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.     

Pax6-dependence of Six3, Eya1 and Dach1 expression during lens and nasal placode induction

The Drosophila eyeless gene plays a central role in fly eye development and controls a subordinate regulatory network consisting of the so, eya and dac genes. All three genes have highly conserved mammalian homologs, suggesting possible conservation of this eye forming regulatory network. sine oculis (so) belongs to the so/Six gene family, and Six3 is prominently expressed in the developing mammalian eye. Eya1 and Dach1 are mammalian homologs of eya and dac, respectively, and although neither Eya1 nor Dach1 knockout mice express prenatal eye defects, possibilities exist for postnatal ocular phenotypes or for functional redundancy between related family members. To examine whether expression relationships analogous to those between ey, so, eya and dac exist in early mammalian oculogenesis, we investigated Pax6, Six3, Eya1 and Dach1 protein expression in murine lens and nasal placode development. Six3 expression in the pre-placode lens ectoderm is initially Pax6-independent, but subsequently both its expression and nuclear localization become Pax6-dependent. Six3, Dach1 and Eya1 nasal expression in pre-placode ectoderm are also initially Pax6-independent, but thereafter become Pax6-dependent. Pax6, Six3, Dach1 and Eya1 are all co-expressed in the developing ciliary marginal zone, a source of retinal stem cells in some vertebrates. An in vitro protein-protein interaction is detected between Six3 and Eya1. Collectively, these findings suggest that the Pax-Eya-Six-Dach network is at best only partly conserved during lens and nasal placode development. However, the findings do not rule out the possibility that such a regulatory network acts at later stages of oculogenesis.     

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.     

Drosophila nemo promotes eye specification directed by the retinal determination gene network

Drosophila nemo (nmo) is the founding member of the Nemo-like kinase (Nlk) family of serine-threonine kinases. Previous work has characterized nmo's role in planar cell polarity during ommatidial patterning. Here we examine an earlier role for nmo in eye formation through interactions with the retinal determination gene network (RDGN). nmo is dynamically expressed in second and third instar eye imaginal discs, suggesting additional roles in patterning of the eyes, ocelli, and antennae. We utilized genetic approaches to investigate Nmo's role in determining eye fate. nmo genetically interacts with the retinal determination factors Eyeless (Ey), Eyes Absent (Eya), and Dachshund (Dac). Loss of nmo rescues ey and eya mutant phenotypes, and heterozygosity for eya modifies the nmo eye phenotype. Reducing nmo also rescues small-eye defects induced by misexpression of ey and eya in early eye development. nmo can potentiate RDGN-mediated eye formation in ectopic eye induction assays. Moreover, elevated Nmo alone can respecify presumptive head cells to an eye fate by inducing ectopic expression of dac and eya. Together, our genetic analyses reveal that nmo promotes normal and ectopic eye development directed by the RDGN.     

Molecular analysis of Drosophila eyes absent mutants reveals features of the conserved Eya domain

The eyes absent (eya) gene is critical to eye formation in Drosophila; upon loss of eya function, eye progenitor cells die by programmed cell death. Moreover, ectopic eya expression directs eye formation, and eya functionally synergizes in vivo and physically interacts in vitro with two other genes of eye development, sine oculis and dachshund. The Eya protein sequence, while highly conserved to vertebrates, is novel. To define amino acids critical to the function of the Eya protein, we have sequenced eya alleles. These mutations have revealed that loss of the entire Eya Domain is null for eya activity, but that alleles with truncations within the Eya Domain display partial function. We then extended the molecular genetic analysis to interactions within the Eya Domain. This analysis has revealed regions of special importance to interaction with Sine Oculis or Dachshund. Select eya missense mutations within the Eya Domain diminished the interactions with Sine Oculis or Dachshund. Taken together, these data suggest that the conserved Eya Domain is critical for eya activity and may have functional subregions within it.     

Activation of Six1 target genes is required for sensory placode formation

In vertebrates, cranial placodes form crucial parts of the sensory nervous system in the head. All cranial placodes arise from a common territory, the preplacodal region, and are identified by the expression of Six1/4 and Eya1/2 genes, which control different aspects of sensory development in invertebrates as well as vertebrates. While So and Eya can induce ectopic eyes in Drosophila, the ability of their vertebrate homologues to induce placodes in non-placodal ectoderm has not been explored. Here we show that Six1 and Eya2 are involved in ectodermal patterning and cooperate to induce preplacodal gene expression, while repressing neural plate and neural crest fates. However, they are not sufficient to induce ectopic sensory placodes in future epidermis. Activation of Six1 target genes is required for expression of preplacodal genes, for normal placode morphology and for placode-specific Pax protein expression. These findings suggest that unlike in the fly where the Pax6 homologue Eyeless acts upstream of Six and Eya, the regulatory relationships between these genes are reversed in early vertebrate placode development.     

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.     

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.     

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.