Mapping of the Lipid-Binding and Stability Properties of the Central Rod Domain of Human Dystrophin

Dystrophin is a cytoskeletal protein that confers resistance to the sarcolemma against the stress of contraction-relaxation cycles by interacting with cytoskeletal and membrane partners. Apart from several proteins, membrane phospholipids are a partner of the central rod domain made up of 24 spectrin-like repeats, separated into sub-domains by four hinges. We previously showed that repeats 1 to 3 bind to membrane anionic phospholipids, while repeats 20 to 24 are not able to do so. We focus here on the phospholipid-binding properties of the major part of the central rod domain, namely, the sub-domain delineated by hinges 2 and 3 comprising 16 repeats ranging from repeat 4 to 19 (R4-19). We designed and produced multirepeat proteins comprising three to five repeats and report their lipid-binding properties as well as their thermal stabilities. When these proteins are mixed with liposomes including the anionic lipid phosphatidylserine, they form stable protein-vesicle complexes as determined by gel-filtration chromatography. The absence of an anionic lipid precludes the formation of such complexes. Spectroscopic analyses by circular dichroism and tryptophan fluorescence show that, while the α-helical secondary structures are not modified by the binding, protein trans conformation leads to the movement of tryptophan residues into more hydrophobic environments. In addition, the decrease in the molar ellipticity ratio at 222/208 nm as observed by circular dichroism indicates that lipid binding reduces the inter-helical interactions of multirepeat proteins, thus suggesting partly “opened” coiled-coil structures. Combining these results with data from our previous studies, we propose a new model of the dystrophin molecule lying along the membrane bilayer, in which the two sub-domains R1-3 and R4-19 interact with lipids and F-actin, while the distal sub-domain R20-24 does not exhibit any interaction. These lipid-binding domains should thus maintain a structural link between cytoskeletal actin and sarcolemma via the membrane phospholipids.     

A triterpene oleanolic acid conjugate with 3-hydroxyflavone derivative as a new membrane probe with two-color ratiometric response

We report on the synthesis by coupling of a triterpenoid oleanolic acid with 4'-diethylamino-3-hydroxyflavone (FE) to produce an environment-sensitive biomembrane probe with two-band ratiometric response in fluorescence emission. The synthesized compound (probe FOT) was tested in a series of model solvents and demonstrated the response to solvent polarity and intermolecular hydrogen bonding very similar to that of parent probe FE. Meantime when incorporated into lipid bilayer membranes, it showed new features differing in response between lipids of different surface charges as well as between glycerophospholipids and sphingomyelin. We observed that in the conditions of coexistence of rafts and non-raft structures the probe is excluded from the rafts.     

The phospholipid headgroup specificity of an atp-dependent calcium pump

We have replaced the lipid associated with a purified calcium transport protein with a series of defined synthetic dioleoyl phospholipids in order to determine the effect of phospholipid headgroup structure on the ATPase activity of the protein. At 37°C the zwitterionic phospholipids (dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) support the highest activity, while a phospholipid with two negative charges (dioleoyl phosphatidic acid) supports an activity which is at least twenty times lower. Dioleoyl phospholipids with a single net negative charge support at intermediate ATPase activity which is not affected by the precise chemical structure of the phospholipid headgroup. The protocol used to determine the phospholipid headgroup specificity of calcium transport protein is novel because it establishes the composition of the lipid in contact with the protein without the need to isolate defined lipid-protein complexes. This allows the lipid specificity to be determined using only very small quantities of test lipids.We also determined the ability of the same phospholipids to support calcium accumulation in reconstituted membranes. Two requirements had to be met. The phospholipid had to support the ATPase activity of the pump protein and it had to form sealed vesicles as determined by electron microscopy. Since a number of phospholipids met those requirements it is clear that in vitro the lipid specificity of the calcium-accumulating system is rather broad.     

Effect of a cholesterol-rich lipid environment on the enzymatic activity of reconstituted hyaluronan synthase

Hyaluronan synthase (HAS) is a unique membrane-associated glycosyltransferase and its activity is lipid dependent. The dependence however is not well understood, especially in vertebrate systems. Here we investigated the functional association of hyaluronan synthesis in a cholesterol-rich membrane-environment. The culture of human dermal fibroblasts in lipoprotein-depleted medium attenuated the synthesis of hyaluronan. The sequestration of cellular cholesterol by methyl-ß-cyclodextrin also decreased the hyaluronan production of fibroblasts, as well as the HAS activity. To directly evaluate the effects of cholesterol on HAS activity, a recombinant human HAS2 protein with a histidine-tag was expressed as a membrane protein by using a baculovirus system, then successfully solubilized, and isolated by affinity chromatography. When the recombinant HAS2 proteins were reconstituted into liposomes composed of both saturated phosphatidylcholine and cholesterol, this provided a higher enzyme activity as compared with the liposomes formed by phosphatidylcholine alone. Cholesterol regulates HAS2 activity in a biphasic manner, depending on the molar ratio of phosphatidylcholine to cholesterol. Furthermore, the activation profiles of different lipid compositions were determined in the presence or absence of cholesterol. Cholesterol had the opposite effect on the HAS2 activity in liposomes composed of phosphatidylethanolamine or phosphatidylserine. Taken together, the present data suggests a clear functional association between HAS activity and cholesterol-dependent alterations in the physical and chemical properties of cell membranes.     

Direct Inhibition of Pumilo Activity by Bam and Bgcn in Drosophila Germ Line Stem Cell Differentiation

The fate of stem cells is intricately regulated by numerous extrinsic and intrinsic factors that promote maintenance or differentiation. The RNA-binding translational repressor Pumilio (Pum) in conjunction with Nanos (Nos) is required for self-renewal, whereas Bam (bag-of-marbles) and Bgcn (benign gonial cell neoplasm) promote differentiation of germ line stem cells in the Drosophila ovary. Genetic analysis suggests that Bam and Bgcn antagonize Pum/Nos function to promote differentiation; however, the molecular basis of this epistatic relationship is currently unknown. Here, we show that Bam and Bgcn inhibit Pum function through direct binding. We identified a ternary complex involving Bam, Bgcn, and Pum in which Bam, but not Bgcn, directly interacts with Pum, and this interaction is greatly increased by the presence of Bgcn. In a heterologous reporter assay to monitor Pum activity, Bam, but not Bgcn, inhibits Pum activity. Notably, the N-terminal region of Pum, which lacks the C-terminal RNA-binding Puf domain, mediates both the ternary protein interaction and the Bam inhibition of Pum function. These studies suggest that, in cystoblasts, Bam and Bgcn may directly inhibit Pum/Nos activity to promote differentiation of germ line stem cells.     

The Full-length Unprocessed Hedgehog Protein Is an Active Signaling Molecule

The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as ∼45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model we show here that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. We were also able to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of hh that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved.     

Evaluation of cytochrome c affinity to anionic phospholipids by means of surface plasmon resonance

We attempted to evaluate the affinity of the anionic phospholipids to cytochrome c by means of surface plasmon resonance (SPR) technique and to correlate it with the cytochrome c active site alterations and peroxidase activity. Our experiments showed a strong interdependence between the phospholipid fatty acid saturation degree, the active site structure alterations and peroxidase activity of the cytochrome c phospholipid complex. Cytochrome c peroxidase activity and Trp59 fluorescence increase in the sequence of phosphatidyl choline (PC) → phosphatidylserine (PS) → cardiolipin (CL) → phosphatidic acid (PA). The association constant (K_a) increased in the sequence PC → PA → PS → CL. The SPR spectroscopy data shows that K_a is independent of lipid saturation degree, but correlates with phospholipid negative charge value.     

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

The functional role of cytochrome (cyt) b₅₅₉ in photosystem II (PSII) was investigated in H22Kα and Y18Sα cyt b₅₅₉ mutants of the cyanobacterium Synechocystis sp. PCC6803. H22Kα and Y18Sα cyt b₅₅₉ mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b₅₅₉ in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b₅₅₉ in H22Kα mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b₅₅₉ in Y18Sα mutant PSII core complexes was shifted to 561 nm. Y18Sα and H22Kα mutant PSII core complexes contained predominately the low potential form of cyt b₅₅₉. The findings lend support to the concept that the redox properties of cyt b₅₅₉ are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b₅₅₉ mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b₅₅₉ in protection of PSII under photoinhibition conditions in vivo.     

Vaccinia Virus F1L Interacts with Bak Using Highly Divergent Bcl-2 Homology Domains and Replaces the Function of Mcl-1

The Bcl-2 family regulates induction of apoptosis at the mitochondria. Essential to this regulation are the interactions between Bcl-2 family members, which are mediated by Bcl-2 homology (BH) domains. Vaccinia virus F1L is a unique inhibitor of apoptosis that lacks significant sequence similarity with the Bcl-2 family and does not contain obvious BH domains. Despite this, F1L inhibits cytochrome c release from mitochondria by preventing Bak and Bax activation. Although F1L constitutively interacts with Bak to prevent Bak activation, the precise mechanism of this interaction remains elusive. We have identified highly divergent BH domains in F1L that were verified by the recent crystal structure of F1L (Kvansakul, M., Yang, H., Fairlie, W. D., Czabotar, P. E., Fischer, S. F., Perugini, M. A., Huang, D. C., and Colman, P. M. (2008) Cell Death Differ. 15, 1564–1571). Here we show that F1L required these BH domains to interact with ectopically expressed and endogenous Bak. The interaction between F1L and Bak was conserved across species, and both F1L and the cellular antiapoptotic protein Mcl-1 required the Bak BH3 domain for interaction. Moreover, F1L replaced Mcl-1 during infection, as the Bak·Mcl-1 complex was disrupted during vaccinia virus infection. In contrast to UV irradiation, vaccinia virus infection did not result in rapid degradation of Mcl-1, consistent with our observation that vaccinia virus did not initiate a DNA damage response. Additionally, Mcl-1 expression prevented Bak activation and apoptosis during infection with a proapoptotic vaccinia virus devoid of F1L. Our data suggest that F1L replaces the antiapoptotic activity of Mcl-1 during vaccinia virus infection by interacting with Bak using highly divergent BH domains.     

Oxidative inactivation of lactonase activity of purified human paraoxonase 1 (PON1)

Paraoxonase1 (PON1), one of HDL-associated antioxidant proteins, is known to lose its activity in vivo systems under oxidative stress. Here, we examined the effect of various oxidants on lactonase activity of PON1, and tried to protect the lactonase activity from oxidative inactivation. Among the oxidative systems tested, the ascorbate/Cu²⁺ system was the most potent in inactivating the lactonase activity of purified PON1; in contrast to a limited role of Fe²⁺, Cu²⁺ (0.05–1.0 µM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.02–0.1 mM). Moreover, Cu²⁺ alone inhibited the lactonase activity at concentrations as low as 1 µM. The ascorbate/Cu²⁺-mediated inactivation of PON1 lactonase activity was prevented by catalase, but not general hydroxyl radical scavengers, suggesting the implication of Cu²⁺-bound hydroxyl radicals in the oxidative inactivation. Compared to arylesterase activity, lactonase activity appears to be more sensitive to Cu²⁺-catalyzed oxidation. Separately, ascorbate/Cu²⁺-mediated inactivation of lactonase activity was prevented by oleic acid as well as phoshatidylcholine. Taken together, our data demonstrate that Cu²⁺-catalyzed oxidation may be a primary factor to cause the decrease of PON1 lactonase activity under oxidative stress and that lactonase activity of PON1 is most susceptible to ascorbate/Cu²⁺ among PON1 activities. In addition, we have showed that radical-induced inactivation of lactonase activity is prevented by some lipids.     

Nitric Oxide Binds to the Proximal Heme Coordination Site of the Ferrocytochrome c/Cardiolipin Complex: FORMATION MECHANISM AND DYNAMICS*

Mammalian mitochondrial cytochrome c interacts with cardiolipin to form a complex (cyt. c/CL) important in apoptosis. Here we show that this interaction leads to structural changes in ferrocytochrome c that leads to an open coordinate site on the central iron, resulting from the dissociation of the intrinsic methionine residue, where NO can rapidly bind (k = 1.2 × 10⁷ m⁻¹ s⁻¹). Accompanying NO binding, the proximal histidine dissociates leaving the heme pentacoordinate, in contrast to the hexacoordinate nitrosyl adducts of native ferrocytochrome c or of the protein in which the coordinating methionine is removed by chemical modification or mutation. We present the results of stopped-flow and photolysis experiments that show that following initial NO binding to the heme, there ensues an unusually complex set of kinetic steps. The spectral changes associated with these kinetic transitions, together with their dependence on NO concentration, have been determined and lead us to conclude that NO binding to cyt. c/CL takes place via an overall scheme comparable to that described for cytochrome c′ and guanylate cyclase, the final product being one in which NO resides on the proximal side of the heme. In addition, novel features not observed before in other heme proteins forming pentacoordinate nitrosyl species, include a high yield of NO escape after dissociation, rapid (<1 ms) dissociation of proximal histidine upon NO binding and its very fast binding (60 ps) after NO dissociation, and the formation of a hexacoordinate intermediate. These features all point at a remarkable mobility of the proximal heme environment induced by cardiolipin.     

Identification of novel oxidized levuglandin D2 in marine red alga and mouse tissue

In animals, the product of cyclooxygenase reacting with arachidonic acid, prostaglandin(PG)H(2), can undergo spontaneous rearrangement and nonenzymatic ring cleavage to form levuglandin(LG)E(2) and LGD(2). These LGs and their isomers are highly reactive γ-ketoaldehydes that form covalent adducts with proteins, DNA, and phosphatidylethanolamine in cells. Here, we isolated a novel oxidized LGD(2) (ox-LGD(2)) from the red alga Gracilaria edulis and determined its planar structure. Additionally, ox-LGD(2) was identified in some tissues of mice and in the lysate of phorbol-12-myristate-13-acetate (PMA)-treated THP-1 cells incubated with arachidonic acid using LC-MS/MS. These results suggest that ox-LGD(2) is a common oxidized metabolite of LGD(2). In the planar structure of ox-LGD(2), H8 and H12 of LGD(2) were dehydrogenated and the C9 aldehyde was oxidized to a carboxylic acid, which formed a lactone ring with the hydrated ketone at C11. These structural differences imply that ox-LGD(2) is less reactive with amines than LGs. Therefore, ox-LGD(2) might be considered a detoxification metabolite of LGD(2).     

Plasminogen activator urokinase expression reveals TRAIL responsiveness and supports fractional survival of cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10/Apo2L) holds promise for cancer therapy as it induces apoptosis in a large variety of cancer cells while exerting negligible toxicity in normal ones. However, TRAIL can also induce proliferative and migratory signaling in cancer cells resistant to apoptosis induced by this cytokine. In that regard, the molecular mechanisms underlying the tumor selectivity of TRAIL and those balancing apoptosis versus survival remain largely elusive. We show here that high mRNA levels of PLAU, which encodes urokinase plasminogen activator (uPA), are characteristic of cancer cells with functional TRAIL signaling. Notably, decreasing uPA levels sensitized cancer cells to TRAIL, leading to markedly increased apoptosis. Mechanistic analyses revealed three molecular events taking place in uPA-depleted cells: reduced basal ERK1/2 prosurvival signaling, decreased preligand decoy receptor 2 (DcR2)-death receptor 5 (DR5) interaction and attenuated recruitment of DcR2 to the death-inducing signaling complex upon TRAIL challenge. These phenomena were accompanied by increased FADD and procaspase-8 recruitment and processing, thus guiding cells toward a caspase-dependent cell death that is largely independent of the intrinsic apoptosis pathway. Collectively, our results unveil PLAU mRNA levels as marker for the identification of TRAIL-responsive tumor cells and highlight a key role of uPA signaling in ‘apoptosis versus survival'' decision-making processes upon TRAIL challenge.     

Autophagy in Drosophila ovaries is induced by starvation and is required for oogenesis

Autophagy, an evolutionarily conserved lysosome-mediated degradation, promotes cell survival under starvation and is controlled by insulin/target of rapamycin (TOR) signaling. In Drosophila, nutrient depletion induces autophagy in the fat body. Interestingly, nutrient availability and insulin/TOR signaling also influence the size and structure of Drosophila ovaries, however, the role of nutrient signaling and autophagy during this process remains to be elucidated. Here, we show that starvation induces autophagy in germline cells (GCs) and in follicle cells (FCs) in Drosophila ovaries. This process is mediated by the ATG machinery and involves the upregulation of Atg genes. We further demonstrate that insulin/TOR signaling controls autophagy in FCs and GCs. The analysis of chimeric females reveals that autophagy in FCs, but not in GCs, is required for egg development. Strikingly, when animals lack Atg gene function in both cell types, ovaries develop normally, suggesting that the incompatibility between autophagy-competent GCs and autophagy-deficient FCs leads to defective egg development. As egg morphogenesis depends on a tightly linked signaling between FCs and GCs, we propose a model in which autophagy is required for the communication between these two cell types. Our data establish an important function for autophagy during oogenesis and contributes to the understanding of the role of autophagy in animal development.     

The need to re-investigate the nature of homoplastic characters: an ontogenetic case study of the 'bracteoles' in Atripliceae (Chenopodiaceae)

Background and Aims

Within Chenopodioideae, Atripliceae have been distinguished by two bracteoles enveloping the female flowers/fruits, whereas in other tribes flowers are described as ebracteolate with persistent perianth. Molecular phylogenetic hypotheses suggest ‘bracteoles’ to be homoplastic. The origin of the bracteoles was explained by successive inflorescence reductions. Flower reduction was used to explain sex determination. Therefore, floral ontogeny was studied to evaluate the nature of the bracteoles and sex determination in Atripliceae.

Methods

Inflorescences of species of Atriplex, Chenopodium, Dysphania and Spinacia oleracea were investigated using light microscopy and scanning electron microscopy.

Key Results

The main axis of the inflorescence is indeterminate with elementary dichasia as lateral units. Flowers develop centripetally, with first the formation of a perianth primordium either from a ring primordium or from five individual tepal primordia fusing post-genitally. Subsequently, five stamen primordia originate, followed by the formation of an annular ovary primordium surrounding a central single ovule. Flowers are either initially hermaphroditic remaining bisexual and/or becoming functionally unisexual at later stages, or initially unisexual. In the studied species of Atriplex, female flowers are strictly female, except in A. hortensis. In Spinacia, female and male flowers are unisexual at all developmental stages. Female flowers of Atriplex and Spinacia are protected by two accrescent fused tepal lobes, whereas the other perianth members are absent.

Conclusions

In Atriplex and Spinacia modified structures around female flowers are not bracteoles, but two opposite accrescent tepal lobes, parts of a perianth persistent on the fruit. Flowers can achieve sexuality through many different combinations; they are initially hermaphroditic, subsequently developing into bisexual or functionally unisexual flowers, with the exception of Spinacia and strictly female flowers in Atriplex, which are unisexual from the earliest developmental stages. There may be a relationship between the formation of an annular perianth primordium and flexibility in floral sex determination.     

Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori

Metamorphosis in insects includes a series of programmed tissue histolysis and remolding processes that are controlled by two major classes of hormones, juvenile hormones and ecdysteroids. Precise pulses of ecdysteroids (the most active ecdysteroid is 20-hydroxyecdysone, 20E), are regulated by both biosynthesis and metabolism. In this study, we show that ecdysone oxidase (EO), a 20E inactivation enzyme, expresses predominantly in the midgut during the early pupal stage in the lepidopteran model insect, Bombyx mori. Depletion of BmEO using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system extended the duration of the final instar larval stage. Ubiquitous transgenic overexpression of BmEO using the Gal4/UAS system induced lethality during the larval–pupal transition. When BmEO was specifically overexpressed in the middle silk gland (MSG), degeneration of MSG at the onset of metamorphosis was blocked. Transmission electron microscope and LysoTracker analyses showed that the autophagy pathway in MSG is inhibited by BmEO ectopic expression. Furthermore, RNA-seq analysis revealed that the genes involved in autophagic cell death and the mTOR signal pathway are affected by overexpression of BmEO. Taken together, BmEO functional studies reported here provide insights into ecdysone regulation of tissue degeneration during metamorphosis.     

SCAR/WAVE-mediated processing of engulfed apoptotic corpses is essential for effective macrophage migration in Drosophila

In vitro studies have shown that SCAR/WAVE activates the Arp2/3 complex to generate actin filaments, which in many cell types are organised into lamellipodia that are thought to have an important role in cell migration. Here we demonstrate that SCAR is utilised by Drosophila macrophages to drive their developmental and inflammatory migrations and that it is regulated via the Hem/Kette/Nap1-containing SCAR/WAVE complex. SCAR is also important in protecting against bacterial pathogens and in wound repair as SCAR mutant embryos succumb more readily to both sterile and infected wounds. However, in addition to driving the formation of lamellipodia in macrophages, SCAR is required cell autonomously for the correct processing of phagocytosed apoptotic corpses by these professional phagocytes. Removal of this phagocytic burden by preventing apoptosis rescues macrophage lamellipodia formation and partially restores motility. Our results show that efficient processing of phagosomes is critical for effective macrophage migration in vivo. These findings have important implications for the resolution of macrophages from chronic wounds and the behaviour of those associated with tumours, because phagocytosis of debris may serve to prolong the presence of these cells at these sites of pathology.     

Alpha-synuclein selectively binds to anionic phospholipids embedded in liquid-disordered domains

Previous studies indicate that binding of α-synuclein to membranes is critical for its physiological function and the development of Parkinson's disease (PD). Here, we have investigated the association of fluorescence-labeled α-synuclein variants with different types of giant unilamellar vesicles using confocal microscopy. We found that α-synuclein binds with high affinity to anionic phospholipids, when they are embedded in a liquid-disordered as opposed to a liquid-ordered environment. This indicates that not only electrostatic forces but also lipid packing and hydrophobic interactions are critical for the association of α-synuclein with membranes in vitro. When compared to wild-type α-synuclein, the disease-causing α-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that the natural association of α-synuclein with membranes is altered in the inherited forms of Parkinson's disease.     

Conserved roles of ems/Emx and otd/Otx genes in olfactory and visual system development in Drosophila and mouse

The regional specialization of brain function has been well documented in the mouse and fruitfly. The expression of regulatory factors in specific regions of the brain during development suggests that they function to establish or maintain this specialization. Here, we focus on two such factors—the Drosophila cephalic gap genes empty spiracles (ems) and orthodenticle (otd), and their vertebrate homologues Emx1/2 and Otx1/2—and review novel insight into their multiple crucial roles in the formation of complex sensory systems. While the early requirement of these genes in specification of the neuroectoderm has been discussed previously, here we consider more recent studies that elucidate the later functions of these genes in sensory system formation in vertebrates and invertebrates. These new studies show that the ems and Emx genes in both flies and mice are essential for the development of the peripheral and central neurons of their respective olfactory systems. Moreover, they demonstrate that the otd and Otx genes in both flies and mice are essential for the development of the peripheral and central neurons of their respective visual systems. Based on these recent experimental findings, we discuss the possibility that the olfactory and visual systems of flies and mice share a common evolutionary origin, in that the conserved visual and olfactory circuit elements derive from conserved domains of otd/Otx and ems/Emx action in the urbilaterian ancestor.