in consent forms and discussion of this possibility are indicated while investigation into the cause of this increased incidence continues, especially if one is administering bevacizumab via the one-piece insulin syringes commonly used by compound pharmacies.

Prosopometamorphopsia and alexia following left splenial corpus callosum infarction: Case report and literature review.

BACKGROUND: Lesions to the posterior section of the corpus callosum, called the splenium, and the immediate area have been separately associated with perceived visual distortions of the face (prosopometamorphopsia) or difficulty reading (alexia). CASE REPORT: This case report describes a right-handed patient who complained of prosopometamorphopsia associated with the lower part of the face and alexia following infarction of the left splenium in the corpus callosum. CONCLUSIONS: The splenium and adjacent retrosplenial cortex facilitate the transfer of visual information and memory function between the two hemispheres of the brain and along the Papez circuit, respectively. We believe that damage to this singular area of the brain could bring about several concurrent yet disparate symptoms, such as the reported prosopometamorphopsia and alexia with this patient.

Systemic Safety of Prolonged Monthly Anti-Vascular Endothelial Growth Factor Therapy for Diabetic Macular Edema: A Systematic Review and Meta-analysis.

IMPORTANCE: Anti-vascular endothelial growth factor (VEGF) therapy is commonly used to treat numerous retinal conditions and appears safe, yet controversy remains regarding systemic safety. OBJECTIVE: To evaluate the systemic safety of intravitreous anti-VEGF injections in high-risk patients with diabetic macular edema (DME) and to investigate separately the subgroup of these patients with the highest level of exposure to anti-VEGF monthly treatment for 2 years. DATA SOURCES: A search of MEDLINE, Cochrane Central Register of Controlled Trials, clincaltrials.gov, and ophthalmology congress abstracts January 1, 1947, to May 19, 2015. STUDY SELECTION: Randomized clinical trials were selected that evaluated monthly anti-VEGF injections for DME for 2 years and reported the outcome measures of cerebrovascular accidents, myocardial infarctions, arteriothrombotic events, and mortality. DATA EXTRACTION AND SYNTHESIS: Two reviewers collected data independently from each study for the meta-analysis. Data were pooled using a fixed-effects model and analyzed from November 6, 2014, to June 28, 2015. Peto odds ratios with 95% CIs were calculated. MAIN OUTCOMES AND MEASURES: Primary end points included cerebrovascular accidents and all-cause mortality in the highest-dose arms. Secondary outcomes included myocardial infarctions, arteriothrombotic events, and vascular-related death. RESULTS: Of 1126 articles reviewed, 598 were removed as duplicate studies and 524, for lack of monthly treatment data for 2 years, leaving 4 studies for the meta-analysis that met the search criteria: 2 trials using monthly aflibercept and 2 using monthly ranibizumab, representing 1328 patients. The primary evaluation (1078 patients) combined the monthly aflibercept and the 0.5-mg ranibizumab arms and yielded an increased risk for death compared with sham and laser treatments (odds ratio [OR], 2.98; 95% CI, 1.44-6.14; P = .003). Analysis including monthly aflibercept and 0.5-mg ranibizumab yielded an increased risk for cerebrovascular accidents (OR, 2.33; 95% CI, 1.04-5.22; P = .04) and vascular death (OR, 2.51; 95% CI, 1.08-5.82; P = .03). No definitive increased risk for myocardial infarctions and arteriothrombotic events was seen with all dose combinations. CONCLUSIONS AND RELEVANCE: In this meta-analysis of anti-VEGF agents for patients with DME, assessment of the highest-level exposure group (those high-risk patients with DME who received 2 years of monthly treatment) revealed a possible increased risk for death and potentially for cerebrovascular accidents. Consideration of total exposure to anti-VEGF agents when treating those at high risk for vascular disease may be important.

A cell-based screening assay to identify pharmaceutical compounds that enhance the regenerative quality of corneal repair.

The goal of this study was to develop and validate a simple but quantitative cell-based assay to identify compounds that might be used pharmaceutically to give tissue repair a more regenerative character. The cornea was used as the model, and some specific aspects of repair in this organ were incorporated into assay design. A quantitative cell-based assay was developed based on transcriptional promoter activity of fibrotic marker genes ACT2A and TGFB2. Immortalized corneal stromal cells (HTK) or corneal epithelial cells (HCLE) were tested and compared to primary corneal stromal cells. Cells were transiently transfected with constructs containing the firefly luciferase reporter gene driven by transcriptional promoters for the selected fibrotic marker genes. A selected panel of seven chemical test compounds was used, containing three known fibrosis inhibitors: lovastatin (LOV), tyrphostin AG 1296 (6,7-dimethoxy-3-phenylquinoxaline) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), and four potential fibrosis inhibitors: 5-iodotubercidin (4-amino-5-iodo-7-(ß-D-ribofuranosyl)-pyrrolo(2,3-d)pyrimidine), anisomycin, DRB (5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole) and latrunculin B. Transfected cells were treated with TGFB2 in the presence or absence of one of the test compounds. To validate the assay, compounds were tested for their direct effects on gene expression in the immortalized cell lines and primary human corneal keratocytes using RT-PCR and immunohistochemistry. Three "hits" were validated LOV, SB203580 and anisomycin. This assay, which can be applied in a high throughput format to screen large libraries of uncharacterized compounds, or known compounds that might be repurposed, offers a valuable tool for identifying new treatments to address a major unmet medical need. Anisomycin has not previously been characterized as antifibrotic, thus, this is a novel finding of the study.

Chondrogenesis in scleral stem/progenitor cells and its association with form-deprived myopia in mice.

PURPOSE: Previously, we demonstrated that scleral stem/progenitor cells (SSPCs) from mice have a chondrogenic differentiation potential, which is stimulated by transforming growth factor-ß (TGF-ß). In the present study, we hypothesized that chondrogenesis in the sclera could be a possible mechanism in myopia development. Therefore, we investigated the association of form-deprivation myopia (FDM) with expressions in mice sclera representing the chondrogenic phenotype: collagen type II (Col2) and α-smooth muscle actin (α-SMA). METHODS: The mRNA levels of α-SMA and Col2 in cultured murine SSPCs during chondrogenesis stimulated by TGF-ß2 were determined by real-time quantitative RT-PCR (qRT-PCR). The expression patterns of α-SMA and Col2 were assessed by immunohistochemistry in a three dimensional pellet culture. In an FDM mouse model, a western blot analysis and immunofluorescence study were used to detect the changes in the α-SMA and Col2 protein expressions in the sclera. In the RPE-choroid complex, qRT-PCR was used to detect any changes in the TGF-ß mRNA expression. RESULTS: The treatment of SSPCs in vitro with TGF-ß2 for 24 h at 1 or 10 ng/ml led to increased levels of both the α-SMA and Col2 expressions. In addition, we observed the formation of cartilage-like pellets from TGF-ß2-treated SSPCs. Both α-SMA and Col2 were expressed in the pellet. In an in-vivo study, the α-SMA and Col2 protein expressions were significantly increased in the sclera of FDM eyes in comparison to contralateral control eyes. Similarly, the levels of TGF-ß in the RPE-choroid complex of an FDM eye were also significantly elevated. CONCLUSION: Based on the concept of stem cells possessing multipotent differentiation potentials, scleral chondrogenesis induced by SSPCs may play a role in myopia development. The increased expressions of the cartilage-associated proteins Col2 and α-SMA during scleral chondrogenesis may be potential markers for myopia development. In addition, the increased levels of TGF-ß mRNA in the RPE-choroid complex might induce the chondrogenic change in the sclera during myopia development.

Injection force comparison of the old and new dexamethasone implant insertion needles in porcine eyes and synthetic sclera.

BACKGROUND AND OBJECTIVE: Determine whether the new dexamethasone intravitreal implant (Ozurdex; Allergan, Irvine, CA) injector needle design can reduce the force needed for insertion when compared to the original needle design. MATERIALS AND METHODS: In vitro testing assessed the force required for insertion from five new-design and five old-design Ozurdex needles on a synthetic test medium and explanted porcine eyes. Maximum penetration force was measured in grams-force, while the total work of the needle was measured in joules. RESULTS: The new design required 29% and 68% less overall work to perform an injection in the synthetic medium (P = .0002) and porcine eyes (P = .009), respectively. The maximum force required to insert the new needle was 25% and 61% that of the old needle in the synthetic medium (P = .001) and porcine eyes (P = .007), respectively. CONCLUSION: The new needle design significantly reduces the force and work needed for insertion, which should improve physician control as well as patient safety and comfort.

Assessment of novel guarded needle to increase patient comfort and decrease injection time during intravitreal injection.

BACKGROUND AND OBJECTIVE: To determine whether a novel guarded 33-gauge injection device can make the intravitreal injection procedure faster and more comfortable for patients. PATIENTS AND METHODS: Single-center, prospective, randomized interventional study. Seventy participants receiving bilateral injections on the same day had one eye injected with the 33-gauge injection device without a speculum and the other eye injected with a standard 30-gauge needle using a speculum. Length of time needed for the injection procedure was assessed, and subjects were asked to complete a questionnaire regarding their comfort level with each device during the injection procedure as well as immediately after, later that evening, and the next day. RESULTS: Intravitreal injections using the 33-gauge injection device were significantly faster, but there was no significant difference in the incidence or levels of pain between the two needle types. CONCLUSION: The 33-gauge injection device may offer advantages over a standard 30-gauge needle when performing an intravitreal injection.

Mechanisms for PDGF, a serum cytokine, stimulating loss of corneal keratocyte crystallins.

PURPOSE: As corneal stromal cells (keratocytes) become activated before transition to the fibroblastic repair phenotype in response to injury (in situ) or serum (in culture), the corneal crystallins, transketolase (TKT) and aldehyde dehydrogenase (ALDH1A1), are lost. The authors previously showed that the serum cytokine platelet-derived growth factor-BB (PDGF), but not transforming growth factor beta2 (TGF-beta2), stimulates TKT loss. The goal of this study was to further define the molecular mechanisms for PDGF-stimulated loss of crystallins to elucidate the pathway for keratocyte activation. METHODS: Freshly isolated rabbit corneal keratocytes were plated in serum-free medium with or without PDGF and/or specific inhibitors of the PDGF-relevant signal pathway components, PDGF receptor, PI3K/AKT, or ras-initiated MAPK proteins. Intracellular TKT protein levels were quantified by immunoblotting. Ubiquitinated TKT levels were assessed by immunoprecipitation, and TKT messenger RNA (mRNA) levels were quantified by quantitative reverse transcription-polymerase chain reaction. RESULTS: PDGF treatment at the same time as inhibition of PDGF receptor, Akt, JNK, and ubiquitin-proteasome pathway prevented PDGF-induced TKT protein loss. In contrast, treatment with PDGF did not affect TKT mRNA levels. CONCLUSIONS: The results suggest that PDGF-stimulated TKT loss is mediated through cross talk between PI3K-independent Akt and JNK. This signaling pathway leads to the degradation of existing TKT protein but does not compromise the accumulation of TKT mRNA. Therefore, cells retain the potential to reaccumulate TKT protein that is enabled by PDGF removal. These findings suggest that targeting PDGF signaling could improve repair outcomes after surgical procedures in the cornea.

Deregulated G1-S control and energy stress contribute to the synthetic-lethal interactions between inactivation of RB and TSC1 or TSC2.

Synthetic lethality is a potential strategy for cancer treatment by specifically promoting the death of cancer cells with particular defects such as the loss of the RB (RB1) tumor suppressor. We previously showed that inactivation of both RB and TSC2 induces synergistic apoptosis during the development of Drosophila melanogaster and in cancer cells. However, the in vivo mechanism of this synthetic-lethal interaction is not clear. Here, we show that synergistic cell death in tissues that have lost the RB and TSC orthologs rbf and dtsc1/gig, respectively, or overexpress Rheb and dE2F1, are correlated with synergistic defects in G1-S control, which causes cells to accumulate DNA damage. Coexpression of the G1-S inhibitor Dap, but not the G2-M inhibitor dWee1, decreases DNA damage and reduces cell death. In addition, we show that rbf and dtsc1 mutant cells are under energy stress, are sensitive to decreased energy levels and depend on the cellular energy stress-response pathway for survival. Decreasing mitochondrial ATP synthesis by inactivating cova or abrogating the energy-stress response by removing the metabolic regulator LKB1 both enhance the elimination of cells lacking either rbf or dtsc1. These observations, in conjunction with the finding that deregulation of TORC1 induces activation of JNK, indicate that multiple cellular stresses are induced and contribute to the synthetic-lethal interactions between RB and TSC1/TSC2 inactivation. The insights gained from this study suggest new approaches for targeting RB-deficient cancers.

Interaction of clusterin and matrix metalloproteinase-9 and its implication for epithelial homeostasis and inflammation.

Uncontrolled increases of matrix metalloproteinase-9 (MMP-9) activity have been causally linked to epithelial barrier disruption and severe symptoms of inflammatory diseases such as dry eye (DE). The data presented here show that the anti-inflammatory, cytoprotective intracellular and extracellular chaperone protein clusterin (CLU) interacts with MMP-9 both inside and outside epithelial cells. CLU bound very strongly to active MMP-9, with an affinity constant K(D) of 2.63 nmol/L. Unexpectedly, CLU had a much higher affinity for pro-MMP-9 than for active MMP-9 or pro-MMP-2, requiring the N-terminal propeptide domain of pro-MMP-9. The significance of the interaction between CLU and MMP-9 was demonstrated by the observation that CLU prevents stress-induced MMP-9 aggregation and inhibits MMP-9 enzymatic activity. Furthermore, CLU inhibited MMP-9-mediated disintegration of the tight junction structure formed between human epithelial cells. Additionally, CLU inhibited enzymatic activities of MMP-2, MMP-3, and MMP-7. Treatment with proinflammatory cytokines, which are known to increase MMP-9 transcription under inflammatory conditions, reduced the expression of CLU in human epithelial cells. Similarly, in a mouse model of human DE, inflammatory stress depleted CLU in the ocular surface epithelium but allowed MMP-9 to prevail therein. The present results thus provide novel insights into previously unrecognized mechanisms by which CLU maintains fluid-epithelial interface homeostasis, thereby preventing the onset of inflammatory conditions, especially where MMP-9 is actively involved.

Conserved RB functions in development and tumor suppression.

The variety of human cancers in which the retinoblastoma protein pRb is inactivated reflects both its broad importance for tumor suppression and its multitude of cellular functions. Accumulating evidence indicates that pRb contributes to a diversity of cellular functions, including cell proliferation, differentiation, cell death, and genome stability. pRb performs these diverse functions through the formation of large complexes that include E2F transcription factors and chromatin regulators. In this review we will discuss some of the recent advances made in understanding the structure and function of pRb as they relate to tumor suppression, and highlight research using Drosophila melanogaster that reveals important, evolutionarily conserved functions of the RB family.

A novel mechanism of increased infections in contact lens wearers.

PURPOSE: It is well documented that contact lens wearers have much higher incidences of corneal infections compared with those of non-contact lens wearers, although the exact cause(s) of this increased susceptibility has not been identified. A distinct subset of mucins (MUCs) is present on the ocular surface, acting to protect the integrity of the corneal epithelium. This study was performed to determine whether multipurpose contact lens solutions (MPCLSs) can cause increased infections in the cornea by destroying the protective cell-bound mucin layer. METHODS: An immortalized human corneal limbal epithelial cell line was treated in the presence of four commonly used MPCLSs or PBS and the expression and release of MUC-16 was assessed. Cells were also cultured with Pseudomonas aeruginosa after MPCLS treatment and internalization of bacteria was assessed by quantitative genomic PCR. Loss of MUC-16 was then correlated with infection rates. RESULTS: Each of the four commonly used MPCLSs examined in this study differentially affected mucin release. The relative effect was correlated with an increase in infection of corneal epithelial cells by P. aeruginosa. CONCLUSIONS: The results of this study are consistent with the hypothesis that MPCLSs cause increased infections in the cornea by destroying the protective cell-bound mucin layer.

RBF and Rno promote photoreceptor differentiation onset through modulating EGFR signaling in the Drosophila developing eye.

The retinoblastoma gene Rb is the prototype tumor suppressor and is conserved in Drosophila. We use the developing fly retina as a model system to investigate the role of Drosophila Rb (rbf) during differentiation. This report shows that mutation of rbf and rhinoceros (rno), which encodes a PHD domain protein, leads to a synergistic delay in photoreceptor cell differentiation in the developing eye disc. We show that this differentiation delay phenotype is caused by decreased levels of different components of the Epidermal Growth Factor Receptor (EGFR) signaling pathway in the absence of rbf and rno. We show that rbf is required for normal expression of Rhomboid proteins and activation of MAP kinase in the morphogenetic furrow (MF), while rno is required for the expression of Pointed (Pnt) and Ebi proteins, which are key factors that mediate EGFR signaling output in the nucleus. Interestingly, while removing the transcription activation function of dE2F1 is sufficient to suppress the synergistic differentiation delay, a mutant form of de2f1 that disrupts the binding with RBF but retains the transcription activation function does not mimic the effect of rbf loss. These observations suggest that RBF has additional functions besides dE2F1 binding that regulates EGFR signaling and photoreceptor differentiation.

Targeting Rb inactivation in cancers by synthetic lethality.

The retinoblastoma protein, pRb, is a key regulator of cell proliferation, differentiation, apoptosis, as well as checkpoint and stress responses. The function of Rb is often inactivated in many types of cancers, a feature that can potentially be used to target this specific subset of cancers. However little is known about how the loss of Rb function can be exploited in cancer therapies. In this review, we overview the functions of Rb, and discuss a genetic screen that led to the finding that inactivation of TSC2 and Rb induces synergistic cell death in both Drosophila developing tissues and human cancer cells. The mechanisms for synergistic cell death involve the accumulation of cellular stress, suggesting that inactivation of TSC2 and chemotherapeutic agents that result in induction of cellular stress can potentially be combined to treat cancers harboring inactivated Rb.

Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness.

Previous work established retinal expression of channelrhodopsin-2 (ChR2), an algal cation channel gated by light, restored physiological and behavioral visual responses in otherwise blind rd1 mice. However, a viable ChR2-based human therapy must meet several key criteria: (i) ChR2 expression must be targeted, robust, and long-term, (ii) ChR2 must provide long-term and continuous therapeutic efficacy, and (iii) both viral vector delivery and ChR2 expression must be safe. Here, we demonstrate the development of a clinically relevant therapy for late stage retinal degeneration using ChR2. We achieved specific and stable expression of ChR2 in ON bipolar cells using a recombinant adeno-associated viral vector (rAAV) packaged in a tyrosine-mutated capsid. Targeted expression led to ChR2-driven electrophysiological ON responses in postsynaptic retinal ganglion cells and significant improvement in visually guided behavior for multiple models of blindness up to 10 months postinjection. Light levels to elicit visually guided behavioral responses were within the physiological range of cone photoreceptors. Finally, chronic ChR2 expression was nontoxic, with transgene biodistribution limited to the eye. No measurable immune or inflammatory response was observed following intraocular vector administration. Together, these data indicate that virally delivered ChR2 can provide a viable and efficacious clinical therapy for photoreceptor disease-related blindness.

Comprehensive gene expression profiling and functional analysis of matrix metalloproteinases and TIMPs, and identification of ADAM-10 gene expression, in a corneal model of epithelial resurfacing.

This study provides a comprehensive expression analysis for the entire matrix metalloproteinase (MMP) gene family during the process of epithelial resurfacing following corneal abrasion injury in the mouse. The mRNA levels for all known MMP genes expressed in mouse, the related enzyme ADAM-10, and the known tissue inhibitors of metalloproteinases (TIMPs) were determined semi-quantitatively by reverse transcriptase-polymerase chain reaction (RT-PCR) in the uninjured epithelium, and in the epithelial tissue resurfacing the abraded area or residing in its periphery at two time points: during the epithelial migration phase and immediately following wound closure. The mRNA levels for MMP-1a, -1b, -9, -10, -12, and -13 as well as TIMP-1 were significantly up-regulated in the migrating corneal epithelium. After wound resurfacing, the mRNA levels for all of these MMPs were down-regulated, although MMP-1a, -1b, and -13 remained significantly elevated in comparison to the uninjured epithelium. The only gene found to be down-regulated was TIMP-3, which occurred throughout the wound-healing process. During resurfacing, MMP-9 was localized to the front of the migrating epithelium, MMP-10 and -13 were localized throughout the migrating epithelium, and MMP-13 could also be found in the periphery. Following epithelial closure, immunoreactive MMPs-9 and -10 became undetectable, but MMP-13 continued to be found throughout the epithelium. Functional analysis of MMP-10 revealed no effects on epithelial migration or cell proliferation. In conclusion, distinct MMP temporal-spatial profiles define the uninjured corneal epithelium and the corneal epithelium at different stages of regeneration. An extensive review of the literature is also provided in the discussion.

Specific killing of Rb mutant cancer cells by inactivating TSC2.

The retinoblastoma (Rb) tumor suppressor is often inactivated in cancers. To identify genes that can be used to specifically target such cancers, we carried out a genetic screen in Drosophila. We identified gig (fly TSC2) and found that inactivation of rbf (fly Rb) and gig synergistically induced cell death. Interestingly, inactivation of TSC2 specifically kills Rb mutant cancer cells under stress conditions, which is correlated with an inhibition of tumor growth. We show that cancer cell killing induced by concomitant inactivation of Rb and TSC2 is mediated by increased cellular stress, including oxidative stress. Inactivation of TSC2 and Rb synergistically induce oxidative stress via increased protein synthesis, inhibited de novo lipid synthesis, and decreased reactive oxygen species scavenger enzyme SOD2 induction.

Correlation between epithelial ingrowth and basement membrane remodeling in human corneas after laser-assisted in situ keratomileusis.

OBJECTIVE: To further investigate the hypothesis that epithelial ingrowth in human corneas after laser-assisted in situ keratomileusis (LASIK) correlates with basement membrane remodeling, as suggested by the presence of matrix metalloproteinase 9 around epithelial cells in the lamellar scar. METHODS: Immunohistochemical analysis and transmission electron microscopy were applied to human postmortem corneas with post-LASIK epithelial ingrowth. RESULTS: Epithelial ingrowth into the flap margin was observed in 8 of 18 corneas (44%). Matrix metalloproteinase 9 immunolocalized around ingrown epithelium in 6 of these 8 corneas (75%). There was a positive correlation between the presence of matrix metalloproteinase 9 at the wound margin and discontinuities in the basement membrane, as determined by laminin and beta(4) integrin immunofluorescence. Transforming growth factor beta2 was present into the stroma of some corneas with epithelial ingrowth and interrupted basement membrane, suggesting some degree of epithelial-stromal interaction. Transmission electron microscopy confirmed large areas of remodeled basement membrane along ingrown epithelial cells. CONCLUSIONS: The neo-basement membrane components underlying the ingrown cells in human corneas with epithelial ingrowth after LASIK appear to be partially disassembled. Epithelial-stromal interaction over time may be related to prolonged wound healing remodeling, which calls into question the stability of the flap.

Retinoblastoma family protein promotes normal R8-photoreceptor differentiation in the absence of rhinoceros by inhibiting dE2F1 activity.

The retinoblastoma gene Rb is a prototype tumor suppressor which is conserved in Drosophila. Although much is known about the roles of Rb in cell proliferation and apoptosis, much less is known about how Rb regulates cell differentiation. Inactivation of Drosophila Rb (rbf) exhibited subtle differentiation defects similar to inactivation of Rb in mice, suggesting the existence of redundant mechanisms in the control of cell differentiation. To test this possibility and to characterize the role of Rbf in cell differentiation during retinal development, we carried out a genetic screen and identified a mutation in rhinoceros (rno), which leads to synergistic differentiation defects in conjunction with rbf inactivation. Characterization of an early differentiation defect, the multiple-R8 phenotype, revealed that this phenotype was caused by limiting amounts of Notch signaling due to reduced expression of the Notch ligand, Delta (Dl). Decreasing the gene dosage of Dl enhanced the multiple-R8 phenotype, while increasing the level of Dl suppressed this phenotype. Interestingly, removal of the transcriptional activation of dE2F1 partially restores Dl expression in rbf,rno mutant clones and suppresses the associated differentiation defects, indicating that this differentiation function of RBF is mediated by its regulation of dE2F1 activity.