Photoreceptor Cell Development Research Articles

NR2E3 loss disrupts photoreceptor cell maturation and fate in human organoid models of retinal development.

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNAseq data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different than those observed in Nr2e3 rodent models. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.

Screening of Chelidonium majus isoquinoline alkaloids reveals berberine and chelidonine as selective ligands for the nuclear receptors RORβ and HNF4α, respectively.

The nuclear receptors hepatocyte nuclear factor 4α (HNF4α) and retinoic acid receptor-related orphan receptor-β (RORβ) are ligand-regulated transcription factors and potential drug targets for metabolic disorders. However, there is a lack of small molecular, selective ligands to explore the therapeutic potential in further detail. Here, we report the discovery of greater celandine (Chelidonium majus) isoquinoline alkaloids as nuclear receptor modulators: Berberine is a selective RORβ inverse agonist and modulated target genes involved in the circadian clock, photoreceptor cell development, and neuronal function. The structurally related chelidonine was identified as a ligand for the constitutively active HNF4α receptor, with nanomolar potency in a cellular reporter gene assay. In human liver cancer cells naturally expressing high levels of HNF4α, chelidonine acted as an inverse agonist and downregulated genes associated with gluconeogenesis and drug metabolism. Both berberine and chelidonine are promising tool compounds to further investigate their target nuclear receptors and for drug discovery.

Particulate Matter Induced Adverse Effects on Eye Development in Zebrafish (Danio rerio) Embryos.

Particulate matter (PM) can cause human diseases, particularly respiratory diseases. Since eyes are directly exposed to the air, they might be directly adversely affected by PM. Therefore, we determined the toxicity caused to eye development by PM using zebrafish (Danio rerio) embryos. The PM-induced embryo toxicity was dependent on dose and time and caused significant morphological defects, reducing the total body length and the total eye area. Reactive oxygen species (ROS) overproduction was confirmed in the PM treatment group, and antioxidant genes (cat and sod2), photoreceptor cell development, pigmentation genes (atoh8, vsx1, and rho), eye-embryogenesis genes (pax6a and pax6b), and eye-lens-development genes (cryaa) were downregulated, while eye-development genes (crybb1) were upregulated. In conclusion, PM had a direct adverse effect on the eyes, and zebrafish embryos can be used as a model to evaluate PM-induced eye toxicity in vivo.

Drosophila Phosphatase of Regenerating Liver Is Critical for Photoreceptor Cell Polarity and Survival during Retinal Development.

Establishing apicobasal polarity, involving intricate interactions among polarity regulators, is key for epithelial cell function. Though phosphatase of regenerating liver (PRL) proteins are implicated in diverse biological processes, including cancer, their developmental role remains unclear. In this study, we explore the role of Drosophila PRL (dPRL) in photoreceptor cell development. We reveal that dPRL, requiring a C-terminal prenylation motif, is highly enriched in the apical membrane of developing photoreceptor cells. Moreover, dPRL knockdown during retinal development results in adult Drosophila retinal degeneration, caused by hid-induced apoptosis. dPRL depletion also mislocalizes cell adhesion and polarity proteins like Armadillo, Crumbs, and DaPKC and relocates the basolateral protein, alpha subunit of Na+/K+-ATPase, to the presumed apical membrane. Importantly, this polarity disruption is not secondary to apoptosis, as suppressing hid expression does not rescue the polarity defect in dPRL-depleted photoreceptor cells. These findings underscore dPRL's crucial role in photoreceptor cell polarity and emphasize PRL's importance in establishing epithelial polarity and maintaining cell survival during retinal development, offering new insights into PRL's role in normal epithelium.

Deciphering the pathogenesis of retinopathy associated with carnitine palmitoyltransferase I deficiency in zebrafish model

Fatty acid oxidation disorders (FAODs) are a group of rare genetic metabolic disorders caused by mutations in genes responsible for transporting and metabolizing fatty acids in the mitochondria. One crucial enzyme involved in this process is carnitine palmitoyltransferase I (CPT1), which transports long-chain fatty acids to the mitochondrial matrix for beta-oxidation. Defects in beta-oxidation enzymes often lead to pigmentary retinopathy; however, the underlying mechanisms are not entirely understood. To investigate FAOD and its impact on the retina, we employed zebrafish as a model organism. Specifically, we used antisense-mediated knockdown strategies to target the cpt1a gene and examined the resulting retinal phenotypes. We demonstrated that the cpt1a MO-injected fish significantly reduced the length of connecting cilia and severely affected photoreceptor cell development. Moreover, our findings highlight that the loss of functional cpt1a disrupted energy homeostasis in the retina, leading to lipid droplet deposition and promoting ferroptosis, which is likely attributed to the photoreceptor degeneration and visual impairments observed in the cpt1a morphants.

Meeting Zn Needs during Medaka Eye Development: Nanoscale Visualization of Retina by Expansion Microscopy.

Fish eyes require high Zn levels to support their early development. Although numerous studies have been conducted on the nutritional and toxic effects of Zn on the eye, the Zn requirement for retinal cell development is still debatable. Moreover, due to the complexity of the retinal structure, it is difficult to clearly visualize each retinal layer and accurately separate cell morphology in vivo by conventional methods. In the present study, we for the first time have achieved nanoscale imaging of retinal anatomy affected by dietary and waterborne Zn exposure by novel expansion microscopy. We demonstrated that the fish retina showed different developmental strategies in response to dietary and aqueous Zn exposures. Excess dietary Zn produced toxicity to retinal photoreceptor cells, resulting in a reduction in cell number and cell area, and this toxicity became severe with biological development. In contrast, waterborne Zn in the natural environment probably failed to meet the Zn requirements of retinal development. Overall, our results indicated that during early development, the Zn requirement of the fish eyes was sensitive, and oversupplementation led to impaired photoreceptor cell development. Our study has provided new perspectives using the powerful and novel expansion microscopy technique in toxicity assessment, enabling ultra-clear visualization of small but complex organ development.

Differential gene expression analysis using RNA sequencing: retinal pigment epithelial cells after exposure to continuous-wave and subthreshold micropulse laser.

Subthreshold micropulse laser (SMPL) is more clinically efficient for the treatment of diabetic macular edema (DME) than the conventional continuous-wave (CW) laser. We aimed to characterize transcriptome changes after the application of these lasers and to compare the transcripts. Human pluripotent stem cell-derived retinal pigment epithelial cells were exposed to laser irradiation. Differentially expressed genes (DEGs), distribution of heat shock protein (Hsp) family, gene expression profile, and gene ontology (GO) enrichment analysis based on RNA sequencing data were investigated at 3 h and 24 h after irradiation. CW laser induced more DEGs than SMPL (1771 vs. 520 genes). The expression of the Hsp family was confirmed in both groups: however, the induction patterns was different for different genes. GO enrichment analysis revealed that CW laser upregulated the expression of DEGs involved in vasculature development (GO: 0001944), related to apoptosis and repair after cell injury whereas SMPL upregulated the expression of DEGs involved in photoreceptor cell maintenance (GO: 0045494), photoreceptor cell development (GO: 0042461), and sensory perception of light stimuli (GO: 0050953). The results provide insights into the genetic responses and may contribute to the understanding of the molecular mechanisms of laser-induced thermal effects.

Biliverdin regulates NR2E3 and zebrafish retinal photoreceptor development

NR2E3 is an orphan nuclear receptor whose loss-of-function causes abnormal retinal photoreceptor development and degeneration. However, despite that many nuclear receptors are regulated by binding of small molecule ligands, biological small molecule ligands regulating NR2E3 have not been identified. Identification of an endogenous NR2E3 ligand might reveal a previously unrecognized component contributing to retinal development and maintenance. Here we report that biliverdin, a conserved green pigment from heme catabolism, regulates NR2E3 and is necessary for zebrafish retinal photoreceptor development. Biliverdin from retinal extracts specifically bound to NR2E3’s ligand-binding domain and induced NR2E3-dependent reporter gene expression. Inhibition of biliverdin synthesis decreased photoreceptor cell populations in zebrafish larvae, and this phenotype was alleviated by exogenously supplied biliverdin. Thus, biliverdin is an endogenous small molecule ligand for NR2E3 and a component necessary for the proper development of photoreceptor cells. This result suggests a possible role of heme metabolism in the regulation of retinal photoreceptor cell development.

Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation.

Stem cell-based cell therapies are considered to be promising treatments for retinal disorders with dysfunction or death of photoreceptors. However, the enrichment of human photoreceptors suitable for transplantation has been highly challenging so far. This study aimed to generate a photoreceptor-specific reporter human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 genome editing, which harbored an enhanced green fluorescent protein (eGFP) sequence at the endogenous locus of the pan photoreceptor marker recoverin (RCVRN). After confirmation of successful targeting and gene stability, three-dimensional retinal organoids were induced from this reporter line. The RCVRN-eGFP reporter faithfully replicated endogenous protein expression of recoverin and revealed the developmental characteristics of photoreceptors during retinal differentiation. The RCVRN-eGFP specifically and steadily labeled photoreceptor cells from photoreceptor precursors to mature rods and cones. Additionally, abundant eGFP-positive photoreceptors were enriched by fluorescence-activated cell sorting, and their transcriptome signatures were revealed by RNA sequencing and data analysis. Moreover, potential clusters of differentiation (CD) biomarkers were extracted for the enrichment of photoreceptors for clinical applications, such as CD133 for the positive selection of photoreceptors. Altogether, the RCVRN-eGFP reporter hiPSC line was successfully established and the first global expression database of recoverin-positive photoreceptors was constructed. These achievements will provide a powerful tool for dynamically monitoring photoreceptor cell development and purification of human photoreceptors, thus facilitating photoreceptor cell therapy for advanced retinal disorders.

Photoreceptor Cell Development Requires CDHR1a Protein Stability Controlled by Siah E3 Ubiquitin Ligase During Zebrafish Eye Development

E3 ubiquitin ligases mediate orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development. Our lab recently discovered that Siah family of E3 ligases plays a role in early ocular morphogenesis, in particular fusion of the optic fissure. Interestingly, Siah ligases are also expressed during photoreceptor development and are predicted to target CDHR1a, a cadherin superfamily of calcium‐dependent cell adhesion molecules and photoreceptor‐specific cadherin. Mutations in this cadherin are associated with inherited retinal dystrophies, such as cone‐rod dystrophy. Using whole mount in situ hybridization and Immuno‐histochemistry we detected Siah gene expression and protein localization in the outer nuclear layer (ONL) and in the retinal ganglion cell layer starting at 3 days‐post fertilization (dpf) until 7dpf. CDHR1a expression and protein localization overlaps with both Siah genes in the outer segment of the retina from 3 until 7dpf. We therefore hypothesized that Siah regulates CDHR1a during photoreceptor development. To test our hypothesis, I first confirmed siah‐mediated targeting of CDHR1a for degradation using a mammalian cell culture, co‐immunoprecipitation followed by western blotting analysis. Second, I created two transgenic zebrafish lines that express Siah1 or a dominant negative (Siah1DRING) under the control of the heat shock promoter. Using the heat shock line to overexpress Siah1 at 2dpf and 2.5dpf, we observed a decrease in the number of developing rods and cones in the retina at 3dpf. The number of retinal ganglion cells, amacrine, bipolar and horizontal cells, however, did not change. In addition, there was a significant reduction of proliferating (EdU+ and PH3+ cells in ONL) cells in the retina. Taken together, our results suggest that Siah ubiquitin ligases may control CDHR1a stability and therefore regulate the photoreceptor cell proliferation and differentiation.Support or Funding InformationThis work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq) under grant number 202970/2014‐0.

Functional and Evolutionary Diversification of Otx2 and Crx in Vertebrate Retinal Photoreceptor and Bipolar Cell Development

Otx family homeoproteins Otx2 and Crx are expressed in photoreceptor precursor cells and bind to the common DNA-binding consensus sequence, but these two proteins have distinct functions in retinal development. To examine the functional substitutability of Otx2 and Crx, we generate knockin mouse lines in which Crx is replaced by Otx2 and vice versa. We find that Otx2 and Crx cannot be substituted in photoreceptor development. Subsequently, we investigate the function of Otx2 in photoreceptor and bipolar cell development. High Otx2 levels induce photoreceptor cell fate but not bipolar cell fate, whereas reduced Otx2 expression impairs bipolar cell maturation and survival. Furthermore, we identify Otx2 and Crx in the lamprey genome by using synteny analysis, suggesting that the last common ancestor of vertebrates possesses both Otx2 and Crx. We find that the retinal Otx2 expression pattern is different between lampreys and mice, suggesting that neofunctionalization of Otx2 occurred in the jawed vertebrate lineage.

Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9.

Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient’s iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient’s c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

Photoreceptor cell development requires prostaglandin signaling in the zebrafish retina

Photoreceptor cells are highly specialized sensory neurons capable of visual phototransduction. The connecting cilia in the retinal photoreceptors link the inner segment to the outer segment and mediate the transport of opsins in vision. Although our previous study demonstrates that Prostaglandin E2 (PGE2) signaling is required for ciliogenesis in zebrafish, its roles in retinal ciliogenesis and photoreceptor cell development remain unknown. Here, we investigated the function of prostaglandin signaling in retina and photoreceptor cell development. We generated zebrafish mutations in prostaglandin endoperoxide synthase 1 (PTGS1) and prostaglandin endoperoxide synthase 2 (PTGS2), two rate-limiting enzymes responsible for prostaglandin production. We found that ptgs2b knockdown in ptgs1-/-ptgs2a-/- double mutants significantly reduced the length of connecting cilia and resulted in severe defects in photoreceptor cell differentiation. Furthermore, mutation in PGE2 transporter Leakytail (Lkt)/ATP-binding cassette transporter 4 (ABCC4) exhibited loss of connecting cilia and outer segment in photoreceptor cells, leading to mislocalization of opsins in the cell bodies of photoreceptors. Together, our findings suggest that PGE2 production and transport are crucial for connecting cilia formation and photoreceptor cell development.

Seven‐In‐Absentia (SINA) Family E3 Ligases in Development and Growth

The RAS signal transduction cascade is a pivotal signaling pathway that controls numerous fundamental cellular processes, such as cell proliferation, differentiation, motility, and apoptosis. Oncogenic K‐RAS pathway activation is detected in approximately 30% of all human cancers. Seven‐IN‐Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway. Underscoring the importance of SINA is its high evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling hub and the downstream signaling “gatekeeper” in the RAS pathway, SIAHs are uniquely positioned to inhibit “undruggable” oncogenic K‐RAS activation that is prevalent in high‐grade metastatic cancer. Thus, it is important to delineate the enzymatic activity, molecular regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the elegant and powerful Drosophila photoreceptor cell development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, suggesting that SINA is an essential gene in development. All other sina mutant alleles exhibit stronger phenotypes than the previously published sina2 and sina3 alleles, providing us with an opportunity to study sinaloss‐of‐function mutant phenotypes. Sequencing analysis of these newly identified mutant alleles reveals the critical roles of several immutatable amino acid residues essential for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we have generated a collection of transgenic fly lines that carry either wild‐type (WT) or dominant negative (DN) DmSINA and human SIAH. The altered DmSINA/hSIAH expression under the control of sev‐, GMR‐, and dpp‐ and salivary gland‐GAL4 drivers revealed the biological consequences of gain of function and/or loss of function of DmSINA/hSIAH1/2 in transmitting RAS signaling in central nervous system (CNS) and peripheral nervous system (PNS) development. Immunofluorescent staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINAWT/DN/SIAHWT/DN in neurons resulted in dramatic changes in neuronal cell fate in the developing eye and notum. Furthermore, immunofluorescent staining of larval salivary glands revealed a critical role for SINA family E3 ligases in regulation of cell shape, focal adhesions, and cellular junctions. The results from these transgenic animals suggested that Drosophila SINA and human SIAH1/SIAH2 are evolutionarily conserved and functionally interchangeable. Through this developmental neurobiology and cellular biology‐based study, we aim to dissect the molecular action of SINA/SIAH1/2 in RAS signaling. Furthermore, we hope to translate these findings to demonstrate the anticancer efficacy of the next‐generation anti‐SIAH‐based anti‐K‐RAS strategy against high‐grade metastatic human cancer in the future.Support or Funding InformationExtramural funding support provided by NIH NIGMS R01 GM069922Z‐05S1 and NCI R01 CA140550.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lessons from KO mice of the genes regulating retinal photoreceptor development and function

Retinal photoreceptor cells, composed of rod and cone photoreceptor cells, work as light detectors through phototransduction. Both rod and cone photoreceptor cells develop cell type-specific unique structures, including ribbon synapses and photosensitive outer segments. These elaborate structures are essential for proper photoreception, phototransduction, and signal transmission to secondary neurons in the retina. Degeneration of photoreceptor cells in human is characteristic in several inherited retinal diseases such as retinitis pigmentosa, macular degeneration, and Usher syndrome. Understanding of molecular mechanisms underlying photoreceptor cell development and maintenance is important for developing treatment for retinal degeneration diseases. We have been investigating molecular mechanisms of photoreceptor cell differentiation, maturation, function, and survival by mainly using mouse molecular genetics. Based on these studies, we are currently attempting to develop a novel neuroprotection method using mouse photoreceptor degeneration models. We will present our recent findings on function and possible neuroprotection of photoreceptor cells.

Abstract 4824: The “gatekeeper” function of Drosophila Seven-IN-Absentia (SINA) E3 ligase and its human homologs, SIAH1 and SIAH2, is highly conserved for proper RAS signal transduction in Drosophila development

Abstract Seven-IN-Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signal transduction cascade. Underscoring the importance of SINA is its high evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling “gatekeeper” in the RAS pathway, we have shown that SIAH is required for oncogenic K-RAS-driven tumorigenesis and metastasis in human pancreatic, lung and breast cancer. Since SIAHs appear to be the ideal drug target to inhibit “undruggable” K-RAS activation, it is important to precisely characterize the activity, regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the elegant and well-established Drosophila development system, we are able to study RAS activation and SINA function under normal physiological conditions. In the developing Drosophila eye, photoreceptor cells are recruited sequentially and acquire their distinctive cell fates through a series of local inductive events. The 800x cell arrays allowed us to dissect the role of SINA/SIAH downstream of RAS activation in photoreceptor cell development. To delineate SINA function, we performed an F1 modifier screen using ethyl methanesulfonate (EMS) and X-ray radiation, isolating 28 novel sina mutant alleles. These mutant alleles exhibit much stronger mutant phenotypes than those of the previously published sina2 and sina3 alleles, suggesting that the sina2 and sina3 alleles are hypomorphic alleles. Sequencing analysis of these sinamutant alleles reveals the functional roles of mutated residues and protein domains. To define SINA/SIAH functional conservation, we have generated a complete panel of transgenic fly models that express either wild-type (WT) or dominant negative (DN) SINA/SIAH. The corresponding UAS-sina/siahGOF/LOF phenotypes have been characterized using sev-, GMR-, dpp- and salivary gland-GAL4 drivers to elucidate the developmental outcomes of altered SINA/SIAH expression upon RAS activation. Ectopic expression of sinaWT/DN/siahWT/DN in neurons resulted in dramatic changes in neuronal cell fate in the developing eye and notum, causing PNS neurodegenerative phenotypes. Our results show that the biological functions of fly SINA and human SIAH1/SIAH2 are evolutionarily conserved and functionally interchangeable. Mechanistic insights and regulatory principles learned from Drosophila can be directly applied to cancer biology to develop and validate next-generation anti-SIAH-based anti-K-RAS and anticancer therapy in the future. Citation Format: Robert E. Van Sciver, Yajun Cao, Atique U. Ahmed, Amy H. Tang. The “gatekeeper” function of Drosophila Seven-IN-Absentia (SINA) E3 ligase and its human homologs, SIAH1 and SIAH2, is highly conserved for proper RAS signal transduction in Drosophila development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4824. doi:10.1158/1538-7445.AM2017-4824

Mfsd2a Is a Transporter for the Essential ω-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development

Eye photoreceptor membrane discs in outer rod segments are highly enriched in the visual pigment rhodopsin and the ω-3 fatty acid docosahexaenoic acid (DHA). The eye acquires DHA from blood, but transporters for DHA uptake across the blood-retinal barrier or retinal pigment epithelium have not been identified. Mfsd2a is a newly described sodium-dependent lysophosphatidylcholine (LPC) symporter expressed at the blood-brain barrier that transports LPCs containing DHA and other long-chain fatty acids. LPC transport via Mfsd2a has been shown to be necessary for human brain growth. Here we demonstrate that Mfsd2a is highly expressed in retinal pigment epithelium in embryonic eye, before the development of photoreceptors, and is the primary site of Mfsd2a expression in the eye. Eyes from whole body Mfsd2a-deficient (KO) mice, but not endothelium-specific Mfsd2a-deficient mice, were DHA-deficient and had significantly reduced LPC/DHA transport in vivo Fluorescein angiography indicated normal blood-retinal barrier function. Histological and electron microscopic analysis indicated that Mfsd2a KO mice exhibited a specific reduction in outer rod segment length, disorganized outer rod segment discs, and mislocalization of and reduction in rhodopsin early in postnatal development without loss of photoreceptors. Minor photoreceptor cell loss occurred in adult Mfsd2a KO mice, but electroretinography indicated visual function was normal. The developing eyes of Mfsd2a KO mice had activated microglia and up-regulation of lipogenic and cholesterogenic genes, likely adaptations to loss of LPC transport. These findings identify LPC transport via Mfsd2a as an important pathway for DHA uptake in eye and for development of photoreceptor membrane discs.

PCB1254 exposure contributes to the abnormalities of optomotor responses and influence of the photoreceptor cell development in zebrafish larvae

Polychlorinated biphenyls (PCBs), a group of highly toxic environmental pollutants, have been report to influence the visual system development in children. However, the underlying mechanism is unclear. The study was aim to investigate the effects of continuous PCBs exposure on optomotor response (OMR) and retinal photoreceptor cell development-related gene expression in zebrafish larvae. The fertilized zebrafish embryos were exposed to PCBs at concentrations of 0.125, 0.25, 0.5, and 1mg/L until 7 days post-fertilization. Control groups with blank and 0.01% methanol were also prepared. OMR test was used to detect the visual behavior. The mRNA expression of the CRX, RHO, SWS1, and SWS2 was assessed by the Quantitative Real-Time PCR. The OMR test showed that the visual behavior of the larvae was most sensitive when the grating spatial frequency was 0.20LP/mm and the moving speed was 25cm/s. Moreover, the proportion of positively swimming fish was significantly reduced in the 0.5 and 1mg/L PCB1254 treatment group (P<0.05) compared with the controls. In addition, the expression of SWS2 was significantly down-regulated in all PCB1254 treatment groups (P<0.05), whereas the decreased expression of the CRX, RHO and SWS1 was found in the 0.5 and 1mg/L PCB1254 groups (P<0.05). This is the first report to demonstrate that continue exposure of zebrafish larvae to PCBs causes photoreceptor cell development-related gene expression changes that lead to OMR behavioral alterations. Analysis of these visual behavioral paradigms may be useful in predicting the adverse effects of toxicants on visual function in fish.

MEF2D Drives Photoreceptor Development through a Genome-wide Competition for Tissue-Specific Enhancers

Organismal development requires the precise coordination of genetic programs to regulate cell fate and function. MEF2 transcription factors (TFs) play essential roles in this process but how these broadly expressed factors contribute to the generation of specific cell types during development is poorly understood. Here we show that despite being expressed in virtually all mammalian tissues, in the retina MEF2D binds to retina-specific enhancers and controls photoreceptor cell development. MEF2D achieves specificity by cooperating with a retina-specific factor CRX, which recruits MEF2D away from canonical MEF2 binding sites and redirects it to retina-specific enhancers that lack the consensus MEF2-binding sequence. Once bound to retina-specific enhancers, MEF2D and CRX co-activate the expression of photoreceptor-specific genes that are critical for retinal function. These findings demonstrate that broadly expressed TFs acquire specific functions through competitive recruitment to enhancers by tissue-specific TFs and through selective activation of these enhancers to regulate tissue-specific genes.

Expression of a single prominin homolog in the embryo of the model chordate Ciona intestinalis

Prominins are a family of pentaspan transmembrane glycoproteins, expressed in various types of cells, including stem and cancer stem cells in mammals. Prominin-1 is critical in generating and maintaining the structure of the photoreceptors in the eye since mutations in the PROM1 gene are associated with retinal and macular degeneration in human. In this study, we identified a single prominin homolog, Ci-prom1/2, in the model chordate the ascidian Ciona intestinalis and characterized Ci-prom1/2 expression profile in relation to photoreceptor differentiation during Ciona embryonic development. In situ hybridization experiments show Ci-prom1/2 transcripts localized in the developing central nervous system, predominantly in photoreceptor cell precursors as early as neurula stage and expression is maintained through larva stage in photoreceptor cells around the simple eye. We also isolated the regulatory region responsible for the specific spatio-temporal expression of the Ci-prom1/2 in photoreceptor cell lineage. Collectively, we report that Ci-prom1/2 is a novel molecular marker for ascidian photoreceptor cells and might represent a potential source to enlarge the knowledge about the function of prominin family in photoreceptor cell evolution and development.