Mutant Eyes Research Articles

Participation of Lens Proteins and miRs in Traumatic and Inheritance Cataract: A Review on Diagnostic and Therapeutic Approaches for Cataract Management.

Traumatic and inherited cataract spiking blindness is caused by accumulated deposition of mutant eye lens protein or lens microarchitecture alteration. A traumatic cataract is a clouding of the eye's natural lens that occurs as a result of physical trauma to the eye. This trauma can be caused by various incidents such as blunt force injury, penetration by a foreign object, or a significant impact on the eye area. Inheritance cataracts or hereditary cataracts are cataracts that are genetically inherited from one or both parents. Complications following cataract surgery encompass various adverse outcomes such as inflammation, infection, bleeding, swelling, drooping eyelid, glaucoma, secondary cataracts, and complete loss of vision. The main purpose of the review is to highlight common pathophysiology associated with traumatic and inherited cataracts. Also, the review discusses diagnosis and treatment strategies for such cataract types by targeting their key pathological hallmarks. γD-crystallin plays a crucial role in maintaining the optical properties of the lens during the life span of an individual. Carbamazepine, Resveratrol, and Myricetin (CRM) are effectively bound at the γD-crystallin binding site and thereby could minimize misfolding and aggregation of γD-crystallin. miR-202, miR-193b, miR-135a, miR365, and miR-376a had the highest levels of abundance in the aqueous humor of individuals diagnosed with cataracts. The validation of these miRs will provide more insights into their functional roles and may be used for diagnostic purposes. The effective CRM combination as a multidrug formulation may postpone both traumatic and inherited cataracts and protect the eye from blindness.

Functional opsin patterning for Drosophila color vision is established through signaling pathways in adjacent object-detection neurons.

Vision is mainly based on two different tasks, object detection and color discrimination, carried out by photoreceptor (PR) cells. The Drosophila compound eye consists of ∼800 ommatidia. Every ommatidium contains eight PR cells, six outer cells (R1-R6) and two inner cells (R7 and R8), by which object detection and color vision are achieved, respectively. Expression of opsin genes in R7 and R8 is highly coordinated through the instructive signal from R7 to R8, and two major ommatidial subtypes are distributed stochastically; pale type expresses Rh3/Rh5 and yellow type expresses Rh4/Rh6 in R7/R8. The homeodomain protein Defective proventriculus (Dve) is expressed in yellow-type R7 and in six outer PRs, and it is involved in Rh3 repression to specify the yellow-type R7. dve mutant eyes exhibited atypical coupling, Rh3/Rh6 and Rh4/Rh5, indicating that Dve activity is required for proper opsin coupling. Surprisingly, Dve activity in R1 is required for the instructive signal, whereas activity in R6 and R7 blocks the signal. Our results indicate that functional coupling of two different neurons is established through signaling pathways from adjacent neurons that are functionally different.

CRISPR/Cas9-mediated knockout of scarlet gene produces eye color mutants in the soybean looper, Chrysodeixis includens.

The CRISPR/Cas9 technology has greatly progressed research on non-model organisms, demonstrating successful applications in genome editing for various insects. However, its utilization in the case of the soybean looper, Chrysodeixis includens, a notable pest affecting soybean crops, has not been explored due to constraints such as limited genomic information and the embryonic microinjection technique. This study presents successful outcomes in generating heritable knockout mutants for a pigment transporter gene, scarlet, in C. includens through CRISPR/Cas9-mediated mutagenesis. The scarlet locus identified in the genome assembly of C. includens consists of 14 exons, with a coding sequence extending for 1,986 bp. Two single guide RNAs (sgRNAs) were designed to target the first exon of scarlet. Microinjection of these two sgRNAs along with the Cas9 protein into fresh embryos resulted in the successful production of variable phenotypes, particularly mutant eyes. The observed mutation rate accounted for about 16%. Genotype analysis revealed diverse indel mutations at the target site, presumably originating from double-strand breaks followed by the nonhomologous end joining repair, leading to a premature stop codon due to frame shift. Single-pair mating of the mutant moths produced G1 offspring, and the establishment of a homozygous mutant strain occurred in G2. The mutant moths exhibited lightly greenish or yellowish compound eyes in both sexes, confirming the involvement of scarlet in pigmentation in C. includens. Notably, the CRISPR/Cas9-mediated genome editing technique serves as a visible phenotypic marker, demonstrating its proof-of-concept applicability in C. includens, as other pigment transporter genes have been utilized as visible markers to establish genetic control for various insects. These results provide the first successful case that the CRISPR/Cas9 method effectively induces mutations in C. includes, an economically important soybean insect pest.

The hAT family hopper transposon exists as highly similar yet discontinuous elements in the Bactrocera tephritid fly genus.

The hAT family transposable element, hopper, was originally discovered as a defective 3120-bp full-length element in a wild-type strain of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), and subsequently a functional 3131-bp element, hopperBdwe, was isolated from a white eye mutant strain. The latter study showed that closely related elements exist in melonfly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae), a closely related subgenus, suggesting that hopper could have a widespread presence in the Bactrocera genus. To further understand the distribution of hopper within and beyond the B. dorsalis species complex, primer pairs from hopperBdwe and its adjacent genomic insertion site were used to survey the presence and relatedness of hopper in five species within the complex and four species beyond the complex. Based on sequence identity of a 1.94 kb internal nucleotide sequence, the closest relationships were with mutated elements from B. dorsalis s.s. and species synonymized with B. dorsalis including B. papayae, B. philippinensis and B. invadens, ranging in identity between 88.4% and 99.5%. Notably, Bactrocera carambolae (Drew & Hancock) (Diptera: Tephritidae), which is most closely related to B. dorsalis beyond the synonymized species, shared hopper identities of 97.3%-99.5%. Beyond the B. dorsalis complex, Z. cucurbitae,Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) and Bactrocera zonata (Saunders) (Diptera: Tephritidae) shared identities of 83.1%-97.1%, while hopper was absent from the Bactrocera oleae (Gmelin) (Diptera: Tephritidae) strain tested. While the functional autonomous hopperBdwe element was not detected in these species, another closely related hopper element isolated from a B. dorsalis genetic sexing strain has an uninterrupted transposase open reading frame. The discontinuous presence of hopper in the Bactrocera genus has implications for its use for genomic manipulation and understanding the phylogenetic relationship of these species.

Embryonic microinjection of ribonucleoprotein complex (Cas9+sgRNA) of whitegene in melon fly, Zeugodacuscucurbitae (Coquillett) (Diptera: Tephritidae) produced whiteeye phenotype.

Melon fly, Zeugodacus cucurbitae (Coquillett) is a major pest of cucurbitaceous crops, and causes substantial yield losses and economic costs. CRISPR/Cas9 is a rapid and effective site-specific genome editing tool for the generation of genetic changes that are stable and heritable. The CRISPR/Cas9 tool uses synthetically designed single guide RNA (sgRNA) that is complementary to the target gene and guides the Cas9 enzyme to perform nuclease activity by making double-strand breaks in the target DNA sequences. This tool can be effectively exploited to improve traits critical for the management of insect pests by targeting specific genes encoding these traits without the need of extensive genetic information. The white gene is an important gene responsible for the transport of body pigment precursor molecules. In this study, we produced effective mutagenesis of the white gene of Z. cucurbitae using the CRISPR/Cas9 tool with double sgRNAto target multiple sites of white to increase the efficiency in the generation of frame-shift mutations resulting in the white eye phenotype in adults. This was achieved through embryonic microinjection of the ribonucleoprotein (RNP) complex in the pre-blastoderm embryo stage 1 h after embryo laying. Our success with the production of a white eye mutant fly by CRISPR/Cas9 mutagenesis is important for the research on gene function and protein-level modifications in melon fly and forms the basis for the development of new genetic control strategies such as precision guided sterile insect technique (pgSIT) for this pest of economic significance.

Bendless is essential for PINK1-Park mediated Mitofusin degradation under mitochondrial stress caused by loss of LRPPRC.

Cells under mitochondrial stress often co-opt mechanisms to maintain energy homeostasis, mitochondrial quality control and cell survival. A mechanistic understanding of such responses is crucial for further insight into mitochondrial biology and diseases. Through an unbiased genetic screen in Drosophila, we identify that mutations in lrpprc2, a homolog of the human LRPPRC gene that is linked to the French-Canadian Leigh syndrome, result in PINK1-Park activation. While the PINK1-Park pathway is well known to induce mitophagy, we show that PINK1-Park regulates mitochondrial dynamics by inducing the degradation of the mitochondrial fusion protein Mitofusin/Marf in lrpprc2 mutants. In our genetic screen, we also discover that Bendless, a K63-linked E2 conjugase, is a regulator of Marf, as loss of bendless results in increased Marf levels. We show that Bendless is required for PINK1 stability, and subsequently for PINK1-Park mediated Marf degradation under physiological conditions, and in response to mitochondrial stress as seen in lrpprc2. Additionally, we show that loss of bendless in lrpprc2 mutant eyes results in photoreceptor degeneration, indicating a neuroprotective role for Bendless-PINK1-Park mediated Marf degradation. Based on our observations, we propose that certain forms of mitochondrial stress activate Bendless-PINK1-Park to limit mitochondrial fusion, which is a cell-protective response.

A mouse model of cone photoreceptor function loss (cpfl9) with degeneration due to a mutation in Gucy2e.

During routine screening of mouse strains and stocks by the Eye Mutant Resource at The Jackson Laboratory for genetic mouse models of human ocular disorders, we identified cpfl9, a mouse model with cone photoreceptor function loss. The mice exhibited an early-onset phenotype that was easily recognized by the absence of a cone-mediated b-wave electroretinography response and by a reduction in rod-mediated photoresponses at four weeks of age. By genetic mapping and high-throughput sequencing of a whole exome capture library of cpfl9, a homozygous 25 bp deletion within exon 11 of the Gucy2e gene was identified, which is predicted to result in a frame shift leading to premature termination. The corresponding protein in human, retinal guanylate cyclase 1 (GUCY2D), plays an important role in rod and cone photoreceptor cell function. Loss-of-function mutations in human GUCY2D cause LCA1, one of the most common forms of Leber congenital amaurosis, which results in blindness at birth or in early childhood. The early loss of cone and reduced rod photoreceptor cell function in the cpfl9 mutant is accompanied by a later, progressive loss of cone and rod photoreceptor cells, which may be relevant to understanding disease pathology in a subset of LCA1 patients and in individuals with cone-rod dystrophy caused by recessive GUCY2D variants. cpfl9 mice will be useful for studying the role of Gucy2e in the retina.

Generation and characterization of Elovl4bknockout zebrafish as a model for Juvenile‐Onset Macular Degeneration

PurposeVery long‐chain polyunsaturated fatty acids (VLC‐PUFAs) are a distinct class of lipids with chain lengths greater than 24 carbons. ELOVL4 is a protein responsible for VLC‐PUFA biosynthesis. However, in patients with autosomal dominant Stargardt 3 disease, the gene encoding ELOVL4 is mutated, and as a result, a dysfunctional, truncated protein is produced instead. It is unclear whether the disease phenotype is caused by truncated protein aggregates, the absence of VLC‐PUFAs, or a combination of these factors. Moreover, homozygous elovl4 ablation is neonatal lethal in mice. This study aimed to delineate the effect of VLC‐PUFA loss on photoreceptor function.MethodsElovl4b knockout zebrafish were generated by creating a deletion mutation in exon 2 of the elovl4b gene using CRISPR‐Cas9. Mutant zebrafish were screened for non‐specific mutations by outcrossing mutant adults with wild‐type fish. Their eyes were isolated, total fatty acids extracted, and the quantity of VLC‐PUFAs determined through gas chromatography and mass spectrometry.ResultsWe found that homozygous Elovl4b mutant zebrafish eyes had altered lipid profiles and a significantly lower abundance of C30 to C34 VLC‐PUFAs compared to age‐matched wild‐type controls. We also predict diminished a‐ and b‐wave amplitudes from ERG recordings – indicative of a loss of photoreceptor function and a thinner outer nuclear layer than controls.ConclusionOur data indicate that the loss of Elovl4b in zebrafish can cause an ocular phenotype comparable to juvenile‐onset macular degeneration. The availability of a homozygous animal model of ELOVL4 deficiency will also allow for further studies on other ELOVL4‐related diseases.

CRISPR-mediated knockout of cardinal and cinnabar eye pigmentation genes in the western tarnished plant bug

The western tarnished plant bug, Lygus hesperus, is a key hemipteran pest of numerous agricultural, horticultural, and industrial crops in the western United States and Mexico. A lack of genetic tools in L. hesperus hinders progress in functional genomics and in developing innovative pest control methods such as gene drive. Here, using RNA interference (RNAi) against cardinal (LhCd), cinnabar (LhCn), and white (LhW), we showed that knockdown of LhW was lethal to developing embryos, while knockdown of LhCd or LhCn produced bright red eye phenotypes, in contrast to wild-type brown eyes. We further used CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) genome editing to generate germline knockouts of both LhCd (Card) and LhCn (Cinn), producing separate strains of L. hesperus characterized by mutant eye phenotypes. Although the cardinal knockout strain Card exhibited a gradual darkening of the eyes to brown typical of the wild-type line later in nymphal development, we observed bright red eyes throughout all life stages in the cinnabar knockout strain Cinn, making it a viable marker for tracking gene editing in L. hesperus. These results provide evidence that CRISPR/Cas9 gene editing functions in L. hesperus and that eye pigmentation genes are useful for tracking the successful genetic manipulation of this insect.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

The role of Ire1 in Drosophila eye pigmentation revealed by an RNase dead allele

Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. Here, we report that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, we found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1H890A homozygous mosaic eyes. Furthermore, the Ire1H890A mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. Our results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation.

Gypenosides Alleviate Cone Cell Death in a Zebrafish Model of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a group of visual disorders caused by mutations in over 70 genes. RP is characterized by initial degeneration of rod cells and late cone cell death, regardless of genetic abnormality. Rod cells are the main consumers of oxygen in the retina, and after the death of rod cells, the cone cells have to endure high levels of oxygen, which in turn leads to oxidative damage and cone degeneration. Gypenosides (Gyp) are major dammarane-type saponins of Gynostemma pentaphyllum that are known to reduce oxidative stress and inflammation. In this project we assessed the protective effect of Gyp against cone cell death in the rpgrip1 mutant zebrafish, which recapitulate the classical pathological features found in RP patients. Rpgrip1 mutant zebrafish were treated with Gyp (50 µg/g body weight) from two-months post fertilization (mpf) until 6 mpf. Gyp treatment resulted in a significant decrease in cone cell death compared to that of untreated mutant zebrafish. A markedly low level of reactive oxygen species and increased expression of antioxidant genes were detected in Gyp-incubated mutant zebrafish eyes compared to that of untreated mutant zebrafish. Similarly, the activities of catalase and superoxide dismutase and the level of glutathione were significantly increased in Gyp-treated mutant zebrafish eyes compared to that of untreated mutant zebrafish. Gyp treatment also decreased endoplasmic reticulum stress in rpgrip1 mutant eyes. Expression of proinflammatory cytokines was also significantly decreased in Gyp-treated mutant zebrafish eyes compared to that of untreated mutant zebrafish. Network pharmacology analysis demonstrated that the promotion of cone cell survival by Gyp is possibly mediated by multiple hub genes and associated signalling pathways. These data suggest treatment with Gyp will benefit RP patients.

Novel Newt Regeneration Genes Regulate Wingless Signaling to Restore Patterning in <i>Drosophila</i> Eye

A fundamental process of regeneration, which varies among animals, recruits conserved signaling pathways to restore missing parts. Only a few animals like newts can repeatedly regenerate lost body parts throughout their lifespan that can be attributed to strategic regulation of conserved signaling pathways by newt’s regeneration tool-kit genes. Lack of genetic tools pose challenges to understand the molecular genetic mechanism of regeneration response of these genes. Here we report use of genetically tractable Drosophila eye model to demonstrate the regeneration potential of a group of unique protein(s) from newt (Notophthalmus viridescens), which when ectopically expressed can significantly rescue missing photoreceptor cells in a Drosophila eye mutant. These newt proteins with signal peptides motifs exhibit non-cell-autonomous rescue properties and their regeneration potential even extends into later stages of fly development. Ectopic expression of these newt genes can rescue eye mutant phenotype by promoting cell proliferation and blocking cell death. These novel newt genes downregulate evolutionarily conserved Wingless (Wg)/Wnt signaling pathway to promote rescue. Modulation of Wg/Wnt signaling levels by using antagonists or agonists of Wg/Wnt signaling pathway in eye mutant background where newt gene(s) is ectopically expressed suggests that Wg signaling acts downstream of newt genes. Our data highlights the regeneration potential of novel newt proteins that regulate conserved pathways to trigger a robust regeneration response in Drosophila model with weak regeneration capability.

The hAT-family transposable element, hopper, from Bactrocera dorsalis is a functional vector for insect germline transformation

BackgroundThe hopper hAT-family transposable element isolated from the Oriental fruit fly, Bactrocera dorsalis, is distantly related to both the Drosophila hobo element and the Activator element from maize. The original 3120 bp hopperBd-Kah element isolated from the Kahuku wild-type strain was highly degenerate and appeared to have a mutated transposase and terminal sequences, while a second 3131 bp element, hopperBd-we, isolated from a white eye mutant strain had an intact transposase reading frame and terminal sequences consistent with function.ResultsThe hopperBd-we element was tested for function by its ability to mediate germline transformation in two dipteran species other than B. dorsalis. This was achieved by creating a binary vector/helper transformation system by linking the hopperBd-we transposase reading frame to a D. melanogaster hsp70 promoter for a heat-inducible transposase helper plasmid, and creating vectors marked with the D. melanogaster mini-white+ or polyubiquitin-regulated DsRed fluorescent protein markers.ConclusionsBoth vectors were successfully used to transform D. melanogaster, and the DsRed vector was also used to transform the Caribbean fruit fly, Anastrepha suspensa, indicating a wide range of hopper function in dipteran species and, potentially, non-dipteran species. This vector provides a new tool for insect genetic modification for both functional genomic analysis and the control of insect populations.

Nf2 fine-tunes proliferation and tissue alignment during closure of the optic fissure in the embryonic mouse eye.

Uveal coloboma represents one of the most common congenital ocular malformations accounting for up to 10% of childhood blindness (~1 in 5000 live birth). Coloboma originates from defective fusion of the optic fissure (OF), a transient gap that forms during eye morphogenesis by asymmetric, ventral invagination. Genetic heterogeneity combined with the activity of developmentally regulated genes suggests multiple mechanisms regulating OF closure. The tumor suppressor and FERM domain protein Neurofibromin 2 (NF2) controls diverse processes in cancer, development and regeneration, via Hippo pathway and cytoskeleton regulation. In humans, NF2 mutations can cause ocular abnormalities, including coloboma, however, its actual role in OF closure is unknown. Using conditional inactivation in the embryonic mouse eye, our data indicate that loss of Nf2 function results in a novel underlying cause for coloboma. In particular, mutant eyes show substantially increased retinal pigmented epithelium (RPE) proliferation in the fissure region with concomitant acquisition of RPE cell fate. Cells lining the OF margin can maintain RPE fate ectopically and fail to transition from neuroepithelial to cuboidal shape. In the dorsal RPE of the optic cup, Nf2 inactivation leads to a robust increase in cell number, with local disorganization of the cytoskeleton components F-actin and pMLC2. We propose that RPE hyperproliferation is the primary cause for the observed defects causing insufficient alignment of the OF margins in Nf2 mutants and failure to fuse properly, resulting in persistent coloboma. Our findings indicate that limiting proliferation particularly in the RPE layer is a critical mechanism during OF closure.

Gypenosides attenuate retinal degeneration in a zebrafish retinitis pigmentosa model

Retinitis pigmentosa (RP) is a collection of heterogenous genetic retinal disorders resulting in cumulative retinal deterioration involving progressive loss of photoreceptors and eventually in total blindness. Oxidative stress plays a central role in this photoreceptor loss. Gypenosides (Gyp) are the main functional component isolated from the climbing vine Gynostemma pentaphyllum and have been shown to defend cells against the effects of oxidative stress and inflammation, providing protection in experimentally-induced optic neuritis. The zebrafish model has been used to investigate a range of human diseases. Previously we reported early retinal degeneration in a mutant zebrafish line carrying a point-nonsense mutation in the retinitis pigmentosa GTPase regulator interacting protein 1 (rpgrip1) gene that is mutated in RP patients. The current study investigated the potential protective effects of Gyp against photoreceptor degeneration in the Rpgrip1 deleted zebrafish. Rpgrip1 mutant zebrafish were treated with 5 μg/ml of Gyp in E3 medium from 6 h post fertilization (hpf) till 1 month post fertilization (mpf). Rpgrip1 mutant zebrafish treated with 5 μg/ml of Gyp showed a significant decrease by 68.41% (p = 0.0002) in photoreceptor cell death compared to that of untreated mutant zebrafish. Expression of antioxidant genes catalase, sod1, sod2, gpx1, gclm, nqo-1 and nrf-2 was significantly decreased in rpgrip1 mutant zebrafish eyes by 61.51%, 77.40%, 60.11%, 81.17%, 72.07%, 78.95% and 85.42% (all p < 0.0001), respectively, when compared to that of wildtype zebrafish; superoxide dismutase and catalase activities, and glutathione levels in rpgrip1 mutant zebrafish eyes were significantly decreased by 87.21%, 21.55% and 96.51% (all p < 0.0001), respectively. There were marked increases in the production of reactive oxygen species (ROS) and malondialdehyde (MDA) by 2738.73% and 510.69% (all p < 0.0001), respectively, in rpgrip1 mutant zebrafish eyes; expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α was also significantly increased by 150.11%, 267.79% and 190.72% (all p < 0.0001), respectively, in rpgrip1 mutant zebrafish eyes, compared to that of wildtype zebrafish. Treatment with Gyp significantly counteracted these effects. This study indicates that Gyp has a potential role in the treatment of RP.

Absolute Quantification of Proteins in the Eye of Drosophila melanogaster.

Absolute (molar) quantification of proteins determines their molar ratios in complexes, networks, and metabolic pathways. MS Western workflow is employed to determine molar abundances of proteins potentially critical for morphogenesis and phototransduction (PT) in eyes of Drosophila melanogaster using a single chimeric 264kDa protein standard that covers, in total, 197 peptides from 43 proteins. The majority of proteins are independently quantified with two to four proteotypic peptides with the coefficient of variation of less than 15%, better than 1000-fold dynamic range and sub-femtomole sensitivity. Here, the molar abundance of proteins of the PT machinery and of the rhabdomere, the photosensitive organelle, is determined in eyes of wild-type flies as well as in crumbs (crb) mutant eyes, which exhibit perturbed rhabdomere morphogenesis.

The Classic Lobe Eye Phenotype of Drosophila Is Caused by Transposon Insertion-Induced Misexpression of a Zinc-Finger Transcription Factor.

Drosophila Lobe (L) alleles were first discovered ∼100years ago as spontaneous dominant mutants with characteristic developmental eye defects. However, the molecular basis for L dominant eye phenotypes has not been clearly understood. A previous work reported identification of CG10109/PRAS40 as the L gene, but subsequent analyses suggested that PRAS40 may not be related to L Here, we revisited the L gene to clarify this discrepancy and understand the basis for the dominance of L mutations. Genetic analysis localized the L gene to Oaz, which encodes a homolog of the vertebrate zinc finger protein 423 (Zfp423) family transcriptional regulators. We demonstrate that RNAi knockdown of Oaz almost completely restores all L dominant alleles tested. Lrev6-3 , a revertant allele of the L2 dominant eye phenotype, has an inframe deletion in the Oaz coding sequence. Molecular analysis of L dominant mutants identified allele-specific insertions of natural transposons (roo[ ]L1 , hopper[ ]L5 , and roo[ ]Lr ) or alterations of a preexisting transposon (L2 -specific mutations in roo[ ]Mohr) in the Oaz region. In addition, we generated additional L2 -reversion alleles by CRISPR targeting at Oaz These new loss-of-function Oaz mutations suppress the dominant L eye phenotype. Oaz protein is not expressed in wild-type eye disc but is expressed ectopically in L2/+ mutant eye disc. We induced male recombination between Oaz-GAL4 insertions and the L2 mutation through homologous recombination. By using the L2 -recombined GAL4 reporters, we show that Oaz-GAL4 is expressed ectopically in L2 eye imaginal disc. Taken together, our data suggest that neomorphic L eye phenotypes are likely due to misregulation of Oaz by spontaneous transposon insertions.

Developmental delay during eye morphogenesis underlies optic cup and neurogenesis defects in mab21l2u517 zebrafish mutants.

Shaping the vertebrate eye requires evagination of the optic vesicles. These vesicles subsequently fold into optic cups prior to undergoing neurogenesis and allocating a population of late progenitors at the margin of the eye. mab21l2 encodes a protein of unknown biological function expressed in the developing optic vesicles, and loss of mab21l2 function results in malformed eyes. The bases of these defects are, however, poorly understood. To further study mab21l2 we used CRISPR/Cas9 to generate a new zebrafish mutant allele (mab21l2u517). We characterized eye morphogenesis and neurogenesis upon loss of mab21l2 function using tissue/cell-type-specific transgenes and immunostaining, in situ hybridization and bromodeoxyuridine incorporation. mab21l2u517 eyes fail to grow properly and display an excess of progenitors in the ciliary marginal zone. The expression of a transgene reporter for the vsx2 gene -a conserved marker for retinal progenitors- was delayed in mutant eyes and accompanied by disruptions in the epithelial folding that fuels optic cup morphogenesis. Mutants also displayed nasal-temporal malformations suggesting asynchronous development along that axis. Consistently, nasal retinal neurogenesis initiated but did not propagate in a timely fashion to the temporal retina. Later in development, mutant retinas did laminate and differentiate. Thus, mab21l2u517 mutants present a complex eye morphogenesis phenotype characterized by an organ-specific developmental delay. We propose that mab21l2 facilitates optic cup development with consequences both for timely neurogenesis and allocation of progenitors to the zebrafish ciliary marginal zone. These results confirm and extend previous analyses supporting the role of mab21l2 in coordinating morphogenesis and differentiation in developing eyes.

Core-clock genes Period 1 and 2 regulate visual cascade and cell cycle components during mouse eye development

The retinas from Period 1 (Per1) and Period 2 (Per2) double-mutant mice (Per1−/−Per2Brdm1) display abnormal blue-cone distribution associated with a reduction in cone opsin mRNA and protein levels, up to 1 year of age. To reveal the molecular mechanisms by which Per1 and Per2 control retina development, we analyzed genome-wide gene expression differences between wild-type (WT) and Per1−/−Per2Brdm1 mice across ocular developmental stages (E15, E18 and P3). All clock genes displayed changes in transcript levels along with normal eye development. RNA-Seq data show major gene expression changes between WT and mutant eyes, with the number of differentially expressed genes (DEG) increasing with developmental age. Functional annotation of the genes showed that the most significant changes in expression levels in mutant mice involve molecular pathways relating to circadian rhythm signaling at E15 and E18. At P3, the visual cascade and the cell cycle were respectively higher and lower expressed compared to WT eyes. Overall, our study provides new insights into signaling pathways -phototransduction and cell cycle- controlled by the circadian clock in the eye during development.