Stage-dependent expression of Pax6 in optic vesicle/cup regulates patterning genes through signaling molecules

Dorso-ventral and proximo-distal axis formation of the optic cup is apparent from early stages of development. Pax6 is initially detectable in the optic vesicle and later shows a distal-high and proximal-low gradient of expression in the retina. To determine the early role of Pax6 in pattern formation of the optic cup, we expressed Pax6 ectopically in the optic vesicle of stages 9-10 chick embryos by in ovo electroporation, which resulted in a small eye-like phenotype. The signaling molecule fibroblast growth factor (FGF)8, which appears to be restricted to the central retina, was increased, whereas bone morphogenetic protein (BMP)4 and Tbx5, two dorsal markers, were down-regulated in Pax6-electroporated eye. Pax6 overexpression also decreased the expression of the ventral marker Vax. Electroporation with a dominant-negative form of Pax6 resulted in a decrease in FGF8 expression, but BMP4 expression was unaffected initially while it was diminished later. Our data suggest a new role for Pax6 in regulating FGF8 and BMP4 expression during pattern formation of the optic cup, and that a Pax6-regulated balance between FGF8 and BMP4 is critical for retinogenesis.     

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.     

Anteroventrally localized activity in the optic vesicle plays a crucial role in the optic development

The vertebrate eye develops from the optic vesicle (OV), a laterally protrusive structure of the forebrain, by a coordinated interaction with surrounding tissues. The OV then invaginates to form an optic cup, and the lens placode develops to the lens vesicle at the same time. These aspects in the early stage characterize vertebrate eye formation and are controlled by appropriate dorsal-ventral coordination. In the present study, we performed surgical manipulation in the chick OV to remove either the dorsal or ventral half and examined the development of the remaining OV. The results show that the dorsal and ventral halves of the OV have a clearly different developmental pattern. When the dorsal half was removed, the remaining ventral OV developed into an entire eye, while the dorsal OV developed to a pigmented vesicle consisting of retinal pigmented epithelium alone. These results indicate that the ventral part of the OV retains the potency to develop the entire eye structure and plays an essential role in proper eye development. In subsequent manipulations of early chick embryos, it was found that only the anterior ventral quadrant of the OV has the potential to develop the entire eye and that no other part of the OV has a similar activity. Fgf8 expression was localized in this portion and no Fgf8 expression was observed within the OV when the ventral OV was removed. These results suggest that the anterior ventral portion of the OV plays a crucial role in the proper development of the eye, possibly generating the dorsal-ventral gradients of signal proteins within the eye primordium.     

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.     

Different roles for Pax6 in the optic vesicle and facial epithelium mediate early morphogenesis of the murine eye

Chimaeric mice were made by aggregating Pax6(-/-) and wild-type mouse embryos, in order to study the interaction between the optic vesicle and the prospective lens epithelium during early stages of eye development. Histological analysis of the distribution of homozygous mutant cells in the chimaeras showed that the cell-autonomous removal of Pax6(-/-) cells from the lens, shown previously at E12.5, is nearly complete by E9.5. Most mutant cells are eliminated from an area of facial epithelium wider than, but including, the developing lens placode. This result suggests a role for Pax6 in maintaining a region of the facial epithelium that has the tissue competence to undergo lens differentiation. Segregation of wild-type and Pax6(-/-) cells occurs in the optic vesicle at E9.5 and is most likely a result of different adhesive properties of wild-type and mutant cells. Also, proximo-distal specification of the optic vesicle (as assayed by the elimination of Pax6(-/-) cells distally), is disrupted in the presence of a high proportion of mutant cells. This suggests that Pax6 operates during the establishment of patterning along the proximo-distal axis of the vesicle. Examination of chimaeras with a high proportion of mutant cells showed that Pax6 is required in the optic vesicle for maintenance of contact with the overlying lens epithelium. This may explain why Pax6(-/-) optic vesicles are inefficient at inducing a lens placode. Contact is preferentially maintained when the lens epithelium is also wild-type. Together, these results demonstrate requirements for functional Pax6 in both the optic vesicle and surface epithelia in order to mediate the interactions between the two tissues during the earliest stages of eye development.     

Function of Rx, but not Pax6, is essential for the formation of retinal progenitor cells in mice

Rx plays a critical role in eye formation. Targeted elimination of Rx results in embryos that do not develop eyes. In this study, we have investigated the expression of Otx2, Six3, and Pax6 in Rx deficient embryos. We find that these genes show normal activation in the anterior neural plate in Rx-/- embryos, but they are not upregulated in the area of the neural plate that would form the primordium of the optic vesicle. In contrast, in homozygous Small eye embryos that lack Pax6 function, Rx shows normal activation in the anterior neural plate and normal upregulation in the optic vesicle/retinal progenitor cells. This suggests that neither Rx expression nor the formation of retinal progenitor cells is dependent on a functional copy of the Pax6 gene, but that Pax6 expression and the formation of the progenitor cells of the optic cup is dependent on a functional copy of the Rx gene.     

The orchestration of mammalian tissue morphogenesis through a series of coherent feed-forward loops

Tissue morphogenesis requires intricate temporal and spatial control of gene expression that is executed through specific gene regulatory networks (GRNs). GRNs are comprised from individual subcircuits of different levels of complexity. An important question is to elucidate the mutual relationship between those genes encoding DNA-binding factors that trigger the subcircuit with those that play major "later" roles during terminal differentiation via expression of specific genes that constitute the phenotype of individual tissues. The ocular lens is a classical model system to study tissue morphogenesis. Pax6 is essential for both lens placode formation and subsequent stages of lens morphogenesis, whereas c-Maf controls terminal differentiation of lens fibers, including regulation of crystallins, key lens structural proteins required for its transparency and refraction. Here, we show that Pax6 directly regulates c-Maf expression during lens development. A 1.3-kb c-Maf promoter with a 1.6-kb upstream enhancer (CR1) recapitulated the endogenous c-Maf expression pattern in lens and retinal pigmented epithelium. ChIP assays revealed binding of Pax6 and c-Maf to multiple regions of the c-Maf locus in lens chromatin. To predict functional Pax6-binding sites, nine novel variants of Pax6 DNA-binding motifs were identified and characterized. Two of these motifs predicted a pair of Pax6-binding sites in the CR1. Mutagenesis of these Pax6-binding sites inactivated transgenic expression in the lens but not in retinal pigmented epithelium. These data establish a novel regulatory role for Pax6 during lens development, link together the Pax6/c-Maf/crystallin regulatory network, and suggest a novel type of GRN subcircuit that controls a major part of embryonic lens development.     

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.     

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.     

Aphakia (ak), a mouse mutation affecting early eye development: Fine mapping,consideration of candidate genes and altered Pax6 and Six3 gene expression pattern

The homozygous mouse mutant aphakia (ak) has been characterized by bilaterally aphakic eyes without a pupil [Varnum DS, Stevens, LC (1968): J Hered 59:147–150]. The mutation was mapped to chromosome 19 [Varnum DS, Stevens, LC (1975): Mouse News Lett 53:35]. Our linkage studies yielded a precise localization of the ak gene 0.6 ± 0.3 cM proximal to the microsatellite marker D19Mit10 and 0.7 ± 0.4 cM distal to D19Mit4 and D19Mit91. No recombination was found with the marker D19Mit9 among 418 backcross offspring tested. The developmental control gene Pax2 mapped 11.0 ± 3.5 cM proximal to ak and is excluded as a candidate gene. Sequence analysis of Fgf8 and Chuk1, which are localized close to the marker D19Mit10, detected no mutations in the ak/ak mutants. Histological analysis of homozygous mutants suggested the arrest of lens development at the lens stalk stage, a transient morphological structure during the formation of the lens vesicle. In the lens remnants, Pax6 and Six3 are expressed, whereas in the persisting lens stalk only Pax6 was detected. The expression pattern of Pax2 appeared normal; Cryaa expression could not be detected. As a consequence of the arrested lens development, other ocular tissues that require for their development information from the intact lens, such as iris, ciliary muscle, retina, and vitreous body, are absent or formed abnormally. Dev. Genet. 23:299–316, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Optic cup morphogenesis requires pre-lens ectoderm but not lens differentiation

The formation of the vertebrate optic cup is a morphogenetic event initiated after the optic vesicle contacts the overlying surface/pre-lens ectoderm. Placodes form in both the optic neuroepithelium and lens ectoderm. Subsequently, both placodes invaginate to form the definitive optic cup and lens, respectively. We examined the role of the lens tissue in inducing and/or maintaining optic cup invagination in ovo. Lens tissue was surgically removed at various stages of development, from pre-lens ectoderm stages to invaginating lens placode. Removal of the pre-lens ectoderm resulted in persistent optic vesicles that initiated neural retinal differentiation but failed to invaginate. In striking contrast, ablation of the lens placode gave rise to optic vesicles that underwent invagination and formed the optic cup. The results suggest that: (1) the optic vesicle neuroepithelium requires a temporally specific association with pre-lens ectoderm in order to undergo optic cup morphogenesis; and (2) the optic cup can form in the absence of lens formation. If ectopic BMP is added, a neural retina does not develop and optic cup morphogenesis fails, although lens formation appears normal. FGF-induced neural retina differentiation in the absence of the pre-lens ectoderm is not sufficient to create an optic cup. We hypothesize the presence of a signal coming from the pre-lens ectoderm that induces the optic vesicle to form an optic cup.