Two distinct mechanisms of Plexin A function in Drosophila optic lobe lamination and morphogenesis.

Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that, in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In Plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of semaphorin function indicates that Semaphorin 1a, acting in a subset of medulla neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A has little effect on the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of Plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles.

Transcriptional control of morphological properties of direction-selective T4/T5 neurons in Drosophila.

In the Drosophila visual system, T4/T5 neurons represent the first stage of computation of the direction of visual motion. T4 and T5 neurons exist in four subtypes, each responding to motion in one of the four cardinal directions and projecting axons into one of the four lobula plate layers. However, all T4/T5 neurons share properties essential for sensing motion. How T4/T5 neurons acquire their properties during development is poorly understood. We reveal that the transcription factors SoxN and Sox102F control the acquisition of properties common to all T4/T5 neuron subtypes, i.e. the layer specificity of dendrites and axons. Accordingly, adult flies are motion blind after disruption of SoxN or Sox102F in maturing T4/T5 neurons. We further find that the transcription factors Ato and Dac are redundantly required in T4/T5 neuron progenitors for SoxN and Sox102F expression in T4/T5 neurons, linking the transcriptional programmes specifying progenitor identity to those regulating the acquisition of morphological properties in neurons. Our work will help to link structure, function and development in a neuronal type performing a computation that is conserved across vertebrate and invertebrate visual systems.

Knock-in mutations of scarecrow, a Drosophila homolog of mammalian Nkx2.1, reveal a novel function required for development of the optic lobe in Drosophila melanogaster.

scarecrow (scro) gene encodes a Drosophila homolog of mammalian Nkx2.1 that belongs to an evolutionally conserved NK2 family. Nkx2.1 has been well known for its role in the development of hypothalamus, lung, thyroid gland, and brain. However, little is known about biological roles of scro. To understand scro functions, we generated two types of knock-in mutant alleles, substituting part of either exon-2 or exon-3 for EGFP (or Gal4) by employing the CRISPR/Cas9 genome editing tool. Using these mutations, we characterized spatio-temporal expression patterns of the scro gene and its mutant phenotypes. Homozygous knock-in mutants are lethal during embryonic and early larval development. In developing embryos, scro is exclusively expressed in the pharyngeal primordia and numerous neural clusters in the central nervous system (CNS). In postembryonic stages, the most prominent scro expression is detected in the larval and adult optic lobes, suggesting that scro plays a role for the development and/or function of this tissue type. Notch signaling is the earliest factor known to act for the development of the optic lobe. scro mutants lacked mitotic cells and Delta expression in the optic anlagen, and showed altered expression of several proneural and neurogenic genes including Delta and Notch. Furthermore, scro mutants showed grossly deformed neuroepithelial (NE) cells in the developing optic lobe and severely malformed adult optic lobes, the phenotypes of which are shown in Notch or Delta mutants, suggesting scro acting epistatic to the Notch signaling. From these data together, we propose that scro plays an essential role for the development of the optic lobe, possibly acting as a regional specification factor.

nox2/cybb Deficiency Affects Zebrafish Retinotectal Connectivity.

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) have been linked to neuronal polarity, axonal outgrowth, cerebellar development, regeneration of sensory axons, and neuroplasticity. However, the specific roles that individual Nox isoforms play during nervous system development in vivo remain unclear. To address this problem, we investigated the role of Nox activity in the development of retinotectal connections in zebrafish embryos. Zebrafish broadly express four nox genes (nox1, nox2/cybb, nox5, and duox) throughout the CNS during early development. Application of a pan-Nox inhibitor, celastrol, during the time of optic nerve (ON) outgrowth resulted in significant expansion of the ganglion cell layer (GCL), thinning of the ON, and a decrease in retinal axons reaching the optic tectum (OT). With the exception of GCL expansion, these effects were partially ameliorated by the addition of H2O2, a key ROS involved in Nox signaling. To address isoform-specific Nox functions, we used CRISPR/Cas9 to generate mutations in each zebrafish nox gene. We found that nox2/cybb chimeric mutants displayed ON thinning and decreased OT innervation. Furthermore, nox2/cybb homozygous mutants (nox2/cybb-/-) showed significant GCL expansion and mistargeted retinal axons in the OT. Neurite outgrowth from cultured zebrafish retinal ganglion cells was reduced by Nox inhibitors, suggesting a cell-autonomous role for Nox in these neurons. Collectively, our results show that Nox2/Cybb is important for retinotectal development in zebrafish.SIGNIFICANCE STATEMENT Most isoforms of NADPH oxidase (Nox) only produce reactive oxygen species (ROS) when activated by an upstream signal, making them ideal candidates for ROS signaling. Nox enzymes are present in neurons and their activity has been shown to be important for neuronal development and function largely by in vitro studies. However, whether Nox is involved in the development of axons and formation of neuronal connections in vivo has remained unclear. Using mutant zebrafish embryos, this study shows that a specific Nox isoform, Nox2/Cybb, is important for the establishment of axonal connections between retinal ganglion cells and the optic tectum.

Retinal determination genes coordinate neuroepithelial specification and neurogenesis modes in the Drosophila optic lobe.

Differences in neuroepithelial patterning and neurogenesis modes contribute to area-specific diversifications of neural circuits. In the Drosophila visual system, two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers, generate neuron subtypes for four ganglia in several ways. Whereas neuroepithelial cells in the medial OPC directly convert into neuroblasts, in an IPC subdomain they generate migratory progenitors by epithelial-mesenchymal transition that mature into neuroblasts in a second proliferative zone. The molecular mechanisms that regulate the identity of these neuroepithelia, including their neurogenesis modes, remain poorly understood. Analysis of Polycomblike revealed that loss of Polycomb group-mediated repression of the Hox gene Abdominal-B (Abd-B) caused the transformation of OPC to IPC neuroepithelial identity. This suggests that the neuroepithelial default state is IPC-like, whereas OPC identity is derived. Ectopic Abd-B blocks expression of the highly conserved retinal determination gene network members Eyes absent (Eya), Sine oculis (So) and Homothorax (Hth). These factors are essential for OPC specification and neurogenesis control. Finally, eya and so are also sufficient to confer OPC-like identity, and, in parallel with hth, the OPC-specific neurogenesis mode on the IPC.

Tumor protein Tctp regulates axon development in the embryonic visual system.

The transcript encoding translationally controlled tumor protein (Tctp), a molecule associated with aggressive breast cancers, was identified among the most abundant in genome-wide screens of axons, suggesting that Tctp is important in neurons. Here, we tested the role of Tctp in retinal axon development in Xenopus laevis We report that Tctp deficiency results in stunted and splayed retinotectal projections that fail to innervate the optic tectum at the normal developmental time owing to impaired axon extension. Tctp-deficient axons exhibit defects associated with mitochondrial dysfunction and we show that Tctp interacts in the axonal compartment with myeloid cell leukemia 1 (Mcl1), a pro-survival member of the Bcl2 family. Mcl1 knockdown gives rise to similar axon misprojection phenotypes, and we provide evidence that the anti-apoptotic activity of Tctp is necessary for the normal development of the retinotectal projection. These findings suggest that Tctp supports the development of the retinotectal projection via its regulation of pro-survival signalling and axonal mitochondrial homeostasis, and establish a novel and fundamental role for Tctp in vertebrate neural circuitry assembly.

Spatio-temporal pattern of neuronal differentiation in the Drosophila visual system: A user's guide to the dynamic morphology of the developing optic lobe.

Visual information processing in animals with large image forming eyes is carried out in highly structured retinotopically ordered neuropils. Visual neuropils in Drosophila form the optic lobe, which consists of four serially arranged major subdivisions; the lamina, medulla, lobula and lobula plate; the latter three of these are further subdivided into multiple layers. The visual neuropils are formed by more than 100 different cell types, distributed and interconnected in an invariant highly regular pattern. This pattern relies on a protracted sequence of developmental steps, whereby different cell types are born at specific time points and nerve connections are formed in a tightly controlled sequence that has to be coordinated among the different visual neuropils. The developing fly visual system has become a highly regarded and widely studied paradigm to investigate the genetic mechanisms that control the formation of neural circuits. However, these studies are often made difficult by the complex and shifting patterns in which different types of neurons and their connections are distributed throughout development. In the present paper we have reconstructed the three-dimensional architecture of the Drosophila optic lobe from the early larva to the adult. Based on specific markers, we were able to distinguish the populations of progenitors of the four optic neuropils and map the neurons and their connections. Our paper presents sets of annotated confocal z-projections and animated 3D digital models of these structures for representative stages. The data reveal the temporally coordinated growth of the optic neuropils, and clarify how the position and orientation of the neuropils and interconnecting tracts (inner and outer optic chiasm) changes over time. Finally, we have analyzed the emergence of the discrete layers of the medulla and lobula complex using the same markers (DN-cadherin, Brp) employed to systematically explore the structure and development of the central brain neuropil. Our work will facilitate experimental studies of the molecular mechanisms regulating neuronal fate and connectivity in the fly visual system, which bears many fundamental similarities with the retina of vertebrates.

Role of En2 in the tectal laminar formation of chick embryos.

The chick optic tectum consists of 16 laminae. Here, we report contribution of En2 to laminar formation in chick optic tecta. En2 is specifically expressed in laminae g-j of stratum griseum et fibrosum superficiale (SGFS). Misexpression of En2 resulted in disappearance of En2-expressing cells from the superficial layers (laminae a-f of SGFS), where endogenous En2 is not expressed. Misexpression of En2 before postmitotic cells had left the ventricular layer indicated that En2-misexpressing cells stopped at the laminae of endogenous En2 expression and that they did not migrate into the superficial layers. Induction of En2 misexpression using a tetracycline-inducible system after the postmitotic cells had reached superficial layers also resulted in disappearance of En2-expressing cells from the superficial layers. Time-lapse analysis showed that En2-misexpressing cells migrated back from the superficial layers towards the middle layers, where En2 is strongly expressed endogenously. Our results suggest a potential role of En2 in regulating cell migration and positioning in the tectal laminar formation.

A conserved transcriptional network regulates lamina development in the Drosophila visual system.

The visual system of insects is a multilayered structure composed externally by the compound eye and internally by the three ganglia of the optic lobe: lamina, medulla and the lobula complex. The differentiation of lamina neurons depends heavily on Hedgehog (Hh) signaling, which is delivered by the incoming photoreceptor axons, and occurs in a wave-like fashion. Despite the primary role of lamina neurons in visual perception, it is still unclear how these neurons are specified from neuroepithelial (NE) progenitors. Here we show that a homothorax (hth)-eyes absent (eya)-sine oculis (so)-dachshund (dac) gene regulatory cassette is involved in this specification. Lamina neurons differentiate from NE progenitors that express hth, eya and so. One of the first events in the differentiation of lamina neurons is the upregulation of dac expression in response to Hh signaling. We show that this dac upregulation, which marks the transition from NE progenitors into lamina precursors, also requires Eya/So, the expression of which is locked in by mutual feedback. dac expression is crucial for lamina differentiation because it ensures repression of hth, a negative regulator of single-minded, and thus dac allows further lamina neuron differentiation. Therefore, the specification of lamina neurons is controlled by coupling the cell-autonomous hth-eya-so-dac regulatory cassette to Hh signaling.

The role of the effector caspases drICE and dcp-1 for cell death and corpse clearance in the developing optic lobe in Drosophila.

In the developing Drosophila optic lobe, cell death occurs via apoptosis and in a distinctive spatio-temporal pattern of dying cell clusters. We analyzed the role of effector caspases drICE and dcp-1 in optic lobe cell death and subsequent corpse clearance using mutants. Neurons in many clusters required either drICE or dcp-1 and each one is sufficient. This suggests that drICE and dcp-1 function in cell death redundantly. However, dying neurons in a few clusters strictly required drICE but not dcp-1, but required drICE and dcp-1 when drICE activity was reduced via hypomorphic mutation. In addition, analysis of the mutants suggests an important role of effecter caspases in corpse clearance. In both null and hypomorphic drICE mutants, greater number of TUNEL-positive cells were observed than in wild type, and many TUNEL-positive cells remained until later stages. Lysotracker staining showed that there was a defect in corpse clearance in these mutants. All the results suggested that drICE plays an important role in activating corpse clearance in dying cells, and that an additional function of effector caspases is required for the activation of corpse clearance as well as that for carrying out cell death.

Ecdysone-dependent and ecdysone-independent programmed cell death in the developing optic lobe of Drosophila.

The adult optic lobe of Drosophila develops from the primordium during metamorphosis from mid-3rd larval stage to adult. Many cells die during development of the optic lobe with a peak of the number of dying cells at 24 h after puparium formation (h APF). Dying cells were observed in spatio-temporal specific clusters. Here, we analyzed the function of a component of the insect steroid hormone receptor, EcR, in this cell death. We examined expression patterns of two EcR isoforms, EcR-A and EcR-B1, in the optic lobe. Expression of each isoform altered during development in isoform-specific manner. EcR-B1 was not expressed in optic lobe neurons from 0 to 6h APF, but was expressed between 9 and 48 h APF and then disappeared by 60 h APF. In each cortex, its expression was stronger in older glia-ensheathed neurons than in younger ones. EcR-B1 was also expressed in some types of glia. EcR-A was expressed in optic lobe neurons and many types of glia from 0 to 60 h APF in a different pattern from EcR-B1. Then, we genetically analyzed EcR function in the optic lobe cell death. At 0 h APF, the optic lobe cell death was independent of any EcR isoforms. In contrast, EcR-B1 was required for most optic lobe cell death after 24 h APF. It was suggested that cell death cell-autonomously required EcR-B1 expressed after puparium formation. ßFTZ-F1 was also involved in cell death in many dying-cell clusters, but not in some of them at 24 h APF. Altogether, the optic lobe cell death occurred in ecdysone-independent manner at prepupal stage and ecdysone-dependent manner after 24 h APF. The acquisition of ecdysone-dependence was not directly correlated with the initiation or increase of EcR-B1 expression.

A temporal mechanism that produces neuronal diversity in the Drosophila visual center.

The brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf), correspond to types of medulla neurons. Here, we examine the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, we searched for transcription factors that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates (Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)). The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. Our results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequentially expressed in NBs.

Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

During neurogenesis in the medulla of the Drosophila optic lobe, neuroepithelial cells are programmed to differentiate into neuroblasts at the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to the lateral regions of the optic lobe, sweeping across the entire neuroepithelial sheet; it is preceded by the transient expression of the proneural gene lethal of scute [l(1)sc] and is thus called the proneural wave. We found that the epidermal growth factor receptor (EGFR) signaling pathway promotes proneural wave progression. EGFR signaling is activated in neuroepithelial cells and induces l(1)sc expression. EGFR activation is regulated by transient expression of Rhomboid (Rho), which is required for the maturation of the EGF ligand Spitz. Rho expression is also regulated by the EGFR signal. The transient and spatially restricted expression of Rho generates sequential activation of EGFR signaling and assures the directional progression of the differentiation wave. This study also provides new insights into the role of Notch signaling. Expression of the Notch ligand Delta is induced by EGFR, and Notch signaling prolongs the proneural state. Notch signaling activity is downregulated by its own feedback mechanism that permits cells at proneural states to subsequently develop into neuroblasts. Thus, coordinated sequential action of the EGFR and Notch signaling pathways causes the proneural wave to progress and induce neuroblast formation in a precisely ordered manner.

A role for juvenile hormone in the prepupal development of Drosophila melanogaster.

To elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, which produce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died at head eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe, downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen or by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-null mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the organization of their optic lobe neuropils. Feeding Met(27) larvae on the JHM diet did not rescue the premature EcR-B1 expression or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the development of the visual system that is likely to result from some cells 'misinterpreting' the ecdysteroid peaks that drive metamorphosis.

Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development.

Notch signaling mediates multiple developmental decisions in Drosophila. In this study, we have examined the role of Notch signaling in Drosophila larval optic lobe development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, our results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells.

Plexin a-semaphorin-1a reverse signaling regulates photoreceptor axon guidance in Drosophila.

While it is well established that Semaphorin family proteins function as axon guidance ligands in invertebrates and vertebrates, several recent studies indicate that the Drosophila Semaphorin-1a (Sema1a), a transmembrane Semaphorin, can also function as a receptor during neural development. The regulator of Sema1a reverse signaling, however, remains unknown. In this study, we show that like Sema1a, the well known Semaphorin receptor Plexin A (PlexA), is required for the proper guidance of photoreceptor (R cell) axons in the Drosophila visual system. Loss of PlexA, like loss of semala, disrupted the association of R-cell growth cones in the optic lobe. Conversely, overexpression of PlexA, like overexpression of sema1a, induced the hyperfasciculation of R-cell axons. Unlike Sema1a, however, the cytoplasmic domain of PlexA is dispensable. Epistasis analysis suggests that PlexA functions upstream of semala. And PlexA and sema1a interact genetically with Rho1. We propose that PlexA regulates Semala reverse signaling in the Drosophila visual system.

Conserved role of the Vsx genes supports a monophyletic origin for bilaterian visual systems.

BACKGROUND: Components of the genetic network specifying eye development are conserved from flies to humans, but homologies between individual neuronal cell types have been difficult to identify. In the vertebrate retina, the homeodomain-containing transcription factor Chx10 is required for both progenitor cell proliferation and the development of the bipolar interneurons, which transmit visual signals from photoreceptors to ganglion cells. RESULTS: We show that dVsx1 and dVsx2, the two Drosophila homologs of Chx10, play a conserved role in visual-system development. DVSX1 is expressed in optic-lobe progenitor cells, and, in dVsx1 mutants, progenitor cell proliferation is defective, leading to hypocellularity. Subsequently, DVSX1 and DVSX2 are coexpressed in a subset of neurons in the medulla, including the transmedullary neurons that transmit visual information from photoreceptors to deeper layers of the visual system. In dVsx mutant adults, the optic lobe is reduced in size, and the medulla is small or absent. These results suggest that the progenitor cells and photoreceptor target neurons of the vertebrate retina and fly optic lobe are ancestrally related. Genetic and functional homology may extend to the neurons directly downstream of the bipolar and transmedullary neurons, the vertebrate ganglion cells and fly lobula projection neurons. Both cell types project to visual-processing centers in the brain, and both sequentially express the Math5/ATO and Brn3b/ACJ6 transcription factors during their development. CONCLUSIONS: Our findings support a monophyletic origin for the bilaterian visual system in which the last common ancestor of flies and vertebrates already contained a primordial visual system with photoreceptors, interneurons, and projection neurons.

Fat/Hippo pathway regulates the progress of neural differentiation signaling in the Drosophila optic lobe.

A large number of neural and glial cell species differentiate from neuronal precursor cells during nervous system development. Two types of Drosophila optic lobe neurons, lamina and medulla neurons, are derived from the neuroepithelial (NE) cells of the outer optic anlagen. During larval development, epidermal growth factor receptor (EGFR)/Ras signaling sweeps the NE field from the medial edge and drives medulla neuroblast (NB) formation. This signal drives the transient expression of a proneural gene, lethal of scute, and we refer to its signal array as the "proneural wave," as it is the marker of the EGFR/Ras signaling front. In this study, we show that the atypical cadherin Fat and the downstream Hippo pathways regulate the transduction of EGFR/Ras signaling along the NE field and, thus, ensure the progress of NB differentiation. Fat/Hippo pathway mutation also disrupts the pattern formation of the medulla structure, which is associated with the regulation of neurogenesis. A candidate for the Fat ligand, Dachsous is expressed in the posterior optic lobe, and its mutation was observed to cause a similar phenotype as fat mutation, although in a regionally restricted manner. We also show that Dachsous functions as the ligand in this pathway and genetically interacts with Fat in the optic lobe. These findings provide new insights into the function of the Fat/Hippo pathway, which regulates the ordered progression of neurogenesis in the complex nervous system.

Gene regulatory networks during the development of the Drosophila visual system.

The Drosophila visual system integrates input from 800 ommatidia and extracts different features in stereotypically connected optic ganglia. The development of the Drosophila visual system is controlled by gene regulatory networks that control the number of precursor cells, generate neuronal diversity by integrating spatial and temporal information, coordinate the timing of retinal and optic lobe cell differentiation, and determine distinct synaptic targets of each cell type. In this chapter, we describe the known gene regulatory networks involved in the development of the different parts of the visual system and explore general components in these gene networks. Finally, we discuss the advantages of the fly visual system as a model for gene regulatory network discovery in the era of single-cell transcriptomics.

Expression patterns of the Egr1 and Egr3 genes during medaka embryonic development.

Egr1 and Egr3 are zinc finger-type transcription factors and known as synaptic activity-inducible immediate-early genes. Egr1 also plays important roles in many aspects of vertebrate development. Egr3 is known as the gene that is related to biological rhythm and muscular development, but its behavior in the central nervous system during development is not clear. We cloned the cDNA of the egr1 and egr3 orthologs in medaka, and examined their expression patterns during embryonic development using whole-mount in situ hybridization. Medaka egr3 was the first cloned egr3 gene in fish. The expression of egr1 mRNA was first detected at 1day post-fertilization (dpf). It was expressed in the whole embryonic body. At 3dpf, the egr1 mRNA was strongly expressed in the telencephalon, diencephalon, hypothalamus, optic tectum, dorsal medulla oblongata, retina, heart, pharynx, and pectoral fin. The expression of egr3 mRNA was first detected at 3dpf. It was expressed in the telencephalon, hypothalamus, optic tectum, and pharynx. By sectioning the whole-mount specimens, expression of both the egr1 and egr3 mRNAs were observed in the telencephalon, hypothalamus, and optic tectum. However, the positions at which the genes were expressed were different.