Brain patterning defects caused by mutations of the twin of eyeless gene in Drosophila melanogaster

The Drosophila Pax6 genes, eyeless (ey) and twin of eyeless (toy), are expressed in both eyes and the brain. Previous studies have demonstrated that ey plays important roles in axonal outgrowth and differentiation of mushroom bodies (MBs), which are centers for associative learning and memory in flies. However, the functional significance of toy in brain development is poorly understood. Here, we describe the expression patterns of TOY, and show that TOY expression partially overlaps with EY expression in the embryonic, larval and adult brains. Mutations of toy perturb brain neuromere formation in the embryonic stages, and result in severe deformation of the MB lobes in pharate adult brains. Moreover, we also analyzed toy functions by gain-of-function experiments, and show that overexpression of toy results in degeneration of MB lobes. Thus, our results demonstrate the importance of toy in embryonic brain patterning as well as in post-embryonic development of the major brain structures such as MBs.     

twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development

The Drosophila Pax-6 gene eyeless (ey) plays a key role in eye development. Here we show tht Drosophila contains a second Pax-6 gene, twin of eyeless (toy), due to a duplication during insect evolution. Toy is more similar to vertebrate Pax-6 proteins than Ey with regard to overall sequence conservation, DNA-binding function, and early expression in the embryo, toy and ey share a similar expression pattern in the developing visual system, and targeted expression of Toy, like Ey, induces the formation of ectopic eyes. Genetic and biochemical evidence indicates, however, that Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey. Toy is therefore required for initiation of ey expression in the embryo and acts through Ey to activate the eye developmental program.     

Differential interactions of eyeless and twin of eyeless with the sine oculis enhancer

Drosophila eye development is under the control of early eye specifying genes including eyeless (ey), twin of eyeless (toy), eyes absent (eya), dachshund (dac) and sine oculis (so). They are all conserved between vertebrates and insects and they interact in a combinatorial and hierarchical network to regulate each other expression. so has been shown to be directly regulated by ey through an eye-specific enhancer (so10). We further studied the regulation of this element and found that both Drosophila Pax6 proteins namely EY and TOY bind and positively regulate so10 expression through different binding sites. By targeted mutagenesis experiments, we disrupted these EY and TOY binding sites and studied their functional involvement in the so10 enhancer expression in the eye progenitor cells. We show a differential requirement for the EY and TOY binding sites in activating so10 during the different stages of eye development. Additionally, in a rescue experiment performed in the so(1) mutant, we show that the EY and TOY binding sites are required for compound eye and ocellus development respectively. Altogether, these results suggest a differential requirement for EY and TOY to specify the development of the two types of adult visual systems, namely the compound eye and the ocellus.     

Early development of the Drosophila mushroom body: the roles of eyeless and dachshund

The mushroom body (MB) is a uniquely identifiable brain structure present in most arthropods. Functional studies have established its role in learning and memory. Here we describe the early embryonic origin of the four neuroblasts that give rise to the mushroom body and follow its morphogenesis through later embryonic stages. In the late embryo, axons of MB neurons lay down a characteristic pattern of pathways. eyeless (ey) and dachshund (dac) are expressed in the progenitor cells and neurons of the MB in the embryo and larva. In the larval brains of the hypomorphic ey(R) strain, we find that beside an overall reduction of MB neurons, one MB pathway, the medial lobe, is malformed or missing. Overexpression of eyeless in MBs under the control of an MB-specific promoter results in a converse type of axon pathway abnormality, i.e. malformation or loss of the dorsal lobe. In contrast, loss of dachshund results in deformation of the dorsal lobe, whereas no lobe abnormalities can be detected following dachshund overexpression. These results indicate that ey and dachshund may have a role in axon pathway selection during embryogenesis.     

Phototaxis and adaptation of the eyeless Drosophila melanogaster line]

An artificial selection on positive and negative phototaxis of eyeless (ey) Drosophila melanogaster line was carried out. Expression of the ey trait in imago was estimating during the selection. The fitness of the "+" and "-" selected lines was evaluated by such components as heat resistance, life span, fertility of flies. The genetic analysis of phototaxis inheritance was carried out. It was found that phototaxis selection results in changes of fertility whereas it doesn't affect other indices of fitness to environmental conditions as well as the ey expression. The main polygenic systems of phototaxis inheritance are located in chromosome 2 and chromosome 3.     

Analysis of twin of eyeless regulation during early embryogenesis in Drosophila melanogaster

The Drosophila Pax6 homolog twin of eyeless (toy) is so far the first zygotically expressed gene involved in eye morphogenesis in Drosophila. The study of its expression during embryogenesis is therefore informative of the initial events of eye development in Drosophila. We have analyzed how the initial expression domain of toy at cellular blastoderm is regulated. We show that the three maternal patterning systems active in the cephalic region (the anterior, terminal and dorsal-ventral systems) cooperate with zygotically activated gap genes to shape the initial expression domain of toy. Whereas Bicoid, Dorsal and Torso signaling synergistically act as activators, Hunchback, Knirps and Decapentaplegic act as repressors.     

Drosophila Pax-6/eyeless is essential for normal adult brain structure and function

A role for the Pax-6 homologue eyeless in adult Drosophila brain development and function is described. eyeless expression is detected in neurons, but not glial cells, of the mushroom bodies, the medullar cortex, the lateral horn, and the pars intercerebralis. Furthermore, severe defects in adult brain structures essential for vision, olfaction, and for the coordination of locomotion are provoked by two newly isolated mutations of Pax-6/eyeless that result in truncated proteins. Consistent with the morphological lesions, we observe defective walking behavior for these eyeless mutants. The implications of these data for understanding postembryonic brain development and function in Drosophila are discussed.     

Influence of genotype on endoreduplication of giant chromosomes and expressivity of character eyeless in Drosophila melanogaster

In Drosophila melanogaster lines LA (low activity), HA (high activity), and Oregon-R (wild type), the effect of genetic background on endoreplication in giant chromosomes of salivary glands, fecundity, and expression of mutation ey was studied. The degree of chromosome polyteny and the number of adult flies in saturated lines eyHA, eyBA, and eyOr were significantly higher than in the original lines. The degree of chromosome polyteny was correlated with fecundity. The expressivity of the ey character was shown to be far lower in lines eyLA, eyHA, and eyOr than in the original eyeless line. In the saturated lines, the eye facets were reduced to a similar degree. All the lines studied displayed clear-cut sexual distinctions in this parameter. In the eyHA line, the coefficients of variation for the degree of chromosome polyteny, fecundity, and expressivity of the ey mutation were much lower than in lines eyeless, eyLA, and eyOr.     

Headless flies produced by mutations in the paralogous Pax6 genes eyeless and twin of eyeless

The two Pax6 gene homologs eyeless and twin of eyeless play decisive early roles in Drosophila eye development. Strong mutants of twin of eyeless or of eyeless are headless, which suggests that they are required for the development of all structures derived from eye-antennal discs. The activity of these genes is crucial at the very beginning of eye-antennal development in the primordia of eye-antennal discs when eyeless is first activated by the twin of eyeless gene product. This activation does not strictly depend on the Twin of eyeless protein, but is temperature-dependent in its absence. Twin of eyeless acts also in parallel to the eyeless gene and exerts functions that are partially redundant with those of Eyeless, while Eyeless is mainly required to prevent early cell death and promote eye development in eye-antennal discs.     

Eye development under the control of SRp55/B52-mediated alternative splicing of eyeless

The genetic programs specifying eye development are highly conserved during evolution and involve the vertebrate Pax-6 gene and its Drosophila melanogaster homolog eyeless (ey). Here we report that the SR protein B52/SRp55 controls a novel developmentally regulated splicing event of eyeless that is crucial for eye growth and specification in Drosophila. B52/SRp55 generates two isoforms of eyeless differing by an alternative exon encoding a 60-amino-acid insert at the beginning of the paired domain. The long isoform has impaired ability to trigger formation of ectopic eyes and to bind efficiently Eyeless target DNA sequences in vitro. When over-produced in the eye imaginal disc, this isoform induces a small eye phenotype, whereas the isoform lacking the alternative exon triggers eye over-growth and strong disorganization. Our results suggest that B52/SRp55 splicing activity is used during normal eye development to control eye organogenesis and size through regulation of eyeless alternative splicing.     

The eyeless mutant gene (e) in the Mexican axolotl (Ambystoma mexicanum) affects pax-6 expression and forebrain axonogenesis.

This study tested the hypothesis that changes in the patterns of pax-6 expression disrupt the anatomy and axonogenesis of the diencephalic areas of the eyeless axolotl. Proper pax-6 expression is necessary for eye and hypothalamus morphogenesis. Since the expression boundaries of pax-6 also provide a permissive environment for axonal outgrowth, an extensive study examining the effects of the eyeless gene (e) in the Mexican axolotl upon pax-6 expression and forebrain axonogenesis was begun. This study used whole embryo in situ hybridization techniques to follow pax-6 expression and whole brain immunocytochemistry to examine axonogenesis and neural differentiation. These studies demonstrated that the mutant gene e in the axolotl alters the response of midanterior neural-plate tissue to signals from the prechordal plate. This response was hypothesized to be a hyper-response to signals (sonic hedgehog?) that suppressed pax-6 expression within the midanterior neural plate and later developmental stages. Alternatively, the affected neuroectoderm of the eyeless embryos may lack competence to express pax-6. Lowered pax-6 expression inhibited eye and forebrain morphogenesis as well as neural axonogenesis and differentiation. Differentiation defects were detected as the suppression of midline dopaminergic neurons within the suprachiasmatic nucleus of eyeless animals. Thus, lowered pax-6 expression by the midanterior neuroectoderm promotes the eyeless condition by inhibiting the role of pax-6 in eye formation. This lowered expression also leads to concurrent alterations in the hypothalamic terrain which disrupt axonogenesis and ultimately promote sterility.     

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.