Early and late changes in Pax6 expression accompany eye degeneration during cavefish development

We have compared Pax6 expression during embryonic development in the eyed surface form (surface fish) and several different eyeless cave forms (cavefish) of the teleost Astyanax mexicanus. Despite lacking functional eyes as adults, cavefish embryos form small optic primordia, which later arrest in development and show various degrees of eye degeneration. The pattern of Pax6 mRNA expression was modified early and late during cavefish development. In early surface fish embryos, two bilateral Pax6 expression domains are present in the anterior neural plate, which extend across the midline and fuse to form the forebrain and optic primordia. In cavefish embryos, these Pax6 domains are diminished in size and remain separated, resulting in an anterior gap in Pax6 expression and presumably the formation of smaller optic primordia. The anterior gap in Pax6 expression was confirmed by double staining for Pax6 and distalless-3 mRNA, which marks the anterior margin of the neural plate and is unaltered in cavefish. Similar anterior gaps in Pax6 expression occurred in independently derived cavefish populations, suggesting that they are important in eye degeneration. Later during surface fish development, Pax6 protein is expressed in the cornea, lens, and ganglion and amacrine cells of the neural retina. Pax6 expression was gradually reduced during cavefish lens development, concomitant with lens arrest and degeneration, and was absent in the corneal epithelium, which does not differentiate in cavefish. In contrast, Pax6 expression in the retinal ganglion and amarcine cells is unmodified in cavefish, despite retarded retinal development. The results suggest that changes in Pax6 expression are involved in the evolution of cavefish eye degeneration.     

Histochemical analysis of extracellular matrix material during embryonic mouse lens morphogenesis in an aphakic strain of mice

Extracellular matrix material (ECM) present during early lens morphogenesis was analyzed histochemically in normal CFW mice and mutant strain aphakia by the Alcian blue 8GX, pH 2.5, Alcian blue 8GX, pH 2.5/periodic acid-Schiff combined, high-iron diamine, and van Gieson methods. At lens placode formation, the optic vesicle basal lamina in both strains was higher in sulfated glycosaminoglycan content than was the ectodermal basal lamina. In the aphakia strain, ECM components were observed intercellularly in the presumptive neural retina and lens rudiment of some specimens. This observation was peculiar to the aphakia strain. At the lens cup stage (10.5 days), the interface ECM became less uniformly dense in the CFW strain, resulting in the formation of a fibrillar structure in the widening interspace area. In contrast, the interface ECM in the mutant strain stained solidly and continuously for acidic materials, particularly sulfated glycosaminoglycans, for a full 2 days longer than in the normal strain. The optic cup and lens rudiment remained closely apposed and intercellular ECM components were observed in these tissues in most mutant specimens throughout these stages. The exact mechanism resulting in these intercellular deposits is unknown, although it is possible that they are either pulled along on the cell surface away from the interface ECM during cell shape changes related to the cell cycle or that they are secreted abnormally due to some disturbed cellular polarity. It is unclear at this time if these abnormalities of the ECM in the aphakia strain play a role in the pathogenesis of the multiple eye anomalies, or if they are a secondary effect of the gene mutation.     

Regressive evolution: ontogeny and genetics of cavefish eye rudimentation

Two hypotheses exist to explain ontogenetic eye reduction in Astyanax cave fish: first, after lens induction by the primordial eye cup, the lens plays the role of a central regulator of eye and retina regression or, second, the retina itself is an independent unit of eye development. A comparative study of five blind cave fish populations and their surface sister form was performed to investigate the differences of ontogenetic eye regression between the cave populations during different stages of development. The study revealed that, in addition to the initial formation of smaller primordia, eye regression is also caused during later ontogeny by different relative growth and specific histological characteristics. Whereas the cave fish lens never properly differentiates, the regressive process of the retina is transitorily interrupted by ongoing differentiation. In the newly-discovered Molino cave population, even visual cells with well-organized outer segments develop, which are secondarily reduced at a later ontogenetic stage. This result shows that the retina and lens are independent developmental units within the eye ball. Presumably, the genetic systems responsible for both show independent inheritance, which is also corroborated by hybrids of F ₂-crosses between the cave and surface fish, in which lens and retina development do not correlate. During ontogeny, the eye size differs between the cave populations. In Pachón cave fish, the relatively large eye size correlates with an ancient introgression from a surface population, which may have delayed eye regression.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 287–296.     

Tissue interactions and lens-forming competence in the outer cornea of larval Xenopus laevis

After lentectomy through the pupillary hole, the outer cornea of larval Xenopus laevis can undergo transdifferentiation to regenerate a new lens. This process is elicited by inductive factor(s) produced by the neural retina and accumulated into the vitreous chamber. During embryogenesis, the outer cornea develops from the outer layer of the presumptive lens ectoderm (PLE) under the influence of the eye cup and the lens. In this study, we investigated whether the capacity of the outer cornea to regenerate a lens is the result of early inductive signals causing lens-forming bias and lens specification of the PLE, or late inductive signals causing cornea formation or both signals. Fragments of larval epidermis or cornea developed from ectoderm that had undergone only one kind of inductive signals, or both kinds of signals, or none of them, were implanted into the vitreous chamber of host larvae. The regeneration potential and the lens-forming transformations of the implants were tested using an antisense probe for pax6 as an earlier marker of lens formation and a monoclonal antibody anti-lens as a definitive indicator of lens cell differentiation. Results demonstrated that the capacity of the larval outer cornea to regenerate a lens is the result of both early and late inductive signals and that either early inductive signals alone or late inductive signals alone can elicit this capacity.     

Otx genes are required for tissue specification in the developing eye

Patterning of the vertebrate eye appears to be controlled by the mutual regulation and the progressive restriction of the expression domains of a number of genes initially co-expressed within the eye anlage. Previous data suggest that both Otx1 and Otx2 might contribute to the establishment of the different eye territories. Here, we have analysed the ocular phenotype of mice carrying different functional copies of Otx1 and Otx2 and we show that these genes are required in a dose-dependent manner for the normal development of the eye. Thus, all Otx1(-/-); Otx2(+/-) and 30% of Otx1(+/-); Otx2(+/-) genotypes presented consistent and profound ocular malformation, including lens, pigment epithelium, neural retina and optic stalk defects. During embryonic development, optic vesicle infolding was severely altered and the expression of pigment epithelium-specific genes, such as Mitf or tyrosinase, was lost. Lack of pigment epithelium specification was associated with an expansion of the prospective neural retina and optic stalk territories, as determined by the expression of Pax6, Six3 and Pax2. Later in development the presumptive pigment epithelium region acquired features of mature neural retina, including the generation of Islet1-positive neurones. Furthermore, in Otx1(-/-); Otx2(+/-) mice neural retina cell proliferation, cell differentiation and apoptotic cell death were also severely affected. Based on these findings we propose a model in which Otx gene products are required for the determination and differentiation of the pigment epithelium, co-operating with other eye patterning genes in the determination of the specialised tissues that will constitute the mature vertebrate eye.     

Noggin producing, MyoD-positive cells are crucial for eye development

A subpopulation of cells expresses MyoD mRNA and the cell surface G8 antigen in the epiblast prior to the onset of gastrulation. When an antibody to the G8 antigen was applied to the epiblast, labeled cells were later found in the ocular primordia and muscle and non-muscle forming tissues of the eyes. In the lens, retina and periocular mesenchyme, G8-positive cells synthesized MyoD mRNA and the bone morphogenetic protein inhibitor Noggin. MyoD expressing cells were ablated in the epiblast by labeling them with the G8 MAb and lysing them with complement. Their ablation in the epiblast resulted in eye defects, including anopthalmia, micropthalmia, altered pigmentation and malformations of the lens and/or retina. The right eye was more severely affected than the left eye. The asymmetry of the eye defects in ablated embryos correlated with differences in the number of residual Noggin producing, MyoD-positive cells in ocular tissues. Exogenously supplied Noggin compensated for the ablated epiblast cells. This study demonstrates that MyoD expressing cells serve as a Noggin delivery system to regulate the morphogenesis of the lens and optic cup.     

Expression of Fibroblast growth factor 19 (Fgf19) during chicken embryogenesis and eye development, compared with Fgf15 expression in the mouse

The normal development of eyes relies on proper signaling through Fibroblast growth factor (FGF) receptors, but the source and identity of cognate ligands have remained largely unknown. We have found that Fgf19 is expressed in the developing chicken retina. In situ hybridization discloses dynamic expression patterns for Fgf19 in the optic vesicle, lens primordia and retinal horizontal cells. Overall expression pattern of Fgf19 during chicken embryogenesis was also examined: Fgf19 is expressed in the regions associated with cranial placodes induction, boundary regions of rhombomeres, somites, specific groups of neural cells in midbrain, hindbrain, and those derived from epibranchial placodes, and the apical ectodermal ridge of limb buds. Expression pattern of the Fgf19-orthologous gene Fgf15 was further examined in the mouse developing eye. Fgf15 is expressed in the optic vesicle, a subset of progenitor cells of neural retina, and emerging ganglion and amacrine cells during retinogenesis.     

Transplantation of the chick early eye primordium into a lensectomized optic cup of a host embryo

Eye primordia of young chick embryos (stage XII) were transplanted into lensectomized optic cups of older embryos (stage XVII) to analyze the influence of the host retina on the degree of morphological differentiation attained by the donor lens. Embryos were sacrificed 24-96 h later. The donor lens primordium showed a differentiation more in correlation with the host eye cup (stage XXIII) after 24-96 h of incubation.     

Requirement for Mab21l2 during development of murine retina and ventral body wall

The mab-21 gene was first identified because of its requirement for ray identity specification in Caenorhabditis elegans. It is now known to constitute a family of genes that are highly conserved from vertebrates to invertebrates, and two homologues Mab21l1 and Mab21l2 have been identified in many species. Here we describe the generation of Mab21l2-deficient mice, which have defects in eye and body wall formation. The mutant mouse eye has a rudimentary retina, as a result of insufficient invagination of the optic vesicle due to deficient proliferation, causing the absence of lens. The defects in optic vesicle development correlate with reduced expression of Chx10, which is also required for retina development; Rx, Lhx2, and Pax6 expression is not significantly affected. We conclude that Mab21l2 expression is essential for optic vesicle growth and formation of the optic cup, its absence causing reduced expression of Chx10. Mutant mice also display abnormal extrusion of abdominal organs, defects in ventral body wall formation, resulting in death in utero at mid-gestational stage. Our results reveal that Mab21l2 plays crucial roles in retina and in ventral body wall formation.     

Role of apoptosis and mitosis during human eye development

The spatial and temporal distribution as well as ultrastructural and biochemical characteristics of apoptotic and mitotic cells during human eye development were investigated in 14 human conceptuses of 4-9 postovulatory weeks, using electron and light microscopy. In the 5th developmental week, apoptotic and mitotic cells were found in the neuroepithelium of the optic cup and stalk, being the most numerous at the borderline between the two layers of the optic cup, and at the place of transition of the optic cup into stalk. They were also found at the region of detachment of the lens pit from the surface ectoderm. In the later developmental stages (the 6th-the 9th week), apoptotic and mitotic cells were observed in the neural retina and the anterior lens epithelium. Throughout all stages examined, mitotic cells were found exclusively adjacent to the lumen either of the intraretinal space or the optic stalk ventricle, or were restricted to the superficial epithelial layer of the lens primordium. Unlike mitotic cells, apoptotic cells occurred throughout the whole width both of the neuroepithelium and the surface epithelium. Ultrastructurally, apoptotic cells were characterised by round- or crescent-shaped condensations of chromatin near the nuclear membrane, while in the more advanced stages of apoptosis by apoptotic bodies. The distribution of caspase-3-positive cells coincided with the location of apoptotic cells described by morphological techniques indicating that the caspase-3-dependent apoptotic pathway operates during the all stages of human eye development. The location of cells positive for anti-apoptotic bcl-2 protein was in accordance with the regions of eye with high mitotic activity, confirming the role of bcl-2 in protecting cells from apoptosis. In the earliest stage of eye development, apoptosis and mitosis might be associated with the sculpturing of the walls of optic cup and stalk, while high mitotic activity along the intraretinal space and optic stalk ventricle indicates its role in the gradual luminal closure. These processes also participate in the detachment of the lens pit epithelium from the surface ectoderm as well as in further closure of the lens vesicle. Later on, both processes seem to be involved in the neural retina differentiation, lens morphogenesis and secondary lens fibre differentiation.     

Effects of exogenous FGF-1 treatment on regeneration of the lens and the neural retina in the newt, Notophthalmus viridescens

Experiments were designed to compare the effects of recombinant newt fibroblast growth factor-1 (rnFGF-1) and recombinant human glial growth factor (rhGGF) on lens and retina regeneration in the eyes of adult newts. Both eyes were retinectomized and lentectomized. Beginning 3 days after the operation, one eye was given either 0.1 microg of rnFGF-1 or 0.1 microg of rhGGF in 1 microl of phosphate-buffered saline (PBS) per injection, three per week. Contralateral operated eyes served as controls and were treated with PBS alone or were not injected. In eyes that were not injected, injected with PBS alone, or with PBS containing rhGGF, regeneration of both the retina and the lens proceeded normally as described in the literature. In these control eyes, the entire retinal pigmented epithelium (RPE) depigmented/dedifferentiated and a retina rudiment formed from which a new retina regenerated by the end of the experiment at day 41 post-operation. Likewise, only a small area of dorsal iris depigmented/dedifferentiated and formed a lens vesicle from which a lens subsequently regenerated. The vitreous remained relatively free of loose cells.In eyes given rnFGF-1, the RPE depigmented/dedifferentiated and formed what appeared to be a retina rudiment but a new retina did not regenerate. Instead, vesicles were seen associated with the retina rudiment. In eyes given rnFGF-1, both the dorsal iris and ventral iris depigmented/dedifferentiated and lens regeneration occurred but the new lenses had abnormal fiber cells and the lens epithelium was very thin or absent. In addition, ectopic lenses usually regenerated in rnFGF-1-treated eyes. An abundance of loose cells were present in the vitreous of rnFGF-1-treated eyes associated largely with the RPE and the dorsal and ventral irises.The results are consistent with the view that the timely expression of FGFs is involved in the depigmentation/dedifferentiation of the RPE and dorsal iris and is necessary for proper regeneration of the lens and neural retina. Continued presence of FGF results in continued and excessive dedifferentiation, resulting in the lack of retina regeneration and abnormal lens regeneration.     

Biochemical and immunological studies of lentoid formation in cultures of embryonic chick neural retina and day-old chick lens epithelium

During long-term cell culture of 8-day embryonic chick neural retina, lentoid bodies containing lens crystallins are developed. Although very low levels of crystallin can be detected in the embryonic neural retina, gross synthesis of each major crystallin class (α, anodal β, cathodal β, and δ) begins only after 12–16 days in culture. This occurs at least 10 days before lentoid bodies can be distinguished by eye. The concentration of each crystallin class was determined during lentoid development in cultures of both neural retina and lens epithelium. The proportions of crystallins in lentoid-containing cultures do not resemble those of embryonic lens fibres. Comparisons between two chick strains (N and Hy-1) differing in their growth rates revealed several differences in the crystallin compositions of lentoid bodies. These differences imply independent quantitative regulation for most or all of the crystallins.     

Genetical and histological studies on a mouse displaying microphthalmia and cataract

A male mouse displaying bilateral microphthalmia and cataract was found among the offspring of pregnant Slc: ICR mouse treated intraperitoneally with 10 mg/kg methylnitrosourea on gestational day 4. This mutant has been maintained by brother-sister mating. By the mating test with normal Slc: ICR mice, this character was revealed to be inherited by an autosomal single recessive gene. Linkage test with the brown locus showed that this mutant gene is linked with the B gene and mapped on chromosome 4. The histological study of the eyes of adult mutant mice revealed various abnormalities all over the eyes, especially in the lens and neural retina. Embryologically, the mutant mice showed persistent connection between the lens vesicle and the surface ectoderm by a cellular stalk, and also the formation of retinal infolding, in the early stages of eye development. Both were considered to be responsible for the consequent abnormal development and degradation of the lens. These results suggest that the mutant mouse we found may be an allele of the dysgenetic lens (dyl) reported by Sanyal and Hawkins.     

Free Radical Enhancer Xenobiotic is an Inducer of Cataract in Rabbit

Free radical enhancers, diquat, paraquat, plumbagin and juglone were used to study the oxy radical-induced damage to the rabbit lens in vitro and in vivo. Each compound caused a 6–8 fold increase in malondialdehyde (MDA) and a 30–55% decrease in reduced glutathione of the lens in vim. These peroxidative and oxidative changes were potentiated in the presence of 100% 0., abolished by N, and prevented by desferal-Mn (III) (DF-Mn) or liposomal superoxide dismutase (LSOD) indicating the involvement of O₂^?.

Diquat injected intravitreally as a single dose (300nmole in 30μl of isotonic saline) in the right eye of a 5-wk-old Dutch belted rabbit, induced early cataract after 24–72h. The lens of the contralateral control eye injected with isotonic saline had no change. In the right eye, O₂,^? and OH -productions were significantly (P < 0.01) higher; O₂-, was about 16 fold higher in the aqueous humor and vitreous humor, and 5 fold in the lens and retina, and OH. was 35 fold higher in the aqueous humor, 2 fold in vitreous humor and 5 fold in the lens and retina as compared to the respective tissues of the control eye. Enhanced lipid peroxidation in the lens was apparent from the higher levels of MDA and formation of aminophospholipid-MDA Schiff-base conjugates.

We propose that cyclic oxidation-reduction of xenobiotics coupled to the endogenous redox systems in the eye, could generate oxy radicals in excessive amounts, triggering cataractogcnesis.     

The stability of the lens-specific Maf protein is regulated by fibroblast growth factor (FGF)/ERK signaling in lens fiber differentiation

Fibroblast growth factor (FGF) signaling is necessary for both proliferation and differentiation of lens cells. However, the molecular mechanisms by which FGFs exert their effects on the lens remain poorly understood. In this study, we show that FGF-2 repressed the expression of lens-specific genes at the proliferative phase in primary cultured lens cells. Using transfected cells, we also found that the activity of L-Maf, a lens differentiation factor, is repressed by FGF/ERK signaling. L-Maf is shown to be phosphorylated by ERK, and introduction of mutations into the ERK target sites on L-Maf promotes its stabilization. The stable L-Maf mutant protein promotes the differentiation of lens cells from neural retina cells. Taken together, these results indicate that FGF/ERK signaling negatively regulates the function of L-Maf in proliferative lens cells and that stabilization of the L-Maf protein is important for lens fiber differentiation.     

The roles of Pax6 in the cornea,retina, and olfactory epithelium of the developing mouse embryo

The roles of Pax6 were investigated in the murine eye and the olfactory epithelium by analysing gene expression and distribution of Pax6(-/-) cells in Pax6(+/+) <--> Pax6(-/-) chimeras. It was found that between embryonic days E10.5 and E16.5 Pax6 is autonomously required for cells to contribute fully not only to the corneal epithelium, where Pax6 is expressed at high levels, but also to the to the corneal stroma and endothelium, where the protein is detected at very low levels. Pax6(-/-) cells contributed only poorly to the neural retina, forming small clumps of cells that were normally restricted to the ganglion cell layer at E16.5. Pax6(-/-) cells in the retinal pigment epithelium could express Trp2, a component of the pigmentation pathway, at E14.5 and a small number went on to differentiate and produce pigment at E16.5. The segregation and near-exclusion of mutant cells from the nasal epithelium mirrored the behaviour of mutant cells in other developmental contexts, particularly the lens, suggesting that common primary defects may be responsible for diverse Pax6-related phenotypes.