Biomedical subjects

Graeme Mardon

Publications and source records attributed to Graeme Mardon.

At least 19 recordsLinked to original sources

PubMed2006

A genetic screen in Drosophila for genes interacting with senseless during neuronal development identifies the importin moleskin.

Senseless (Sens) is a conserved transcription factor required for normal development of the Drosophila peripheral nervous system. In the Drosophila retina, sens is necessary and sufficient for differentiation of R8 photoreceptors and interommatidial bristles (IOBs). When Sens is expressed in undifferentiated cells posterior to the morphogenetic furrow, ectopic IOBs are formed. This phenotype was used to identify new members of the sens pathway in a dominant modifier screen. Seven suppressor and three enhancer complementation groups were isolated. Three groups from the screen are the known genes Delta, lilliputian, and moleskin/DIM-7 (msk), while the remaining seven groups represent novel genes with previously undefined functions in neural development. The nuclear import gene msk was identified as a potent suppressor of the ectopic interommatidial bristle phenotype. In addition, msk mutant adult eyes are extremely disrupted with defects in multiple cell types. Reminiscent of the sens mutant phenotype, msk eyes demonstrate reductions in the number of R8 photoreceptors due to an R8 to R2,5 fate switch, providing genetic evidence that Msk is a component of the sens pathway. Interestingly, in msk tissue, the loss of R8 fate occurs earlier than with sens and suggests a previously unidentified stage of R8 development between atonal and sens.

Animals↗

PubMed2006

Genome-wide identification of direct targets of the Drosophila retinal determination protein Eyeless.

The discovery of direct downstream targets of transcription factors (TFs) is necessary for understanding the genetic mechanisms underlying complex, highly regulated processes such as development. In this report, we have used a combinatorial strategy to conduct a genome-wide search for novel direct targets of Eyeless (Ey), a key transcription factor controlling early eye development in Drosophila. To overcome the lack of high-quality consensus binding site sequences, phylogenetic shadowing of known Ey binding sites in sine oculis (so) was used to construct a position weight matrix (PWM) of the Ey protein. This PWM was then used for in silico prediction of potential binding sites in the Drosophila melanogaster genome. To reduce the false positive rate, conservation of these potential binding sites was assessed by comparing the genomic sequences from seven Drosophila species. In parallel, microarray analysis of wild-type versus ectopic ey-expressing tissue, followed by microarray-based epistasis experiments in an atonal (ato) mutant background, identified 188 genes induced by ey. Intersection of in silico predicted conserved Ey binding sites with the candidate gene list produced through expression profiling yields a list of 20 putative ey-induced, eye-enriched, ato-independent, direct targets of Ey. The accuracy of this list of genes was confirmed using both in vitro and in vivo methods. Initial analysis reveals three genes, eyes absent, shifted, and Optix, as novel direct targets of Ey. These results suggest that the integrated strategy of computational biology, genomics, and genetics is a powerful approach to identify direct downstream targets for any transcription factor genome-wide.

Animals↗

PubMed2006

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.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

Keeping an eye on the fly genome.

With its unique structure and dynamic development, the Drosophila eye has been a powerful genetic model system for studying molecular mechanisms of cell fate specification and differentiation. Hundreds of genes that function in a complex genetic network controlling this process have been identified during the past two decades. To further advance our understanding of the molecular mechanisms of eye development, it is increasingly important to place the current genetic pathway into a whole-genome perspective. Here, we review emerging technologies and strategies that will help to achieve this goal, including generation of a complete mutant set in Drosophila, genome-wide transcription factor target identification, and systematic studies of gene function aided by computational biology.

Animals↗

PubMed2005

Whole-mount analysis reveals normal numbers of dopaminergic neurons following misexpression of alpha-Synuclein in Drosophila.

Previously published reports have suggested that misexpression of alpha-Synuclein in the Drosophila central nervous system causes neurodegeneration and progressive age-dependent locomotor dysfunction similar to pathologic and clinical manifestations of Parkinson's disease. The number of dopaminergic (DA) neurons in these studies was assessed using immunohistochemistry with an anti-tyrosine hydroxylase antibody on sequential paraffin sections of fly brains. In contrast, we do not observe any DA cell loss in alpha-Synuclein expressing fly brains when using whole-mount immunohistochemistry as an assay. Our results suggest that the DA cell loss observed with misexpression of alpha-Synuclein is not fully penetrant under a variety of experimental conditions and that this may complicate interpretation of such experiments.

Aging↗

PubMed2005

Dual regulation and redundant function of two eye-specific enhancers of the Drosophila retinal determination gene dachshund.

Drosophila eye development is controlled by a conserved network of retinal determination (RD) genes. The RD genes encode nuclear proteins that form complexes and function in concert with extracellular signal-regulated transcription factors. Identification of the genomic regulatory elements that govern the eye-specific expression of the RD genes will allow us to better understand how spatial and temporal control of gene expression occurs during early eye development. We compared conserved non-coding sequences (CNCSs) between five Drosophilids along the approximately 40 kb genomic locus of the RD gene dachshund (dac). Our analysis uncovers two separate eye enhancers in intron eight and the 3' non-coding regions of the dac locus defined by clusters of highly conserved sequences. Loss- and gain-of-function analyses suggest that the 3' eye enhancer is synergistically activated by a combination of eya, so and dpp signaling, and only indirectly activated by ey, whereas the 5' eye enhancer is primarily regulated by ey, acting in concert with eya and so. Disrupting conserved So-binding sites in the 3' eye enhancer prevents reporter expression in vivo. Our results suggest that the two eye enhancers act redundantly and in concert with each other to integrate distinct upstream inputs and direct the eye-specific expression of dac.

3' Untranslated Regions↗

PubMed2004

Regulation of R7 and R8 differentiation by the spalt genes.

Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of the developing ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt was previously proposed to promote R7 and R8 terminal differentiation. Here we show that spalt is required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta0.5, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specification or differentiation in the larval disc but promotes terminal differentiation during pupation. We show that spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. We demonstrate that spalt and senseless are part of a genetic network, which regulates rhodopsin 6 and rhodopsin 1. Taken together, our results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8 differentiation during pupal stages, including the regulation of rhodopsin expression.

Animals↗

PubMed2004

Structure-function analysis of the Drosophila retinal determination protein Dachshund.

Dachshund (Dac) is a highly conserved nuclear protein that is distantly related to the Ski/Sno family of corepressor proteins. In Drosophila, Dac is necessary and sufficient for eye development and, along with Eyeless (Ey), Sine oculis (So), and Eyes absent (Eya), forms the core of the retinal determination (RD) network. In vivo and in vitro experiments suggest that members of the RD network function together in one or more complexes to regulate the expression of downstream targets. For example, Dac and Eya synergize in vivo to induce ectopic eye formation and they physically interact through conserved domains. Dac contains two highly conserved domains, named DD1 and DD2, but no function has been assigned to either of them in an in vivo context. We performed structure-function studies to understand the relationship between the conserved domains of Dac and the rest of the protein and to determine the function of each domain during development. We show that only DD1 is essential for Dac function and while DD2 facilitates DD1, it is not absolutely essential in spite of more than 500 million years of conservation. Moreover, the physical interaction between Eya and DD2 is not required for the genetic synergy between the two proteins. Finally, we show that DD1 also plays a central role for nuclear localization of Dac.

Amino Acid Sequence↗

PubMed2004

Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress.

Mutations in the gene parkin in humans (PARK2) are responsible for a large number of familial cases of autosomal-recessive Parkinson disease. We have isolated a Drosophila homolog of human PARK2 and characterized its expression and null phenotype. parkin null flies have 30% lower mass than wild-type controls which is in part accounted for by a reduced cell size and number. In addition, these flies are infertile, show significantly reduced longevity, and are unable to jump or fly. Rearing mutants on paraquat, which generates toxic free radicals in vivo, causes a further reduction in longevity. Furthermore, loss of parkin results in progressive degeneration of most indirect flight muscle (IFM) groups soon after eclosion, accompanied by apoptosis. However, parkin mutants have normal neuromuscular junction recordings during the third larval instar stage, suggesting that larval musculature is intact and that parkin is required only in pupal and adult muscle. parkin flies do not show an age-dependent dopaminergic neuron loss in the brain, even after aging adults for 3 weeks. Nevertheless, degeneration of IFMs demonstrates the importance of parkin in maintaining specific cell groups, perhaps those with a high-energy demand and the concomitant production of high levels of free radicals. parkin mutants will be a valuable model for future analysis of the mechanisms of cell and tissue degeneration.

Amino Acid Sequence↗

PubMed2004

Senseless represses nuclear transduction of Egfr pathway activation.

The Epidermal growth factor receptor (Egfr) pathway controls cell fate decisions throughout phylogeny. Typically, binding of secreted ligands to Egfr on the cell surface initiates a well-described cascade of events that ultimately invokes transcriptional changes in the nucleus. In contrast, the mechanisms by which autocrine effects are regulated in the ligand-producing cell are unclear. In the Drosophila eye, Egfr signaling, induced by the Spitz ligand, is required for differentiation of all photoreceptors except for R8, the primary source of Spitz. R8 differentiation is instead under the control of the transcription factor Senseless. We show that high levels of Egfr activation are incompatible with R8 differentiation and describe the mechanism by which Egfr signaling is actively prevented in R8. Specifically, Senseless does not affect cytoplasmic transduction of Egfr activation, but does block nuclear transduction of Egfr activation through transcriptional repression of pointed, which encodes the nuclear effector of the pathway. Thus, Senseless promotes normal R8 differentiation by preventing the effects of autocrine stimulation by Spitz. An analogous relationship exists between Senseless and Egfr pathway orthologs in T-lymphocytes, suggesting that this mode of repression of Egfr signaling is conserved.

Animals↗

PubMed2004

Senseless is required for pupal retinal development in Drosophila.

Drosophila sensory organs are specified by a family of proneural genes which induce the expression of several common targets. One such target is senseless, which encodes a zinc finger transcription factor. We analyzed the function of senseless during pupal retinal development and found that senseless is required for recruitment of both cone and pigment cells, the pupal-derived ommatidial support cells. We also found that Senseless is expressed in neural precursors shortly after the larval-pupal transition and is both necessary and sufficient for interommatidial bristle development. Furthermore, senseless is the primary target of achaete and scute during interommatidial bristle development. We also identified several differences between the development of interommatidial bristles and other macrochaete. In particular, EGFR signaling is not required for interommatidial bristle development, nor is positive feedback regulation of proneural genes by senseless. A model for interommatidial bristle specification is presented.

Animals↗

PubMed2004

Genetic control of retinal specification and determination in Drosophila.

The Drosophila compound eye has long served as an outstanding model system to study many processes, including cell fate specification, cell division, cell growth and cell death. In addition, exploring the molecular basis of eye specification in Drosophila has identified a set of nuclear factors that trigger the conversion of a group of multipotent epithelial cells into eye primordia. These nuclear factors act in complex networks to regulate retinal specification and appear to be conserved throughout phylogeny. Finally, evidence suggests that these nuclear networks have been co-opted to specify cell fates in other tissues. We review the latest developments in the field of retinal specification in Drosophila and discuss several future directions that remain open for investigation.

Animals↗

PubMed2003

Eyes absent represents a class of protein tyrosine phosphatases.

The Eyes absent proteins are members of a conserved regulatory network implicated in the development of the eye, muscle, kidney and ear. Mutations in the Eyes absent genes have been associated with several congenital disorders including the multi-organ disease bronchio-oto-renal syndrome, congenital cataracts and late-onset deafness. On the basis of previous analyses it has been shown that Eyes absent is a nuclear transcription factor, acting through interaction with homeodomain-containing Sine oculis (also known as Six) proteins. Here we show that Eyes absent is also a protein tyrosine phosphatase. It does not resemble the classical tyrosine phosphatases that use cysteine as a nucleophile and proceed by means of a thiol-phosphate intermediate. Rather, Eyes absent is the prototype for a class of protein tyrosine phosphatases that use a nucleophilic aspartic acid in a metal-dependent reaction. Furthermore, the phosphatase activity of Eyes absent contributes to its ability to induce eye formation in Drosophila.

Amino Acid Sequence↗

PubMed2003

Drosophila retinal homeobox (drx) is not required for establishment of the visual system, but is required for brain and clypeus development.

The possibility that mechanisms of retinal determination may be similar between vertebrates and Drosophila has been supported by the observations that Pax6/eyeless genes are necessary and sufficient for retinal development. These studies suggest that the function of other gene families, operating during early eye development, might also be conserved. One candidate is the retinal homeobox (Rx) family of transcription factors. Vertebrate Rx is expressed in the prospective eye and forebrain and is required for eye morphogenesis, retinal precursor appearance, and normal forebrain development, indicating that it is an essential regulator of early eye and brain formation. Here, we test the hypothesis that Drosophila Rx (drx) is required for adult and larval eye development. We have isolated a drx null allele and demonstrate that the mutant compound eye and larval visual system is not detectably abnormal. However, we find that drx is required for development of a central brain structure, the ellipsoid body, suggesting that Rx function in the brain may be conserved. Finally, we characterize a novel anterior head phenotype and demonstrate that drx is required for clypeus development. Thus, our data suggest that drx may be required for the regulation of genes involved in brain morphogenesis and clypeus precursor development. We propose that differences in insect and vertebrate eye development may be explained by changes in gene regulation and/or the tissue of origin for eye precursor cells.

Alleles↗

PubMed2003

Mechanism of hedgehog signaling during Drosophila eye development.

Although Hedgehog (Hh) signaling is essential for morphogenesis of the Drosophila eye, its exact link to the network of tissue-specific genes that regulate retinal determination has remained elusive. In this report, we demonstrate that the retinal determination gene eyes absent (eya) is the crucial link between the Hedgehog signaling pathway and photoreceptor differentiation. Specifically, we show that the mechanism by which Hh signaling controls initiation of photoreceptor differentiation is to alleviate repression of eya and decapentaplegic (dpp) expression by the zinc-finger transcription factor Cubitus interruptus (Ci(rep)). Furthermore, our results suggest that stabilized, full length Ci (Ci(act)) plays little or no role in Drosophila eye development. Moreover, while the effects of Hh are primarily concentration dependent in other tissues, hh signaling in the eye acts as a binary switch to initiate retinal morphogenesis by inducing expression of the tissue-specific factor Eya.

Animals↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2002

Cloning and expression of medaka Dachshund.

The nuclear factor dachshund (dac) is a regulator of retinal cell fate determination in Drosophila. We have cloned a Dachshund homologue of a lower vertebrate, the teleost medaka (Oryzias latipes). Sequence comparison reveals high similarity of medaka Dachshund (OlDach) to the known homologues in higher vertebrates and Drosophila. OlDach is first expressed at early somitogenesis stages in the otic placode territory and forming somites. Subsequently, expression is detected in the retina, specific regions of the central nervous system, pancreas and the finbuds.

Amino Acid Sequence↗