Genetische Zerlegung peripherer Pfade im visuellen System von Drosophila

Die visuellen Systeme von Vertebraten und Invertebraten weisen Ähnlichkeiten in den ersten Schritten visueller Informationsverarbeitung auf. Im menschlichen Gehirn werden zum Beispiel die Modalitäten Farbe, Form und Bewegung separat in parallelen neuronalen Pfaden verarbeitet. Dieses grundlegende Merkmal findet sich auch bei der Fliege Drosophila melanogaster, welche eine ähnliche Trennung in farbsensitive und (farbenblinde) bewegungssensitive Pfade aufweist, die durch zwei verschiedene Gruppen von Photorezeptoren (dem R1-6 und dem R7/8 System) determiniert werden. Fliegen haben ein hoch organisiertes visuelles System, welches durch die repetitive, retinotope Organisation von vier Neuropilen charakterisiert ist: Dies sind die Lamina, die Medulla, die Lobula und die Lobulaplatte. Jedes einzelne besteht aus Kolumnen, die denselben Satz von Nervenzellen enthalten. In der Lamina formen Axonbündel von sechs Photorezeptoren R1-6, die auf denselben Bildpunkt blicken, Säulen, die als Cartridges bezeichnet werden. Diese sind die funktionellen visuellen „sampling units“ und sind mit vier Typen von Interneuronen erster Ordnung assoziiert, die von R1-6 den gleichen Input erhalten: L1, L2, L3 und die Amakrinzellen (amc, mit ihrem postsynaptischen Partner T1). Diese stellen parallele Pfade dar, die auf anatomischer Ebene im Detail untersucht wurden; jedoch ist wenig über ihre funktionelle Rolle bei der Verarbeitung für das Verhalten relevanter Information bekannt, z.B. hinsichtlich der Blickstabilisierung, der visuellen Kurskontrolle oder der Fixation von Objekten. Die Verfügbarkeit einer Vielfalt von neurogenetischen Werkzeugen für die Struktur-Funktionsanalyse bei Drosophila ermöglicht es, erste Schritte in Richtung einer genetischen Zerlegung des visuellen Netzwerks zu unternehmen, das Bewegungs- und Positionssehen vermittelt. In diesem Zusammenhang erwies sich die Wahl des Effektors als entscheidend. Überraschenderweise wurde festgestellt, dass das clostridiale Tetanus-Neurotoxin die Photorezeptorsynapsen adulter Drosophila Fliegen nicht blockiert, hingegen irreversible Schäden bei Expression während deren Entwicklung verursacht. Aus diesem Grund wurde das dominant-negative shibire Allel shits1, welches sich als geeigneter erwies, zur Blockierung der Lamina Interneurone verwendet, um die Notwendigkeit der jeweiligen Pfade zu analysieren. Um festzustellen, ob letztere auch hinreichend für das gleiche Verhalten waren, wurde für die umgekehrte Strategie die Tatsache ausgenutzt, daß die Lamina Interneurone Histaminrezeptoren exprimieren, die vom ort Gen kodiert werden. Die spezifische Rettung der ort Funktion in definierten Pfaden im mutanten Hintergrund ermöglichte festzustellen, ob sie für eine bestimmte Funktion hinreichend waren. Diese neurogenetischen Methoden wurden mit der optomotorischen Reaktion und dem objektinduzierten Orientierungsverhalten als Verhaltensmaß kombiniert, um folgende Fragen innerhalb dieser Doktorarbeit zu beantworten: (a) Welche Pfade stellen einen Eingang in elementare Bewegungsdetektoren dar und sind notwendig und/oder hinreichend für die Detektion gerichteter Bewegung? (b) Gibt es Pfade, die spezifisch Reaktionen auf unidirektionale Bewegung vermitteln? (c) Welche Pfade sind notwendig und/oder hinreichend für das objektinduzierte Orientierungsverhalten? Einige grundlegende Eigenschaften des visuellen Netzwerks konnten dabei aufgedeckt werden: Die zwei zentralen Cartridge Pfade, die von den großen Monopolarzellen L1 und L2 repräsentiert werden, haben eine Schlüsselfunktion bei der Bewegungsdetektion. Über ein breites Spektrum von Reizbedingungen hinweg sind die beiden Subsysteme redundant und können Bewegung unabhängig voneinander verarbeiten. Um eine Beeinträchtigung des Systems festzustellen, wenn nur einer der beiden Pfade intakt ist, muß dieses an die Grenzen seiner Leistungsfähigkeit gebracht werden. Bei niedrigem Signal/Rauschverhältnis, d.h. bei geringem Musterkontrast oder geringer Hintergrundbeleuchtung, hat der L2 Pfad eine höhere Sensitivität. Bei mittlerem Musterkontrast sind beide Pfade auf die Verarbeitung unidirektionaler Bewegung in entgegengesetzten Reizrichtungen spezialisiert. Im Gegensatz dazu sind weder der L3, noch der amc/T1 Pfad notwendig oder hinreichend für die Detektion von Bewegungen. Während der erstere Positionsinformation für Orientierungsverhalten zu verarbeiten scheint, nimmt der letztere eine modulatorische Rolle bei mittlerem Kontrast ein. Es stellte sich heraus, daß das Orientierungsverhalten noch robuster als das Bewegungssehen ist und möglicherweise auf einem weniger komplizierten Mechanismus beruht, da dieser keinen nichtlinearen Vergleich der Signale benachbarter visueller „sampling units“ benötigt. Die Fixation von Objekten setzt nicht grundsätzlich das Bewegungssehen voraus, allerdings verbessert die Detektion von Bewegung die Fixation von Landmarken, im besonderen, wenn diese schmal sind oder einen geringen Kontrast aufweisen.Vertebrate and invertebrate visual systems exhibit similarities in early stages of visual processing. For instance, in the human brain, the modalities of color, form and motion are separately processed in parallel neuronal pathways. This basic property is also found in the fly Drosophila melanogaster which has a similar division in color- sensitive and (color blind) motion-sensitive pathways that are determined by two distinct subsets of photoreceptors (the R1-6 and the R7/8 system, respectively). Flies have a highly organized visual system that is characterized by its repetitive, retinotopic organization of four neuropils: the lamina, the medulla, the lobula and the lobula plate. Each of these consists of columns which contain the same set of neurons. In the lamina, axon bundles of six photoreceptors R1-6 that are directed towards the same point in space form columnar structures called cartridges. These are the visual sampling units and are associated with four types of first-order interneuron that receive common input from R1-6: L1, L2, L3 and the amacrine cells (amc, together with their postsynaptic partner T1). They constitute parallel pathways that have been studied in detail at the anatomical level. Little is known, however, about their functional role in processing behaviorally relevant information, e.g. for gaze stabilization, visual course control or the fixation of objects. The availability of a variety of neurogenetic tools for structure-function analysis in Drosophila allowed first steps into the genetic dissection of the neuronal circuitry mediating motion and position detection. In this respect, the choice of the effector turned out to be crucial. Surprisingly, it was found that the clostridial tetanus neurotoxin failed to block mature Drosophila photoreceptor synapses, but caused irreversible damage when expressed during their development. Therefore, the dominant-negative shibire allele shits1 which turned out to be better suited was used for blocking lamina interneurons and thereby analyzing the necessity of the respective pathways. To determine whether the latter were also sufficient for the same behavioral task, the inverse strategy was developed, based on the fact that lamina interneurons express histamine receptors encoded by the ort gene. The specific rescue of ort function in defined channels in an otherwise mutant background allowed studying their sufficiency in a given task. Combining these neurogenetic methods with the optomotor response and object induced orientation behavior as behavioral measures, the aim of the present thesis was to answer the following questions: (a) Which pathways feed into elementary motion detectors and which ones are necessary and/or sufficient for the detection of directional motion? (b) Do pathways exist which specifically mediate responses to unidirectional motion? (c) Which pathways are necessary and/or sufficient for object induced orientation behavior? Some basic properties of the visual circuitry were revealed: The two central cartridge pathways, represented by the large monopolar cells L1 and L2, are key players in motion detection. Under a broad range of stimulatory conditions, the two subsystems are redundant and are able to process motion independently of each other. To detect an impairment when only one of the pathways is intact, one has to drive the system to its operational limits. At low signal to noise ratios, i.e. at low pattern contrast or low background illumination, the L2 pathway has a higher sensitivity. At intermediate pattern contrast, both pathways are specialized in mediating responses to unidirectional motion of opposite stimulus direction. In contrast, neither the L3, nor the amc/T1 pathway is necessary or sufficient for motion detection. While the former may provide position information for orientation, the latter has a modulatory role at intermediate pattern contrast. Orientation behavior turned out to be even more robust than motion vision and may utilize a less sophisticated mechanism, as it does not require a nonlinear comparison of signals from neighboring visual sampling units. The position of objects is processed in several redundant pathways, involving both receptor subsystems. The fixation of objects does not generally require motion vision. However, motion detection improves the fixation of landmarks, especially when these are narrow or have a reduced contrast

Differential potencies of effector genes in adult Drosophila

The GAL4/UAS gene expression system in Drosophila has been crucial in revealing the behavioral significance of neural circuits. Transgene products that block neurotransmitter release and induce cell death have been proved to inhibit neural function powerfully. Here we compare the action of the five effector genes shibirets1, Tetanus toxin light chain (TNT), reaper, Diphtheria toxin A-chain (DTA), and inwardly rectifying potassium channel (Kir2.1) and show differences in their efficiency depending on the target cells and the timing of induction. Specifically, effectors blocking neuronal transmission or excitability led to adultinduced paralysis more efficiently than those causing cell ablation. We contrasted these differential potencies in adult to their actions during development. Furthermore, we induced TNT expression in the adult mushroom bodies. In contrast to the successful impairment in short-term olfactory memory by shibirets1, adult TNT expression in the same set of cells did not lead to any obvious impairment. Altogether, the efficiency of effector genes depends on properties of the targeted neurons. Thus, we conclude that the selection of the appropriate effector gene is critical for evaluating the function of neural circuits

Binary cell fate decisions and fate transformation in the Drosophila larval eye

Controlling cellular diversity requires a complex interplay of transcription factors. Using the Drosophila larval eye as genetic model we identify distinct mechanisms of how binary cell fate decisions are made, how sensory receptor gene expression is regulated and how cell fate identity is switched during metamorphosis. We show that the transcription factor Senseless fulfills three temporally and functionally separable roles in the same cells by (1) initiating a binary cell fate decision by controlling the cell fate determinants Spalt and Seven-up, (2) suppressing apoptosis during metamorphosis and (3) promoting Rhodopsin expression after metamorphosis. We further show that the transcription factor Hazy provides is required for early embryonic PR differentiation and that maintained Hazy expression is essential for Rhodopsin expression. Hazy provides a third function during metamorphosis by repressing Sens in one PR-subtype allowing it to undergo apoptotic cell death. We identified a novel mode of Rhodopsin regulation in which the highly conserved RCSI motif is dispensable for expression, demonstrating that the regulation of the Rhodopsin promoter is distinct in different visual organs. Our findings provide a unique example of how the same regulators control very distinct key aspects of development at distinct stages

Distinct Roles for Two Histamine Receptors (hclA and hclB) at the Drosophila Photoreceptor Synapse

Histamine (HA) is the photoreceptor neurotransmitter in arthropods, directly gating chloride channels on large monopolar cells (LMCs), postsynaptic to photoreceptors in the lamina. Two histamine-gated channel genes that could contribute to this channel in Drosophila are hclA (also known as ort) and hclB (also known as hisCl1), both encoding novel members of the Cys-loop receptor superfamily. Drosophila S2 cells transfected with these genes expressed both homomeric and heteromeric histamine-gated chloride channels. The electrophysiological properties of these channels were compared with those from isolated Drosophila LMCs.HCLAhomomershad nearly identicalHA sensitivity to the native receptors (EC 50 = 25 µM). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states (~25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors were more sensitive to HA (EC50 = 14 and 1.2µM, respectively), with much smaller single-channel conductances (~4 pS). Null mutations of hclA (ortUS6096) abolished the synaptic transients in the electroretinograms (ERGs). Surprisingly, theERG“on” transients in hclB mutants transients were approximately twofold enhanced, whereas intracellular recordings from their LMCs revealed altered responses with slower kinetics. However, HCLB expression within the lamina, assessed by both a GFP (green fluorescent protein) reporter gene strategy and mRNA tagging, was exclusively localized to the glia cells, whereas HCLA expression was confirmed in the LMCs. Our results suggest that the native receptor at the LMC synapse is an HCLA homomer, whereas HCLB signaling via the lamina glia plays a previously unrecognized role in shaping the LMC postsynaptic response

Sens is required for Rh5-PR identity and acts in parallel with Otd.

<p>(A, B) Rh5 and Rh6 expression in wild-type and <i>sens^E2</i> mutant PRs during embryonic stage 17, stained with anti-Rh6 (green), anti-Rh5 (blue) and anti-Elav (red); z-projection of confocal sections. No Rh5 expression was seen in <i>sens^E2</i> mutants and all the cells were marked by Rh6. (C, D) Otd expression in wild-type and <i>sens^E2</i> mutant PRs during embryonic stage 15 stained with anti-Kr (green) and anti-Otd (red). Both in wild-type and <i>sens^E2</i> mutants, all the PR nuclei expresses Otd, showing that Otd was not affected in <i>sens^E2</i> mutant; z-projection of confocal sections. (E, F, G) Sens expression (red) in <i>otd^uvi</i>, <i>sal¹⁶</i> and <i>svp^E22</i> mutant PRs during embryonic stage 12. Staining against FasII or Hazy (green), shows that Sens expression was not affected in these mutants; z-projection of confocal sections.</p

Role of Sens in the transformation of larval eye into the adult eyelet.

<p>(A, B) Rh6 expression in wild-type and <i>sens^RNAi</i> (<i>lGMR</i>-Gal4; UAS-<i>Dcr2</i>/UAS-<i>sens^RNAi</i>) adult eyelets, stained with anti-Rh6 (red) and anti-Elav (green). In all <i>lGMR</i>-Gal4; UAS-<i>Dcr2</i>/UAS-<i>sens^RNAi</i> animals, the eyelet was absent (inset: high magnification of eyelet position). (C, C′) Rh6 expression (red) in <i>sens^RNAi</i> when <i>p35</i> was ectopically expressed in the eyelet to keep the cells alive (UAS-<i>sens^RNAi</i>/<i>lGMR</i>-Gal4; UAS-<i>p35</i>/UAS-<i>Dcr2</i>), stained with anti-Chp (green), and anti-elav (blue); z-projection of confocal sections. No Rh6 expression was found in the eyelet. (D, D′) Sens expression (red) in the eyelet when a dominant-negative form of EcR was ectopically expressed in Rh5-PRs (<i>rh5</i>-Gal4/UAS-<i>EcR^DN</i>) and stained with anti-Chp (green) and anti-Elav (blue); z-projection of confocal sections. Sens was expressed in all four eyelet cells. (E) A model describing the role of Sens during different developmental stages.</p

Rescue of the <i>hazy^−/−</i> mutant phenotype in the larval eye.

<p>(A) Heat shock mediated rescue of <i>hazy^−/−</i> mutant at different stages during development and consequence for Rhodopsin expression. White bar indicates no Rhodopsin expression, while black bar indicates Rhodopsin expression. Red arrowhead marks the stage at which heat shock was given. (B–G) All panels show larval eyes stained with anti-Elav (red), anti-Rh6 (green) and anti-Rh5 (blue); z-projection of confocal sections. (B, C) Heat shocks were performed three times at embryonic stage 12. At the L1 stage, Rh5 and Rh6 expression was detected (B), while at L2 stage, Rh5 and Rh6 expression was lost (C). Heat shocks performed at stage 13 (D), stage 15 (E) and at L1 stage (F) did not result in a rescue of Rh5 and Rh6 expression in the larval eyes at L1 in (D, E) and L2 in (F). Heat shocks at embryonic stage 12 and at L2 stage restores Rh5 and Rh6 expression in L3 larvae (G).</p

Role of Hazy in the adult eyelet.

<p>(A, B) Wild-type and <i>hazy^−/−</i> mutant eyelets were stained against Sens (red), Rh6 (green) and Elav (blue); z-projection of confocal sections. In <i>hazy^−/−</i> mutant, the eyelet consisted of 12 cells and all of them expressed Sens, while Rh6 expression was restricted to four cells. (C, D) <i>rh6</i>-GFP and <i>rh6^ΔRCSI</i>-GFP eyelets, stained with anti-Rh6 (red) and anti-GFP (green); z-projection of confocal sections. GFP expression was still observed in the eyelets of <i>rh6^ΔRCSI</i>-GFP. (E) UAS-<i>hazy</i> was expressed under the control of <i>chp^4.5</i>-Gal4 in a <i>hazy^−/−</i> null background (<i>hazy^−/−; chp^4.5</i>-Gal4/UAS-<i>hazy</i>) and the adult eyelet was analyzed for Elav (red) and Rh6 (green); z-projection of confocal sections. Normal number of Rh6 expressing PRs was found in <i>hazy^−/−; chp^4.5</i>-Gal4/UAS-<i>hazy</i> animals. (F) Hazy expression was assessed in <i>sens^RNAi</i> when <i>p35</i> was ectopically expressed in the eyelet to keep the cells alive (UAS-<i>sens^RNAi</i>/<i>lGMR</i>-Gal4; UAS-<i>p35</i>/UAS-<i>Dcr2</i>) and stained with anti-Hazy (red), anti-Chp (green) and anti-Elav (blue). Eyelet consists of 12 cells and Hazy was expressed in all the cells in UAS-<i>sens^RNAi</i>/<i>lGMR</i>-Gal4;UAS-<i>p35</i>/UAS-<i>Dcr2</i> animals; z-projection of confocal sections. (G) Hazy expression (red) when a dominant-negative form of EcR was ectopically expressed in Rh5-PRs (<i>rh5</i>-Gal4/UAS-<i>EcR^DN</i>) in the eyelet and stained with anti-Chp (green) and anti-Elav (blue); z-projection of confocal sections. Eyelet consists of four cells and Hazy was expressed in all PR cells. (H) A Model describing the role of Hazy in the larval eye and the adult eyelet.</p

Pulsed Sens expression during precursor development.

<p>(A–F) Schematic representation of Sens and Sal expression during stage 11 and stage 12 in wild-type embryonic PRs. (A′–F′) Sens expression (red) from stage 11 to 15 in wild-type embryonic PRs stained with anti-FasII (green) and anti-Sal (blue); single confocal sections are shown. (A′, A″) Sens staining in mid stage 11 is detected in two cells. (B′, B″) In late stage 11, Sens staining is detected in three cells. (C′, C″, C‴) Sens staining in early stage 12 in four cells, co-expressed with Sal in two cells. (D′, D″, D‴) At mid stage 12, all cells express Sal, co-expression with Sens is found in three cells (high, arrow) and one cell (low, arrowhead). (E′, E″, E‴) Sens staining in late stage 12, all cells express Sal, co-expression with Sens is restricted to two cells (high) and very low residual expression is detected in the remaining two cells. (F′, F″, F‴) No Sens expression is seen at stage 15, while Sal expression is observed in all four cells.</p