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We describe that Sox11, a member of the group C of the Sox transcription factor family, is critically required during the morphogenetic processes of early eye development, and that lack of Sox11 results in ocular anterior segment dysgenesis (ASD). Sox11 -deficient mice show a persistent lens stalk, a delay in lens formation, and the phenotypes of Peters' anomaly and microphthalmia at birth. In addition, the optic fissure does not close in the anterior halves of the eyes resulting in anterior coloboma. The delay in lens formation is associated with a reduced mitotic activity in the lens placode during its invagination into the optic cup. No changes in Pax6 expression are observed in the developing eyes of Sox11 −/− mice, whereas the expression of Sox11 is reduced in optic cup, optic vesicle and lens placode of Pax6 + / − embryos and in the optic vesicle of Pax6 −/− mice. Transfection experiments show an increase in Sox11 expression when higher doses of Pax6 are present. Considerably smaller amounts of BMP7 are expressed in lens and optic cup of Sox11 −/− mice as compared to their wild-type littermates. We conclude that Sox11 is required during separation of the lens vesicle from the surface ectoderm and the closure of the anterior optic fissure. The expression of Sox11 in early eye development is under control of Pax6, and changes in BMP7-signalling appear to be involved in the effects of Sox11 on anterior eye development. Our findings suggest that SOX11 might similarly be involved in the pathogenesis of ASD in human patients.

The similarity between the cephalopod lens and the teleost (vertebrate) lens can be considered an optical example of convergent evolution. However, the embryology and ultrastructure of the cephalopod lens appear to be different from that of vertebrates, and perhaps unique to the animal kingdom. Using light and scanning electron microscopy, the morphogenesis of the squid (Loligo opalescens) lens is characterized. Results indicate that the posterior lens primordium appears first during development and is derived from cellular processes which extend from a middle group, group 2, of lentigenic (ectodermal) cells. The processes extend from the basal aspect of the lentigenic cells, project down into the optic vesicle during early stages of development, and fuse to form the posterior lens primordium. During later stages, the processes extend from surrounding lentigenic cells and are applied to the stalk of the lens, where they form bud-shaped protrusions. Once applied to the lens, the processes form lens elements that later fuse into plate-like elements evident in later-staged embryo and adult lenses. The anterior lens primordium is derived from an anterior group, group 1, of lentigenic cells, during later stages of development. Lentigenic processes extend from these lentigenic cells and are laid down in a circumferential fashion to form the anterior lens cap. As in the posterior lens, evidence indicates that the anterior lens elements fuse to form plate-like elements. The ultrastructure and morphogenesis of the cephalopod lens is discussed and contrasted with other strategies of lens development.

Ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signaling molecules. Together, these molecules regulate cell proliferation, apoptosis and specify retinal fate. In the zebrafish (Danio rerio), hmx1 is a homeobox transcription factor implicated in eye and brain development. Hmx1 transcripts were detected in the nasal retina and lens as well as otic vesicles and pharyngeal arches by 24-32 hpf. Before this stage, transcripts were more uniformly expressed in the optic vesicle. Knockdown of hmx1 led to microphthalmia. Delayed withdrawal of retinal progenitors from the cell cycle resulting in retarded retinal differentiation was observed in morphant. The retina and brain also showed an increased cell death at 24 hpf. The polarized expression of hmx1 to the nasal part in the zebrafish retina strongly suggested an involvement in the nasal-temporal patterning. However, the key patterning genes tested so far were not regulated by hmx1. Altogether, these results suggest an important role for hmx1 in retinogenesis. (C) 2012 Elsevier Ltd. All rights reserved.

Invagination of the optic vesicle to form the optic cup results in the formation of two apposed layers of neuroepithelium which follow divergent developmental pathways. Changes in the expression of cell surface molecules may be either the cause or result of important inductive signals during this process. We have used immunological reagents to study the expression of two molecules in the rat: the neural cell adhesion molecule N-CAM, and a cell membrane-associated protein which is specific for pigment epithelium in the adult, RET-PE2. Both N-CAM and RET-PE2 are present in both layers of the optic cup at embryonic age E13, but they become restricted to inner retina and pigment epithelium, respectively, by E17 and maintain that pattern of expression in the normal adult. Culture of pigment epithelial cells results in the reexpression of N-CAM and the continued expression of RET-PE2. Western blotting reveals that the size and relative proportions of the 180- and 140-kDa N-CAM molecules synthesized by rat pigment epithelial cells in vitro differ from that made by rat brain, retina and liver. Embryonic RPE N-CAM contains the sulfated carbohydrate recognized by the HNK-1 antibody, but this epitope was not present on N-CAM synthesized by cultured RPE cells. The reexpression of an embryonic antigen when placed in culture suggests that pigment epithelial cells retain some degree of plasticity in the adult state.

We used two different methodologies to examine at what stage development retinal positional specificity is established and which molecules are responsible. The first goal was achieved by removing parts of the presumptive temporal primary optic vesicle at stage 11 (40 to 45 hr of incubation) and fate mapping of tissue with presumptive nasal properties that shifted into the wound during the events of wound-healing. Participation of the shifted tissue in the healing resulted in assembly of a temporal retina with mosaic-like projection properties, as examined by retrograde double staining of the retinal ganglion cells from the optic tectum. In addition to cells with normal temporal–rostral projections, clusters of ganglion cells with nasal-like projection identities appeared labelled within the temporal hemiretina. The number of clusters increased with the amount of resected tissue, and by almost complete ablation of the presumptive temporal anlage, a temporal hemiretina with predominantly nasal retinotectal specificity was created. These neuroanatomical results suggested that neuroepithelial cells had fixed nasal and temporal positional specificities at stage 11. To examine differences in the cells derived of either half of the eye cup, we performed biochemical one-and two-dimensional gel electrophoresis of the hemianlagen at stage 11. In addition, incorporation of 35 S-methionin into newly synthesized peptides was investigated. Both techniques revealed the exclusive expression of one major and three less-abundant proteins within the presumptive nasal anlage. The most abundant of these proteins has a molecular weight of about 40 kDa and is clearly distinguishable both in gel electrophoresis and autoradiography. The asymmetric protein patterns had disappeared when the retina was analysed with the same methods at the more advanced embryonic days E4 and E6. The asymmetry in the expression of proteins in the retinal primordium may be the biochemical correlate of an early positional specification of the retinal neuroepithelium. The difference in the protein expression may explain that mixing the positionally specified cells of either origins results in projection mosaics.

Ascorbate is found in high concentrations in the aqueous, lens and retina of a variety of animal species. Several functions have been proposed for this, including a vital role in aqueous secretion. We investigated the possible presence of ascorbate in the early embryonic chicken eye as a marker for the differentiation of the ciliary epithelium. Embryonic chicken eyes were processed for histochemical demonstration of ascorbate by silver staining. A strongly positive reaction was found in the interstitial matrix between the lens rudiment and the optic vesicle of the 2-day chicken embryo. By 3 days, with lens and optic cup separating from each other, the silver grains were concentrated in the vitreous cavity, primarily in a band-like configuration between the lens equator and the rim of the optic cup, which is the presumptive ciliary epithelium. A weaker reaction was seen in the lens vesicle and primitive neural retina. During the second and third day of incubation the optic vesicle is just beginning to expand. The appearance of ascorbic acid in the newly formed vitreous cavity at this time may indicate its importance in the biosynthesis of the matrix that is filling the expanding optic vesicle.

The importance of differential epithelial spreading in the early stages of chick lens placode invagination in the chick has been demonstrated experimentally in two ways. Generalized swelling of the head caused by immersion in 50% ethanol and restricting the freedom of ectoderm in the eye region to spread by means of ligatures produce both precocious and exaggerated invagination. Inequality of spreading between the placode and surrounding ectoderm in normal development is caused by the adhesion of the former to the optic vesicle, while the ectoderm is free to spread and move over underlying tissues. Folds, i.e. invaginations, form at the boundary between the placode and its surroundings.

An in ovo application of the microfilament-disrupting drug cytochalasin B was used to study the consequences of a block to microfilament contraction during normal development of the chick eye. Three well-defined patterns of abnormality emerged. Their interpretation was possible using results of a study of the normal process made with a contraction medium (Maloney and Wakely, in press) similar to that used in studies of muscle physiology to induce muscle contraction in vitro. Combination of the two techniques may provide a powerful tool for the analysis of microfilament-influenced morphogenesis. From our study, normal microfilament contraction is necessary for accurate positioning of the optic vesicle relative to head ectoderm, prior to formation of the interspace and induction. During invagination of the lens and optic vesicle, shape of the vesicle is influenced by the shape change of the invaginating lens, the action of the ‘rim’ microfilaments and the spatial and temporal interactions of these factors. Fine control of the shape of the invaginating lens is achieved by the interaction of its inherent tendency to invaginate, and the shape of the optic cup, governed by microfilament contraction. In closure of the lens vesicle it is essential that contraction occurs at a particular stage in the process, to co-ordinate effects of tissue growth and relative shifts in the positioning of the tissues involved. Microfilaments in chick eye morphogenesis thus have a vital role in co-ordination of the temporal and spatial interactions of the tissues and forces involved, as well as direct effects on tissue shape.

The mesenchyme which surrounds the avian embryonic eye and forms all periocular skeletal and connective tissues, including the orbit and part of the cornea, is derived from two sources, the ectodermal neural crest and the mesoderm. Due to difficulties in distinguishing cells of these separate origins, the precise contributions of each to periocular tissues has not been defined. By transplanting labeled neural creast or mesoderm cells into unlabeled host embryos we have been able to catalogue first the migratory patterns of these populations and then their precise derivatives. Some donor tissues were labeled with [3H]thymidine; in other cases embryonic quail cells, which contain a replicating heterochromatin marker, were grafted into chick hosts. As the optic vesicle forms, its caudal (future temporal) surface is contacted by a sparse population of mesodermal mesenchyme; the rest of the eye is closely surrounded by superficial ectoderm. Shortly thereafter neural crest cells migrate over the caudal and rostral surfaces of the eye, forming the maxillary and frontonasal processes as well as presumptive scleral and choroidal cells. Neural crest cells form all of the skeletal and connective tissues adjacent to the medial, fnasal, ienrior, and lateral parts of the eye, including the endothelial and stromal cells of the cornea and much of the orbit. Most of the tissues behind the eye are derived from mesoderm, with the exception of the squamosal (temporal) bone. The myofibers of extrinsic ocular muscles are mesodermal, but connective tissues associated with these muscles are of neural crest origin. The ciliary muscles are formed from neural crest cells. Periocular vascular endothelial cells are all mesodermal, but peri-vascular connective tissues, including associated smooth muscles cells, are of neural crest origin. By performing heterotopic transplantations, for example replacing the chick prosencephalic neural crest with quail metencephalic crest, we have proved that neural crest cells from other regions of the head can mimic the development of periocular crest cells. This proves that the environment through which these cells migrate plays an essential role in establishing the timing and spatial patterns of their movement. Included in the environment are the basement membranes associated with epithelial tissues, such as the optic vesicle and superficial ectoderm. We have described several instances in which basement membranes underlying these epithelia become tightly apposed and appear to act as barriers to cell migration. The patterns of basement membrane apposition and subsequent separation are described and correlated with patterns of mesenchymal cell migrations.

Immunofluorescence with anti-crystallin antisera was done on the eye rudiments of mouse embryos of a congenitally anophthalmic strain. In a few of the embryos a lens vesicle formed, which, although much smaller than in controls and delayed in development, appeared morphologically normal. α-Crystallin could be identified in the cells of the rudiments. In addition the cells looked cytologically similar to those of the normal lens vesicle; they were elongated and the nuclei displayed interkinetic nuclear migration. Therefore, the surface ectoderm of the anophthalmia mouse can differentiate into a lens vesicle that appears biochemically and cytologically normal. This indicates that the optic vesicle in this mutant is capable of inducing a lens, while the surface ectoderm can respond to the stimulus in a normal fashion.

The function of microfilaments in early eye morphogenesis in the chick was investigated using an ATP-containing contraction medium to induce contraction of intracellular actomyosin systems. Invagination of the optic vesicle broadly similar to that seen in normal optic cup formation was seen when the treated eyes were at early stages, before the normal onset of optic cup invagination or lens placode formation. In older embryos where optic cup formation had begun the cupping process was actually reversed. In contrast neither the presumptive lens epithelium of the earliest embryos nor the fully formed lens placodes invaginated in response to the medium, although almost completely closed lens vesicles completed their closure. These results suggest that while optic cup formation is at least partly the result of active microfilament contraction the initial invagination of the presumptive lens is brought about by some other mechanism.

At 11 days of embryonic development of the rat there is a close association between presumptive lens ectoderm and optic vesicle. A space of 5·7±0·9 μm which separates the two tissues at this stage of development contains cytoplasmic processes and fibrils. The fibrils are probably mostly collagenous since some are seen to extend from the basel laminae and others have characteristic collagen banding patterns. The cytoplasmic processes have thick and thin components. The thin processes commonly arise from thick processes and are more numerous. In some cases the processes have been shown to bridge the interspace and make contact with the adjacent tissue.