The conditional medaka mutation eyeless uncouples patterning and morphogenesis of the eye

In early vertebrate eye development, the retinal anlage is specified in the anterior neuroectoderm. During neurulation, the optic vesicles evaginate from the lateral wall of the prosencephalon. Here we describe the temperature-sensitive mutation eyeless in the Japanese medakafish. Marker gene analysis indicates that, whereas, specification of two retinal primordia and proximodistal patterning takes place in the mutant embryo, optic vesicle evagination does not occur and subsequent differentiation of the retinal primordia is not observed. The mutation eyeless thus uncouples patterning and morphogenesis at early steps of retinal development. Temperature-shift experiments indicate a requirement for eyeless activity prior to optic vesicle evagination. Cell transplantation shows that eyeless acts cell autonomously.     

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.     

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.     

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.     

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.     

Molecular cloning and embryonic expression of Xenopus Six homeobox genes

Six genes are vertebrate homologues of the homeobox-containing gene sine oculis, which plays an essential role in controlling Drosophila compound eye development. Here we report the identification and expression patterns of all three subfamilies of Xenopus Six genes. Two Six2 subfamily genes (Six1, Six2) showed very similar expression patterns in cranial ganglia, otic placodes and the eyes. Non-neural expression of Six1 and Six2 was observed with mesodermal head mesenchyme, somites and their derivatives, the muscle anlagen of the embryonic trunk. In addition, Six2 expression was also found with mesenchyme associated with the developing stomach and pronephros. Expression of Six3 subfamily genes (Six3.1, Six3.2, Six6.1, and Six6.2) was restricted to the developing head, where expression was especially observed in derivatives of the forebrain (eyes, optic stalks, the hypothalamus and pituitary gland). Interestingly, expression of all Six3 subfamily members but Six6.2 was also found with the pineal gland primordium and the tegmentum. Expression of Six4 subfamily genes (Six4.1, Six4.2) was present in the developing visceral arches, placodal derivatives (otic vesicle, olfactory system), head mesenchyme and the eye. The observed dynamic expression patterns are largely conserved between lower and higher vertebrates and imply important roles of Six family genes not only in eye formation and myogenesis, but also in the development of the gut, the kidney and of placode-derived structures.     

Zebrafish blowout provides genetic evidence for Patched1-mediated negative regulation of Hedgehog signaling within the proximal optic vesicle of the vertebrate eye

In this study, we have characterized the ocular defects in the recessive zebrafish mutant blowout that presents with a variably penetrant coloboma phenotype. blowout mutants develop unilateral or bilateral colobomas and as a result, the retina and retinal pigmented epithelium are not contained within the optic cup. Colobomas result from defects in optic stalk morphogenesis whereby the optic stalk extends into the retina and impedes the lateral edges of the choroid fissure from meeting and fusing. The expression domain of the proximal optic vesicle marker pax2a is expanded in blowout at the expense of the distal optic vesicle marker pax6, suggesting that the initial patterning of the optic vesicle into proximal and distal territories is disrupted in blowout. Later aspects of distal optic cup formation (i.e. retina development) are normal in blowout mutants, however. Positional cloning of blowout identified a nonsense mutation in patched1, a negative regulator of the Hedgehog pathway, as the underlying cause of the blowout phenotype. Expanded domains of expression of the Hedgehog target genes patched1 and patched2 were observed in blowout, consistent with a loss of Patched1 function and upregulation of Hedgehog pathway activity. Moreover, colobomas in blowout could be suppressed by pharmacologically inhibiting the Hedgehog pathway with cyclopamine, and maximal rescue occurred when embryos were exposed to cyclopamine between 5.5 and 13 hours post-fertilization. These observations highlight the critical role that Hedgehog pathway activity plays in mediating patterning of the proximal/distal axis of the optic vesicle during the early phases of eye development and they provide genetic confirmation for the integral role that patched1-mediated negative regulation of Hedgehog signaling plays during vertebrate eye development.     

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.     

Candidate gene screen in the red flour beetle Tribolium reveals six3 as ancient regulator of anterior median head and central complex development

Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, we use the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and we also identified several genes that had not been implicated in this process before. Furthermore, we show that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, we demonstrate that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly.