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Mutations disrupting neuronal connectivity in the Drosophila visual system
Abstract
The photoreceptor neurons (R cells) of the Drosophila compound eye elaborate a precise array of neuronal connections in the brain. These projections exhibit target specificity and create topographic maps (retinotopy). We have screened histologically for mutations disrupting R cell connectivity in developing tissue. Eighty mutations were isolated from over 6000 ethylmethane sulfonate-mutagenized lines. Characterization of these mutations included genetic mosaic analysis to determine whether the gene is required in the retina or in the optic ganglia. Most mutations were found to affect connectivity indirectly by disrupting development more generally in the eye or brain. Genes were identified as candidates for playing direct roles in R cell connectivity by affecting axonal outgrowth (eddy), target recognition (limbo and nonstop), and retinotopy (limbo).
... Genetic studies in Caenorhabditis elegans and Drosophila have been an effective means of identifying evolutionarily conserved molecular regulators of axonal growth cone guidance. So far, these studies have identified components of the four major guidance cue systems (the netrins, slits, semaphorins, and ephrins) and a variety of morphogens [1][2][3][4][5][6][7][8][9][10][11][12][13]. ...
... One of the areas where axons make a major decision on their projection path is the embryonic midline [1][2][3][4][5][6][7][8][9][10][11][12][13]. Embryonic midline cells are the main source of secreted midline guidance cues encoded by the netrin and slit genes [2,5,7,[9][10][11][12][13]. ...
... sequence tag (EST) clones AT02763 and GH15753 near the 5'-end of the gene are still present even in the largest deletion, tutl 4 . With this in mind, we mobilized the tutl k14703 P-element insertions to generate null mutation of the turtle gene ( Figure 1A). ...
Neuronal growth cones follow specific pathways over long distances in order to reach their appropriate targets. Research over the past 15 years has yielded a large body of information concerning the molecules that regulate this process. Some of these molecules, such as the evolutionarily conserved netrin and slit proteins, are expressed in the embryonic midline, an area of extreme importance for early axon pathfinding decisions. A general model has emerged in which netrin attracts commissural axons towards the midline while slit forces them out. However, a large number of commissural axons successfully cross the midline even in the complete absence of netrin signaling, indicating the presence of a yet unidentified midline attractant.
The evolutionarily conserved Ig proteins encoded by the turtle/Dasm1 genes are found in Drosophila, Caenorhabditis elegans, and mammals. In Drosophila the turtle gene encodes five proteins, two of which are diffusible, that are expressed in many areas, including the vicinity of the midline. Using both molecular null alleles and transgenic expression of the different isoforms, we show that the turtle encoded proteins function as non-cell autonomous axonal attractants that promote midline crossing via a netrin-independent mechanism. turtle mutants also have either stalled or missing axon projections, while overexpression of the different turtle isoforms produces invasive neurons and branching axons that do not respect the histological divisions of the nervous system.
Our findings indicate that the turtle proteins function as axon guidance cues that promote midline attraction, axon branching, and axonal invasiveness. The latter two capabilities are required by migrating axons to explore densely packed targets.
... Here, we report our identification of nonstop (not) from a screen of DUBs involved in BC migration. not encodes the Drosophila USP22 orthologue (Martin et al., 1995) and is best known as the enzymatic component of the histone H2B DUB module of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex (Koutelou et al., 2010;Lee et al., 2011;Zhang et al., 2008). Histone modifications such as acetylation and ubiquitination modulate the accessibility of genomic loci to transcriptional machinery, with ubiquitination being associated with both activation and repression . ...
... not/USP22 plays essential roles during embryogenesis in Drosophila and mammals (Li et al., 2017;Lin et al., 2012) as well as in neural development and lineage specification (Kosinsky et al., 2015). In the Drosophila nervous system, not loss of function is associated with defects in the migration of a subset of glial cells to their appropriate position in the developing optic lobe and subsequent targeting of photoreceptor axons in the lamina (Martin et al., 1995;Poeck et al., 2001). The underlying mechanisms are not fully understood, but it has recently been suggested that this role may be mediated in part by a SAGA-independent role of Not in deubiquitinating and stabilizing the actin regulator Scar (Cloud et al., 2019). ...
Polarization of the actin cytoskeleton is vital for the collective migration of cells in vivo. During invasive border cell migration in Drosophila, actin polarization is directly controlled by the Hippo signaling complex, which resides at contacts between border cells in the cluster. Here, we identify, in a genetic screen for deubiquitinating enzymes involved in border cell migration, an essential role for nonstop/USP22 in the expression of Hippo pathway components expanded and merlin. Loss of nonstop function consequently leads to a redistribution of F-actin and the polarity determinant Crumbs, loss of polarized actin protrusions, and tumbling of the border cell cluster. Nonstop is a component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex, but SAGA’s histone acetyltransferase module, which does not bind to expanded or merlin, is dispensable for migration. Taken together, our results uncover novel roles for SAGA-independent nonstop/USP22 in collective cell migration, which may help guide studies in other systems where USP22 is necessary for cell motility and invasion.
... CG4166 was previously named nonstop or not (22). IX-14 1 /not 1 transheterozygotes were 100% lethal and died prior to eclosion from pupal cases, demonstrating the genetic interaction between invadolysin and nonstop. ...
... nonstop is a homozygous pupal lethal mutation so named because of its 'nonstop' axon guidance phenotype, as it is required for the correct migration of the R1-R6 axons (22,23). The nonstop gene encodes a DUB, or deubiquitinating enzyme--a ubiquitin specific protease (23). ...
Identification of components essential to chromosome structure and behaviour remains a vibrant area of study. We have previously shown that invadolysin is essential in Drosophila, with roles in cell division and cell migration. Mitotic chromosomes are hypercondensed in length, but display an aberrant fuzzy appearance. We additionally demonstrated that in human cells, invadolysin is localized on the surface of lipid droplets, organelles that store not only triglycerides and sterols but also free histones H2A, H2Av and H2B. Is there a link between the storage of histones in lipid droplets and the aberrantly structured chromosomes of invadolysin mutants? We have identified a genetic interaction between invadolysin and nonstop, the de-ubiquitinating protease component of the SAGA (Spt-Ada-Gcn5-acetyltransferase) chromatin-remodelling complex. invadolysin and nonstop mutants exhibit phenotypic similarities in terms of chromosome structure in both diploid and polyploid cells. Furthermore, IX-14(1)/not(1) transheterozygous animals accumulate mono-ubiquitinated histone H2B (ubH2B) and histone H3 tri-methylated at lysine 4 (H3K4me3). Whole mount immunostaining of IX-14(1)/not(1) transheterozygous salivary glands revealed that ubH2B accumulates surprisingly in the cytoplasm, rather than the nucleus. Over-expression of the Bre1 ubiquitin ligase phenocopies the effects of mutating either the invadolysin or nonstop genes. Intriguingly, nonstop and mutants of other SAGA subunits (gcn5, ada2b and sgf11) all suppress an invadolysin-induced rough eye phenotype. We conclude that the abnormal chromosome phenotype of invadolysin mutants is likely the result of disrupting the histone modification cycle, as accumulation of ubH2B and H3K4me3 is observed. We further suggest that the mislocalization of ubH2B to the cytoplasm has additional consequences on downstream components essential for chromosome behaviour. We therefore propose that invadolysin plays a crucial role in chromosome organization via its interaction with the SAGA complex.
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
... Further to the requirement for Scny, a second H2B ubiquitin protease, Nonstop, also plays a role in fruit fly development. [33][34][35] First identified as the result of a screen for mutations that affect neuronal connectivity in the brain, Nonstop expression in glia was subsequently found to be required for the migration of these cells into the axonal projection field. 34,35 Nonstop is the fly orthologue of yeast Ubp8, a component of the SAGA complex required for the activation of certain stress-inducible genes ( Table 1). ...
... [33][34][35] First identified as the result of a screen for mutations that affect neuronal connectivity in the brain, Nonstop expression in glia was subsequently found to be required for the migration of these cells into the axonal projection field. 34,35 Nonstop is the fly orthologue of yeast Ubp8, a component of the SAGA complex required for the activation of certain stress-inducible genes ( Table 1). 10,33 demonstrated that Nonstop may affect glial migration as part of the SAGA complex, as mutations in genes encoding other components of SAGA also disrupt axonal projections to varying extents. ...
The reversible ubiquitylation of histone H2B has long been implicated in transcriptional activation and gene silencing. However, many questions regarding its regulation and effects on chromatin structure remain unanswered. In addition, while several studies have uncovered an involvement of this modification in the control of certain developmental processes, a more general understanding of its requirement is lacking. Herein, we present a broad overview of the pathways known to be regulated by H2B ubiquitylation, while drawing parallels between findings in disparate organisms, in order to facilitate continued delineation of its spatiotemporal role in development. Finally, we integrate the findings of recent studies into how H2B ubiquitylation affects chromatin, and cast an eye over emerging areas for future research.
... Furthermore, it can facilitate the observation of processes such as axonal outgrowth and targeting in intact living specimens over time (Chalfie et al., 1994). One promising system for the molecular and genetic study of axonal outgrowth and targeting is the visual system of the fruit fly Drosophila melanoguster (Martin et al., 1995). The adult Drosophila compound eye is made up of approx. ...
... R-cell development has been extensively studied and a large number of molecular and genetic tools are available for use in this system (Zipursky and Rubin, 1994 ). The ability to visualize these axons in unfixed tissue over time, would provide another useful tool for studying the formation of this projection pattern in wild-type animals, as well as in animals containing mutations that disrupt this projection pattern (Martin et al., 1995). Transgenic Drosophila were created that express gfp in all developing R-cells and thc'ir J~IUJIS. ...
Imaging a fluorophore in a living tissue presents several unique problems. The fluorescence from the labeled cell(s) may be weak, the labeled cells may be buried deep within tissue and the presence of a fluorophore may render the cells photo-sensitive. Two-photon laser-scanning microscopy (TPLSM) offers several advantages in meeting these challenges. We show that TPLSM provides greater sensitivity, better resolution and less photo-bleaching, as compared to confocal laser-scanning microscopy. The dramatically reduced photo-bleaching makes it possible to image cells continuously for long periods of time. Therefore, TPLSM allows a safer and higher-resolution means of imaging living cells labeled with a variety of fluorophores, including green fluorescent protein.
... The molecular mechanisms underlying the assembly of selective synaptic connections are currently under intense scrutiny. Recent genetic approaches in the worm (Hedgecock et al., 1990; McIntire et al., 1992) and the fly (Seeger et al., 1993; Van Vactor et al., 1993; Martin et al., 1995; Kania et al., 1995) have identified some of the genes that direct specific patterns of axon growth. Characterization of these genes and their products is revealing a variety of molecules involved in axon guidance and the assembly of the nervous system. ...
... These range from molecules that attract or repel growth cones from a distance, such as the netrins (Kennedy et al., 1994; Colamarino and Tessier-Lavigne, 1995; Matthes et al., 1995), through a group of cell surface molecules involved in growth cone steering such as fasciclin II (Grenningloh et al., 1991; Lin and Goodman, 1994; Chiba et al., 1995), to molecules that interact with the cytoskeleton and may function in the dynamics of growth cone shape changes required for directional growth of axons (Tanaka and Sabry, 1995). Much of the work in Drosophila is focused on the embryo, where a wide array of immunological, genetic and molecular probes have been developed for visualizing specific aspects of neuronal anatomy (Seeger et al., 1993; Van Vactor et al., 1993; Martin et al., 1995; Salzberg et al., 1994; Kania et al., 1995). These methods have allowed detailed examination of axon pathfinding, both within the embryonic CNS (Seeger et al., 1993; Nose et al., 1992) and in the periphery (Kania et al., 1995; Van Vactor et al., 1993). ...
Mutations in an 8 kDa (8x10(3) Mr) cytoplasmic dynein light chain disrupt sensory axon trajectories in the imaginal nervous system of Drosophila. Weak alleles are behaviorally mutant, female-sterile and exhibit bristle thinning and bristle loss. Null alleles are lethal in late pupal stages and alter neuronal anatomy within the imaginal CNS. We utilized P[Gal4] inserts to examine the axon projections of stretch receptor neurons and an engrailed-lacZ construct to characterize the anatomy of tactile neurons. In mutant animals both types of sensory neurons exhibited altered axon trajectories within the CNS, suggesting a defect in axon pathfinding. However, the alterations in axon trajectory did not prevent these axons from reaching their normal termination regions. In the alleles producing these neuronal phenotypes, expression of the cytoplasmic dynein 8 kDa light chain gene is completely absent. These results demonstrate a new function for the cytoplasmic dynein light chain in the regulation of axonogenesis and may provide a point of entry for studies of the role of cellular motors in growth cone guidance.
... To our knowledge, this study identifies for the first time a cell-surface receptor on R-cell axons that plays an essential and specific role in mediating the exit of R-cell axons and WG membrane from the eye disc. In several previous studies [24][25][26], forward genetic screens were performed to search for key players that control R-cell axonal projections. Although these studies led to the identification of a number of important genes required for R-cell axonal guidance and layer-specific target selection in the optic lobe, no R-cell surface receptor that specifically controls the exit of R-cell axons from the eye disc was uncovered from these genetic screens. ...
Coordinated development of neurons and glia is essential for the establishment of neuronal circuits during embryonic development. In the developing Drosophila visual system, photoreceptor (R cell) axons and wrapping glial (WG) membrane extend from the eye disc through the optic stalk into the optic lobe. Extensive studies have identified a number of genes that control the establishment of R-cell axonal projection pattern in the optic lobe. The molecular mechanisms directing the exit of R-cell axons and WG membrane from the eye disc, however, remain unknown. In this study, we show that integrins are required in R cells for the extension of R-cell axons and WG membrane from the eye disc into the optic stalk. Knockdown of integrins in R cells but not WG caused the stalling of both R-cell axons and WG membrane in the eye disc. Interfering with the function of Rhea (i.e. the Drosophila ortholog of vertebrate talin and a key player of integrin-mediated adhesion), caused an identical stalling phenotype. These results support a key role for integrins on R-cell axons in directing R-cell axons and WG membrane to exit the eye disc.
... There are several Drosophila mutants identified displaying various defects in the targeting of the photoreceptor axons to the optic ganglion, including mistargeting, hypo-and hyper-innervation, and clumping of axons (Martin et al., 1995;Kaminker et al., 2002;Fan et al., 2005). Other aspects of axonal development such as growth cone morphology, axon growth, or cell-cell interactions, were also affected in many mutants (Berger et al., 2008). ...
The development of the wild type Drosophila compound eye involves stereotypical targeting of photoreceptor axons to the specific layers of the optic ganglion, medulla and lamina, in the third instar larvae. To test the hypothesis that ubiquitin ligases play an important role during retinal axon targeting we have examined the patterns of axon targeting in the developing eye of the retina aberrant in pattern (rap/fzr) mutants. Rap/Fzr is a homolog of mammalian Cdh1, an activator of anaphase promoting complex (APC), a multi-subunit E3 ubiquitin ligase, regulating the cell cycle progression. Previous work has shown that Rap/Fzr is required during eye development for proper cell cycle regulation, glia differentiation and pattern formation. It was also necessary for proper neuromuscular junction development and circadian rhythms. Our results show that Rap/Fzr is required for proper retinal axon targeting in the developing eye. Using ro-tau-lacZ , we show that the R2-R5 axons fail to stop in the lamina and mis-target to the medulla levels. Also, mosaic analyses experiments using FLP-FRT and GAL4-UAS techniques show that Rap/Fzr functions in a cell autonomous manner. To test for possible role of other signalling molecules and interactions with Rap/Fzr, we have examined rap/fzr axon projection phenotypes in double mutant combinations with the RGS protein, locomotion defective ( loco ) mutants and a scaffolding protein, Liprin-α. Our studies suggest that Rap/Fzr is required for proper axon targeting during Drosophila visual system development, and the phenotype is enhanced in double mutants with either loco or Liprin-α. These results are consistent with other mammalian studies reporting a role of Cdh1 in axon growth and targeting and provides further insights into neuronal functions of the ubiquitin ligase APC/C Cdh1 .
Highlights
Loss of rap/fzr in the third instar Drosophila eye disc leads to photoreceptor axon overgrowth
Overexpression of rap/fzr leads to photoreceptor axon leads to axon shortening and clumping
Loss of Loco P452 leads to photoreceptor overgrowth
Double mutants of rap and loco or rap and Liprin-α show axon enhancement of the axon targeting defects in the Drosophila third instar larvae eye imaginal discs.
... Non-stop is a critical mediator of retinal axon guidance and important for glial cell survival (Martin et al., 1995;Weake et al., 2008). Interestingly, abnormal ubiquitin signaling in the nervous system contributes to a number of genetic and spontaneous neurological and retinal diseases, including SCA3, SCAR16, Alzheimer's, Parkinson's, ALS, and Huntington's disease (Campello et al., 2013a;Campello et al., 2013b;Mohan et al., 2014a;Petrucelli and Dawson, 2004). ...
Ataxin-7 (Atxn7), a subunit of the SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the amino terminus of Atxn7 anchors the Non-stop deubiquitinase to the complex. To understand the consequences of Atxn7-dependent regulation of Non-stop, we sought substrates for Non-stop and discovered the deubiquitinase, dissociated from SAGA, interacts with Arp2/3 and WAVE regulatory complexes (WRC). Protein levels of WRC subunit s uppressor of extracellular cA MP r eceptor (cAR) (SCAR) are regulated by a constant ubiquitination/proteasomal degradation mechanism. Loss of Atxn7 frees Non-stop from SAGA, leading to increased Non-stop interaction with SCAR and also increased SCAR protein levels. A Non-stop enzymatic pocket mutation that increases binding to ubiquitin increased interaction with SCAR, while an enzymatic pocket mutation reducing binding to ubiquitin also reduced binding to SCAR. Loss of Non-stop increased polyubiquitination of SCAR and reduced SCAR protein levels although SCAR protein levels were rescued by protease inhibition. Dependent on conserved W RC interacting r eceptor s equences (WIRS), Non-stop overexpression increased SCAR protein levels and directed subcellular localization of SCAR, leading to decreased cell area and decreased number of protrusions. In vivo , heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR to produce F actin, but heterozygous mutation of SCAR did not significantly rescue retinal axon mistargeting upon knockdown of Atxn7.
Summary
SAGA subunits Ataxin-7 and Non-stop regulate stability and subcellular localization of WRC subunit SCAR. Loss of Ataxin-7 increases, while loss of Non-stop decreases, SCAR protein levels and F-actin network assembly.
... Embryos were collected using standard apple juice plates with yeast. The non-stop 02069 stock (P{ry +t7.2 = PZ} not 02069 ry 506 /TM6B, r CB Tb + ) was provided by the Bloomington Drosophila Stock Center (BL11553) (Martin et al. 1995;Poeck et al. 2001). The ada2b 1 fly stock was provided by Matthias Mannervik (Qi et al. 2004). ...
The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex is a transcriptional coactivator that contains four different modules of subunits. The intact SAGA complex has been well characterized for its function in transcription regulation and development. However, little is known about the roles of individual modules within SAGA and whether they have any SAGA-independent functions. Here we demonstrate that the two enzymatic modules of Drosophila SAGA are differently required in oogenesis. Loss of the histone acetyltransferase (HAT) activity blocks oogenesis, while loss of the H2B deubiquitinase (DUB) activity does not. However, the DUB module regulates a subset of genes in early embryogenesis, and loss of the DUB subunits causes defects in embryogenesis. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that both the DUB and HAT modules bind most SAGA target genes even though many of these targets do not require the DUB module for expression. Furthermore, we found that the DUB module can bind to chromatin and regulate transcription independently of the HAT module. Our results suggest that the DUB module has functions within SAGA and independent functions.
... Mutations in other SAGA subunits result in lethality in different stages of larval development, most probably due to residual maternal load of mRNAs for these subunits. For example, ada2b and nonstop homozygotes die as pupae, gcn5 homozygotes die as third instar larvae, and saf6 and wda homozygotes die as second instar larvae [40,49,57,62,63]. To compare sf3b5 EY12579 flies with other SAGA mutants, we sought to determine the developmental stage at which homozygous sf3b5 EY12579 flies die. ...
The interaction between splicing factors and the transcriptional machinery provides an intriguing link between the coupled processes of transcription and splicing. Here, we show that two components of the SF3B complex that forms part of the U2 small nuclear ribonucleoprotein particle (snRNP), SF3B3 and SF3B5, are also subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex in Drosophila melanogaster. Whereas SF3B3 had previously been identified as a human SAGA subunit, SF3B5 had not been identified as a component of SAGA in any species. We show that SF3B3 and SF3B5 bind to SAGA independent of RNA, and interact with multiple SAGA subunits including Sgf29 and Spt7 in a yeast two-hybrid assay. Through analysis of sf3b5 mutant flies, we show that SF3B5 is necessary for proper development and cell viability, but not for histone acetylation. Although SF3B5 does not appear to function in SAGA's histone modifying activities, SF3B5 is still required for expression of a subset of SAGA-regulated genes independent of splicing. Thus, our data support an independent function of SF3B5 in SAGA's transcription coactivator activity that is separate from its role in splicing.
... In a control experiment, we demonstrated that the expression of nsyb-GFP does not perturb the normal pattern of axonal projections in the Drosophila visual system. In the adult visual system of the fly, the crystalline array of photoreceptors and the spatially ordered projection of axons to the optic lobe provides a sensitive readout for perturbations in the development of a sensory map (Simon et al., 1991;Martin et al., 1995). We crossed UAS-nsyb-GFP flies to flies carrying the GMR-Gal4 transgene that is strongly expressed in the visual system (Hay et al., 1994). ...
... Several molecular cues have been identifi ed that are required for the correct targeting of R1 -R6 in the lamina and R7/R8 in the medulla. For example, the ubiquitin-specifi c protease Nonstop is required for the development of the glial cells that provide the initial R1 -R6 target (Martin et al., 1995;Poeck et al., 2001). On R1 -R6 growth cones, the receptor tyrosine phosphatase PTP69D is required for correct targeting (Garrity et al., 1999). ...
Abstract Visual systems have a rich history as model systems for the discovery and understanding of basic principles underlying neuronal connectivity. The compound eyes of insects consist of up to thousands of small unit eyes that are connected by photoreceptor axons to set up a visual map in the brain. The photoreceptor axon terminals thereby represent neighboring points seen in the environment in neighboring synaptic units in the brain. Neural superposition is a special case of such a wiring principle, where photoreceptors from different unit eyes that receive the same input converge upon the same synaptic units in the brain. This wiring principle is remarkable, because each photoreceptor in a single unit eye receives different input and each individual axon, amongst thousands others in the brain, must be sorted together with those few axons that have the same input. Key aspects of neural superposition have been described as early as 1907. Since then neuroscientists, evolutionary and developmental biologists have been fascinated how such a complicated wiring principle could evolve, how it is genetically encoded, and how it is developmentally realized. In this review article, we will discuss current ideas about the evolutionary origin and developmental program of neural superposition. Our goal is to identify in what way the special case of neural superposition can help us answer more general questions about the evolution and development of genetically 'hard-wired' synaptic connectivity in the brain.
... In this study, we analyzed the zygotic function of abi in the context of the developing fly visual system, and dissected the molecular regulation of WAVE activity by Abi in vivo. The developing Drosophila visual system has served as a good model to identify and study factors, including cytoskeletal regulators that control axonal growth and axonal targeting (Martin et al., 1995;Berger et al., 2008). The adult fly eye is a compound eye comprising ∼750 individual eye units called ommatidia (Ting and Lee, 2007). ...
A tight spatial-temporal coordination of F-actin dynamics is crucial for a large variety of cellular processes that shape cells. The Abelson interactor (Abi) has a conserved role in Arp2/3-dependent actin polymerization, regulating Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE). In this paper, we report that Abi exerts nonautonomous control of photoreceptor axon targeting in the Drosophila visual system through WAVE. In abi mutants, WAVE is unstable but restored by reexpression of Abi, confirming that Abi controls the integrity of the WAVE complex in vivo. Remarkably, expression of a membrane-tethered WAVE protein rescues the axonal projection defects of abi mutants in the absence of the other subunits of the WAVE complex, whereas cytoplasmic WAVE only slightly affects the abi mutant phenotype. Thus complex formation not only stabilizes WAVE, but also provides further membrane-recruiting signals, resulting in an activation of WAVE.
... R axons from the developing retina are guided to specific retinotopic positions in the brain. They appear to respond to guidance cues in their local environment Martin et al., 1995). We investigated the possibility that the optic lobe glia provide positional information to ingrowing R axons. ...
During the development of the Drosophila visual system, photoreceptor (retinal) axons (R axons) project retino-topically to their targets in the optic lobes. The establishment of this precise pattern of connections does not depend on interactions between adjacent axon bundles, suggesting that R axons rely on environmental signals for proper pathfinding. Glial cells that are located along the R-axon trajectory are likely candidates to provide guidance cues for R-axon navigation. This study defines the origin of lamina glia (L glia), and demonstrates that L glia migrate into the lamina over a considerable distance. Glia are located in positions at which the R axons make critical growth choices. In the absence of cues from the eye, several classes of glia migrate to their final positions within the optic lobe anlage and begin to differentiate. Our results are consistent with a role for the glia in providing guidance cues to the R axons.
... Guidance molecules along the pathway are thought to provide the information necessary for a growth cone to navigate to its target (reviewed in Goodman and Shatz, 1993; Holt and Harris, 1993). While genetics has been successfully employed to study invertebrate axon guidance (reviewed in Goodman and Shatz, 1993; Holt and Harris, 1993), including in the visual system (Martin et al., 1995), the identification of molecules that function in vertebrate axon guidance has been achieved mostly using in vitro assays. While several candidate molecules have been identified (for example, see Reichardt and Tomaselli, 1991; Kennedy et al., 1994), the in vivo function of these molecules has been difficult to demonstrate. ...
We have isolated mutants in the zebrafish Danio rerio that have defects in axonal connectivity between the retina and tectum. 5-day-old fish larvae were screened by labeling retinal ganglion cells with DiI and DiO and observing their axonal projections to and on the tectum.
82 mutations, representing 13 complementation groups and 6 single allele loci, were found that have defects in retinal ganglion cell axon pathfinding to the tectum. These pathfinding genes fall into five classes, based on the location of pathfinding errors between eye and tectum. In Class I mutant larvae (belladonna, detour, you-too, iguana, umleitung, blowout) axons grow directly to the ipsilateral tectal lobe after leaving the eye. Class II mutant larvae (chameleon, bashful) have ipsilaterally projecting axons and, in addition, pathfinding mistakes are seen within the eye. In Class III mutant larvae (esrom, tilsit, tofu) fewer axons than normal cross the midline, but some axons do reach the contralateral tectal lobe. Class IV mutant larvae (boxer, dackel, pinscher) have defects in axon sorting after the midline and retinal axons occasionally make further pathfinding errors upon reaching the contralateral tectal lobe. Finally, Class V mutant larvae (bashful, grumpy, sleepy, cyclops, astray) have anterior-posterior axon trajectory defects at or after the midline.
The analysis of these mutants supports several conclusions about the mechanisms of retinal axon pathfinding from eye to tectum. A series of sequential cues seems to guide retinal axons to the contralateral tectal lobe. Pre-existing axon tracts seem not to be necessary to guide axons across the midline. The midline itself seems to play a central role in guiding retinal axons. Axons in nearby regions of the brain seem to use different cues to cross the ventral midline. Mutant effects are not all- or-none, as misrouted axons may reach their target, and if they do, they project normally on the tectum. The retinotectal pathfinding mutants reveal important choice points encountered by neuronal growth cones as they navigate between eye and tectum.
... So far, large-scale genetic screens in animals have only been possible for Drosophila melanogaster and Caenorhabditis elegans (e.g., Nüsslein-Volhard and Wieschaus, 1980). In these organisms, mutant screens have recently uncovered genes that are important for axon guidance and neuronal target recognition (Hedgecock et al., 1985;Kunes et al., 1993;Seeger et al., 1993;van Vactor et al., 1993;Martin et al., 1995). Before the advent of zebrafish genetics, screens of a similar dimension could not be carried out on a vertebrate. ...
A systematic search for mutations affecting the retinotectal projection in zebrafish larvae was performed, as part of the large-scale Tübingen screen for homozygous diploid mutants in embryonic development. 2,746 inbred lines (F2 families) from males mutagenized with ethylnitroso urea were screened. In wild-type larvae, developing retinal axons travel along a stereotyped route to the contralateral optic tectum. Here, their terminals form a highly ordered retinotopic map. To detect deviations from this pattern, an axon tracing assay was developed that permits screening of large numbers of mutagenized fish. Two fluorescent tracer dyes (DiI and DiO) were injected at opposite poles of the eyes of day-5 aldehyde-fixed larvae. 12 hours later, retinal axons were labelled over their entire length, and could be observed through the intact skin. The assay procedure (aldehyde fixation, mounting, injection of dyes, microscopic analysis) took about 1 minute per fish. In total, 125,000 individual fish larvae were processed.
During the screen, 114 mutations in approx. 35 genes were discovered. For the mutants subjected to complementation testing, the number of alleles per locus ranges from 1 to 15. The mutations affect distinct steps in the retinotectal pathway, from pathfinding between eye and tectum to map formation along the dorsal-ventral and the anterior-posterior axis of the tectum. Mutations that disturb axon pathfinding to the tectum for the most part do not disrupt retinotopic mapping, and vice versa. The majority of the mutants display associated defects in other tissues and die before day 10. These mutants provide new tools for studying the formation of neuronal maps. The results of this screen show that a large-scale genetic approach can be applied to relatively late and circum-scribed developmental processes in the vertebrate brain.
... Thus, there has remained a lack of cell type specific markers whose use is simple and reliable. Furthermore, given the recent emphasis on understanding the role of gene expression and the environment in the development and function of the Dvosophilu brain (Davis, 1993(Davis, , 1996Heisenberg et al., 1995: Technau, 1984: DeZazzo and Tully, 1995Kunes and Steller, 1993;Prokop and Technau, 1994;Martin et al., 1995;Buchanan and Be-nzer, 1993;Ferveur et al., 1995;Sentry et al., 1994;O'Dell et al., 1995), the availability of such markers is critical for significant progress in the future. ...
The patterns of gene expression in the Drosophila brain were studied by using the lacZ reporter gene carried on an enhancer detector element. From the analysis of serial sections of the heads of 6000 enhancer detector lines, reporter gene expression in some lines was found to generally follow boundaries established by cell type or anatomy, revealing distinct patterns of lacZ expression restricted to the lamina, the medulla, mushroom bodies, antennal lobes, or other anatomical subdivisions. About 15% of the lines showed ubiquitous expression in most or all head tissues and 25% of the lines showed expression throughout the CNS. Another quarter of the lines showed widespread expression in the CNS, with large regions of the brain showing expression. This suggests that the majority of detected genes are expressed with little spatial specificity. The expression patterns produced by 12 different insertions at the rutabaga locus were found to be extremely similar in the brain and offer strong evidence that the enhancer detector elements generally report the activity of an adjacent gene. Only 15% of the lines were judged to have relatively specific expression in one brain region, including those with preferential or specific expression in the mushroom bodies, antennal lobes, lamina, medulla, etc. The cytological insertion sites for elements showing preferential mushroom body expression were found to be dispersed in the genome at approximately 50 different chromosomal regions. In addition to providing a broad picture of the transcriptional activity in the Drosophila brain, these enhancer detector lines offer access to interesting new genes and form a novel collection of lines in which identifiable brain cells are marked in a reproducible way.
... In the retinotectal example, it is proposed that ganglion axons stop growing in response to a threshold of repulsive activity (Baier and Bonhoffer, 1992; Holland et al., 1996). Candidate molecules for guidance and stop signals have been identified through either in vitro (Stahl et al., 1990; Ullrich et al., 1995) or in vivo (White et al., 1992; Phillis et al., 1993; Seeger et al., 1993; Martin et al., 1995; Callahan et al., 1996) approaches. When the biological significance of these molecules is tested in the corresponding null mutants, however, the resulting phenotypes in general are surprisingly mild and variable (Whitlock, 1993). ...
gigas is a lethal mutant that differentiates enlarged cells, including the nucleus. This trait manifests only after the completion of the mitotic program. We have taken advantage of this phenotype to test in vivo the capacity of normal target cells to arrest the growth of mutant sensory axons. Single neuron connectivity changes have been analyzed in mosaics after horseradish peroxidase retrograde tracings. A mutant mechanoreceptor neuron, growing over a genetically normal substrate, contacts its normal target, and in addition projects to novel areas of the CNS. The mutant axon does terminate its growth eventually, and the new additional targets that are reached correspond to mechanoreceptor domains in other ganglia, indicating that this territorial constraint is operational in the mutant. gigas neurons maintain their stereotyped profile and represent an expanded version of the normal branching pattern. The ultrastructure of the invading projections does not reveal gliotic or necrotic reactions from the new cell contacts. The functional consequences of the connectivity changes produced by the mutant mechanoreceptors have been studied in grooming behavior. Mosaic flies carrying a single gigas mechanoreceptor show modified, albeit context-coherent, grooming responses after stimulation of the mutant bristle, whereas the response from neighboring normal sensory neurons remains unchanged. All of these experiments indicate that target recognition and growth arrest are two dissectible processes of neural development, and they highlight the autonomous features of the growth cone during pathfinding.
... The success of each of these steps involves coordination of intercellular associations, some temporary and some permanent, which are facilitated in part by the selective expression of cell adhesion molecules. Complementary genetic (Seeger et al., 1993; Van Vactor et al., 1993; Salzberg et al., 1994; Kania et al., 1995; Kolodziej et al., 1995; Martin et al., 1995; Schmucker et al., 1997; Holmes et al., 1998), biochemical (Bonhoeffer and Huf, 1985; Luo et al., 1993; Serafini et al., 1994; Colamarino and TessierLavigne, 1995; Drescher et al., 1995) and molecular (Patel et al., 1987; Bieber et al., 1989; Grenningloh et al., 1991; Kolodkin et al., 1993; Harris et al., 1996; Mitchell et al., 1996) analyses have revealed some of the genes and molecules that direct neuronal pathfinding and connectivity. Analysis of development of the motoneurons of the Drosophila larva has proved particularly revealing and has enabled the characterization of an array of proteins including those involved in adhesion, anti-adhesion, attraction and repulsion, as well as signal transduction molecules with specific roles in neuronal development (reviewed in Goodman and Shatz, 1993; Chiba and Keshishian, 1996; Garrity and Zipursky, 1996; Goodman, 1996; Kolodkin, 1996; Tessier-Lavigne and Goodman, 1996; Brunner and O'Kane, 1997). ...
Previous studies demonstrated that Fasciclin II and Beaten path are necessary for regulating cell adhesion events that are important for motoneuron development in Drosophila. We observe that the cell adhesion molecule Fasciclin II and the secreted anti-adhesion molecule Beaten path have additional critical roles in the development of at least one set of sensory organs, the larval visual organs. Taken together, phenotypic analysis, genetic interactions, expression studies and rescue experiments suggest that, in normal development, secretion of Beaten path by cells of the optic lobes allows the Fasciclin II-expressing larval visual organ cells to detach from the optic lobes as a cohesive cell cluster. Our results also demonstrate that mechanisms guiding neuronal development may be shared between motoneurons and sensory organs, and provide evidence that titration of adhesion and anti-adhesion is critical for early steps in development of the larval visual system.
A hallmark of aging is loss of differentiated cell identity. Aged Drosophila midgut differentiated enterocytes (ECs) lose their identity, impairing tissue homeostasis. To discover identity regulators, we performed an RNAi screen targeting ubiquitin-related genes in ECs. Seventeen genes were identified, including the deubiquitinase Non-stop (CG4166). Lineage tracing established that acute loss of Non-stop in young ECs phenocopies aged ECs at cellular and tissue levels. Proteomic analysis unveiled that Non-stop maintains identity as part of a Non-stop identity complex (NIC) containing E(y)2, Sgf11, Cp190, (Mod) mdg4, and Nup98. Non-stop ensured chromatin accessibility, maintaining the EC-gene signature, and protected NIC subunit stability. Upon aging, the levels of Non-stop and NIC subunits declined, distorting the unique organization of the EC nucleus . Maintaining youthful levels of Non-stop in wildtype aged ECs safeguards NIC subunits, nuclear organization, and suppressed aging phenotypes. Thus, Non-stop and NIC, supervise EC identity and protects from premature aging.
The histone acetyltransferase Gcn5 is conserved throughout eukaryotes where it functions as part of large multi-subunit transcriptional coactivator complexes that stimulate gene expression. Here, we describe how studies in the model insect Drosophila melanogaster have provided insight into the essential roles played by Gcn5 in the development of multicellular organisms. We outline the composition and activity of the four different Gcn5 complexes in Drosophila: the Spt-Ada-Gcn5 Acetyltransferase (SAGA), Ada2a-containing (ATAC), Ada2/Gcn5/Ada3 transcription activator (ADA), and Chiffon Histone Acetyltransferase (CHAT) complexes. Whereas the SAGA and ADA complexes are also present in the yeast Saccharomyces cerevisiae, ATAC has only been identified in other metazoa such as humans, and the CHAT complex appears to be unique to insects. Each of these Gcn5 complexes is nucleated by unique Ada2 homologs or splice isoforms that share conserved N-terminal domains, and differ only in their C-terminal domains. We describe the common and specialized developmental functions of each Gcn5 complex based on phenotypic analysis of mutant flies. In addition, we outline how gene expression studies in mutant flies have shed light on the different biological roles of each complex. Together, these studies highlight the key role that Drosophila has played in understanding the expanded biological function of Gcn5 in multicellular eukaryotes. This article is part of a Special Issue entitled: Gcn5: the quintessential histone acetyltransferase.
Atxn7, a subunit of SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the Atxn7 amino terminus anchors Non-stop, a deubiquitinase, to the complex. To understand the scope of Atxn7-dependent regulation of Non-stop, substrates of the deubiquitinase were sought. This revealed Non-stop, dissociated from Atxn7, interacts with Arp2/3 and WAVE regulatory complexes (WRC), which control actin cytoskeleton assembly. There, Non-stop countered polyubiquitination and proteasomal degradation of WRC subunit SCAR. Dependent on conserved WRC interacting receptor sequences (WIRS), Non-stop augmentation increased protein levels, and directed subcellular localization, of SCAR, decreasing cell area and number of protrusions. In vivo, heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR, but heterozygous mutation of SCAR did not significantly rescue knockdown of Atxn7.
Thesis (Ph. D.)--University of Washington, 1999 The diffusible gas nitric oxide (NO) can act as an intracellular messenger in the nervous system, primarily by activating isoforms of soluble guanylate cyclase (sGC). This stimulates the synthesis of cyclic 3 ',5'-cyclic guanosine monophosphate (cGMP), a second messenger molecule with several intercellular targets. NO and cGMP have been implicated in the regulation of both vertebrate and invertebrate neural development. The presented work investigates the role of the NO/cGMP signaling pathway in the developing visual system of the fruitfly, Drosophila melanogaster. I show that the molecular components of this system are present in the Drosophila nervous system during both larval life and metamorphosis. The photoreceptors express a NO-sensitive sGC during the first half of metamorphosis, following retinal axon outgrowth but prior to synaptic assembly. At the same time, retinal targets in the optic lobe label with an antibody to nitric oxide synthase (NOS). Using an in vitro culture technique, I demonstrate that pharmacological inhibition of the NO/cGMP pathway during the period of NO sensitivity disrupts the retinal projection pattern in the optic lobe, and leads to the growth of photoreceptors beyond their normal targets. Using genetic mutants, I show that expression of the Drosophila sGC alpha subunit is required for NO-sensitive cyclase activity in the developing photoreceptors. The retinal projection pattern in these mutants appears undisturbed, however, the developing visual system has an increased sensitivity to NOS inhibition in vitro. As adults these mutants display no positive phototaxis, a defect that is rescued with the heat shock-induced expression of the wild-type alpha subunit during the first half of metamorphosis. Extracellular recordings from the retinas of mutant adults reveal an asynchrony and overall decrease in the response of optic lobe cells to synaptic input from the photoreceptors. This physiological defect can also be rescued with heat-shock expression of the sGC alpha subunit during metamorphosis. I conclude that NO and cGMP are required to maintain the retinal axons in the vicinity of their future post-synaptic targets prior to synaptogenesis. If this pathway is pharmacologically or genetically compromised, proper retinal innervation of the optic lobe fails to occur.
The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2 and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop and sgf11 larval optic lobes using affinity-purification of KASH-EGFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia.
This chapter considers recent progress on eye development. It describes the structure and development of the visual system, and the genetic strategies used to identify genes controlling its development. It then discusses the molecular basis of a number of different processes regulating individual steps in visual system development.
This chapter describes the developmental, cellular and molecular mechanisms involved in establishing neural connectivity in the adult Drosophila visual system. It concerns itself primarily with the projections of photoreceptor axons into the optic lobe, with an emphasis on the more recent literature.
The idea that differential gene expression is critical to the establishment of different cell identities is well-worn into the psyches of all scientists who think about the cellular dynamics of development. Equally commonplace is the idea that temporally and spatially dynamic gene expression is quite often regulated at the level of transcription initiation. More exotic forms of regulation are also well-known, including mRNA splicing, mRNA and protein localization, protein-protein interactions and protein modification. Most recently, it has become apparent that specific alteration of protein stability is a widely used mechanism for controlling the dynamics of important cellular regulators. For example, the levels of cyclins and transcription factors are controlled by specifically targeted protein degradation via the ubiquitin/proteasome pathway.
This chapter explains optic lobe structure, its development, visual behavior and along with this, it focuses on two behavioral domains: phototaxis and optomotor response. The optic lobes underneath the compound eyes are the main computation stations of visual information in the fly head. Their overall structure is conserved in the Diptera and other insect orders. The chapter also contains glimpses across the species fence. The characteristic features of neuronal structure, including cell body position, number, level, and width of dendritic and terminal arborization are explored in detail. The chapter presents predominantly small field neurons that connect lamina and medulla, may serve as a relatively simple example of neuronal complexity in the optic lobes. The evaluation of extensive collections of enhancer trap lines indicates that such mutants may not be easy to find by conventional means.
The life cycle of a protein consists of highly regulated stages of synthesis, maturation, activity, and degradation. The last stage of this cycle frequently occurs through the ubiquitin-proteasome system, which tags and destroys proteins in the cell. Work in recent years regarding the ubiquitin-proteasome system has extended into the field of neurobiology, where the system is critical for proper neuronal function. In this review, we summarize existing knowledge regarding the ubiquitin-proteasome pathway and recount recent studies that frame its importance in neuronal development and synaptic plasticity. Furthermore, we discuss the evidence for protein degradation in neuropathologies, concentrating on neurodegenerative disorders characterized by ubiquitin-rich protein aggregates. We conclude by surveying ongoing drug discovery efforts directed at the ubiquitin-proteasome pathway. Although the current focus of potential proteasomal drugs is on cancer, the prevalence of this pathway in neuronal function makes it a tantalizing target for future central nervous system therapeutics.
The ubiquitin–proteasome system (UPS) is responsible for clearing most soluble proteins in the cytoplasm and nucleus. Recent
studies reveal that the UPS function is critical for maintaining synaptic plasticity and transmission and that the UPS dysfunction
is associated with axonal degeneration and impaired synaptic transmission. In this chapter, we will focus on the role of the
UPS in synapses and the association of UPS impairment with neurological disorders. Since protein misfolding causes several
neurological disorders that show synaptic dysfunction during the early stages of disease, understanding the involvement of
the synaptic UPS in neurological disorders may help determine effective strategies for treating neurological disorders caused
by the accumulation of misfolded proteins.
The lozenge locus is genetically complex, containing two functionally distinct units, cistrons A and B, that influence the structure of
the compound eye. Extreme mutations of either cistron produce adult phenotypes that share similarities and that have striking
differences. We have analyzed the expression of several developmentally important eye genes including boss, scabrous, rhomboid, seven-up, and Bar in lozenge mutant backgrounds representing both cistrons. This analysis follows the progressive recruitment of photoreceptor neurons
during eye development and has confirmed that the initial development of photoreceptors is normal up to the five cell precluster
stage (R8, R2/5 and R3/4). However, when lozenge is mutant, further eye development is perturbed. As cells R1, R6 and R7 are recruited, patterns of gene expression for seven-up and Bar become abnormal. We have also characterized the expression of two different enhancer trap alleles of lozenge. The lozenge product(s) appear to be first expressed in the eye disc in undifferentiated cells shortly after the five cell precluster forms.
Then, as distinct cells are recruited to a fate, lozenge expression persists and is refined in those cells. Our data suggests that lozenge functions in cone cells and pigment cells as well as in specific glia. With respect to photoreceptor neurons, lozenge biases the developmental potential of cells R1, R6 and R7, by directly influencing the expression of genes important for establishing
cell fate.
To identify genes necessary for establishing connections in the Drosophila sensory nervous system, we designed a screen for mutations affecting development of the larval visual system. The larval visual system has a simple and stereotypic morphology, can be recognized histologically by a variety of techniques, and is unnecessary for viability. Therefore, it provides an opportunity to identify genes involved in all stages of development of a simple, specific neuronal connection. By direct observation of the larval visual system in mutant embryos, we identified 24 mutations affecting its development; 13 of these are larval visual system-specific. These 13 mutations can be grouped phenotypically into five classes based on their effects on location, path or morphology of the larval visual system nerves and organs. These mutants and phenotypic classifications provide a context for further analysis of neuronal development, pathfinding and target recognition.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1996. Includes bibliographical references. by Michael H. Brodsky. Ph.D.
BEACH proteins comprise an evolutionarily conserved family characterized by the presence of a BEACH (Beige and Chediak-Higashi) domain of unknown function. They have been shown to play a role in a number of important cellular processes, ranging from cytokinesis to synaptic transmission, and implicated in human diseases, such as Chediak-Higashi Syndrome and cancer. Analysis of several BEACH proteins suggests that they may be involved in membrane trafficking; however, little insight has been gained into their molecular mechanism of function. We identified Drosophila Beach1 in a gain-of-function screen: beach1 overexpression in the photoreceptors drastically alters their growth cone morphology. In a subsequent genetic modifier screen, I identified rabll as a strong enhancer of the beach1 eye overexpression phenotype. Rabll is a small GTPase, which has been shown to regulate the delivery of vesicles and cargo to the plasma membrane via both the recycling and the biosynthetic pathways. Although beach1 loss-of-function mutants exhibit no obvious phenotypes, a sensitized background of a rabll mutant revealed a requirement for beach1 during development and in bristle extension. (cont.) I also found that Beach1 functionally antagonizes Rab11 at the neuromuscular junction by suppressing the rabll synaptic overgrowth phenotype. Subcellular fractionation and double-labeling experiments suggest that these proteins may function in the same subcellular compartment; however, further experiments are needed to determine whether Beach1 and Rab11 interact directly, function in the same protein complex, or closely cooperate in the same molecular pathway. The interaction I found between Beach1 and Rab11 suggests a mechanism by which other BEACH proteins may be involved in vesicle trafficking. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005. Includes bibliographical references.
Photoreceptors undergoing target selection in the optic lobe of Drosophila express a nitric oxide sensitive soluble guanylate cyclase (sGC). At the same time, cells in the target region of the optic lobe express nitric oxide synthase (NOS). Pharmacological inhibition of NOS, NO or sGC leads to disruption of the retinal projection pattern in vitro , and the extension of individual retinal axons beyond their appropriate targets. The disruptive effects of NOS inhibition in vitro are prevented by adding a cGMP analog. Mutations in the sGC alpha subunit gene, Gcα1 , reduce sGC expression and attenuate NO-sensitive retinal cGMP production in the visual system. Although the retinal projection pattern is undisturbed in Gcα1 mutants, they lack positive phototaxis as adults, suggesting inappropriate connections exist between the photoreceptors and optic lobe interneurons in these flies. Preliminary results show that heat-shock expression of wild-type Gcα1 during metamorphosis can restore positive phototaxis in severe Gcα1 mutants. These in vivo results support the in vitro findings that NOS and sGC activity are required to promote the appropriate retinal innervation of the optic lobe.
The optic lobes comprise approximately half of the fly's brain. In four major synaptic ganglia, or neuropils, the visual input from the compound eyes is received and processed for higher order visual functions like motion detection and color vision. A common characteristic of vertebrate and invertebrate visual systems is the point-to-point mapping of the visual world to synaptic layers in the brain, referred to as visuotopy. Vision requires the parallel extraction of numerous parameters in a visuotopic manner. Consequently, the optic neuropils are arranged in columns and perpendicularly oriented synaptic layers that allow for the selective establishment of synapses between columnar neurons. How this exquisite synaptic specificity is established during approximately 100 hours of brain development is still poorly understood. However, the optic lobe contains one of the best characterized brain structures in any organism-both anatomically and developmentally. Moreover, numerous molecules and their function illuminate some of the basic mechanisms involved in brain wiring. The emerging picture is that the development of the visual system of Drosophila is (epi-)genetically hard-wired; it supplies the emerging fly with vision without requiring neuronal activity for fine tuning of neuronal connectivity. Elucidating the genetic and cellular principles by which gene activity directs the assembly of the optic lobe is therefore a fascinating task and the focus of this chapter.
Recent analyses have shed light on the roles of genes involved in early events of eye cell determination and the spatiotemporal control of differentiation within the eye field. These genes function at sequential steps in the programming, initiation, or progression of differentiation, highlighting an elegant orchestration of gene activities to achieve this striking developmental event. Progress has been made in the study of the coordination between cell cycle control and cell differentiation, as well as in the genetic control of morphogenetic movements within the developing eye disc.
The giant fiber system (GFS) is a simple network of neurons that mediates visually elicited escape behavior in Drosophila. The giant fiber (GF), the major component of the system, is a large, descending interneuron that relays visual stimuli to the motoneurons that innervate the tergotrochanteral jump muscle (TTM) and dorsal longitudinal flight muscles (DLMs). Mutations in the neural transcript from the shaking-B locus abolish the behavioral response by disrupting transmission at some electrical synapses in the GFS. This study focuses on the role of the gene in the development of the synaptic connections. Using an enhancer-trap line that expresses lacZ in the GFs, we show that the neurons develop during the first 30 hr of metamorphosis. Within the next 15 hr, they begin to form electrical synapses, as indicated by the transfer of intracellularly injected Lucifer yellow. The GFs dye-couple to the TTM motoneuron between 30 and 45 hr of metamorphosis, to the peripherally synapsing interneuron that drives the DLM motoneurons at approximately 48 hr, and to giant commissural interneurons in the brain at approximately 55 hr. Immunocytochemistry with shaking-B peptide antisera demonstrates that the expression of shaking-B protein in the region of GFS synapses coincides temporally with the onset of synaptogenesis; expression persists thereafter. The mutation shak-B2, which eliminates protein expression, prevents the establishment of dye coupling shaking-B, therefore, is essential for the assembly and/or maintenance of functional gap junctions at electrical synapses in the GFS.
Mutations in the Drosophila gene dreadlocks (dock) disrupt photoreceptor cell (R cell) axon guidance and targeting. Genetic mosaic analysis and cell-type-specific expression of dock transgenes demonstrate dock is required in R cells for proper innervation. Dock protein contains one SH2 and three SH3 domains, implicating it in tyrosine kinase signaling, and is highly related to the human proto-oncogene Nck. Dock expression is detected in R cell growth cones in the target region. We propose Dock transmits signals in the growth cone in response to guidance and targeting cues. These findings provide an important step for dissection of signaling pathways regulating growth cone motility.
Drosophila retinal axons trigger both the proliferation of their targets, the lamina neurons, as well as the final differentiation and migration of the lamina glia. To date, the molecular basis of these interactions has remained unclear. We have identified a new gene, lamina ancestor (lama). Both the lamina's neural and glial progenitors express lama, even though these cells have very different developmental origins. Expression of lama is down-regulated once the precursors begin their differentiation programs. Loss of function mutants are viable and fertile, and appear to have normally developed visual systems. lama encodes a novel protein that is 74% identical to its D. virilis homologue.
Systematic genetic screens have been powerful tools in identifying genes responsible for axon guidance in fruitflies and nematodes. This approach has now been extended to the study of axon guidance and the formation of topographic neuronal connections in the vertebrate brain. A systematic genetic screen was used to identify genes responsible for precise axon pathfinding and targeting in the retinotectal system of the zebrafish (Danio rerio). Over 30 genes were found that affect either: (1) retinal axon pathfinding to the contralateral tectal lobe; or (2) the topographic connection between the eye and the tectum. The zebrafish retinotectal mutants represent a new resource for the study of axon guidance in the vertebrate brain.
Encoding visual information requires a complex neuronal network. Recently, genes regulating early tissue specification, the growth of retinal target structures, the connectivity of photoreceptor axons, and mirror-image retinal symmetry in Drosophila have been identified. The insights gained from studying visual system development in flies promise to inform our understanding of similar processes in vertebrates.
A neuroanatomical screening of a collection of P-element mutagenized flies has been carried out with the aim of finding new mutants affecting the optic lobe of the adult brain in Drosophila melanogaster. We have identified a new gene that is involved in the development of the adult axon array in the optic ganglia and in the ommatidia assembly. We have named this locus visual system disorganizer (vid). Reversional mutagenesis demonstrated that the vid mutant was the result of a P-element insertion in the Drosophila genome and allowed us to generate independent alleles, some of which resulted in semilethality, like the vid original mutant, while the others were completely lethal. A genetic somatic mosaic analysis indicated that the vid gene is required in the eye for its normal development by inductive effects. This analysis also suggests an inductive effect of the vid gene on the distal portion of the optic lobe, particularly the lamina and the first optic chiasma. Moreover, the absence of mutant phenotype in the proximal region of the optic ganglia, including the medulla, the second optic chiasma, and the lobula complex underlying mosaic eyes, is suggestive of an autonomously acting mechanism of the vid gene in the optic lobe. The complete or partial lethality generated by different mutations at the vid locus suggests that this gene's role may not be limited to the visual system, but may also affect a vital function during Drosophila development.
To identify novel genes and to isolate tagged mutations in known genes that are required for the development of the peripheral nervous system (PNS), we have screened a novel collection of 2460 strains carrying lethal or semilethal P element insertions on the third chromosome. Monoclonal antibody 22C10 was used as a marker to visualize the embryonic PNS. We identified 109 mutant strains that exhibited reproducible phenotypes in the PNS. Cytological and genetic analyses of these strains indicated that 87 mutations affect previously identified genes: tramtrack (n = 18 alleles), string (n = 15), cyclin A (n = 13), single-minded (n = 13), Delta (n = 9), neuralized (n = 4), pointed (n = 4), extra macrochaetae (n = 4), prospero (n = 3), tartan (n = 2), and pebble (n = 2). In addition, 13 mutations affect genes that we identified recently in a chemical mutagenesis screen designed to isolate similar mutants: hearty (n = 3), dorsotonals (n = 2), pavarotti (n = 2), sanpodo (n = 2), dalmatian (n = 1), missensed (n = 1), senseless (n = 1), and sticky ch1 (n = 1). The remaining nine mutations define seven novel complementation groups. The data presented here demonstrate that this collection of P elements will be useful for the identification and cloning of novel genes on the third chromosome, since >70% of mutations identified in the screen are caused by the insertion of a P element. A comparison between this screen and a chemical mutagenesis screen undertaken earlier highlights the complementarity of the two types of genetic screens.
We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover approximately 25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven "super-contigs" that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the alphagamma element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms.
The arrival of retinal axons in the brain of Drosophila triggers the assembly of glial and neuronal precursors into a 'neurocrystalline' array of lamina synaptic 'cartridges'. Hedgehog, a secreted protein, is an inductive signal delivered by retinal axons for the initial steps of lamina differentiation. In the development of many tissues, Hedgehog acts in a signal relay cascade via the induction of secondary secreted factors. Here we show that lamina neuronal precursors respond directly to Hedgehog signal reception by entering S-phase, a step that is controlled by the Hedgehog-dependent transcriptional regulator Cubitus interruptus. The terminal differentiation of neuronal precursors and the migration and differentiation of glia appear to be controlled by other retinal axon-mediated signals. Thus retinal axons impose a program of developmental events on their postsynaptic field utilizing distinct signals for different precursor populations.
The major axon tracts in the embryonic CNS ofDrosophila are organised in a simple, ladder-like pattern. Each neuromere contains two commissures which connect the contra-lateral sides and two longitudinal connectives which connect the different neuromeres along the anterior-posterior axis. The commissures form in close association with only few cells located at the CNS midline. The formation of longitudinal connectives depends in part on the presence of specific lateral glial cells.
To unravel the genes underlying the formation of the embryonic CNS axon pattern, we conducted a saturating F2 EMS mutagenesis, screening for mutations, which disrupt this process. The analyses of the identified mutations lead to a simple sequential model on axon pattern formation in embryonic CNS.
Using monoclonal and polyclonal antibodies as differentiation markers, we have found that the eight photoreceptors of the Drosophila ommatidium differentiate in a fixed sequence. The foundation photoreceptor, R8, expresses neural antigens first. The paired photoreceptors R2/5 are next to express, followed by the pair R3/4, followed by the pair R1/6; R7 is the final photoreceptor to differentiate. From previous studies it is known that Drosophila photoreceptors use local, positional cues to select their identities. Together with the morphological picture of ommatidial development, the sequential order of photoreceptor differentiation demonstrated here suggests that these cues may be encoded in the particular combination of cells an undetermined cell finds itself in contact with.
Due to its small size Drosophila melanogaster can conveniently be used in screening experiments for anatomical brain mutants. A simple method has been designed which allows to process up to 20 identifiable flies as a single preparation in a standard histology routine. Anatomical brain mutants are very frequent. Over 60 mutants were obtained from the inspection of about 3000 brains. So far genetic variations of brain structure fall into 4 classes: (1) “low fidelity” mutants in which brains are less precisely built; (2) “brain shape” mutants with globally or partially reduced brains; (3) “architectonic” mutants which show constructional defects mainly in the repetitive structures of the brain and (4) “vacuolar” mutants with globular “holes” in certain areas of the brain. These mutant classes obviously reflect different aspects of brain development like cell proliferation (2), “wiring” (3) and cell death (4). Some of the mutants may prove to be useful in anatomical, physiological or genetic brain research.
The Drosophila major wing nerve collects axons from the anterior margin sensory organs. Using the flp recombinase to make clones, I show that all glia present on this nerve are clonally related to wing epithelial cells. Glial cells arise only from regions that also give rise to sensory organs and migrate along the nerve following the direction taken by axons. As in vertebrates, wing glial cells start migrating at a stage at which axons are growing. The migration of wing glial cells is affected by two mutations altering axonogenesis, fused and Notch, which suggests that the two processes are tightly associated.
The unc-6 gene is required for the guidance of pioneer axons and migrating cells along the body wall in C. elegans. In mutants, dorsal and ventral migrations are disrupted, but longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by two independent transposon insertions. Based on genomic and cDNA sequencing, the gene encodes a novel laminin-related protein, UNC-6 (591 amino acids). The N-terminus is homologous to the N-termini (i.e., domains V1, V-1, V-2, and V-3) of laminin subunits, while the C-terminus is a unique domain. We propose that UNC-6 is a component of an extracellular matrix cue that guides dorsoventral migrations on the epidermis.
We have examined the generation and development of glial cells in the first optic ganglion, the lamina, of Drosophila melanogaster. Previous work has shown that the growth of retinal axons into the developing optic lobes induces the terminal cell divisions that generate the lamina monopolar neurons. We investigated whether photoreceptor ingrowth also influences the development of lamina glial cells, using P element enhancer trap lines, genetic mosaics and birthdating analysis. Enhancer trap lines that mark the differentiating lamina glial cells were found to require retinal innervation for expression. In mutants with only a few photoreceptors, only the few glial cells near ingrowing axons expressed the marker. Genetic mosaic analysis indicates that the lamina neurons and glial cells are readily separable, suggesting that these are derived from distinct lineages. Additionally, BrdU pulse-chase experiments showed that the cell divisions that produce lamina glia, unlike those producing lamina neurons, are not spatially or temporally correlated with the retinal axon ingrowth. Finally, in mutants lacking photoreceptors, cell divisions in the glial lineage appeared normal. We conclude that the lamina glial cells derive from a lineage that is distinct from that of the L-neurons, that glia are generated independently of photoreceptor input, and that completion of the terminal glial differentiation program depends, directly or indirectly, on an inductive signal from photoreceptor axons.
The decapentaplegic (dpp) gene in Drosophila melanogaster encodes a TGF-beta-like signalling molecule that is expressed in a complex and changing pattern during development. One of dpp's contributions is to proximal-distal outgrowth of the adult appendages, structures derived from the larval imaginal disks. Appendage specific mutations of dpp fall in a 20 kb interval 3' to the known dpp transcripts. Here, we directly test the hypothesis that these mutations define an extended 3' cis-regulatory region. By analysis of germ-line transformants expressing a reporter gene, we show that sequences from this portion of the gene, termed the dppdisk region, are capable of directing expression comparable to that defined by RNA in situ hybridization. We localize two intervals of the dppdisk region that appear to account for much of the dpp spatial pattern in imaginal disks and discuss the positions of these important elements in terms of the genetics of dpp. Finally, we provide evidence to suggest that one of our constructs expresses beta-galactosidase in the early imaginal disk primordia in the embryo, at approximately the time when they are set aside from surrounding larval epidermal tissues. Thus, dpp may be involved directly in the determination of the imaginal disks.
The catalogue of data presented here form many systems demonstrates that multiple mechanisms are involved in the formation of topographic maps. We are not yet in a position to explain why a particular mechanism appears to dominate in some situations and not in others. Certain generalizations can be made, however. First, at least some form of chemospecificity can be invoked to help explain connectivity in all of the experiments we have cited. Often, the differential identities of a population of neurons can be reflected in an orderly pattern of axon outgrowth and in the actively maintained preservation of neighbor relations as the axons grow toward their targets; such orderly arrangements are not obligatory, but, where present, they facilitate the speedy establishment of orderly maps when the axons reach their target nuclei. Within a terminal zone, chemospecific cues may dominate and constrain a given axon to terminate in a specific location, but axon-axon interactions commonly supercede chemospecific matching. At least two forms of axon-axon interaction occur, one based on some sort of biochemical properties related to the axon's embryological identity and another based on the axons' electrical activity. Tasks for the future are to identify the cellular bases of each of these mechanisms and to understand the situations in which each is manifested.
A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates of Drosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development.
We report the identification of RK2, a glial-specific homeodomain protein. RK2 is localized to the nucleus of virtually all embryonic and imaginal glial cells, with the exception of midline glia. Embryos mutant for the gene encoding RK2 are embryonic lethal but normal for early gliogenesis (birth, initial divisions and migration of glia) and axonogenesis (neuronal pathfinding and fasciculation). However, later in development, there are significantly fewer longitudinal glia that are spatially disorganized; in addition, there is a slight disorganization of axon fascicles and a defective nerve cord condensation. This suggests that RK2 is not required for early glial determination, but rather for aspects of glial differentiation or function that are required for embryonic viability.
In vertebrates, commissural axons pioneer a circumferential pathway to the floor plate at the ventral midline of the embryonic spinal cord. Floor plate cells secrete a diffusible factor that promotes the outgrowth of commissural axons in vitro. We have purified from embryonic chick brain two proteins, netrin-1 and netrin-2, that each possess commissural axon outgrowth-promoting activity, and we have also identified a distinct activity that potentiates their effects. Cloning of cDNAs encoding the two netrins shows that they are homologous to UNC-6, a laminin-related protein required for the circumferential migration of cells and axons in C. elegans. This homology suggests that growth cones in the vertebrate spinal cord and the nematode are responsive to similar molecular cues.
The development of the adult visual system of Drosophila requires the establishment of precise retinotopic connections between retinal photoreceptor cell axons and their synaptic partners in the optic lobe of the brain. To assess the role of axon-axon interactions in retinal axon guidance, we used genetic methods to disrupt the normal spatiotemporal order of retinal axon ingrowth. We examined retinal axon projections to the developing first optic ganglion, the lamina, in two mutants in which reduced numbers of ommatidia develop in the eye imaginal disk. We find that in the developing lamina of these mutants, sine oculis and Ellipse, retinal axons project to proper dorsoventral positions despite the absence of the usual array of neighboring retinal axons. In a second approach, we examined animals that were somatic mosaics for the mutation, glass. In glass- animals, retinal axons project aberrantly and the larval optic nerve is absent. We find that in the developing lamina of glass mosaic animals, wild-type retinal axons project to proper dorsoventral positions despite the misrouted projections of neighboring glass- retinal axons. In addition, wild-type retinal axons project normally in the absence of the larval optic nerve, indicating that the latter is not an essential pioneer for retinal axon navigation. Our observations support the proposal that axon fascicles can make at least some pathfinding decisions independently of other retinal axon fascicles. We suggest that positional guidance cues that might label axon pathways and target destinations contribute to retinotopic pattern formation in the Drosophila visual system.
Using monoclonal and polyclonal antibodies as differentiation markers, we have found that the eight photoreceptors of the Drosophila ommatidium differentiate in a fixed sequence. The foundation photoreceptor, R8, expresses neural antigens first. The paired photoreceptors R2/5 are next to express, followed by the pair R3/4, followed by the pair R1/6; R7 is the final photoreceptor to differentiate. From previous studies it is known that Drosophila photoreceptors use local, positional cues to select their identities. Together with the morphological picture of ommatidial development, the sequential order of photoreceptor differentiation demonstrated here suggests that these cues may be encoded in the particular combination of cells an undetermined cell finds itself in contact with.
The methods currently used to impregnate nerve tissue with reduced silver fall broadly into two classes: methods of block impregnation and the so-called silver-on-the-slide techniques. In the earlier block impregnation procedures, such as those of Golgi, Cajal, and Bielschowsky, and their modifications, whole pieces of tissue are treated with silver solutions and only afterward sectioned and mounted. In the silver-on-the-slide methods the material is embedded and sectioned first, and then the sections are stained after they have been mounted on slides. Block-impregnation methods are discussed elsewhere in this volume (see Chap. 9) and need not be considered further here. Procedures for impregnating mounted sections, usually of paraffin wax-embedded material, are numerous, but they can be subdivided roughly according to the type of silver compound used as the impregnator. This is generally either an inorganic silver salt, most often silver nitrate, or a silver-protein complex, such as protargol.
The larval and early pupal development of the optic lobes in Drosophila is described qualitatively and quantitatively using [3H]thymidine autoradiography on 2-μm plastic sections. The optic lobes develop from 30–40 precursor cells present in each hemisphere of the freshly hatched larva. During the first and second larval instars, these cells develop to neuroblasts arranged in two epithelial optic anlagen. In the third larval instar and in the early pupa these neuroblasts generate the cells of the imaginal optic lobes at discrete proliferation zones, which can be correlated with individual visual neuropils.
The different neuropils as well as the repetitive elements of each neuropil are generated in a defined temporal sequence. Cells of the medulla are the first to become postmitotic with the onset of the third larval instar, followed by cells of the lobula complex and finally of the lamina at about the middle of the third instar. The elements of each neuropil connected to the most posterior part of the retina are generated first, elements corresponding to the most anterior retina are generated last.
The proliferation pattern of neuroblasts into ganglion mother cells and ganglion cells is likely to include equal as well as unequal divisions of neuroblasts, followed by one or two generations of ganglion mother cells. For the lamina the proliferation pattern and its temporal coordination with the differentiation of the retina are shown.
The eyes and optic lobes of adult Drosophila melanogaster comprise a highly organized system of interconnected neurons. The eye and optic lobe primordia are physically separate during the embryonic and larval stages of development, and these tissues do not come into contact until the third larval instar, as a consequence of axons growing from the receptor cells of the developing eyes to the primordial optic lobes. After this contact, the axons of the eyes arrange themselves into their complex and orderly adult pattern. Simultaneously, the optic lobe cells begin elaborating axons which organize into their precise adult array. One question posed by this system is: Does cellular pattern formation in either the eyes or optic lobes depend on eye-brain interactions, or do the two tissues organize autonomously? To answer this question, mutations were found which cause abnormal ommatidial array in the eyes and which also perturb the normal adult axon array in the optic lobes. By means of X ray-induced somatic recombination and by genetically controlled mitotic chromosome loss (gynandromorph formation), flies mosaic for genotypically mutant and normal tissue were constructed. Analysis of the neuronal array in mosaic flies in which eye and optic lobe tissue differed genotypically showed that the axon array phenotype of the optic lobe depends on the genotype of the eye tissue innervating that lobe, while the eye phenotype does not depend on optic lobe genotype. Thus, the axonal organization of the D. melanogaster optic lobe has been shown to depend on the transmission of information from the eyes to the optic lobes.
THE axons of photoreceptors in arthropods project in a precisely ordered manner from the retina to the lamina. As retina and lamina consist of two-dimensional arrays of structural units (the ommatidia and the cartridges, respectively), a particular unit of origin and a specific target unit can be defined uniquely for each axon. In a number of species, such as water fleas1, crabs2 and locusts3, all axons originating from an ommatidium terminate in the same cartridge, where they form synapses onto laminar neurones. How is a particular target cartridge selected by the retinular axons as they grow into the laminar anlage during embryogenesis? The adult pattern of projections could arise in several ways. For example, each ommatidium and each cartridge could have unique chemical labels varying with or according to their positions in their respective domains. Input and target could then be matched by a chemoaffinity mechanism such as that originally proposed by Sperry4 for the formation of vertebrate retinotectal connections. A second possibility is that retinular fibres do not recognise any specific labels in the laminar cells, but grow into this region in the appropriate spatio-temporal order to sequentially fill available sites in a pre-formed laminar array. In a variation of this second mechanism the lamina would be unstructured at the time of arrival of retinular axons and the axons would impose a pattern on the lamina by sequentially recruiting immature laminar neurones to form the regular array of cartridges. These possibilities have been tested by deleting ommatidia at specified developmental stages in Daphnia magna, a small branchiopod crustacean with a total of 22 ommatidia and 22 cartridges1. The observations reported here support the last hypothesis, that the order of retinular axon ingrowth determines the retino-laminar projections in this organism.
Enzyme marker techniques allow the identification of the genotype of internal tissues in Drosophila genetic mosaics. These methods were used to derive fate maps for the nervous system and several other internal organs. The fate maps were compared to other cell lineage studies and to the fate mapping results on neurological mutants. The mosaic data for the nervous system also indicate that only a few blastoderm cells (3–10) give rise to each major ganglion.
Pattern formation in the Drosophila retina proceeds by the recruitment of cells, along a morphogenetic front, into a lattice. At the advancing front, marked by a dorso-ventral furrow in the eye imaginal disc, cells are organized into ommatidial precursors, each containing cells destined to become photoreceptors 2, 3, 4, 5, and 8. Behind the front, a mitotic wave produces photoreceptors 1, 6, and 7, plus the remaining cells needed to complete the ommatidia. During the third larval instar, the front sweeps anteriorly across the eye disc, leaving a highly ordered pattern in its wake. Preceding the dorso-ventral furrow is a groove that bisects the eye disc into dorsal and ventral halves and presumably plays a role in establishing the equatorial symmetry line. Cell lineage plays little role in pattern formation in the eye. Genetic mosaics show that the cells of each ommatidium are not derived from a single mother cell; the cells appear to be recruited at random at the morphogenetic front. Similarly, the mirror symmetry above and below the equator is not established by a clonal mechanism; a single clone can contribute cells to ommatidia on both sides of the equator.
Tenascin, an extracellular matrix protein, is expressed in an unusually restricted pattern during embryogenesis and has been implicated in a variety of morphogenetic phenomena. To directly assess the function of tenascin in vivo, we generated mutant mice in which the tenascin gene was nully disrupted by replacing it with the lacZ gene. In mutant mice, lacZ was expressed in place of tenascin, and no tenascin product was detected. Homozygous mutant mice were, however, obtained in accordance with Mendelian laws, and both females and males produced offspring normally. No anatomical or histological abnormalities were detected in any tissues, and no major changes were observed in distribution of fibronectin, laminin, collagen, and proteoglycan. The existence of these mutant mice, lacking tenascin yet phenotypically normal, casts doubt on the theory that tenascin plays and essential role in normal development.
In the newly cellularized Drosophila embryo, progress through the cell cycle is regulated at the G2-M transition. We have examined cell-cycle regulation later in Drosophila development, in a group of postembryonic neuronal precursors. The S-phase precursor cells, which generate photoreceptor target neurons (lamina neurons) in the central nervous system, are not present in the absence of photoreceptor innervation. Here we report that axons selectively approach G1-phase precursors. Without axon ingrowth, lamina precursors do not enter their final S phase and by several criteria, arrest in the preceding G1 phase. These findings provide evidence that at this stage in development the control of cell division can occur at the G1-S transition.
The outgrowth of single axons through different cellular environments requires distinct sets of genes in the nematode C. elegans. Three genes are required for the pioneering circumferential outgrowth of identified motor neuron axons between the lateral hypodermal cell membrane and the basal lamina. Three other genes are required for the longitudinal outgrowth of these axons along preexisting axon bundles as well as for the fasciculation of axons within these neuron bundles. Five additional genes are required for circumferential outgrowth, longitudinal outgrowth, and fasciculation; mutations in three of these genes disrupt axon ultrastructure, suggesting that they function in axon formation rather than in axon guidance.
The development of the Drosophila R7 photoreceptor cell is determined by a specific inductive interaction between the R8 photoreceptor cell and a single neighboring precursor cell. This process is mediated by bride of sevenless (boss), a cell-surface bound ligand, and the sevenless (sev) tyrosine kinase receptor. The boss ligand is expressed specifically on the surface of the R8 cell, whereas the sev receptor is expressed on 5 cells contacting the developing R8 cell and other cells not in contact with R8. By altering the spatial and temporal expression of boss, we demonstrate that sev-expressing cells that do not contact R8 can assume an R7 cell fate. By contrast, the sev-expressing precursor cells to the R1-R6 photoreceptor cells that do contact R8 are nonresponsive to the inductive cue. Using the rough and Nspl mutations, we demonstrate that an early commitment to an R1-R6 cell fate blocks the pathway of sev activation in these cells.
The development of neurons and virtually all other cell types in the organism depends upon interactions with molecules in their environment. Studics of individual cell types have revealed tremendous diversity in the molecules that regulate the development of cells in the nervous system. These include chemotropic and trophic factors [e.g. nerve growth-factor (NGF) and brain derived neurotrophic factor (BDNF)], cell adhesion molecules [e.g. the neural cell adhesion molecule (NCAM) and N-cadherin], and molecules secreted into the extracellular matrix (ECM) [e.g. laminin (LN) and fibronectin (FN)]. Each class of molecule has now been shown to influence major steps in the development of the nervous system, including neuronal survival, determination, and migration; axonal growth and guidance; synapse formation; and glial differentiation. As molecules in the ECM influence all of these events and can be used to illustrate many of the principles derived from studies of the other classes of molecules, this review focuses upon constituents of the ECM and their receptors. The role of the ECM in neural development has recently been reviewed in this series (Sanes 1989). This review focuses on cellular and molecular themes not emphasized in the previous one and includes examples of recent work on nonneural systems that illustrate probable future directions for research in the nervous system.
Recent reviews on the composition and function of the ECM and its receptors include those of Hynes (1990), Hemler (1990), Kishimoto et al (1989), Plow & Ginsberg (1989), Burgeson (1988), Buck & Horowitz (1987), McDonald (1988), Ruoslahti (1988, 1989), Fessler & Fessler (1989), and Erickson & Bourdon (1989). Reviews focusing on aspects of ECM function in neural development include those by Lander (1989), Sanes (1989), and Edgar (1989). Because of space limitations, only representative examples and references are cited in this review.
We have examined the influence of retinal innervation on the development of target neurons in the first optic ganglion, the lamina, of D. melanogaster. Mitotically active lamina precursor cells (LPCs), which normally produce lamina neurons, are absent in mutants that lack retinal innervation, while other proliferative centers appear unaffected. Reducing the number of innervating photoreceptor axons results in fewer mitotic LPCs. In glass mutants photoreceptors project to abnormal locations and LPCs are found adjacent to these aberrant projections. We conclude that the arrival of photoreceptor axons in the larval brain initiates, directly or indirectly, cell division to produce lamina neurons. Our results provide an explanation for how the synchronous development of these two interacting systems is coordinated.
The glass gene encodes a zinc finger protein required for normal photoreceptor cell development in Drosophila. We show that glass transcripts are present in the third-instar eye-imaginal disc starting in the morphogenetic furrow and extending to the posterior margin of the disc; glass protein is detected in the nuclei of all cells in this region. We also show that glass encodes a site-specific DNA-binding protein. A 27-bp glass-binding site can confer glass-dependent expression on a reporter gene in developing photoreceptor cells, the particular subset of glass-expressing cells known to require glass function. This specificity may represent a regulation of glass protein activity after cells are recruited to the photoreceptor cell fate.
The rhodopsin genes of Drosophila melanogaster are expressed in nonoverlapping subsets of photoreceptor cells within the insect visual system. Two of these genes, Rh3 and Rh4, are known to display complementary expression patterns in the UV-sensitive R7 photoreceptor cell population of the compound eye. In addition, we find that Rh3 is expressed in a small group of paired R7 and R8 photoreceptor cells at the dorsal eye margin that are apparently specialized for the detection of polarized light. In this paper we present a detailed characterization of the cis-acting requirements of both Rh3 and Rh4. Promoter deletion series demonstrate that small regulatory regions (less than 300 bp) of both R7 opsin genes contain DNA sequences sufficient to generate their respective expression patterns. Individual cis-acting elements were further identified by oligonucleotide-directed mutagenesis guided by interspecific sequence comparisons. Our results suggest that the Drosophila rhodopsin genes share a simple bipartite promoter structure, whereby the proximal region constitutes a functionally equivalent promoter "core" and the distal region determines cell-type specificity. The expression patterns of several hybrid rhodopsin promoters, in which all or part of the putative core regions have been replaced with the analogous regions of different rhodopsin promoters, provide additional evidence in support of this model.
During pupation, long-range order is imposed on the autonomously developing ommatidia which compose the Drosophila eye. To accomplish this, eight additional cell types arise: the primary, secondary, and tertiary pigment cells, and the four cells that form the bristle. These cells form an interweaving lattice between ommatidia. The lattice is refined when excess cells are removed to bring neighboring ommatidia into register. Recent evidence suggests that in larval development, local contacts direct cell fate. The same appears to be true during pupal development: the contacts a cell makes predict the cell type it will become. Cells which contact the anterior or posterior cone cells in an ommatidium invariably become primary pigment cells. Cells which contact primary pigment cells from different ommatidia become secondary and tertiary pigment cells. Bristle development is in several ways distinct from ommatidial development. The four cells of each bristle group appear to be immediate descendents of a single founder cell. During their early differentiation, they do not make stereotyped contacts with surrounding ommatidial cells, but do make particular contacts within the bristle group. And unlike the surrounding ommatidia, differentiation of the bristles radiates from the center of the eye to the edges. As cells are removed during two stages of programmed cell death, the bristles are brought into their final position. When all cells in the lattice have achieved their final position, a second stage of retinal development begins as structures specific to each cell type are produced. This paper follows these various stages of pupal development, and suggests how local cell-cell contacts may produce the cells needed for a functional retina.
We have analyzed the cis-acting regulatory sequences of the ninaE gene. This gene encodes the major Drosophila melanogaster opsin, the protein component of the primary chromophore of photo-receptor cells R1-R6 of the adult eye. DNA fragments containing the start point of transcription of the ninaE gene were fused to either the Escherichia coli chloramphenicol acetyltransferase or lacZ (beta-galactosidase) gene and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the ninaE gene to confer the ninaE pattern of expression. Fragments containing between 2.8 kb and 215 bp of the sequences upstream of the start of transcription plus the first 67 bp of the untranslated leader were able to direct nearly wild-type expression. We have identified three separable control regions in the ninaE promoter. The first, which has the properties of an enhancer element, is located between nucleotides -501 and -219. The removal of this sequence had little effect on promoter function; this sequence appears to be redundant. However, it appears to be able to substitute for the second control region which is located between nucleotides -215 and -162, and which also affects the level of output from this promoter. Removal of these two control regions resulted in a 30-fold decrease in expression; however tissue specificity was not affected. The third control region, located downstream from nucleotide -120, appears to be absolutely necessary for promoter function in the absence of the first two regulatory sequences. Examination of larvae containing fusion genes expressing beta-galactosidase suggests that the ninaE gene is also expressed in a subset of cells in the larval photoreceptor organ.
The mechanisms involved in producing the vast number of specific connections in the nervous system are unknown. As nerve cells grow during development and seek out their targets there must be some sort of molecular cues for axonal guidance and final target recognition. One strategy for identifying such factors is to locate the genes which code for or control the expression of the molecules. To identify such factors is to locate the genes which code for or control the expression of the molecules. To identify these genes, we have chosen a simple network of eight neuones in Drosophila melanogaster, comprising the giant fibre (GF) system, in which we can detect the effect of mutations on connectivity. We report here the isolation of an x-linked, recessive mutation, bendless (ben), which deletes one process of the GF while leaving other portions functionally unaltered.
The Drosophila anachronism (ana) locus controls the proliferation of neuroblasts, neuronal stem cells that give rise to the central nervous system. In ana mutants, quiescent postembryonic central brain and optic lobe neuroblasts enter S phase precociously. ana encodes a novel secreted protein of 474 amino acids that is expressed not in the affected neuroblasts, but rather in a subclass of neighboring glial cells. These studies argue for an important role for glia in negatively regulating proliferation of neuronal precursor cells, thereby controlling the timing of postembryonic neurogenesis.
Through a systematic genetic screen, we have identified 55 mutations that affect the development of the PNS of Drosophila embryos. These mutations specify 13 novel and 5 previously characterized genes and define new phenotypes for 2 other known genes. Five classes of mutant phenotypes were identified in the screen: gain of neurons, loss of neurons, abnormal position of chordotonal neurons, aberrant neuronal trajectories, and abnormal morphology of neurons. Phenotypic analyses of mutations identified in this study revealed three novel aspects of PNS development. First, we have identified a novel gene that may be required to define glial versus neuronal cell identity. Second, our data indicate that neuronal migration plays an important role in pattern formation in the embryonic PNS. Third, we have identified mutations that cause a lack of sensory organs, but unlike mutations in proneural genes, do not affect the formation of sensory organ precursors. These genes may be required for key aspects of neuronal differentiation. Our studies suggest that approximately 70 essential genes are required for proper PNS development in Drosophila embryos.
A stepwise morphogenetic program of cell division and cell fate determination generates the precise neuronal architecture of the visual centers of the Drosophila brain. Here, we show that the assembly of the target structure for ingrowing retinal axons involves cell-cell interactions mediated by the secreted product of the wingless (wg) gene. wg, expressed in two symmetrical domains of the developing brain, is required to induce and maintain the expression of the secreted decapentaplegic (dpp) gene product in adjacent domains. wg and dpp function are required for target field neurons to adopt their proper fates and to send axons into the developing target structure. These observations implicate a cascade of diffusible signaling molecules in patterning the visual centers of the Drosophila brain.
The guidance of axons to their targets in the developing nervous system is believed to involve diffusible chemotropic factors secreted by target cells. Floor plate cells at the ventral midline of the spinal cord secrete a diffusible factor or factors that promotes the outgrowth of spinal commissural axons and attracts these axons in vitro. Two membrane-associated proteins isolated from brain, netrin-1 and netrin-2, possess commissural axon outgrowth-promoting activity. We show here that netrin-1 RNA is expressed by floor plate cells, whereas netrin-2 RNA is detected at lower levels in the ventral two-thirds of the spinal cord, but not the floor plate. Heterologous cells expressing recombinant netrin-1 or netrin-2 secrete diffusible forms of the proteins and can attract commissural axons at a distance. These results show that netrin-1 is a chemotropic factor expressed by floor plate cells and suggest that the two netrin proteins guide commissural axons in the developing spinal cord.
Neural-cell adhesion molecules (N-CAMs) are members of the immunoglobulin superfamily mediating homo- and heterophilic cell-cell interactions. N-CAM exists in various isoforms which are generated by alternative splicing. During embryonic development, N-CAMs are expressed in derivatives of all three germ layers, whereas in the adult animal they are predominantly present in neural tissue. Processes like neurulation, axonal outgrowth, histogenesis of the retina and development of the olfactory system are correlated with the regulated expression of N-CAMs. We show here that N-CAM-deficient mice generated by gene targeting appear healthy and fertile, but adult mutants show a 10% reduction in overall brain weight and a 36% decline in size of the olfactory bulb. N-CAM deficiency coincides with almost total loss of protein-bound alpha-(2,8)-linked polysialic acid, a carbohydrate structure thought to be correlated with neural development and plasticity. The animals showed deficits in spatial learning when tested in the Morris water maze, whereas activity and motor abilities appeared normal.
We have identified a set of retinal basal glia, designated RBG cells, in the axon layer of the developing Drosophila eye disc. In vivo pulse labeling with bromodeoxyuridine shows that these cells originate in the optic stalk and migrate into the disc. In mutants lacking photoreceptor axons, RBG cells accumulate in the optic stalk, but do not invade the disc. The association of RBG cells with photoreceptor axons, their origin in the optic stalk, and their migration into the retina are in common with the behavior of astrocytes in the developing mammalian retina.
N-CAM is abundantly expressed in the nervous system in the form of numerous structural variants with characteristic distribution patterns and functional properties. N-CAM-180, the variant having the largest cytoplasmic domain, is expressed by all neurons. The N-CAM-180-specific exon 18 has been deleted to generate homozygous mice unable to express this N-CAM form. The most conspicuous mutant phenotype was in the olfactory bulb, where granule cells were both reduced in number and disorganized. In addition, precursors of these cells were found to be accumulated at their origin in the subependymal zone at the lateral ventricle. Analysis of the mutant in this region suggests that the mutant phenotype involves a defect in cell migration, possibly through specific loss of the polysialylated form of N-CAM-180, which is expressed in the migration pathway. Subtle but distinct abnormalities also were observed in other regions of the brain.
The two serotonergic HSN motor neurons of the nematode Caenorhabditis elegans innervate the vulval muscles and stimulate egg laying by hermaphrodites. By analyzing mutant and laser-operated animals, we find that both epithelial cells of the developing vulva and axons of the ventral nerve cord are required for HSN axonal guidance. Vulval precursor cells help guide the growth cone of the emerging HSN axon to the ventral nerve cord. Vulval cells also cause the two HSN axons to join the ventral nerve cord in two separate fascicles and to defasciculate from the ventral nerve cord and branch at the vulva. The axons of either the PVP or PVQ neurons are also necessary for the HSN axons to run in two separate fascicles within the ventral nerve cord. Our observations indicate that the outgrowth of the HSN axon is controlled in multiple ways by both neuronal and nonneuronal cells.
Growth cones in developing nervous systems encounter a sequence of extracellular cues during migration. In theory, a growth cone can navigate by selectively expressing or activating surface receptor(s) that recognize extracellular cues appropriate to each migratory phase. Using the simple Caenorhabditis elegans nervous system, we attempted to demonstrate that path selection by migrating growth cones can be predictably altered by ectopic expression of a single receptor. The unc-5 gene of C. elegans encodes a unique receptor of the immunoglobulin superfamily (UNC-5), required cell-autonomously to guide growth cone and mesodermal cell migrations in a dorsal direction on the epidermis. We report here that the UNC-5 receptor induces dorsally oriented axon trajectories when ectopically expressed in the touch receptor neurons which normally extend pioneer axons longitudinally or ventrally on the epidermis. These errant trajectories depend on unc-6, which encodes a putative epidermal path cue, just as normal dorsally oriented axon trajectories do (such as those of certain motor neurons), suggesting that UNC-5 acts to reorient the touch cell growth cones by using its normal guidance mechanisms. These results support previous evidence that UNC-5 and UNC-6 play instructive rules in guiding growth cone migrations on the epidermis in C. elegans, and indicate that pioneering growth cones, which normally migrate in different directions, may use equivalent intracellular signalling mechanisms for guidance.
The bendless (ben) mutation of Drosophila alters synaptic connectivity between a subset of CNS neurons. Here, we show that ben also causes morphological abnormalities within the visual system, suggesting that ben functions in a number of different developmental processes. We show that the ben gene encodes a protein which is closely related to ubiquitin-conjugating enzymes and that a missense mutation in the highly conserved active site region is associated with the ben mutation. High levels of ben expression are restricted to the nervous system during development. These results suggest a role for ubiquitin-mediated protein modification in nervous system development, including, but not exclusive to, the regulation of synaptic connectivity.
We have used electron-microscopic studies, bromodeoxyuridine (BrdU) incorporation and antibody labeling to characterize the development of the Drosophila larval photoreceptor (or Bolwig's) organ and the optic lobe, and have investigated the role of Notch in the development of both. The optic lobe and Bolwig's organ develop by invagination from the posterior procephalic region. After cells in this region undergo four postblastoderm divisions, a total of approximately 85 cells invaginate. The optic lobe invagination loses contact with the outer surface of the embryo and forms an epithelial vesicle attached to the brain. Bolwig's organ arises from the ventralmost portion of the optic lobe invagination, but does not become incorporated in the optic lobe; instead, its 12 cells remain in the head epidermis until late in embryogenesis when they move in conjunction with head involution to reach their final position alongside the pharynx. Early, before head involution, the cells of Bolwig's organ form a superficial group of 7 cells arranged in a 'rosette' pattern and a deep group of 5 cells. Later, all neurons move out of the surface epithelium. Unlike adult photoreceptors, they do not form rhabdomeres; instead, they produce multiple, branched processes, which presumably carry the photopigment. Notch is essential for two aspects of the early development of the visual system. First, it delimits the number of cells incorporated into Bolwig's organ. Second, it is required for the maintenance of the epithelial character of the optic lobe cells during and after its invagination.
We have constructed a series of strains to facilitate the generation and analysis of clones of genetically distinct cells in developing and adult tissues of Drosophila. Each of these strains carries an FRT element, the target for the yeast FLP recombinase, near the base of a major chromosome arm, as well as a gratuitous cell-autonomous marker. Novel markers that carry epitope tags and that are localized to either the cell nucleus or cell membrane have been generated. As a demonstration of how these strains can be used to study a particular gene, we have analyzed the developmental role of the Drosophila EGF receptor homolog. Moreover, we have shown that these strains can be utilized to identify new mutations in mosaic animals in an efficient and unbiased way, thereby providing an unprecedented opportunity to perform systematic genetic screens for mutations affecting many biological processes.
We performed a large-scale screen for mutations that affect the development of CNS axon pathways in the Drosophila embryo. We screened embryos from over 13,500 balanced lines and saved over 250 mutant lines whose phenotypes suggest possible defects in growth cone guidance. Here we focus on two new genes: commissureless (comm) and roundabout (robo). Mutations in comm lead to an absence of nearly all CNS axon commissures, such that growth cones that normally project across the midline instead now extend only on their own side. Mutations in robo lead to the opposite misrouting, such that some growth cones that normally extend only on their own side instead now project across the midline. The phenotypes of these two genes suggest that they may encode components of attractive and repulsive signaling systems at the midline that either guide growth cones across the midline or keep them on their own side.
In each abdominal hemisegment of the Drosophila embryo, an array of 30 muscle fibers is innervated by about 34 motoneurons in a highly stereotyped and cell-specific fashion. To begin to elucidate the molecular basis of neural specificity in this system, we conducted a genetic screen for mutations affecting neuromuscular connectivity. We focus on 5 genes required for specific aspects of pathway (beaten path, stranded, and short stop) and target (walkabout and clueless) recognition. The different classes of mutant phenotypes suggest that neural specificity is controlled by a hierarchy of molecular mechanisms: motoneurons are guided toward the correct region of mesoderm, in many cases navigating a series of choice points along the way; they then display an affinity for a particular domain of neighboring muscles; and finally, they recognize their specific muscle target from within this domain.
The development of the optic lobe
- Meinertzhagen
Meinertzhagen, I. A., and Hanson, T. E. (1993). The development of
the optic lobe. In The Development of Drosophila melanogaster, M.
Bate and A. M. Arias, eds. (Cold Spring Harbor, New York: Cold Spring
Harbor Laboratory Press), pp. 1363-1492.