Coordinated regulation of dorsal bone morphogenetic protein 4 and ventral Sonic hedgehog signaling specifies the dorso‐ventral polarity in the optic vesicle and governs ocular morphogenesis through fibroblast growth factor 8 upregulation

Dorsal and ventral specification in the early optic vesicle plays a crucial role in vertebrate ocular morphogenesis, and proper dorsal‐ventral polarity in the optic vesicle ensures that distinct structures develop in separate domains within the eye primordium. The polarity is determined progressively during development by coordinated regulation of extraocular dorsal and ventral factors. In the present study, we cultured discrete portions of embryonic chick brains by preparing anterior cephalon, anterior dorsal cephalon and anterior ventral cephalon, and clearly demonstrate that bone morphogenetic protein 4 (BMP4) and Sonic hedgehog (Shh) constitute a dorsal‐ventral signaling system together with fibroblast growth factor 8 (FGF8). BMP4 and Shh upregulate Tbx5 and Pax2, as reported previously, and at the same time Shh downregulates Tbx5, while BMP4 affects Pax2 expression to downregulate similarly. Shh induces Fgf8 expression in the ventral optic vesicle. This, in turn, determines the distinct boundary of the retinal pigmented epithelium and the neural retina by suppressing Mitf expression. The lens develops only when signals from both the dorsal and ventral regions come across together. Inverted deposition of Shh and BMP4 signals in organ‐cultured optic vesicle completely re‐organized ocular structures to be inverted. Based on these observations we propose a novel model in which the two signals govern the whole of ocular development when they encounter each other in the ocular morphogenic domain.     

Generation of a second eye by embryonic transplantation of the antero‐ventral hemicephalon

Vertebrate ocular morphogenesis requires proper dorso‐ventral polarity within the optic vesicle, and loss of dorso‐ventral polarity results in failure of optic cup formation and domain specification, as shown by a reverse transplantation of the optic vesicle. We have shown previously that the ocular development depends not only on the signal within the antero‐ventral optic vesicle but also on the extraocular signals. In the present study, using embryonic transplantation of a discrete portion of the embryonic chick brain, we demonstrate formation of a second eye from the antero‐ventral hemicephalon when it was transplanted in the antero‐dorsal hemicephalon of the host embryo. The transplant consists of an antero‐ventral quadrant of the optic vesicle and the surrounding part of the anterior cephalon. The original dorso‐ventral polarity of the transplant was once cancelled and re‐established in accordance with that of the host embryo. Neither dorsal nor ventral cephalic halves in isolation did not develop into entire eye structures under the culture condition; the dorsal halves developed merely into the retinal pigmented epithelium and the ventral halves into the neural retina alone. The present study clearly suggests that extraocular dorsal and ventral signals counterbalance each other to specify the polarity of the optic vesicle.     

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.     

FGF-mediated induction of ciliary body tissue in the chick eye

Upon morphogenesis, the simple neuroepithelium of the optic vesicle gives rise to four basic tissues in the vertebrate optic cup: pigmented epithelium, sensory neural retina, secretory ciliary body and muscular iris. Pigmented epithelium and neural retina are established through interactions with specific environments and signals: periocular mesenchyme/BMP specifies pigmented epithelium and surface ectoderm/FGF specifies neural retina. The anterior portions (iris and ciliary body) are specified through interactions with lens although the molecular mechanisms of induction have not been deciphered. As lens is a source of FGF, we examined whether this factor was involved in inducing ciliary body. We forced the pigmented epithelium of the embryonic chick eye to express FGF4. Infected cells and their immediate neighbors were transformed into neural retina. At a distance from the FGF signal, the tissue transitioned back into pigmented epithelium. Ciliary body tissue was found in the transitioning zone. The ectopic ciliary body was never in contact with the lens tissue. In order to assess the contribution of the lens on the specification of normal ciliary body, we created optic cups in which the lens had been removed while still pre-lens ectoderm. Ciliary body tissue was identified in the anterior portion of lens-less optic cups. We propose that the ciliary body may be specified at optic vesicle stages, at the same developmental stage when the neural retina and pigmented epithelium are specified and we present a model as to how this could be accomplished through overlapping BMP and FGF signals.     

Sonic hedgehog is involved in formation of the ventral optic cup by limiting Bmp4 expression to the dorsal domain

Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal–distal and dorsal–ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, we utilized the Smoothened (Smo) conditional knockout (CKO) mouse line. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. We analyzed expression of various genes such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. Our data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventral optic cup morphology.     

Extraocular dorsal signal affects the developmental fate of the optic vesicle and patterns the optic neuroepithelium

Dorsal-ventral (DV) specification in the early optic vesicle plays a crucial role in the proper development of the eye. To address the questions of how DV specification is determined and how it affects fate determination of the optic vesicle, isolated optic vesicles were cultured either in vitro or in ovo. The dorsal and ventral halves of the optic vesicle were fated to develop into retinal pigment epithelium (RPE) and neural retina, respectively, when they were separated from each other and cultured. In optic vesicles treated with collagenase to remove the surrounding tissues, the neuroepithelium gave rise to cRax expression but not Mitf, suggesting that surrounding tissues are necessary for RPE specification. This was also confirmed in in ovo explant cultures. Combination cultures of collagenase-treated optic vesicles with either the dorsal or ventral part of the head indicated that head-derived factors have an important role in the fate determination of the optic vesicle: in the optic vesicles co-cultured with the dorsal part of the head Mitf expression was induced in the neuroepithelium, while the ventral head portion did not have this effect. The dorsal head also suppressed Pax2 expression in the optic vesicle. These observations indicate that factors from the dorsal head portion have important roles in the establishment of DV polarity within the optic vesicle, which in turn induces the patterning and differentiation of the neural retina and pigment epithelium.     

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.     

Temporal and spatial effects of Sonic hedgehog signaling in chick eye morphogenesis

Proper dorsal--ventral pattern formation of the optic cup is essential for vertebrate eye morphogenesis and retinotectal topographic mapping. Previous studies have suggested that midline tissue-derived Sonic hedgehog (Shh) molecules play critical roles in establishing the bilateral eye fields and in determining the proximal--distal axis of the eye primordium. Here, we have examined the temporal requirements for Shh during the optic vesicle to optic cup transition and after early optic cup formation in chick embryos. Both misexpressing Shh by virus and blocking Shh activity by antibodies resulted in disruption of ventral ocular tissues. Decreasing endogenous Shh signals unexpectedly revealed a sharp morphological boundary subdividing dorsal and ventral portions of the optic cup. In addition, Shh signals differentially influenced expression patterns of genes involved in ocular tissue specification (Pax6, Pax2, and Otx2) and dorsal--ventral patterning (cVax) within the ventral but not dorsal optic cup. Ectopic Shh suppressed expression of Bone Morphogenetic Protein 4 (BMP4) in the dorsal retina, whereas reducing endogenous Sonic hedgehog activity resulted in a ventral expansion of BMP4 territory. These results demonstrate that temporal requirements for Shh signals persist after the formation of the optic cup and suggest that the early vertebrate optic primordium may be subdivided into dorsal and ventral compartments. We propose a model in which ventrally derived Shh signals and dorsally restricted BMP4 signals act antagonistically to regulate the growth and specification of the optic primordium.     

The role of cell death during morphogenesis of the mammalian eye

Serial sections of embryonic rat eyes were stained with hematoxylin and eosin, quantified (by counting pycnotic and viable nuclei), reproduced by camera lucida on wax plates, and moulded into reconstructions in order to study the normal progression of cellular death during morphogenesis. At least nine distinct necrotic loci (A through I) can be distinguished. Immediately following contact between the retina and surface ectoderm (day 11) degenerating cells were observed in (A) the ventral extent of the optic vesicle, beginning in the mid-retinal primordium and continuing ventrally in the optic stalk, (B) in the rostral optic stalk base, and (C) in the surface ectoderm encircling the early lens placode. No degeneration was observed in the dorsal half of the presumptive retina, in the entire pigment epithelium, or in the lens placode proper. During day 11.5 the lens placode thickens and forms a degenerating locus (D) in its ventral portion opposite the underlying pycnotic zone in the retina (A). During day 12 the ventral pycnotic zone (A) divides into two subunits (A₁ and A₂). Invagination of the lens displaces its marginal and ventral components (C and D) so that they come to occupy the lens pore area and presumptive corneal epithelium. Simultaneous invagination of the retinal rudiment juxtaposes the pigment epithelium which concurrently forms a necrotic area (E) adjacent ventrally to that in the retina (A₁). Degeneration appears in the caudal optic stalk (I). The density of viable cells decreases adjacent to pycnotic areas in the retina and pigment epithelium and increases within these death centers. During day 13 the optic fissure forms within the subunits of the ventral pycnotic zone (A₁ and A₂). Degenerations are seen in the dorsal optic stalk (F) and in the walls of the optic fissure (G and H). Throughout these stages necrosis appears only in those portions of the eye rudiment where invagination is either retarded or completely absent. In part, these observations suggest that cell death serves (1) to retard or inhibit invagination within death centers, (2) to integrate the series of invaginations which mould the dorsal optic cup and optic fissure, (3) to assist formation of the pigment epithelium monolayer, and (4) to orient the lens vesicle within the eye cup. The spatio-temporal relationship between necrotic loci suggests that pycnotic cells in the retina may influence their production in the lens and pigment epithelium. Preliminary observations on the mouse, pig, and human substantiate those on the rat.     

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.     

Compartments and organising boundaries in the Drosophila eye: the role of the homeodomain Iroquois proteins.

The Drosophila eye is patterned by a dorsal-ventral organising centre mechanistically similar to those in the fly wing and the vertebrate limb bud. Here we show how this organising centre in the eye is initiated - the first event in retinal patterning. Early in development the eye primordium is divided into dorsal and ventral compartments. The dorsally expressed homeodomain Iroquois genes are true selector genes for the dorsal compartment; their expression is regulated by Hedgehog and Wingless. The organising centre is then induced at the interface between the Iroquois-expressing and non-expressing cells at the eye midline. It was previously thought that the eye develops by a mechanism distinct from that operating in other imaginal discs, but our work establishes the importance of lineage compartments in the eye and thus supports their global role as fundamental units of patterning.     

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.