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.     

The homeodomain of Eyeless regulates cell growth and antagonizes the paired domain-dependent retinal differentiation function

Pax6 and its Drosophila homolog Eyeless (Ey) play essential roles during eye development. Ey/Pax6 contains two distinct DNA binding domains, a Paired domain (PD) and a Homeodomain (HD). While Ey/Pax6 PD is required for the expression of key regulators of retinal development, relatively little is known about the HD-dependent Ey function. In this study, we used the UAS/GAL4 system to determine the functions of different Ey domains on cell growth and on retinal development. We showed that Ey can promote cell growth, which requires the HD but not the PD. In contrast, the ability of Ey to activate Ato expression and induce ectopic eye formation requires the PD but not the HD. Interestingly, deletion of the HD enhanced Ey-dependent ectopic eye induction while overexpression of the HD only Ey forms antagonizes ectopic eye induction. These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction. We further show the third helix of the Ey HD can directly interact with the RED subdomain in Ey PD and that deletion of the HD increased the binding of Ey PD to its target. These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects. Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.     

Patterning function of homothorax/extradenticle in the thorax of Drosophila

In Drosophila, the morphological diversity is generated by the activation of different sets of active developmental regulatory genes in the different body subdomains. Here, we have investigated the role of the homothorax/extradenticle (hth/exd) gene pair in the elaboration of the pattern of the anterior mesothorax (notum). These two genes are active in the same regions and behave as a single functional unit. We find that their original uniform expression in the notum is downregulated during development and becomes restricted to two distinct, alpha and betasubdomains. This modulation appears to be important for the formation of distinct patterns in the two subdomains. The regulation of hth/exd expression is achieved by the combined repressing functions of the Pax gene eyegone (eyg) and of the Dpp pathway. hth/exd is repressed in the body regions where eyg is active and that also contain high levels of Dpp activity. We also present evidence for a molecular interaction between the Hth and the Eyg proteins that may be important for the patterning of the alpha subdomain.     

The Pax-homeobox gene eyegone is involved in the subdivision of the thorax of Drosophila

The eyegone (eyg) gene is known to be involved in the development of the eye structures of Drosophila. We show that eyg and its related gene, twin of eyegone (toe), are also expressed in part of the anterior compartment of the adult mesothorax (notum). We report experiments concerning the role of these genes in the notum. In the absence of eyg function the anterior-central region does not develop, whereas ectopic activity of either eyg or toe induces the formation of the anterior-central pattern in the posterior or lateral region of the notum. These results demonstrate that eyg and toe play a role in the genetic subdivision of the notum, although the experiments indicate that eyg exerts the principal function. However, by itself the Eyg product cannot induce the formation of notum patterns; its thoracic function requires co-expression with the Iroquois (Iro) genes. We show that the restriction of eyg activity to the anterior-central region of the wing disc is achieved by the antagonistic regulatory activities of the Iro and pnr genes, which promote eyg expression, and those of the Hh and Dpp pathways, which act as repressors. We argue that eyg is a subordinate gene of the Iro genes, and that pnr mediates their thoracic patterning function. The activity of eyg gives rise to a new notum subdivision that acts upon the pre-extant one generated by the Iro genes and pnr. As a result the notum becomes subdivided into four distinct genetic domains.     

Hipk promotes photoreceptor differentiation through the repression of Twin of eyeless and Eyeless expression

Organogenesis is a complex developmental process, which requires tight regulation of selector gene expression to specify individual organ types. The Pax6 homolog Eyeless (Ey) is an example of such a factor and its expression pattern reveals it is dynamically controlled during development. Ey?s paralog Twin of eyeless (Toy) induces its expression during embryogenesis, and the two genes are expressed in nearly identical patterns during the larval stages of development. While Ey must be expressed to initiate retinal specification, it must subsequently be repressed behind the morphogenetic furrow to allow for neuronal differentiation. Thus far, a few factors have been implicated in this repression including the signaling pathways Hedgehog (Hh) and Decapentaplegic (Dpp), and more recently downstream components of the retinal determination gene network (RDGN) Sine oculis (So), Eyes absent (Eya), and Dachshund (Dac). Homeodomain-interacting protein kinase (Hipk), a conserved serine–threonine kinase, regulates numerous factors during tissue patterning and development, including the Hh pathway. Using genetic analyses we identify Hipk as a repressor of both Toy and Ey and show that it may do so, in part, through Hh signaling. We also provide evidence that Ey repression is a critical step in ectopic eye development and that Hipk plays an important role in this process. Because Ey repression within the retinal field is a critical step in eye development, we propose that Hipk is a key link between eye specification and patterning.     

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.