Prenatal development of the microphthalmic eye in the golden hamster

Prenatal development of the eye in a microphthalmic hamster strain (“anophthalmic white”) is compared with established normal developmental periods. The mutant eye primordium is first distinguished at an average of ten gestational days (Period 6) by an incompletely invaginated optic cup, uniformly pseudostratified outer neuroepithelial layer and widely separated margins of the optic fissure. The outer layer of the mutant cup subsequently becomes abnormally thickened, especially posteriorly and midventrally, and, except in a few eyes with localized imperfect fusion, the optic fissure is unfused at twelve days (Period 9), by which time fusion is normally complete. At 13 to 15 days (Periods 10–11) the fissure is unfused or irregularly fused in regions of variable location and extent. The occurrence of fissure fusion with concomitant loss of continuity between inner and outer epithelial layers is generally restricted to expanded anterior regions in 14–16 day (Periods 11–12) eyes. The presence of presumptive neural retina in the outer layer of the cup characterizes the mutant eye; and to varying degrees, in day 13–16 eyes, the presumptive neural retina (1) provides persistent continuity between the two cup layers, (2) forms both fused and unfused margins of the optic fissure, and (3) extends into an outer position of the optic cup. As early as 13 days (Period 10), nerve fibers are present in the outer layer of the cup, and by the last prenatal and first postnatal days (Period 12), ectopic nerve fiber bundles are widely distributed.     

The role of bone morphogenetic proteins in the differentiation of the ventral optic cup

The ventral region of the chick embryo optic cup undergoes a complex process of differentiation leading to the formation of four different structures: the neural retina, the retinal pigment epithelium (RPE), the optic disk/optic stalk, and the pecten oculi. Signaling molecules such as retinoic acid and sonic hedgehog have been implicated in the regulation of these phenomena. We have now investigated whether the bone morphogenetic proteins (BMPs) also regulate ventral optic cup development. Loss-of-function experiments were carried out in chick embryos in ovo, by intraocular overexpression of noggin, a protein that binds several BMPs and prevents their interactions with their cognate cell surface receptors. At optic vesicle stages of development, this treatment resulted in microphthalmia with concomitant disruption of the developing neural retina, RPE and lens. At optic cup stages, however, noggin overexpression caused colobomas, pecten agenesis, replacement of the ventral RPE by neuroepithelium-like tissue, and ectopic expression of optic stalk markers in the region of the ventral retina and RPE. This was frequently accompanied by abnormal growth of ganglion cell axons, which failed to enter the optic nerve. The data suggest that endogenous BMPs have significant effects on the development of ventral optic cup structures.     

Ectopic Pax2 expression in chick ventral optic cup phenocopies loss of Pax2 expression

Pax2 is essential for the development of the urogenital system, neural tube, otic vesicle, optic cup and optic tract [Dressler, G.R., Deutsch, U., et al., 1990. PAX2, a new murine paired-box-containing gene and its expression in the developing excretory system. Development 109 (4), 787-795; Nornes, H.O., Dressler, G.R., et al., 1990. Spatially and temporally restricted expression of Pax2 during murine neurogenesis. Development 109 (4), 797-809; Eccles, M.R., Wallis, L.J., et al., 1992. Expression of the PAX2 gene in human fetal kidney and Wilms’ tumor. Cell Growth Differ 3 (5), 279-289]. Within the visual system, a loss-of-function leads to lack of choroid fissure closure (known as a coloboma), a loss of optic nerve astrocytes, and anomalous axonal pathfinding at the optic chiasm [Favor, J., Sandulache, R., et al., 1996. The mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney. Proc. Natl. Acad. Sci. U. S. A. 93 (24), 13870-13875; Torres, M., Gomez-Pardo, E., et al., 1996. Pax2 contributes to inner ear patterning and optic nerve trajectory. Development 122 (11), 3381-3391]. This study is directed at determining the effects of ectopic Pax2 expression in the chick ventral optic cup past the normal developmental period when Pax2 is found. In ovo electroporation of Pax2 into the chick ventral optic cup results in the formation of colobomas, a condition typically associated with a loss of Pax2 expression. While the overexpression of Pax2 appears to phenocopy a loss of Pax2, the mechanism of the failure of choroid fissure closure is associated with a cell fate switch from ventral retina and retinal pigmented epithelium (RPE) to an astrocyte fate. Further, ectopic expression of Pax2 in RPE appears to have non-cell autonomous effects on adjacent RPE, creating an ectopic neural retina in place of the RPE.     

Development of dorsal-ventral polarity in the optic vesicle and its presumptive role in eye morphogenesis as shown by embryonic transplantation and in ovo explant culturing

Dorsal and ventral specification in the early optic vesicle appears to play a crucial role in the proper development of the eye. In the present study, we performed embryonic transplantation and organ culturing of the chick optic vesicle in order to investigate how the dorsal-ventral (D-V) polarity is established in the optic vesicle and what role this polarity plays in proper eye development. The left optic vesicle was cut and transplanted inversely in the right eye cavity of host chick embryos. This method ensured that the D-V polarity was reversed while the anteroposterior axis remained normal. The results showed that the location of the choroid fissure was altered from the normal (ventral) to ectopic positions as the embryonic stage of transplantation progressed from 6 to 18 somites. At the same time, the shape of the optic vesicle and the expression patterns of Pax2 and Tbx5, marker genes for ventral and dorsal regions of the optic vesicle, respectively, changed concomitantly in a similar way. The crucial period was between the 8- and 14-somite stages, and during this period the polarity seemed to be gradually determined. In ovo explant culturing of the optic vesicle showed that the D-V polarity and choroid fissure formation were already specified by the 10-somite stage. These results indicate that the D-V polarity of the optic vesicle is established gradually between 8- and 14-somite stages under the influence of signals derived from the midline portion of the forebrain. The presumptive signal(s) appeared to be transmitted from proximal to distal regions within the optic vesicle. A severe anomaly was observed in the development of optic vesicles reversely transplanted around the 10-somite stage: the optic cup formation was disturbed and subsequently the neural retina and pigment epithelium did not develop normally. We concluded that establishment of the D-V polarity in the optic vesicle plays an essential role in the patterning and differentiation of the neural retina and pigment epithelium.     

The temporal requirement for vitamin A in the developing eye: mechanism of action in optic fissure closure and new roles for the vitamin in regulating cell proliferation and adhesion in the embryonic retina

Mammalian eye development requires vitamin A (retinol, ROL). The role of vitamin A at specific times during eye development was studied in rat fetuses made vitamin A deficient (VAD) after embryonic day (E) 10.5 (late VAD). The optic fissure does not close in late VAD embryos, and severe folding and collapse of the retina is observed at E18.5. Pitx2, a gene required for normal optic fissure closure, is dramatically downregulated in the periocular mesenchyme in late VAD embryos, and dissolution of the basal lamina does not occur at the optic fissure margin. The addition of ROL to late VAD embryos by E12.5 restores Pitx2 expression, supports dissolution of the basal lamina, and prevents coloboma, whereas supplementation at E13.5 does not. Surprisingly, ROL given as late as E13.5 completely prevents folding of the retina despite the presence of an open fetal fissure, showing that coloboma and retinal folding represent distinct VAD-dependent defects. Retinal folding due to VAD is preceded by an overall reduction in the percentage of cyclin D1 positive cells in the developing retina, (initially resulting in retinal thinning), as well as a dramatic reduction in the cell adhesion-related molecules, N-cadherin and β-catenin. Reduction of retinal cell number combined with a loss of the normal cell-cell adhesion proteins may contribute to the collapse and folding of the retina that occurs in late VAD fetuses.     

Change in the developmental fate of the chick optic vesicle from the neural retina to the telencephalon

The forebrain develops into the telencephalon, diencephalon, and optic vesicle (OV). The OV further develops into the optic cup, the inner and outer layers of which develop into the neural retina and retinal pigmented epithelium (RPE), respectively. We studied the change in fate of the OV by using embryonic transplantation and explant culture methods. OVs excised from 10-somite stage chick embryos were freed from surrounding tissues (the surface ectoderm and mesenchyme) and were transplanted back to their original position in host embryos. Expression of neural retina-specific genes, such as Rax and Vsx2 (Chx10), was downregulated in the transplants. Instead, expression of the telencephalon-specific gene Emx1 emerged in the proximal region of the transplants, and in the distal part of the transplants close to the epidermis, expression of an RPE-specific gene Mitf was observed. Explant culture studies showed that when OVs were cultured alone, Rax was continuously expressed regardless of surrounding tissues (mesenchyme and epidermis). When OVs without surrounding tissues were cultured in close contact with the anterior forebrain, Rax expression became downregulated in the explants, and Emx1 expression became upregulated. These findings indicate that chick OVs at stage 10 are bi-potential with respect to their developmental fates, either for the neural retina or for the telencephalon, and that the surrounding tissues have a pivotal role in their actual fates. An in vitro tissue culture model suggests that under the influence of the anterior forebrain and/or its surrounding tissues, the OV changes its fate from the retina to the telencephalon.     

Early embryonic interaction of retinal pigment epithelium and mesenchymal tissue induces conversion of pigment epithelium to neural retinal fate in the silver mutation of the Japanese quail

The neural retina and retinal pigment epithelium (RPE) diverge from the optic vesicle during early embryonic development. They originate from different portions of the optic vesicle, the more distal part developing as the neural retina and the proximal part as RPE. As the distal part appears to make contact with the epidermis and the proximal part faces mesenchymal tissues, these two portions would encounter different environmental signals. In the present study, an attempt has been made to investigate the significance of interactions between the RPE and mesenchymal tissues that derive from neural crest cells, using a unique quail mutant silver (B/B) as the experimental model. The silver mutation is considered to affect neural crest-derived tissues, including the epidermal melanocytes. The homozygotes of the silver mutation have abnormal eyes, with double neural retinal layers, as a result of aberrant differentation of RPE to form a new neural retina. Retinal pigment epithelium was removed from early embryonic eyes (before the process began) and cultured to see whether it expressed any phenotype characteristic of neural retinal cells. When RPE of the B/B mutant was cultured with surrounding mesenchymal tissue, neural retinal cells were differentiated that expressed markers of amacrine, cone or rod cells. When isolated RPE of the B/B mutant was cultured alone, it acquired pigmentation and did not show any property characteristic of neural retinal cells. The RPE of wild type quail always differentiated to pigment epithelial cells. In the presence of either acidic fibroblast growth factor (aFGF) or basic FGF (bFGF), the RPE of the B/B mutant differentiated to neural retinal cells in the absence of mesenchymal tissue, but the RPE of wild type embryos only did so in the presence of 10–40 times as much aFGF or bFGF. These observations indicate that genes responsible for the B/B mutation are expressed in the RPE as well as in those cells that have a role in the differentiation of neural crest cells. They further suggest that development of the neural retina and RPE is regulated by some soluble factor(s) that is derived from or localized in the surrounding embryonic mesenchyme and other ocular tissues, and that FGF may be among possible candidates.     

The retinal pigment epithelium of the eye regulates the development of scleral cartilage

The majority of vertebrate species have a layer of hyaline cartilage within the fibrous sclera giving an extra degree of support to the eyeball. In chicks, this is seen as a cuplike structure throughout the scleral layer. However, the mechanisms that control the development of scleral cartilage are largely unknown.Here we have studied the phases of scleral cartilage development and characterised expression profiles of genes activated during the cartilage differentiation programme. CART1 and SOX9, the earliest markers of pre-committed cartilage, are expressed in the mesenchyme surrounding the optic cup. Later AGGRECAN, a matrix protein expressed during chondrocyte differentiation, is also expressed. The expression of these genes is lost following early removal of the optic cup, suggesting a role for this tissue in inducing scleral cartilage. By grafting young retinal pigment epithelium (RPE) and retina into cranial mesenchyme in vivo, it was found that RPE alone has the ability to induce cartilage formation.There are some exceptions within the vertebrates where scleral cartilage is not present; one such example is the placental mammals. However, we found that the cartilage differentiation pathway is initiated in mice as seen by the expression of Cart1 and Sox9, but expression of the later cartilage marker Aggrecan is weak. Furthermore, cartilage forms in mouse peri-ocular mesenchyme micromass culture. This suggests that the process halts in vivo before full differentiation into cartilage, but that murine scleral mesenchyme has retained the potential to make cartilage in vitro.RA, Wnts and Bmps have been linked to the cartilage development process and are expressed within the developing RPE. We find that RA may have a role in early scleral cartilage development but is not likely to be the main factor involved.These data reveal the course of scleral cartilage formation and highlight the key role that the optic cup plays in this process. The driving element within the optic cup is almost certainly the retinal pigmented epithelium.     

Developmental changes in the subcellular localization of R-cadherin in chick retinal pigment epithelium

 It has been reported that in the chick embryonic retina, N-cadherin first appears at the very early stages and is subsequently substituted by R-cadherin at the middle to late stages of development. To examine the role of R-cadherin in the morphogenesis of chick retinal pigment epithelium (RPE), the distribution of this adhesion molecule was studied by immunofluorescence cytochemistry and immunoelectron microscopy from embryonic day (E) 6 to hatching. R-cadherin immunoreactivity was detected at E6, and was strongest at E12–13. During these stages, R-cadherin was expressed uniformly on the lateral plasma membranes of RPE cells in contact with each other. Thereafter, R-cadherin immunoreactivity was markedly decreased, with intense immunoreactivity restricted to zonulae adherentes in latero-apical regions at E16. R-cadherin immunoreactivity was no longer detectable in the newly hatched chick RPE, even though morphologically well developed zonulae adherentes were present in latero-apical regions. No immunoreactivity was detected on the apical side facing the neural retina or on the basal side facing the basal lamina at any stage of development. These findings indicate that R-cadherin plays an important role as a major cadherin subtype in the morphogenesis of chick embryo RPE, and is involved initially in non-specific cell-cell adhesions, and subsequently in the formation and maintenance of developing zonulae adherentes. Accepted: 11 April 1997     

Establishment of the scleral cartilage in the chick.

This study was undertaken to investigate the establishment of the scleral cartilage in the chick embryo. Johnston et al. (1974) has demonstrated that most of the cells of the scleral cartilage originate in the cranial neural crest. By means of a series of chorioallantoic grafts of pigmented retina, and its adherent periocular mesenchyme from stage 11 to 25, the present experiments show that the cranial neural crest cells arrive at the eye in sufficient numbers to form cartilage by stage 14. Pigmented retina, denuded of mesenchyme, from stage 16 embryos implanted into the head of stage 13 embryos induces cartilage formation in head mesenchyme. However, neither pigmented retina nor spinal cord could induce cartilage formation in chorioallantoic mesenchyme. Combination grafts of cranial neural crest and presumptive optic vesicle developed neural tissue, pigmented retina, and in some cases sclera-like cartilage. Thus, periorbital mesenchyme, derived largely from cranial neural crest, at about stage 14 develops the scleral cartilage in response to induction by the pigmented retina.     

Electron microscopic observations on the retinal pigment epithelium and choroid in the domestic pig (Sus scrofa)

Summary The morphology of the retinal pigment epithelium (RPE) and adjacent choroid has been investigated by electron microscopy in the domestic pig. The RPE consists of a single layer of cells which are columnar posteriorly but become cuboidal and even squamous moving peripherally in the fundus. The cells of the RPE layer regardless of location display basal (scleral) infoldings and apical (vitreal) processes and are joined laterally by junctional complexes. Throughout the retina the epithelial cells are rich in smooth endoplasmic reticulum and mitochondria but less so in rough endoplasmic reticulum and polysomes. The epithelial nucleus is vesicular and basally located. In the superior fundus an area of the RPE is very lightly pigmented and richer in lysosomes than is this layer in the inferior and peripheral fundus. The choroid overlying this area is also lightly pigmented and contains much collagen in a lamellar arrangement. This region may represent a vestigial tapetum fibrosum. Bruch's membrane is slightly thicker posteriorly but is everywhere seen to have a pentalaminate substructure. The choriocapillaris is a single layer of large capillaries which show numerous fenestrations facing the RPE. In the superior fundus the choriocapillaris is also highly fenestrated facing the choroid.     

Induction of retinal regeneration in vivo by growth factors

We have previously reported that basic fibroblast growth factor (bFGF) can induce retinal regeneration in the stage 22-24 chicken embryo. The present study was undertaken to identify the cellular source of the regenerate and to determine whether other growth factors also elicit regeneration in this animal model. Polymer implants containing bFGF were inserted into eyes of chicken embryos immediately after extirpation of the neural retina. The retinal pigment epithelium (RPE) was left intact. Evaluation by light microscopy revealed that in bFGF-treated eyes the new neural retina arose by transdifferentiation of the entire RPE layer. Differentiation of the new neural retina occurred in a sequence similar to that of normal development but proceeded in a reverse (vitread) direction. All retinal laminae had differentiated by Day 15. However, the regenerate displayed reversed polarity, with photoreceptors closest to the lens. The RPE, pecten, and optic nerve were absent. Focal areas of degeneration in the retinal regenerate became evident for the first time on Day 10. Retinal regeneration was also observed after treatment with higher doses of acidic fibroblast growth factor, but not with nerve growth factor-beta, transforming growth factor-beta 1, insulin, or insulin-like growth factors I or II. These results raise the possibility that FGFs may play a role in retinal differentiation during development.     

A complex choreography of cell movements shapes the vertebrate eye

Optic cup morphogenesis (OCM) generates the basic structure of the vertebrate eye. Although it is commonly depicted as a series of epithelial sheet folding events, this does not represent an empirically supported model. Here, we combine four-dimensional imaging with custom cell tracking software and photoactivatable fluorophore labeling to determine the cellular dynamics underlying OCM in zebrafish. Although cell division contributes to growth, we find it dispensable for eye formation. OCM depends instead on a complex set of cell movements coordinated between the prospective neural retina, retinal pigmented epithelium (RPE) and lens. Optic vesicle evagination persists for longer than expected; cells move in a pinwheel pattern during optic vesicle elongation and retinal precursors involute around the rim of the invaginating optic cup. We identify unanticipated movements, particularly of central and peripheral retina, RPE and lens. From cell tracking data, we generate retina, RPE and lens subdomain fate maps, which reveal novel adjacencies that might determine corresponding developmental signaling events. Finally, we find that similar movements also occur during chick eye morphogenesis, suggesting that the underlying choreography is conserved among vertebrates.     

Complicated colobomatous microphthalmia in the microphthalmic (mi/mi) mouse

A study of the development of the eye in the cinnamon mouse, homozygous for the gene for microphthalmia (mi), has shown that the microphthalmia is due to failure of secondary vitreous formation associated with a coloboma. The retina is dystrophic but there is a residual population of large ganglion cells and the optic nerve also contains ganglion cells. All these ganglion cells have cytoplasm similar to the retinal ganglion cells in the normal controls. It is postulated that they communicate with axons in the optic nerve. In addition, the outer epithelial layer of the eye cup, which normally becomes pigmented, forms retinal tissue in the homozygous mouse and this is also true of the dorsal part of the eyestalk near the eye.     

A newly established mutant strain with mild-type ocular coloboma (retinochoroidal coloboma without microphthalmia) in albino mice

BACKGROUND: A complicated malformation of the fundus accompanied by typical ocular coloboma was detected in albino fatty liver Shionogi (FLS) mice. We elucidated a new type of 3-dimensional anomalous structure inside the eye in this mouse strain. METHODS: The fundi of FLS mice aged 1, 3, 5, and 20 weeks were observed intensively, both macroscopically and by light microscopy. For the prenatal study, coronal serial sections of eyes of FLS embryos were examined by light microscopy on gestation day (GD) 15.0. RESULTS: The frequency of ocular coloboma was almost 70% in FLS mice, and the inheritance mode of this anomaly is suggested to be autosomal recessive with incomplete penetrance. Stereoscopic observation and light microscopy revealed that the mice had characteristic fundus features at any age during the postnatal period. Following ectopic ciliary epithelia, the surface of the retina protruded like a roof, and on the opposite side of the "roof," a translucent membrane without retinal tissue and choroidal tissue was also consistently detected in the inferior part of the fundus. On GD 15.0, the inner layer and the outer layer were not normally fused at the optic fissure, where a part of the outer layer was absent and the irregular fold of the inner layer was conspicuous in the colobomatous eye of the FLS embryo. CONCLUSIONS: The characteristics of the ocular coloboma in FLS mice are thought to be similar to a mild-type malformation in humans. These ocular defects seem to be situated along the failed fetal optic fissure.     

Patterning the optic neuroepithelium by FGF signaling and Ras activation.

During vertebrate embryogenesis, the neuroectoderm differentiates into neural tissues and also into non-neural tissues such as the choroid plexus in the brain and the retinal pigment epithelium in the eye. The molecular mechanisms that pattern neural and non-neural tissues within the neuroectoderm remain unknown. We report that FGF9 is normally expressed in the distal region of the optic vesicle that is destined to become the neural retina, suggesting a role in neural patterning in the optic neuroepithelium. Ectopic expression of FGF9 in the proximal region of the optic vesicle extends neural differentiation into the presumptive retinal pigment epithelium, resulting in a duplicate neural retina in transgenic mice. Ectopic expression of constitutively active Ras is also sufficient to convert the retinal pigment epithelium to neural retina, suggesting that Ras-mediated signaling may be involved in neural differentiation in the immature optic vesicle. The original and the duplicate neural retinae differentiate and laminate with mirror-image polarity in the absence of an RPE, suggesting that the program of neuronal differentiation in the retina is autonomously regulated. In mouse embryos lacking FGF9, the retinal pigment epithelium extends into the presumptive neural retina, indicating a role of FGF9 in defining the boundary of the neural retina.