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.     

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.     

Six3 activation of Pax6 expression is essential for mammalian lens induction and specification

The homeobox gene Six3 regulates forebrain development. Here we show that Six3 is also crucial for lens formation. Conditional deletion of mouse Six3 in the presumptive lens ectoderm (PLE) disrupted lens formation. In the most severe cases, lens induction and specification were defective, and the lens placode and lens were absent. In Six3-mutant embryos, Pax6 was downregulated, and Sox2 was absent in the lens preplacodal ectoderm. Using ChIP, electrophoretic mobility shift assay, and luciferase reporter assays, we determined that Six3 activates Pax6 and Sox2 expression. Misexpression of mouse Six3 into chick embryos promoted the ectopic expansion of the ectodermal Pax6 expression domain. Our results position Six3 at the top of the regulatory pathway leading to lens formation. We conclude that Six3 directly activates Pax6 and probably also Sox2 in the PLE and regulates cell autonomously the earliest stages of mammalian lens induction.     

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.     

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.     

Segregation of lens and olfactory precursors from a common territory: cell sorting and reciprocity of Dlx5 and Pax6 expression

Cranial placodes are focal regions of columnar epithelium next to the neural tube that contribute to sensory ganglia and organs in the vertebrate head, including the olfactory epithelium and the crystalline lens of the eye. Using focal dye labelling within the presumptive placode domain, we show that lens and nasal precursors arise from a common territory surrounding the anterior neural plate. They then segregate over time and converge to their final positions in discrete placodes by apparently directed movements. Since these events closely parallel the separation of eye and antennal primordia (containing olfactory sensory cells) from a common imaginal disc in Drosophila, we investigated whether the vertebrate homologues of Distalless (Dll) and Eyeless (Ey), which determine antennal and eye identity in the fly, play a role in segregation of lens and nasal precursors in the chick. Dlx5 and Pax6 are initially co-expressed by future lens and olfactory cells. As soon as presumptive lens cells acquire columnar morphology all Dlx family members are down-regulated in the placode, while Pax6 is lost in the olfactory region. Lens precursor cells that express ectopic Dlx5 never acquire lens-specific gene expression and are excluded from the lens placode to cluster in the head ectoderm. These results suggest that the loss of Dlx5 is required for cells to adopt a lens fate and that the balance of Pax6 and Dlx expression regulates cell sorting into appropriate placodal domains.     

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.     

BMP7 acts in murine lens placode development

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation.     

Cell-autonomous involvement of Mab21l1 is essential for lens placode development

The mab-21 gene was first identified because of its requirement for ray identity specification in Caenorhabditis elegans. It is now known to constitute a family of genes that are highly conserved from vertebrates to invertebrates, and two homologs, Mab21l1 and Mab21l2, have been identified in many species. We describe the generation of Mab21l1-deficient mice with defects in eye and preputial gland formation. The mutant mouse eye has a rudimentary lens resulting from insufficient invagination of the lens placode caused by deficient proliferation. Chimera analyses suggest that the lens placode is affected in a cell-autonomous manner, although Mab21l1 is expressed in both the lens placode and the optic vesicle. The defects in lens placode development correlate with delayed and insufficient expression of Foxe3, which is also required for lens development, while Maf, Sox2, Six3 and PAX6 levels are not significantly affected. Significant reduction of Mab21l1 expression in the optic vesicle and overlying surface ectoderm in Sey homozygotes indicates that Mab21l1 expression in the developing eye is dependent upon the functions of Pax6 gene products. We conclude that Mab21l1 expression dependent on PAX6 is essential for lens placode growth and for formation of the lens vesicle; lack of Mab21l1 expression causes reduced expression of Foxe3 in a cell-autonomous manner.     

Chordate betagamma-crystallins and the evolutionary developmental biology of the vertebrate lens

Several animal lineages, including the vertebrates, have evolved sophisticated eyes with lenses that refract light to generate an image. The nearest invertebrate relatives of the vertebrates, such as the ascidians (sea squirts) and amphioxus, have only basic light detecting organs, leading to the widely-held view that the vertebrate lens is an innovation that evolved in early vertebrates. From an embryological perspective the lens is different from the rest of the eye, in that the eye is primarily of neural origin while the lens derives from a non-neural ectodermal placode which invaginates into the developing eye. How such an organ could have evolved has attracted much speculation. Recently, however, molecular developmental studies of sea squirts have started to suggest a possible evolutionary origin for the lens. First, studies of the Pax, Six, Eya and other gene families have indicated that sea squirts have areas of non-neural ectoderm homologous to placodes, suggesting an origin for the embryological characteristics of the lens. Second, the evolution and regulation of the betagamma-crystallins has been studied. These form one of the key crystallin gene families responsible for the transparency of the lens, and regulatory conservation between the betagamma-crystallin gene in the sea squirt Ciona intestinalis and the vertebrate visual system has been experimentally demonstrated. These data, together with knowledge of the morphological, physiological and gene expression similarities between the C. intestinalis ocellus and vertebrate retina, have led us to propose a hypothesis for the evolution of the vertebrate lens and integrated vertebrate eye via the co-option and combination of ancient gene regulatory networks; one controlling morphogenetic aspects of lens development and one controlling the expression of a gene family responsible for the biophysical properties of the lens, with the components of the retina having evolved from an ancestral photoreceptive organ derived from the anterior central nervous system.