Mouse Eye Development Research Articles

Role of INK4a locus in normal eye development and cataract genesis

The murine INK4a locus encodes the critical tumor suppressor proteins, p16 INK4a and p19 ARF . Mice lacking both p16 INK4a and p19 ARF ( INK4a −/−) in their FVB/NJ genetic backgrounds developed cataracts and microophthalmia. Histopathologically, INK4a −/− mice showed defects in the developmental regression of the hyaloid vascular system (HVS), retinal dysplasia, and cataracts with numerous vacuolations, closely resembling human persistent hyperplastic primary vitreous (PHPV). Ocular defects, such as retinal fold and abnormal migration of lens fiber cells, were observed as early as embryonic day (E) 15.5, thereby resulting in the abnormal differentiation of the lens. We also found that ectopic expression of p16 INK4a resulted in the induction of γF-crystallin, suggesting an important role of INK4a locus during mouse eye development, and also providing insights into the potential genetic basis of human cataract genesis.

Severe Defects in Proliferation and Differentiation of Lens Cells in Foxe3 Null Mice

During mouse eye development, the correct formation of the lens occurs as a result of reciprocal interactions between the neuroectoderm that forms the retina and surface ectoderm that forms the lens. Although many transcription factors required for early lens development have been identified, the mechanism and genetic interactions mediated by them remain poorly understood. Foxe3 encodes a winged helix-forkhead transcription factor that is initially expressed in the developing brain and in the lens placode and later restricted exclusively to the anterior lens epithelium. Here, we show that targeted disruption of Foxe3 results in abnormal development of the eye. Cells of the anterior lens epithelium show a decreased rate of proliferation, resulting in a smaller than normal lens. The anterior lens epithelium does not properly separate from the cornea and frequently forms an unusual, multilayered tissue. Because of the abnormal differentiation, lens fiber cells do not form properly, and the morphogenesis of the lens is greatly affected. The abnormally differentiated lens cells remain irregular in shape, and the lens becomes vacuolated. The defects in lens development correlate with changes in the expression of growth and differentiation factor genes, including DNase II-like acid DNase, Prox1, p57, and PDGFalpha receptor. As a result of abnormal lens development, the cornea and the retina are also affected. While Foxe3 is also expressed in a distinct region of the embryonic brain, we have not observed abnormal development of the brain in Foxe3(-/-) animals.

Searching for biomarkers of developmental toxicity with microarrays: normal eye morphogenesis in rodent embryos

Gene expression arrays reveal the potential linkage of altered gene expression with specific adverse effects leading to disease phenotypes. But how closely do microarray data reflect early physiological or pharmacological measures that predict toxic event(s)? To explore this issue, we have undertaken experiments in early mouse embryos exposed to various teratogens during neurulation stages with the aim of correlating large-scale changes in gene expression across the critical period during exposure. This study reports some of the large-scale changes in gene expression that can be detected in the optic rudiment of the developing mouse and rat embryo across the window of development during which the eye is exceedingly sensitive to teratogen-induced micro-/anophthalmia. Microarray analysis was performed on RNA from the headfold or ocular region at the optic vesicle and optic cup stages when the ocular primordium is enriched for Pax-6, a master control gene for eye morphogenesis. Statistical selection of differentially regulated genes and various clustering techniques identified groups of genes in upward or downward trajectories in the normal optic primordium during early eye development in mouse and rat species. We identified 165 genes with significant differential expression during eye development, and a smaller subset of 58 genes that showed a tight correlation between mouse–rat development. Significantly over-represented functional categories included fatty acid metabolism (up-regulated) and glycolysis (down-regulated). From studies such as these that benchmark large-scale gene expression during normal embryonic development, we may be able to identify the panel of biomarkers that best correlate with species differences and the risks for developmental toxicity.

The Basic Helix-Loop-Helix Leucine Zipper Transcription Factor Mitf Is Conserved in Drosophila and Functions in Eye Development

The MITF protein is a member of the MYC family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors and is most closely related to the TFE3, TFEC, and TFEB proteins. In the mouse, MITF is required for the development of several different cell types, including the retinal pigment epithelial (RPE) cells of the eye. In Mitf mutant mice, the presumptive RPE cells hyperproliferate, abnormally express the retinal transcriptional regulator Pax6, and form an ectopic neural retina. Here we report the structure of the Mitf gene in Drosophila and demonstrate expression during embryonic development and in the eye-antennal imaginal disc. In vitro, transcriptional regulation by Drosophila Mitf, like its mouse counterpart, is modified by the Eyeless (Drosophila Pax6) transcription factor. In vivo, targeted expression of wild-type or dominant-negative Drosophila Mitf results in developmental abnormalities reminiscent of Mitf function in mouse eye development. Our results suggest that the Mitf gene is the original member of the Mitf-Tfe subfamily of bHLH-Zip proteins and that its developmental function is at least partially conserved between vertebrates and invertebrates. These findings further support the common origin of the vertebrate and invertebrate eyes.

Zebrafish rx3 and mab21l2 are required during eye morphogenesis

Two alleles of an eyeless mutant, chokh ( chk), were identified in ongoing zebrafish F 3 mutagenesis screens. Morphologically, chk mutants can be identified at 15 h post-fertilization by the failure of optic primordia to evaginate from the forebrain. The chk phenotype appears specific, as marker genes in the forebrain, midbrain, and pineal are expressed in normal temporal, spatial, and circadian patterns. Sequence analysis of the chk alleles revealed nonsense or missense mutations in the rx3 homeobox. Rx genes encode paired-type homeodomain transcription factors known to be key regulators of eye development in mouse, medaka, Xenopus, and zebrafish . To uncover novel Rx targets, we analyzed the expression of multiple eye development genes in chk. We find that expression of mab21l2, mab21l1 and rx2 are specifically absent in the eye field of chk embryos. Knockdown of Mab21l2 by antisense morpholino microinjections partially phenocopies the rx3 mutation, leading to microphthalmia, incomplete eye maturation, and dramatic increases in apoptotic eye progenitors. We propose that mab21l2 is an early downstream effector of rx3 and is critical for survival of eye progenitors.

Analysis of mouse eye development with chimeras and mosaics

Analysis of experimental mouse chimeras (chimaeras) and mosaics provides a means of investigating patterning and differentiation within the developing mammalian eye. Chimeric and mosaic mice carry two or more genetically distinct cell populations and extend the repertoire of analytical tools available to the geneticist. Here we review the impact these techniques have had on our understanding of eye organogenesis. Chimeras and mosaics are routinely used to investigate cell lineages, patterns of growth and gene function, and provide a means to clear analytical hurdles that otherwise limit standard genetic approaches. In particular, chimeras are used to investigate the roles of genes in tissues that do not develop in conventional mutant or knock-out mice, to test whether genes act cell autonomously or non-autonomously in different tissues and to dissect tissue-tissue interactions in less tractable, complex systems. Chimeras, in which cells of different genetic composition are mixed at a fine-scale cellular level, may provide qualitatively different data from mosaic mice with conditional knockouts. The uses of chimeras, Cre-loxP mosaics and in vitro tissue recombination for study of ocular organogenesis are compared. Wider use of mosaics and chimeras should provide further insights into eye development.

Systematic Analysis of E-, N- and P-cadherin Expression in Mouse Eye Development

Cadherins are a family of Ca2+-dependent cell adhesion molecules. Through their homophilic binding interactions, cadherins play important roles in tissue formation and maintenance during development. Here the authors compare the expression patterns of the three classical cadherins, E-, N- and P-cadherin, during mouse eye development from embryonic day 9.5 (E9.5) to adult. It was found that: (1) The expression patterns of N- and P-cadherin are mutually exclusive in most ocular tissues during development. N-cadherin mRNA is detected specifically in the lens placode during lens induction at E9.5, and is absent in the rest of the surface ectodermal tissues. In contrast, P-cadherin is expressed in the surface ectoderm but not in the lens vesicle. N-cadherin is expressed continuously in the lens pit, lens vesicle, and in the epithelial cells and newly differentiating fiber cells of the mature lens. P-cadherin is expressed in the epithelial cells of the cornea, eyelids and Harderian gland. Reciprocal expression patterns of N-and P-cadherins are also seen during retinal development. N-cadherin is initially expressed in both the inner and outer layers of the optic cup at E9.5. N-cadherin expression persists in the inner layer as it develops into neural retina, but is turned off in the outer layer where the cells differentiate into retinal pigment epithelial (RPE) cells and express P-cadherin. Reciprocal patterns of expression are also seen in the ciliary epithelium. N-cadherin is expressed in the inner layer and P-cadherin in the outer layer of the ciliary epithelium. (2) E- and P-cadherins are epithelial cadherins. Their expression patterns in the eye are not identical. Both cadherins are found in the epithelia of the cornea, eyelid and Harderian gland. In contrast, lens epithelial cells express E- but not P-cadherin, and RPE cells express P- but not E-cadherin. (3) In addition to its high expression in surface ectoderm-derived tissues, E-cadherin mRNA was also detected in some of the retinal ganglion neurons at postnatal day 14 (P14). E-cadherin expression in the neural retina has not been reported before. This study shows that cell fate determination in the eye occurs in conjunction with distinct changes in the patterns of cadherin gene expression.

The Arf tumor suppressor gene promotes hyaloid vascular regression during mouse eye development

A key tumor suppressor mechanism that is disrupted frequently in human cancer involves the ARF and p53 genes. In mouse fibroblasts, the Arf gene product responds to abnormal mitogenic signals to activate p53 and trigger either cell cycle arrest or apoptosis. Recent evidence indicates that Arf also has p53-independent functions that may contribute to its tumor suppressor activity. Using Arf(-/-) and p53(-/-) mice, we have discovered a p53-independent requirement for Arf in the developmental regression of the hyaloid vascular system (HVS) in the mouse eye. Arf is expressed in the vitreous of the eye and is induced before HVS regression in the first postnatal week. In the absence of Arf, failed HVS regression causes a pathological process that resembles persistent hyperplastic primary vitreous, a developmental human eye disease thought to have a genetic basis. These findings demonstrate an essential and unexpected role for Arf during mouse eye development, provide insights into the potential genetic basis for persistent hyperplastic primary vitreous, and indicate that Arf regulates vascular regression in a p53-independent manner. The latter finding raises the possibility that Arf may function as a tumor suppressor at least in part by regulating tumor angiogenesis.

Nestin expression during mouse eye and lens development

Nestin is an intermediate filament protein, which is expressed predominantly in the developing central nervous system and skeletal muscles. In situ hybridization revealed that mouse nestin mRNA is in the optic stalk at 9.0 days post coitus (dpc) and in the lens vesicle at 10.0 dpc. From 14.5 dpc onward, nestin transcripts appear in lens fibers and neuroretina. Immunohistochemistry showed that nestin protein appears in the optic stalk at 9.5 dpc and in the posterior lens epithelium at 10.5 dpc. By 12.5 dpc, it is found in the lens, neuroretina, and optic stalk as well as in developing extrinsic ocular muscle, and it localizes in lens epithelium, optic disc, and optic nerve from 14.5 dpc to postnatal day 1. In adult eye, nestin protein appears in the optic nerve.

Co-localization of FAM and AF-6, the mammalian homologues of Drosophila faf and canoe, in mouse eye development

The Drosophila fat facets and canoe genes regulate non-neural cell fate decisions during ommatidium formation. We have shown previously that the FAM (fat facets in mouse) de-ubiquitinating enzyme regulates the function of AF-6, (mammalian canoe homologue), in the MDCK epithelial cell line ( Taya et al., 1998. The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme. J. Cell Biol. 142, 1053–1062). We report here that the expression of the FAM and AF-6 proteins overlaps extensively in the mouse eye from embryogenesis to maturity, especially in the non-neural epithelia including the retinal pigment epithelium, subcapsular epithelium of the lens and corneal epithelium. Expression is not limited to the epithelia however, as FAM and AF-6 also co-localize during lens fibre development as well as in sub-populations of the neural retina.

Isolation of a Drosophila homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development.

Vertebrate and invertebrate eye development require the activity of several evolutionarily conserved genes. Among these the Pax-6 genes play a major role in the genetic control of eye development. Mutations in Pax-6 genes affect eye development in humans, mice, and Drosophila, and misexpression of Pax-6 genes in Drosophila can induce ectopic eyes. Here we report the identification of a paired-like homeobox gene, DRx, which is also conserved from flies to vertebrates. Highly conserved domains in the Drosophila protein are the octapeptide, the identical homeodomain, the carboxyl-terminal OAR domain, and a newly identified Rx domain. DRx is expressed in the embryo in the procephalic region and in the clypeolabrum from stage 8 on and later in the brain and the central nervous system. Compared with eyeless, the DRx expression in the embryo starts earlier, similar to the pattern in vertebrates, where Rx expression precedes Pax-6 expression. Because the vertebrate Rx genes have a function during brain and eye development, it was proposed that DRx has a similar function. The DRx expression pattern argues for a conserved function at least during brain development, but we could not detect any expression in the embryonic eye primordia or in the larval eye imaginal discs. Therefore DRx could be considered as a homolog of vertebrate Rx genes. The Rx genes might be involved in brain patterning processes and specify eye fields in different phyla.

Multiple functions for Pax6 in mouse eye and nasal development.

Mouse embryos, homozygous for the small eye (Sey) mutation die soon after birth with severe facial abnormalities that result from the failure of the eyes and nasal cavities to develop. Mutations in the Pax6 gene are responsible for the Sey phenotype. As a general disruption of eye and nasal development occurs in the homozygous Sey embryos, it is unclear, from the mutant phenotype alone, which tissues require functional Psx6. To examine the roles for Pax6 in eye and nasal development we produced chimeric mouse embryos composed of wild-type and Sey mutant cells. In these embryos we found that mutant cells were excluded from both the lens and nasal epithelium. Both of these tissues were smaller, and in some cases absent, in chimeras with high proportions of mutant cells. The morphology of the optic cup was also severely affected in these chimeras; mutant cells were excluded from the retinal pigmented epithelium and did not intermix with wild-type cells in other regions. The evidence shows that Pax6 has distinct roles in the nasal epithelium and the principal tissue components of the embryonic eye, acting directly and cell autonomously in the optic cup and lens. We suggest that Pax6 may promote cell surface changes in the optic cup and control the fate of the ectoderm from which the lens and nasal epithelia are derived.

OTIS and the communication industry: risks and benefits

Gene expression arrays reveal the potential linkage of altered gene expression with specific adverse effects leading to disease phenotypes. But how closely do microarray data reflect early physiological or pharmacological measures that predict toxic event(s)? To explore this issue, we have undertaken experiments in early mouse embryos exposed to various teratogens during neurulation stages with the aim of correlating large-scale changes in gene expression across the critical period during exposure. This study reports some of the large-scale changes in gene expression that can be detected in the optic rudiment of the developing mouse and rat embryo across the window of development during which the eye is exceedingly sensitive to teratogen-induced micro-/anophthalmia. Microarray analysis was performed on RNA from the headfold or ocular region at the optic vesicle and optic cup stages when the ocular primordium is enriched for Pax-6, a master control gene for eye morphogenesis. Statistical selection of differentially regulated genes and various clustering techniques identified groups of genes in upward or downward trajectories in the normal optic primordium during early eye development in mouse and rat species. We identified 165 genes with significant differential expression during eye development, and a smaller subset of 58 genes that showed a tight correlation between mouse–rat development. Significantly over-represented functional categories included fatty acid metabolism (up-regulated) and glycolysis (down-regulated). From studies such as these that benchmark large-scale gene expression during normal embryonic development, we may be able to identify the panel of biomarkers that best correlate with species differences and the risks for developmental toxicity.

Developmental patterns of two α1(IX) collagen mRNA isoforms in mouse

Northern blot hybridization, reverse-transcription polymerase chain reaction (RT-PCR), and RNase protection assays were used to examine the expression of two alpha 1(IX) collagen mRNA species (long and short form) in developing mouse tissues. Furthermore, in situ hybridization was used to identify cells expressing the Col9a1 gene during eye development. The results indicate that during embryonic development eye and heart preferentially express the short form; lung and cartilage express the long form; whereas liver expresses a very low level of long form alpha 1(IX) mRNA which can only be detected by RT-PCR. In situ hybridization demonstrated that at 10.5 day postcoitum (d.p.c.), the alpha 1(IX) collagen mRNAs were first expressed in optic cup (neural ectoderm) but not in lens vesicle (surface ectoderm). By 13.5 d.p.c., the cells that express the alpha 1(IX) mRNA progressively were concentrated toward the anterior part of the neural retina. By 16.5-18.5 d.p.c., the hybridization signals were found exclusively in the inner non-pigmented layer of the presumptive ciliary epithelium. As ciliary epithelial cells become well differentiated 3 weeks after birth, cells expressing the Col9a1 gene were limited to the junction between mature ciliary folds and the neural retina. No hybridization signal could be detected in ocular tissues of mouse older than 6 weeks. It is of interest to note that a hybridization signal was not detected in cornea at the various developmental stages examined, suggesting that mouse cornea does not significantly express alpha 1(IX) mRNA during embryonic development. This differs from that of chick cornea development. In summary, the expression of the Col9a1 gene shows a temporospatial pattern throughout mouse eye development.(ABSTRACT TRUNCATED AT 250 WORDS)