Small Eye Phenotype Research Articles

Apoptotic and Proliferative Defects Characterize Ocular Development in a Microphthalmic BMP Model

Vision is critically dependent on ocular size, which is regulated by environmental and genetic factors. Mutation of human Growth and Differentiation Factor 6 (GDF6) or zebrafish gdf6a results in a spectrum of small eye phenotypes (microphthalmia, anophthalmia, and coloboma). However, current models do not explain their etiology fully. As such, analyses of apoptosis and cell cycle regulation were undertaken in a zebrafish gdf6a mutant. Microarray analysis was performed at 2 days after fertilization to uncover novel gdf6a-dependent cell cycle regulators. Altered expression of Gdf6a targets was confirmed by in situ hybridization, and resulting changes in cell proliferation were assessed by phosphohistone H3 immunohistochemistry. Analysis of apoptosis was evaluated through activated Caspase 3 immunohistochemistry and chemical inhibitors of cell death. Reduced numbers of retinal progenitor cells are observed at 24 hours post fertilization (hpf), resulting in microphthalmic eyes in gdf6a(-/-) embryos. At 28 hpf, a wave of apoptosis occurs; however, apoptosis inhibition does not rescue eye size, indicating a limited contribution. Mutants display altered proliferation and expression levels of cell cycle regulators, including members of the forkhead box i (foxi) transcription factor family expressed in the ciliary marginal zone. Notably, inhibition of foxi2 in gdf6a(-/-) embryos further reduces eye size. These data support a model whereby the gdf6a(-/-)-induced microphthalmia is based on early regulation of retinal progenitor cell number, and later by regulation of proliferation in the ciliary marginal zone. Foxi genes represent downstream effectors of Gdf6a function in the CMZ required for eye size determination.

The small eye phenotype in the EPIC-Norfolk eye study: prevalence and visual impairment in microphthalmos and nanophthalmos

ObjectiveTo describe the prevalence and phenotypic characteristics of small eyes in the European Prospective Investigation of Cancer (EPIC)-Norfolk Eye Study.DesignCommunity cross-sectional study.SettingEast England population (Norwich, Norfolk and surrounding area).Participants8033 participants...

Defining small eye phenotypes

Defining small eye phenotypes

Differential requirement for the N-terminal catalytic domain of the DNA polymerase ε p255 subunit in the mitotic cell cycle and the endocycle

In Drosophila, the 255kDa catalytic subunit (dpolεp255) and the 58kDa subunit of DNA polymerase ε (dpolεp58) have been identified. The N-terminus of dpolεp255 carries well-conserved six DNA polymerase subdomains and five 3′→5′ exonuclease motifs as observed with Polε in other species. We here examined roles of dpolεp255 during Drosophila development using transgenic fly lines expressing double stranded RNA (dsRNA). Expression of dpolεp255 dsRNA in eye discs induced a small eye phenotype and inhibited DNA synthesis, indicating a role in the G1–S transition and/or S-phase progression of the mitotic cycle. Similarly, expression of dpolεp255 dsRNA in the salivary glands resulted in small size and endoreplication defects, demonstrating a critical role in endocycle progression. In the eye disc, defects induced by knockdown of dpolεp255 were rescued by overexpression of the C-terminal region of dpolεp255, indicating that the function of this non-catalytic domain is conserved between yeast and Drosophila. However, this was not the case for the salivary gland, suggesting that the catalytic N-terminal region is crucial for endoreplication and its defect cannot be complemented by other DNA polymerases. In addition, several genetic interactants with dpolεp255 including genes related to DNA replication such as RFC, DNA primase, DNA polη, Mcm10 and Psf2 and chromatin remodeling such as Iswi were also identified.

Significance of the Dynamic Expression of Pax6 and Dlx2 in the Postnatal Periventricular Zone Cells

Significance of the Dynamic Expression of Pax6 and Dlx2 in the Postnatal Periventricular Zone Cells

Drosophila Ribosomal Protein Mutants Control Tissue Growth Non-Autonomously via Effects on the Prothoracic Gland and Ecdysone

The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycEJP). We demonstrated that the suppression of cycEJP by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycEJP is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously.

Knockdown of the Zebrafish Ortholog of the Retinitis Pigmentosa 2 (RP2) Gene Results in Retinal Degeneration

The authors investigated the expression and function of the zebrafish ortholog of the retinitis pigmentosa 2 (RP2) gene. Zebrafish RP2 (ZFRP2) cDNA was isolated from adult eye mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Gene expression was examined by RT-PCR. The deduced peptide sequence was aligned with RP2 orthologues from different species. Translational suppression (knockdown) of zebrafish RP2 was carried out by antisense morpholino-injection. The phenotype of ZFRP2 knockdown morphants was characterized by immunohistology and histology. Human wild-type and mutant RP2 mRNAs were coinjected with ZFRP2 morpholinos to test whether human RP2 mRNA could rescue ZFRP2 knockdown phenotypes. ZFRP2 encodes a protein of 376 amino acids containing an N-terminal tubulin folding cofactor C-like domain and a C-terminal nucleoside diphosphate kinase-like domain. It shares 63% to 65% amino acid identity with human, mouse and bovine RP2. RP2 is expressed at the earliest stages of zebrafish development and persists into adulthood. Knockdown of RP2 in zebrafish causes a curved body axis and small eye phenotype, associated with increased cell death throughout the retina. Human wild-type RP2 mRNA could rescue the body curvature phenotype of ZFRP2 morphants, and the eye size of the resultant morphants was significantly increased over that of morphants in which ZFRP2 had been depleted. Zebrafish RP2 is widely expressed throughout development. ZFRP2 knockdown caused retinal degeneration in zebrafish. Human RP2 could partially rescue the small eye phenotype of ZFRP2 morphants.

A male with unilateral microphthalmia reveals a role for TMX3 in eye development.

Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

The Chromatin-Remodeling Protein Osa Interacts With CyclinE in Drosophila Eye Imaginal Discs

Coordinating cell proliferation and differentiation is essential during organogenesis. In Drosophila, the photoreceptor, pigment, and support cells of the eye are specified in an orchestrated wave as the morphogenetic furrow passes across the eye imaginal disc. Cells anterior of the furrow are not yet differentiated and remain mitotically active, while most cells in the furrow arrest at G(1) and adopt specific ommatidial fates. We used microarray expression analysis to monitor changes in transcription at the furrow and identified genes whose expression correlates with either proliferation or fate specification. Some of these are members of the Polycomb and Trithorax families that encode epigenetic regulators. Osa is one; it associates with components of the Drosophila SWI/SNF chromatin-remodeling complex. Our studies of this Trithorax factor in eye development implicate Osa as a regulator of the cell cycle: Osa overexpression caused a small-eye phenotype, a reduced number of M- and S-phase cells in eye imaginal discs, and a delay in morphogenetic furrow progression. In addition, we present evidence that Osa interacts genetically and biochemically with CyclinE. Our results suggest a dual mechanism of Osa function in transcriptional regulation and cell cycle control.

Identification, Characterization, and Effects ofXenopus laevisPNAS-4 Gene on Embryonic Development

Apoptosis plays an important role in embryonic development. PNAS-4 has been demonstrated to induce apoptosis in several cancer cells. In this study, we cloned Xenopus laevis PNAS-4 (xPNAS-4), which is homologous to the human PNAS-4 gene. Bioinformatics analysis for PNAS-4 indicated that xPNAS-4 shared 87.6% identity with human PNAS-4 and 85.5% with mouse PNAS-4. The phylogenetic tree of PNAS-4 protein was also summarized. An analysis of cellular localization using an EGFP-fused protein demonstrated that xPNAS-4 was localized in the perinuclear region of the cytoplasm. RT-PCR analysis revealed that xPNAS-4, as a maternally expressed gene, was present in all stages of early embryo development. Whole-mount in situ hybridization showed that xPNAS-4 was mainly expressed in ectoderm and mesoderm. Furthermore, microinjection of xPNAS-4 mRNA in vivo caused developmental defects manifesting as a small eye phenotype in the Xenopous embryos, and as a small eye or one-eye phenotype in developing zebrafish embryos. In addition, embryos microinjected with xPNAS-4 antisense morpholino oligonucleotides (MOs) exhibited a failure of head development and shortened axis.

Zebrafish Rpgr is required for normal retinal development and plays a role in dynein-based retrograde transport processes

Mutations in the human RPGR gene cause one of the most common and severe forms of inherited retinal dystrophy, but the function of its protein product remains unclear. We have identified two genes resembling human RPGR (ZFRPGR1, ZFRPGR2) in zebrafish (Danio rerio), both of which are expressed within the nascent and adult eye as well as more widely during development. ZFRPGR2 appears to be functionally orthologous to human RPGR, because it encodes similar protein isoforms (ZFRPGR2(ORF15), ZFRPGR2(ex1-17)) and causes developmental defects similar to other ciliary proteins, affecting gastrulation, tail and head development after morpholino-induced knockdown (translation suppression). These defects are consistent with a ciliary function and were rescued by human RPGR but not by RPGR mutants causing retinal dystrophy. Unlike mammals, RPGR knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. Developmental abnormalities in the eye included lamination defects, failure to develop photoreceptor outer segments and a small eye phenotype, associated with increased cell death throughout the retina. These defects could be rescued by expression of wild-type but not mutant forms of human RPGR. ZFRPGR2 knockdown also resulted in an intracellular transport defect affecting retrograde but not anterograde transport of organelles. ZFRPGR2 is therefore necessary both for the normal differentiation and lamination of the retina and to prevent apoptotic retinal cell death, which may relate to its proposed role in dynein-based retrograde transport processes.

Polyhomeotic has a tumor suppressor activity mediated by repression of Notch signaling

Polycomb Group (PcG) proteins silence critical developmental genes and modulate cell proliferation. Using the Drosophila melanogaster eye as a model system, we show that cells with mutations in the gene locus (ph) that encodes the PcG protein Polyhomeotic (PH) overproliferate and lose both the ability to differentiate and their normal polarity. They invade the neighboring tissues and, when combined with an activated form of the Ras proto-oncogene, they trigger the formation of metastases. PcG proteins bind to multiple genes in the Notch pathway and control their transcription as well as Notch signaling. The massive cell-autonomous overproliferation of ph mutant cell clones can be rescued by ectopic expression of a dominant negative form of Notch or by RNA interference (RNAi)-mediated repression of Notch. Conversely, overexpression of ph induces a small-eye phenotype that is rescued by activation of Notch signaling. These data show that ph is a tumor suppressor locus that controls cellular proliferation by silencing multiple Notch signaling components.

Transcription factor directed differentiation of embryonic stem cells in vitro

OBJECTIVE: We hypothesize that Pax6 and Six3 transcription factors (TF) that play roles critical in formation of the vertebrate ocular lens, may also facilitate directed differentiation of embryonic stem cells (ESC) to generate lens tissue in vitro. DESIGN: Mammalian lens placode development is regulated by key TFs, Pax6, a paired box gene in conjunction with the homeobox gene Six3. Loss-of-function mutations of PAX6 results in the ocular defects aniridia and anophthalmia in humans and the Small eye (Sey) phenotype in mice which includes diminished lens development and cataracts. We therefore chose to test whether Pax6 and Six3 can direct ESC differentiation to a lens fate in vitro. MATERIALS AND METHODS: G4 mouse ESCs and H1 NIH human ESCs were used in these studies. Plasmids or lentiviral vectors for Pax6 and Six3 were employed for transfection and infection of cultured mESC or hESC. Post transduction cultured cells were followed for 7 or 14 days and analyzed by immuncytochemistry, RT-PCR and protein analysis. RESULTS: We observed that transduction of mouse or human ESC with either Pax6 or Six3 genes drives differentiation of ESCs along the lens forming pathway. This was apparent from a near 40-fold increase in the number of γ-crystallin immunoreactive colonies, a lens terminal antigen, in Pax6 or Six3 transduced ESC colonies after 14 days. Furthermore, γ-crystallin expressing colonies formed distinct multicellular rosettes. Additional lens proteins such as Prox1 were also upregulated in the experimental cultures. Expression of other markers associated with lens development and differentiation such as Bmp7, Foxe3, Mab2111, Gja8, α-crystallin and β-crystallin was confirmed by RT-PCR and Western blot analysis. CONCLUSIONS: The lens endogenous TFs, Pax6 and Six3, can program mouse and human ESCs to similar fates in vitro. This suggests a potential route for using specific TFs to direct the efficient differentiation of ESCs to desired tissue fates in vitro.

Mutation in a Novel Connexin-like Gene (Gjf1) in the Mouse Affects Early Lens Development and Causes a Variable Small-Eye Phenotype

The purpose of the study was the characterization of the novel small-eye mutant Aey12 in the mouse. The eyes of the mutants were described morphologically and histologically and by in situ hybridization. The homozygotes were viable and fully fertile, which identifies Aey12 as a new microphthalmia phenotype in the mouse, different from Maf or Pax6 mutants. Histologic analysis indicated the presence of the lens vesicle; however, the primary fiber cells did not elongate properly. Genome-wide linkage analysis mapped the mutation to the proximal region of chromosome 10 between the markers D10Mit206 and D10Mit189. Among the positional candidate genes, one EST (expressed sequence tag), D230044M03Rik, encodes a connexin-like protein. A G-->T point mutation was identified at cDNA position 96, resulting in an R32Q amino acid exchange in a transmembrane domain. The mutation leads to a loss of an SsiI restriction site, which is present in five wild-type mouse strains (102, C3H, C57BL/6, DBA, and JF1). The gene is expressed in the posterior part of the lens vesicle, where the primary fiber elongation starts. In the mutants, the expression pattern of Pax6, Prox1, Six3, and Crygd are modified, but not the pattern of Pax2. The mutated mouse gene belongs to the family of connexin-encoding genes (gene symbols Gja-Gje). Together with its rat and human homologues, it defines a new subgroup, referred to as Gjf1. The mouse mutant described herein offers a new functional candidate gene for microphthalmia-related disorders at the corresponding locus on human chromosome 6, area q24.

Mechanism of Small Heat Shock Protein Function in Vivo: A KNOCK-IN MOUSE MODEL DEMONSTRATES THAT THE R49C MUTATION IN αA-CRYSTALLIN ENHANCES PROTEIN INSOLUBILITY AND CELL DEATH

alphaA-crystallin (Cryaa/HSPB4) is a small heat shock protein and molecular chaperone that prevents nonspecific aggregation of denaturing proteins. Several point mutations in the alphaA-crystallin gene cause congenital human cataracts by unknown mechanisms. We took a novel approach to investigate the molecular mechanism of cataract formation in vivo by creating gene knock-in mice expressing the arginine 49 to cysteine mutation (R49C) in alphaA-crystallin (alphaA-R49C). This mutation has been linked with autosomal dominant hereditary cataracts in a four-generation Caucasian family. Homologous recombination in embryonic stem cells was performed using a plasmid containing the C to T transition in exon 1 of the cryaa gene. alphaA-R49C heterozygosity led to early cataracts characterized by nuclear opacities. Unexpectedly, alphaA-R49C homozygosity led to small eye phenotype and severe cataracts at birth. Wild type littermates did not show these abnormalities. Lens fiber cells of alphaA-R49C homozygous mice displayed an increase in cell death by apoptosis mediated by a 5-fold decrease in phosphorylated Bad, an anti-apoptotic protein, but an increase in Bcl-2 expression. However, proliferation measured by in vivo bromodeoxyuridine labeling did not decline. The alphaA-R49C heterozygous and homozygous knock-in lenses demonstrated an increase in insoluble alphaA-crystallin and alphaB-crystallin and a surprising increase in expression of cytoplasmic gamma-crystallin, whereas no changes in beta-crystallin were observed. Co-immunoprecipitation analysis showed increased interaction between alphaA-crystallin and lens substrate proteins in the heterozygous knock-in lenses. To our knowledge this is the first knock-in mouse model for a crystallin mutation causing hereditary human cataract and establishes that alphaA-R49C promotes protein insolubility and cell death in vivo.

The myeloid leukemia factor interacts with COP9 signalosome subunit 3 in Drosophila melanogaster

The human myeloid leukemia factor 1 (hMLF1) gene was first identified as an NPM-hMLF1 fusion gene produced by chromosomal translocation. In Drosophila, dMLF has been identified as a protein homologous to hMLF1 and hMLF2, which interacts with various factors involved in transcriptional regulation. However, the precise cellular function of dMLF remains unclear. To generate further insights, we first examined the behavior of dMLF protein using an antibody specific to dMLF. Immunostaining analyses showed that dMLF localizes in the nucleus in early embryos and cultured cells. Ectopic expression of dMLF in the developing eye imaginal disc using eyeless-GAL4 driver resulted in a small-eye phenotype and co-expression of cyclin E rescued the small-eye phenotype, suggesting the involvement of dMLF in cell-cycle regulation. We therefore analyzed the molecular mechanism of interactions between dMLF and a dMLF-interacting protein, dCSN3, a subunit of the COP9 signalosome, which regulates multiple signaling and cell-cycle pathways. Biochemical and genetic analyses revealed that dMLF interacts with dCSN3 in vivo and glutathione S-transferase pull-down assays revealed that the PCI domain of the dCSN3 protein is sufficient for this to occur, possibly functioning as a structural scaffold for assembly of the COP9 signalosome complex. From these data we propose the possibility that dMLF plays a negative role in assembly of the COP9 signalosome complex.

Eye Development under the control of SRp55/B52-Mediated Alternative Splicing of eyeless

The genetic programs specifying eye development are highly conserved during evolution and involve the vertebrate Pax-6 gene and its Drosophila melanogaster homolog eyeless (ey). Here we report that the SR protein B52/SRp55 controls a novel developmentally regulated splicing event of eyeless that is crucial for eye growth and specification in Drosophila. B52/SRp55 generates two isoforms of eyeless differing by an alternative exon encoding a 60-amino-acid insert at the beginning of the paired domain. The long isoform has impaired ability to trigger formation of ectopic eyes and to bind efficiently Eyeless target DNA sequences in vitro. When over-produced in the eye imaginal disc, this isoform induces a small eye phenotype, whereas the isoform lacking the alternative exon triggers eye over-growth and strong disorganization. Our results suggest that B52/SRp55 splicing activity is used during normal eye development to control eye organogenesis and size through regulation of eyeless alternative splicing.

Isolation of Xenopus HIF-prolyl 4-hydroxylase and rescue of a small-eye phenotype caused by Siah2 over-expression

Hypoxia is an important physiological condition during embryonic development. Hypoxia-inducible factor (HIF) is the mediator of hypoxic response of cells. The prolyl hydroxylase (PHD) of HIF plays a key role in stabilizing of HIF and the oxygen homeostasis of organisms. In this study, we isolated two PHD proteins, PHD45 and PHD28, and characterized them during the embryonic development of Xenopus laevis, which is an excellent model for embryonic development because of the ease of embryonic manipulation and the feasibility of transgenesis. Based on amino acid sequences, Xenopus PHD45 and PHD28 were homologous with human PHD2 and PHD3, respectively. In embryonic development, PHD45 expression was complementary to that of PHD28. xHIF-1α protein level was at a maximum around stage 20 when expression of PHD45 disappeared, while expression of PHD28 reached a maximum at stage 20, suggesting that PHD28 is inducible by HIF-1α. Recently, Siah2 was found to be an ubiquitin ligase of PHD proteins and to regulate degradation of PHD proteins. Over-expression of xSiah2 decreased PHD45 but not PHD28 and caused the small-eye phenotype of Xenopus. Additional over-expression of PHD47 rescued the abnormality caused by xSiah2, suggesting that the level of expression or activity of PHD proteins is important to the maintenance of homeostasis in embryonic development.

Regulation of the activity of the tumor suppressor PTEN by thioredoxin in Drosophila melanogaster

Human Thioredoxin-1 (hTrx-1) is a small redox protein with a molecular weight of 12 kDa that contains two cysteine residues found in its catalytic site. HTrx-1 plays an important role in cell growth, apoptosis, and cancer patient prognosis. Recently, we have demonstrated that hTrx-1 binds to the C2 domain of the human tumor suppressor, PTEN, in a redox dependent manner. This binding leads to the inhibition of PTEN lipid phosphatase activity in mammalian tissue culture systems. In this study, we show that over-expression of hTrx-1 in Drosophila melanogaster promotes cell growth and proliferation during eye development as measured by eye size and ommatidia size. Furthermore, hTrx-1 rescues the small eye phenotype induced by the over-expression of PTEN. We demonstrate that this rescue of the PTEN-induced eye size phenotype requires cysteine-218 in the C2 domain of PTEN. We also show that hTrx-1 over-expression results in increased Akt phosphorylation in fly head extracts supporting our observations that the hTrx-1-induced eye size increase results from the inhibition of PTEN activity. Our study confirms the redox regulation of PTEN through disulfide bond formation with the hTrx-1 in Drosophila and suggests conserved mechanisms for thioredoxins and their interactions with the phosphatidylinositol-3-kinase signaling pathway in humans and fruit flies.

Heat shock factor 1 is required for constitutive Hsp70 expression and normal lens development in embryonic zebrafish

Heat shock factors (HSFs) are the major transcription factors responsible for heat-induced upregulation of heat shock protein (Hsp) genes. All three mammalian HSFs (HSF1, HSF2, HSF4) have also been shown to be required for normal mammalian development. It is currently unknown if HSFs play similarly important roles during normal development of non-mammalian vertebrates. In the present study, a morpholino modified antisense oligonucleotide (MO) approach targeted against hsf1 mRNA (hsf1-MO) was used to examine the requirement of HSF1 in zebrafish development. Embryos depleted of HSF1 displayed a reproducible small eye phenotype characterized by an immature lens and a disorganized retinal structure. These defects were strikingly similar to those observed when constitutive, lens specific Hsp70 expression was reduced through the microinjection of MO targeting hsp70. The data suggest that HSF1 is involved in regulating constitutive lens specific expression of hsp70 in the embryonic zebrafish. This conclusion is supported by a marked reduction in Hsp70 protein in hsf1-MO injected embryos. Microinjection of MO targeted to hsf2 mRNA (hsf2-MO) did not result in a small eye phenotype in a significant number of embryos. These data also suggest that HSF1 and HSF2 play distinct roles in non-mammalian vertebrates, similarly to what has been demonstrated previously in mouse.