Characterization of mouse Dach2, a homologue of Drosophila dachshund

The Drosophila genes eyeless, eyes absent, sine oculis and dachshund cooperate as components of a network to control retinal determination. Vertebrate homologues of these genes have been identified and implicated in the control of cell fate. We present the cloning and characterization of mouse Dach2, a homologue of dachshund. In situ hybridization studies demonstrate Dach2 expression in embryonic nervous tissues, sensory organs and limbs. This pattern is similar to mouse Dach1, suggesting a partially redundant role for these genes during development. In addition, we determine that Dach2 expression in the forebrain of Pax6 mutants and dermamyotome of Pax3 mutants is not detectably altered. Finally, genetic mapping experiments place mouse Dach2 on the X chromosome between Xist and Esx1. The identification of human DACH2 sequences at Xq21 suggests a possible role for this gene in Allan-Herndon syndrome, Miles-Carpenter syndrome, X-linked cleft palate and/or Megalocornea.     

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.     

Redeployment of the Six genes in evolution

It has become clear that during evolution, efficient molecular mechanisms are used over and over again to achieve various patterning tasks. The Six gene story illustrates a new aspect of the molecular conservation during embryogenesis. Members of the Six gene family have been identified on the basis of sequence homology with Drosophila sine oculis gene, which acts within a network of genes including eyeless (Pax family), eyes absent (Eya family) and dachshund (Dach family) to trigger compound eye organogenesis. Some aspects of the regulatory complex operating in Drosophila appear to be conserved during vertebrate eye patterning, but also for other differentiation processes. In this regard, Six1 is required nonetheless during myogenesis, but also for kidney, thymus, inner ear, nose, lacrimal and salivary gland organogenesis. These phenotypes are reminiscent of those previously described for Eya and Pax mutants, suggesting a functional link between these factors during mammalian organogenesis.     

Identification of functional sine oculis motifs in the autoregulatory element of its own gene, in the eyeless enhancer and in the signalling gene hedgehog

In Drosophila, the sine oculis (so) gene is important for the development of the entire visual system, including Bolwig's organ, compound eyes and ocelli. Together with twin of eyeless, eyeless, eyes absent and dachshund, so belongs to a network of genes that by complex interactions initiate eye development. Although much is known about the genetic interactions of the genes belonging to this retinal determination network, only a few such regulatory interactions have been analysed down to the level of DNA-protein interactions. Previous work in our laboratory identified an eye/ocellus specific enhancer of the sine oculis gene that is directly regulated by eyeless and twin of eyeless. We further characterized this regulatory element and identified a minimal enhancer fragment of so that sets up an autoregulatory feedback loop crucial for proper ocelli development. By systematic analysis of the DNA-binding specificity of so we identified the most important nucleotides for this interaction. Using the emerging consensus sequence for SO-DNA binding we performed a genome-wide search and have thereby been able to identify eyeless as well as the signalling gene hedgehog as putative targets of so. Our results strengthen the general assumption that feedback loops among the genes of the retinal determination network are crucial for proper development of eyes and ocelli.     

The Drosophila homeobox gene optix is capable of inducing ectopic eyes by an eyeless-independent mechanism

optix is a new member of the Six/so gene family from Drosophila that contains both a six domain and a homeodomain. Because of its high amino acid sequence similarity with the mouse Six3 gene, optix is considered to be the orthologous gene from Drosophila rather than sine oculis, as previously believed. optix expression was detected in the eye, wing and haltere imaginal discs. Ectopic expression of optix leads to the formation of ectopic eyes suggesting that optix has important functions in eye development. Although optix and sine oculis belong to the same gene family (Six/so) and share a high degree of amino acid sequence identity, there are a number of factors which suggest that their developmental roles are different: (1) the expression patterns of optix and sine oculis are clearly distinct; (2) sine oculis acts downstream of eyeless, whereas optix is expressed independently of eyeless; (3) sine oculis functions synergistically with eyes absent in eye development whereas optix does not; (4) ectopic expression of optix alone, but not of sine oculis can induce ectopic eyes in the antennal disc. These results suggest that optix is involved in eye morphogenesis by an eyeless-independent mechanism.     

Molecular interaction and synergistic activation of a promoter by Six,Eya, and Dach proteins mediated through CREB binding protein

Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation. The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation. To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, we examined the molecular interactions among the encoded proteins. Eya interacted directly with Six but never with Dach. Dach transactivated a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins. Transactivation by Eya and Dach was repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding. Recruitment of CBP to the immobilized chromatin DNA template was dependent on FLAG-Dach and GAL4-Eya3. These results indicate that CBP is a mediator of the interaction between Eya and Dach. Contrary to our expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3. Dach also binds to naked DNA with lower affinity. The conserved DD1 domain is responsible for binding to DNA. Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein. Our results demonstrated that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach. This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis.     

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.     

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.     

Molecular cloning and expression of the human and mouse homologues of the Drosophila dachshund gene

Recent genetic analysis of the Drosophila dachshund (dac) gene has established that dac encodes a novel nuclear protein that is involved in both eye and leg development. In the Drosophila eye, dac expression appears to be controlled by the product of the eyeless/Pax6 gene. In order to analyze the Pax6 pathway in vertebrates we have isolated and characterized the cDNA and genomic clones corresponding to the human and mouse homologues of Drosophila dac. A full-length human cDNA encoding dachshund (DACH) encodes the 706 amino acids protein with predicted molecular weight of 73 kDa. A 109 amino acid domain located at the N-terminus of the DACH showed significant sequence and secondary structure homologies to the ski/sno oncogene products. Northern blot analysis found human DACH predominantly in adult kidney, heart, and placenta, with less expression detected in the brain, lung, skeletal muscle and pancreas. A panel of human cell lines was studied and most notably a large proportion of neuroblastomas expressed DACH mRNA. Mouse Dach encodes a protein of 751 amino acids with predicted molecular weight of 78 kDa that is 95% identical to the human DACH. RNase protection analysis showed the highest Dach mRNA expression in the adult mouse kidney and lung, whereas lower expression was detected in the brain and testis. RT/PCR analysis readily detected Dach mRNA in the adult mouse cornea and retina. Dach mRNA expression in the mouse E11.5 embryo was observed primarily in the fore and hind limbs, as well as in the somites. Received: 9 February 1999 / Accepted: 19 April 1999     

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.     

Mouse Dach2 mutants do not exhibit gross defects in eye development or brain function

Drosophila dachshund is a critical regulator of eye, brain, and limb formation. Vertebrate homologs, Dach1 and Dach2, are expressed in the developing retina, brain, and limbs, suggesting functional conservation of the dachshund/Dach gene family. Dach1 mutants die postnatally, but exhibit grossly normal development. Here we report the generation of Dach2 mutant mice. Although deletion of Dach2 exon 1 results in abrogation of RNA expression, Dach2 mutants are viable and fertile. Histochemical analysis reveals grossly normal Dach2 mutant eye development. In addition, a battery of neurological assays failed to yield significant differences in behavior between Dach2 mutants and controls. We discuss these findings in the light of published observations of DACH2 mutations in the human population. Finally, to test the functional conservation hypothesis, we generated Dach2; Dach1 double mutant mice. Dach double mutants die after birth, similar to Dach1 homozygotes. However, unlike Drosophila dachshund mutants that lack eyes and exhibit leg truncations, the eyes and limbs of Dach double mutants are present, suggesting differences between Dach and dachshund gene function during embryonic eye and limb formation.     

Control of Drosophila eye specification by Wingless signalling

Organ formation requires early specification of the groups of cells that will give rise to specific structures. The Wingless protein plays an important part in this regional specification of imaginal structures in Drosophila, including defining the region of the eye-antennal disc that will become retina. We show that Wingless signalling establishes the border between the retina and adjacent head structures by inhibiting the expression of the eye specification genes eyes absent, sine oculis and dachshund. Ectopic Wingless signalling leads to the repression of these genes and the loss of eyes, whereas loss of Wingless signalling has the opposite effects. Wingless expression in the anterior of wild-type discs is complementary to that of these eye specification genes. Contrary to previous reports, we find that under conditions of excess Wingless signalling, eye tissue is transformed not only into head cuticle but also into a variety of inappropriate structures.     

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.     

Parallel reduction in expression of the eye development gene hedgehog in separately derived cave populations of the amphipod Gammarus minus

Caves provide excellent settings to examine evolutionary questions. Subterranean environments are characterized by similar and consistent conditions. Cave-adapted species often share characteristics such as diminished pigmentation, elongated limbs and reduced or absent eyes. Relatively little is known about the evolution and development of troglomorphic traits in invertebrates. In this study, we compare expression of the eye development genes hedgehog, pax6, sine oculis and dachshund in individuals from multiple independently derived cave populations of the amphipod Gammarus minus. hedgehog expression was significantly reduced in cave populations, compared to genetically related surface populations. Interestingly, no differences were found in pax6, sine oculis or dachshund expression. Because hedgehog-related genes are also involved in eye reduced in Astyanax mexicanus, these genes may be consistent targets of evolution during cave adaptation. These results provide support for the hypothesis of genomic 'hotspots' of evolution and allow comparison of adaptive mechanisms among diverse animals in subterranean environments.