Jens Rister

Dissection of the Peripheral Motion Channel in the Visual System of Drosophila melanogaster

In the eye, visual information is segregated into modalities such as color and motion, these being transferred to the central brain through separate channels. Here, we genetically dissect the achromatic motion channel in the fly Drosophila melanogaster at the level of the first relay station in the brain, the lamina, where it is split into four parallel pathways (L1-L3, amc/T1). The functional relevance of this divergence is little understood. We now show that the two most prominent pathways, L1 and L2, together are necessary and largely sufficient for motion-dependent behavior. At high pattern contrast, the two pathways are redundant. At intermediate contrast, they mediate motion stimuli of opposite polarity, L2 front-to-back, L1 back-to-front motion. At low contrast, L1 and L2 depend upon each other for motion processing. Of the two minor pathways, amc/T1 specifically enhances the L1 pathway at intermediate contrast. L3 appears not to contribute to motion but to orientation behavior.

The Neural Substrate of Spectral Preference in Drosophila

Drosophila vision is mediated by inputs from three types of photoreceptor neurons; R1-R6 mediate achromatic motion detection, while R7 and R8 constitute two chromatic channels. Neural circuits for processing chromatic information are not known. Here, we identified the first-order interneurons downstream of the chromatic channels. Serial EM revealed that small-field projection neurons Tm5 and Tm9 receive direct synaptic input from R7 and R8, respectively, and indirect input from R1-R6, qualifying them to function as color-opponent neurons. Wide-field Dm8 amacrine neurons receive input from 13-16 UV-sensing R7s and provide output to projection neurons. Using a combinatorial expression system to manipulate activity in different neuron subtypes, we determined that Dm8 neurons are necessary and sufficient for flies to exhibit phototaxis toward ultraviolet instead of green light. We propose that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to projection neurons, which then provide output to higher visual centers.

Flies lacking all synapsins are unexpectedly healthy but are impaired in complex behaviour

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine‐regulate neurotransmitter release by phosphorylation‐dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock‐out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock‐out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified ‘model’ synapse of the larval nerve muscle preparation revealed no difference between wild‐type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild‐type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.

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 adult‐induced 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. J. Comp. Neurol. 498:194–203, 2006.

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. HCLA homomers had nearly identical HA sensitivity to the native receptors (EC50 = 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 1.2 μm, respectively), with much smaller single-channel conductances (∼4 pS). Null mutations of hclA (ortUS6096) abolished the synaptic transients in the electroretinograms (ERGs). Surprisingly, the ERG “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.

Opposite Feedbacks in the Hippo Pathway for Growth Control and Neural Fate

Introduction: A finite number of signaling pathways are repurposed during animal development to regulate an extraordinary array of cellular decisions. Elucidating context-specific mechanisms is crucial for understanding how cellular diversity is generated and for defining potential avenues of pathway misregulation during disease. The Hippo tumor suppressor pathway has been primarily studied in growth control where it inhibits the oncogenic transcriptional coactivator Yorkie (Yki) (YAP/TAZ in vertebrates). The Hippo pathway also functions in nongrowth contexts such as postmitotic fate specification. In the Drosophila visual system, R8 photoreceptor neurons terminally differentiate into one of two alternative subtypes that express either blue-light–sensitive Rhodopsin5 (Rh5) or green-light–sensitive Rhodopsin6 (Rh6). These mutually exclusive cell fates are established by the Hippo pathway kinase warts and the growth regulator gene melted, which repress each other’s expression. However, the mechanisms underlying the context-specific use of the Hippo pathway in postmitotic fate decisions remain unclear. Context-specific regulation by the Hippo signaling in postmitotic photoreceptors. The Hippo pathway uses negative feedback through its transcriptional effector Yki for homeostatic control of proliferation. In Drosophila eyes, two alternative fates of blue- versus green-sensitive R8 photoreceptors are regulated by antagonism between the growth regulator Melted and the Hippo pathway. Contrary to the growth mechanism, Yki positive feedback and a cell-type–restricted transcription factor network promote repurposing of the Hippo pathway for binary fate decisions. Methods: To define the regulatory mechanisms of Hippo-dependent cell fate decisions in Drosophila photoreceptor neurons, we used a combination of genetic epistasis analyses, in vivo cis-regulatory studies, a candidate gene RNA interference screen, and cell culture–based transcription assays Results: We show that the transcriptional output of the Hippo pathway in photoreceptor differentiation, as in cell proliferation, is mediated through the factors Yki and Scalloped. In contrast to growth control, where Yki limits its own activity by negative feedback, we identify two Yki positive-feedback mechanisms: In blue-sensitive Rh5 photoreceptors, Yki represses its own negative regulator warts, downstream of melted; Yki also promotes melted expression, which subsequently represses warts to further promote Yki function. Yki cooperates with the transcription factors Orthodenticle (Otd) and Traffic Jam (Tj) to promote melted expression and Rh5 photoreceptor fate. Otd and Tj, othologs of the mammalian OTX/CRX and MAF/NRL transcription factors, form an evolutionarily conserved transcriptional module for generating photoreceptor subtype diversity. We also show that the transcription factors Senseless and Pph13 create a permissive environment for Warts/Hippo signaling to promote the alternative “default” green-sensitive Rh6 fate. Hence, Hippo pathway function integrates with four cell-type–restricted transcription factors, each promoting genetically different aspects of R8 subtypes, such that Yki activity ultimately coordinates the binary fate decision between blue- and green-sensitive photoreceptors. Discussion: This work illustrates how molecular signaling pathways can adopt context-specific regulation. Yki positive feedback in the photoreceptor fate decision is opposite to the negative feedback found in Hippo growth control. These distinct network-level feedback mechanisms provide context-appropriate functions: homeostasis to fine-tune growth regulation and an all-or-nothing fate decision to ensure robust differentiation of sensory neuron subtypes. Altering network-level systems properties, such as positive or negative feedback, within biochemically conserved pathways may be broadly used to co-opt signaling networks for use in cellular contexts as distinct as proliferation and terminal differentiation. Complexity and Diversity Complex organisms must produce and maintain an extraordinary diversity of cell and tissue types with a limited number of genes and molecular pathways. Cells accomplish this by reusing the same signaling networks at different times, in different tissues, and for different purposes, yet how this context-specificity is achieved is poorly understood. Jukam et al. (1238016, published online 29 August) show how a set of genes that function in cell and tissue growth can be used again in nondividing fly photoreceptor neurons to ensure that flies develop appropriate sensitivity to both blue and green light. The Hippo pathway undergoes a regulatory change—from negative to positive feedback—that requires a tissue-specific transcription factor network. This network uses evolutionarily conserved regulatory factors whose mutations in humans result in degenerative retinal diseases. The context-appropriate positive feedback in flies ensures an all-or-nothing fate decision necessary to establish a functional visual system. Hippo directs cell differentiation and fate through context- and tissue-specific feedback and transcription networks. Signaling pathways are reused for multiple purposes in plant and animal development. The Hippo pathway in mammals and Drosophila coordinates proliferation and apoptosis via the coactivator and oncoprotein YAP/Yorkie (Yki), which is homeostatically regulated through negative feedback. In the Drosophila eye, cross-repression between the Hippo pathway kinase LATS/Warts (Wts) and growth regulator Melted generates mutually exclusive photoreceptor subtypes. Here, we show that this all-or-nothing neuronal differentiation results from Hippo pathway positive feedback: Yki both represses its negative regulator, warts, and promotes its positive regulator, melted. This postmitotic Hippo network behavior relies on a tissue-restricted transcription factor network—including a conserved Otx/Orthodenticle-Nrl/Traffic Jam feedforward module—that allows Warts-Yki-Melted to operate as a bistable switch. Altering feedback architecture provides an efficient mechanism to co-opt conserved signaling networks for diverse purposes in development and evolution.

Establishing and maintaining gene expression patterns: insights from sensory receptor patterning.

In visual and olfactory sensory systems with high discriminatory power, each sensory neuron typically expresses one, or very few, sensory receptor genes, excluding all others. Recent studies have provided insights into the mechanisms that generate and maintain sensory receptor expression patterns. Here, we review how this is achieved in the fly retina and compare it with the mechanisms controlling sensory receptor expression patterns in the mouse retina and in the mouse and fly olfactory systems.

Deciphering the genome's regulatory code: The many languages of DNA

The generation of patterns and the diversity of cell types in a multicellular organism require differential gene regulation. At the heart of this process are enhancers or cis‐regulatory modules (CRMs), genomic regions that are bound by transcription factors (TFs) that control spatio‐temporal gene expression in developmental networks. To date, only a few CRMs have been studied in detail and the underlying cis‐regulatory code is not well understood. Here, we review recent progress on the genome‐wide identification of CRMs with chromatin immunoprecipitation of TF‐DNA complexes followed by microarrays (ChIP‐on‐chip). We focus on two computational approaches that have succeeded in predicting the expression pattern driven by a CRM either based on TF binding site preferences and their expression levels, or quantitative analysis of CRM occupancy by key TFs. We also discuss the current limits of these methods and highlight some of the key problems that have to be solved to gain a more complete understanding of the structure and function of CRMs.

Regional Modulation of a Stochastically Expressed Factor Determines Photoreceptor Subtypes in the Drosophila Retina

Stochastic mechanisms are sometimes utilized to diversify cell fates, especially in nervous systems. In the Drosophila retina, stochastic expression of the PAS-bHLH transcription factor Spineless (Ss) controls photoreceptor subtype choice. In one randomly distributed subset of R7 photoreceptors, Ss activates Rhodopsin4 (Rh4) and represses Rhodopsin3 (Rh3); counterparts lacking Ss express Rh3 and repress Rh4. In the dorsal third region of the retina, the Iroquois Complex transcription factors induce Rh3 in Rh4-expressing R7s. Here, we show that Ss levels are controlled in a binary on/off manner throughout the retina yet are attenuated in the dorsal third region to allow Rh3 coexpression with Rh4. Whereas the sensitivity of rh3 repression to differences in Ss levels generates stochastic and regionalized patterns, the robustness of rh4 activation ensures its stochastic expression throughout the retina. Our findings show how stochastic and regional inputs are integrated to control photoreceptor subtype specification in the Drosophila retina.

Single-base pair differences in a shared motif determine differential Rhodopsin expression

Broad versus restricted expression Color vision in fruit flies requires the restricted expression of light-sensing rhodopsins with different wavelength sensitivities in subsets of photoreceptors. However, all photoreceptors express factors that transduce and amplify the visual signal. Rister et al. found that the distinct expression patterns are determined by a highly tunable regulatory motif. Genes that are broadly expressed have a palindromic variant of the motif. Spatially restricted rhodopsin genes display single-base-pair changes that alter the symmetry of the palindrome and are critical for subtype-specific expression. These findings on the differential regulation of gene expression in fly photoreceptors have implications for the evolution of neuronal subtype diversity. Science, this issue p. 1258 Single–base pair changes in a transcription factor binding motif modulate cell type–specific gene expression. The final identity and functional properties of a neuron are specified by terminal differentiation genes, which are controlled by specific motifs in compact regulatory regions. To determine how these sequences integrate inputs from transcription factors that specify cell types, we compared the regulatory mechanism of Drosophila Rhodopsin genes that are expressed in subsets of photoreceptors to that of phototransduction genes that are expressed broadly, in all photoreceptors. Both sets of genes share an 11–base pair (bp) activator motif. Broadly expressed genes contain a palindromic version that mediates expression in all photoreceptors. In contrast, each Rhodopsin exhibits characteristic single-bp substitutions that break the symmetry of the palindrome and generate activator or repressor motifs critical for restricting expression to photoreceptor subsets. Sensory neuron subtypes can therefore evolve through single-bp changes in short regulatory motifs, allowing the discrimination of a wide spectrum of stimuli.