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.     

Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes.

We have cloned a chick homologue of Drosophila dachshund (dac), termed Dach1. Dach1 is the orthologue of mouse and human Dac/Dach (hereafter referred to as Dach1). We show that chick Dach1 is expressed in a variety of sites during embryonic development, including the eye and ear. Previous work has demonstrated the existence of a functional network and genetic regulatory hierarchy in Drosophila in which eyeless (ey, the Pax6 orthologue), eyes absent (eya), and dac operate together to regulate Drosophila eye development, and that ey regulates the expression of eya and dac. We find that in the developing eye of both chick and mouse, expression domains of Dach1 overlap with those of Pax6, a gene required for normal eye development. Similarly, in the developing ear of both mouse and chick, Dach1 expression overlaps with the expression of another Pax gene, Pax2. In the mouse, Dach1 expression in the developing ear also overlaps with the expression of Eya1 (an eya homologue). Both Pax2 and Eya1 are required for normal ear development. Our expression studies suggest that the Drosophila Pax-eya-dac regulatory network may be evolutionarily conserved such that Pax genes, Eya1, and Dach1 may function together in vertebrates to regulate neural development. To address the further possibility that a regulatory hierarchy exists between Pax, Eya, and Dach genes, we have examined the expression of mouse Dach1 in Pax6, Pax2 and Eya1 mutant backgrounds. Our results indicate that Pax6, Pax2, and Eya1 do not regulate Dach1 expression through a simple linear hierarchy.     

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.     

Expression of evolutionarily conserved eye specification genes during Drosophila embryogenesis

Eye specification in Drosophila is thought be controlled by a set of seven nuclear factors that includes the Pax6 homolog, Eyeless. This group of genes is conserved throughout evolution and has been repeatedly recruited for eye specification. Several of these genes are expressed within the developing eyes of vertebrates and mutations in several mouse and human orthologs are the underlying causes of retinal disease syndromes. Ectopic expression in Drosophila of any one of these genes is capable of inducing retinal development, while loss-of-function mutations delete the developing eye. These nuclear factors comprise a complex regulatory network and it is thought that their combined activities are required for the formation of the eye. We examined the expression patterns of four eye specification genes, eyeless (ey), sine oculis (so), eyes absent (eya), and dachshund (dac) throughout all time points of embryogenesis and show that only eyeless is expressed within the embryonic eye anlagen. This is consistent with a recently proposed model in which the eye primordium acquires its competence to become retinal tissue over several time points of development. We also compare the expression of Ey with that of a putative antennal specifying gene Distal-less (Dll). The expression patterns described here are quite intriguing and raise the possibility that these genes have even earlier and wide ranging roles in establishing the head and visual field.     

Isolation of three zebrafish dachshund homologues and their expression in sensory organs, the central nervous system and pectoral fin buds

Drosophila dachshund (dac) interacts with sine oculis (so), eyes absent (eya) and eyeless (ey) to control compound eye development. We have cloned three zebrafish dac homologues, dachA, dachB and dachC, which are expressed widely, in distinct but overlapping patterns. Expression of all three is found in sensory organs, the central nervous system and pectoral fin buds. dachA is also expressed strongly in the somites and dachC in the neural crest and pronephros. These expression domains overlap extensively with those of zebrafish pax, eya and six family members, the homologues of Drosophila ey, eya and so, respectively. This is consistent with the proposal that Dach, Eya, Six and Pax family members may form networks, similar to that found in the fly eye, in the development of many vertebrate organs.     

Mutations in the Drosophila mitochondrial tRNA amidotransferase, bene/gatA, cause growth defects in mitotic and endoreplicating tissues

Rapid larval growth is essential in the development of most metazoans. In this article, we show that bene, a gene previously identified on the basis of its oogenesis defects, is also required for larval growth and viability. We show that all bene alleles disrupt gatA, which encodes the Drosophila homolog of glutamyl-tRNA(Gln) amidotransferase subunit A (GatA). bene alleles are now referred to as gatA. GatA proteins are highly conserved throughout eukaryotes and many prokaryotes. These enzymes are required for proper translation of the proteins encoded by the mitochondrial genome and by many eubacterial genomes. Mitotic and endoreplicating tissues in Drosophila gatA loss-of-function mutants grow slowly and never achieve wild-type size, and gatA larvae die before pupariation. gatA mutant eye clones exhibit growth and differentiation defects, indicating that gatA expression is required cell autonomously for normal growth. The gatA gene is widely expressed in mitotic and endoreplicating tissues.     

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.     

Utilization ofDrosophila eye to probe the functions of two mammalian serine/threonine kinases, Snk and HsHPK

Here we report a quick functional analysis of two mammalian serine/threonine kinases, a serum inducible kinase (Snk) and Homo sapiens hepatoma protein kinase (HsHPK), using Drosophila eye as a model system. We generated transgenic fly lines carrying constructs of both kinases under control of the GAL upstream activating sequence (UAS). Each UAS line was then crossed to a line in which GAL4 expression was driven by one of the following promoters, eyeless (ey), glass or decapentaplegic. Thus, different kinase mutants can be ectopically expressed in a promoter-dependent manner. We observed that the ectopic expression of either the wild-type or active form of Snk driven by the glass promoter resulted in a rough-eye phenotype. Nevertheless, the ectopic expression of HsHPK under the control of the ey promoter resulted in a small-eye phenotype. The results of this study demonstrated that ectopic expression of these two mammalian genes could be achieved by the regulation of Drosophila promoters. In addition, the effects of these ectopically expressed genes on eye development could be an implication of their functions with respect to cell proliferation and differentiation. Thus, Drosophila eye, with the powerful genetic tools and vast information on eye development available, can be a useful system to probe the functions of mammalian genes in the postgenome era.     

The Drosophila Jak kinase hopscotch is required for multiple developmental processes in the eye.

Jak kinases are critical signaling components in hematopoiesis. While a large number of studies have been conducted on the roles of Jak kinases in the hematopoietic cells, much less is known about the requirements for these tyrosine kinases in other tissues. We have used loss of function mutations in the Drosophila Jak kinase Hopscotch (Hop) to determine the role of Hop in eye development. We find that Hop is required for cell proliferation/survival in the eye imaginal disc, for the differentiation of photoreceptor cells, and for the establishment of the equator and of ommatidial polarity. These results indicate that hop activity is required for multiple developmental processes in the eye, both cell-autonomously and nonautonomously.     

Eyeless collaborates with Hedgehog and Decapentaplegic signaling in Drosophila eye induction

eyeless (ey) is a key regulator of the eye development pathway in Drosophila. Ectopic expression of ey can induce the expression of several eye-specification genes (eya, so, and dac) and induce eye formation in multiple locations on the body. However, ey does not induce eye formation everywhere where it is ectopically expressed, suggesting that EY needs to collaborate with additional factors for eye induction. We examined ectopic eye induction by EY in the wing disc and found that eye induction was spatially restricted to the posterior compartment and the anterior-posterior (A/P) compartmental border, suggesting a requirement for both HH and DPP signaling. Although EY in the anterior compartment induced dpp and dac, these were not sufficient for eye induction. Coexpression experiments show that EY needs to collaborate with high level of HH and DPP to induce ectopic eye formation. Ectopic eye formation also requires the activation of an eye-specific enhancer of the endogenous hh gene.