RESUMEN
Organisms respond to dietary and environmental challenges by altering the molecular composition of their glycerolipids and glycerophospholipids (GPLs), which may favorably adjust the physicochemical properties of lipid membranes. However, how lipidome changes affect the membrane proteome and, eventually, the physiology of specific organs is an open question. We addressed this issue in Drosophila melanogaster, which is not able to synthesize sterols and polyunsaturated fatty acids but can acquire them from food. We developed a series of semisynthetic foods to manipulate the length and unsaturation of fatty acid moieties in GPLs and singled out proteins whose abundance is specifically affected by membrane lipid unsaturation in the Drosophila eye. Unexpectedly, we identified a group of proteins that have muscle-related functions and increased their abundances under unsaturated eye lipidome conditions. In contrast, the abundance of two stress response proteins, Turandot A and Smg5, is decreased by lipid unsaturation. Our findings could guide the genetic dissection of homeostatic mechanisms that maintain visual function when the eye is exposed to environmental and dietary challenges.
Asunto(s)
Drosophila , Proteoma , Animales , Proteoma/genética , Drosophila melanogaster/genética , Lipidómica , Ácidos Grasos , GlicerofosfolípidosRESUMEN
In human languages, a palindrome reads the same forward as backward (e.g., 'madam'). In regulatory DNA, a palindrome is an inverted sequence repeat that allows a transcription factor to bind as a homodimer or as a heterodimer with another type of transcription factor. Regulatory palindromes are typically imperfect, that is, the repeated sequences differ in at least one base pair, but the functional significance of this asymmetry remains poorly understood. Here, we review the use of imperfect palindromes in Drosophila photoreceptor differentiation and mammalian steroid receptor signaling. Moreover, we discuss mechanistic explanations for the predominance of imperfect palindromes over perfect palindromes in these two gene regulatory contexts. Lastly, we propose to elucidate whether specific imperfectly palindromic variants have specific regulatory functions in steroid receptor signaling and whether such variants can help predict transcriptional outcomes as well as the response of individual patients to drug treatments.
Asunto(s)
Regulación de la Expresión Génica , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Secuencia de Bases , Humanos , Mamíferos , Factores de Transcripción/genéticaRESUMEN
Color vision in Drosophila melanogaster is based on the expression of five different color-sensing Rhodopsin proteins in distinct subtypes of photoreceptor neurons. Promoter regions of less than 300 base pairs are sufficient to reproduce the unique, photoreceptor subtype-specific rhodopsin expression patterns. The underlying cis-regulatory logic remains poorly understood, but it has been proposed that the rhodopsin promoters have a bipartite structure: the distal promoter region directs the highly restricted expression in a specific photoreceptor subtype, while the proximal core promoter region provides general activation in all photoreceptors. Here, we investigate whether the rhodopsin promoters exhibit a strict specialization of their distal (subtype specificity) and proximal (general activation) promoter regions, or if both promoter regions contribute to generating the photoreceptor subtype-specific expression pattern. To distinguish between these two models, we analyze the expression patterns of a set of hybrid promoters that combine the distal promoter region of one rhodopsin with the proximal core promoter region of another rhodopsin. We find that the function of the proximal core promoter regions extends beyond providing general activation: these regions play a previously underappreciated role in generating the non-overlapping expression patterns of the different rhodopsins. Therefore, cis-regulatory motifs in both the distal and the proximal core promoter regions recruit transcription factors that generate the unique rhodopsin patterns in a combinatorial manner. We compare this combinatorial regulatory logic to the regulatory logic of olfactory receptor genes and discuss potential implications for the evolution of rhodopsins.
Asunto(s)
Visión de Colores/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Fotorreceptoras de Invertebrados/metabolismo , Regiones Promotoras Genéticas , Rodopsina/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Evolución Molecular , Femenino , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Fotorreceptoras de Invertebrados/clasificación , Células Fotorreceptoras de Invertebrados/citología , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Rodopsina/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Drosophila melanogaster is a popular model organism to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies, aging, light-induced damage, or dietary deficiencies. Large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which led to the discovery of key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins, including the absolute (molar) quantities of 43 proteins in the eye of adult male Drosophila reared on standard laboratory food. This work provides a generic and expandable resource for further genetic, pharmacological, and dietary studies.
RESUMEN
Vitamin A deficiency can cause human pathologies that range from blindness to embryonic malformations. This diversity is due to the lack of two major vitamin A metabolites with very different functions: the chromophore 11-cis-retinal (vitamin A aldehyde) is a critical component of the visual pigment that mediates phototransduction, while the signaling molecule all-trans-retinoic acid regulates the development of various tissues and is required for the function of the immune system. Since animals cannot synthesize vitamin A de novo, they must obtain it either as preformed vitamin A from animal products or as carotenoid precursors from plant sources. Due to its essential role in the visual system, acute vitamin A deprivation impairs photoreceptor function and causes night blindness (poor vision under dim light conditions), while chronic deprivation results in retinal dystrophies and photoreceptor cell death. Chronic vitamin A deficiency is the leading cause of preventable childhood blindness according to the World Health Organization. Due to the requirement of vitamin A for retinoic acid signaling in development and in the immune system, vitamin A deficiency also causes increased mortality in children and pregnant women in developing countries. Drosophila melanogaster is an excellent model to study the effects of vitamin A deprivation on the eye because vitamin A is not essential for Drosophila development and chronic deficiency does not cause lethality. Moreover, genetic screens in Drosophila have identified evolutionarily conserved factors that mediate the production of vitamin A and its cellular uptake. Here, we review our current knowledge about the role of vitamin A in the visual system of mammals and Drosophila melanogaster. We compare the molecular mechanisms that mediate the uptake of dietary vitamin A precursors and the metabolism of vitamin A, as well as the consequences of vitamin A deficiency for the structure and function of the eye.
Asunto(s)
Visión Ocular/fisiología , Deficiencia de Vitamina A/fisiopatología , Vitamina A/metabolismo , Animales , Drosophila melanogaster/metabolismo , Mamíferos/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Retinaldehído/metabolismo , Tretinoina/metabolismo , Percepción Visual/fisiología , Vitamina A/fisiología , Deficiencia de Vitamina A/metabolismoRESUMEN
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.
Asunto(s)
Drosophila/citología , Regulación de la Expresión Génica , Retina/citología , Opsinas de Bastones/metabolismo , Células Receptoras Sensoriales/citología , Animales , Diferenciación Celular , Ojo Compuesto de los Artrópodos/citología , Ojo Compuesto de los Artrópodos/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ratones , Mucosa Olfatoria/citología , Mucosa Olfatoria/metabolismo , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Opsinas de Bastones/genética , Células Receptoras Sensoriales/metabolismo , Transcripción GenéticaRESUMEN
The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. We show that interplay of two transcription factors Senseless (Sens) and Hazy control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. Our findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner.
Asunto(s)
Linaje de la Célula/genética , Proteínas de Drosophila/genética , Ojo/crecimiento & desarrollo , Metamorfosis Biológica/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Diferenciación Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Ecdisona/biosíntesis , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Larva/crecimiento & desarrollo , Larva/metabolismo , Proteínas Nucleares/metabolismo , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Factores de Transcripción/metabolismoRESUMEN
During terminal differentiation of the mammalian retina, transcription factors control binary cell fate decisions that generate functionally distinct subtypes of photoreceptor neurons. For instance, Otx2 and RORß activate the expression of the transcriptional repressor Blimp-1/PRDM1 that represses bipolar interneuron fate and promotes rod photoreceptor fate. Moreover, Otx2 and Crx promote expression of the nuclear receptor Nrl that promotes rod photoreceptor fate and represses cone photoreceptor fate. Mutations in these four transcription factors cause severe eye diseases such as retinitis pigmentosa. Here, we show that a post-mitotic binary fate decision in Drosophila color photoreceptor subtype specification requires ecdysone signaling and involves orthologs of these transcription factors: Drosophila Blimp-1/PRDM1 and Hr3/RORß promote blue-sensitive (Rh5) photoreceptor fate and repress green-sensitive (Rh6) photoreceptor fate through the transcriptional repression of warts/LATS, the nexus of the phylogenetically conserved Hippo tumor suppressor pathway. Moreover, we identify a novel interaction between Blimp-1 and warts, whereby Blimp-1 represses a warts intronic enhancer in blue-sensitive photoreceptors and thereby gives rise to specific expression of warts in green-sensitive photoreceptors. Together, these results reveal that conserved transcriptional regulators play key roles in terminal cell fate decisions in both the Drosophila and the mammalian retina, and the mechanistic insights further deepen our understanding of how Hippo pathway signaling is repurposed to control photoreceptor fates for Drosophila color vision.
RESUMEN
While the proteome of an organism is largely determined by the genome, the lipidome is shaped by a poorly understood interplay of environmental factors and metabolic processes. To gain insights into the underlying mechanisms, we analyzed the impacts of dietary lipid manipulations on the ocular proteome of Drosophila melanogaster . We manipulated the lipidome with synthetic food media that differed in the supplementation of an equal amount of saturated or polyunsaturated triacylglycerols. This allowed us to generate flies whose eyes had a highly contrasting length and unsaturation of glycerophospholipids, the major lipid class of biological membranes, while the abundance of other membrane lipid classes remained unchanged. By bioinformatically comparing the resulting ocular proteomic trends and contrasting them with the impacts of vitamin A deficiency, we identified ocular proteins whose abundances are differentially affected by lipid saturation and unsaturation. For instance, we unexpectedly identified a group of proteins that have muscle-related functions and increase their abundances in the eye upon lipidome unsaturation but are unaffected by lipidome saturation. Moreover, we identified two differentially lipid-responsive proteins involved in stress responses, Turandot A and Smg5, whose abundances decrease with lipid unsaturation. Lastly, we discovered that the ocular lipid class composition is robust to dietary changes and propose that this may be a general homeostatic feature of the organization of eukaryotic tissues, while the length and unsaturation of fatty acid moieties is more variable to compensate environmental challenges. We anticipate that these insights into the molecular responses of the Drosophila eye proteome to specific lipid manipulations will guide the genetic dissection of the mechanisms that maintain visual function when the eye is exposed to dietary challenges.
RESUMEN
The Drosophila melanogaster eye is a popular model to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies. For instance, the Drosophila eye has been used to investigate the impacts of ageing and environmental stresses such as light-induced damage or dietary deficiencies. Moreover, large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which includes key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins he adult Drosophila melanogaster eye and provide a generic and expandable resource for further genetic, pharmacological, and dietary studies.
RESUMEN
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.
Asunto(s)
Genoma/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN/métodos , Animales , Inmunoprecipitación de Cromatina , Biología Computacional , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción/metabolismoRESUMEN
The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, we took advantage of the fact that Drosophila melanogaster predominantly requires vitamin A for vision, but not for development or survival. We analyzed the impacts of vitamin A deficiency on the morphology, the lipidome, and the proteome of the Drosophila eye. We found that chronic vitamin A deprivation damaged the light-sensing compartments and caused a dramatic loss of visual pigments, but also decreased the molar abundance of most phototransduction proteins that amplify and transduce the visual signal. Unexpectedly, vitamin A deficiency also decreased the abundances of specific subunits of mitochondrial TCA cycle and respiratory chain components but increased the levels of cuticle- and lens-related proteins. In contrast, we found no apparent effects of vitamin A deficiency on the ocular lipidome. In summary, chronic vitamin A deficiency decreases the levels of most components of the visual signaling pathway, but also affects molecular pathways that are not vision-specific and whose mechanistic connection to vitamin A remains to be elucidated.
Asunto(s)
Proteínas de Drosophila , Deficiencia de Vitamina A , Animales , Drosophila , Proteínas de Drosophila/genética , Drosophila melanogaster , Fototransducción/fisiología , Proteoma , Vitamina ARESUMEN
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.
Asunto(s)
Conducta Animal/fisiología , Percepción de Color/fisiología , Percepción de Movimiento/fisiología , Sistema Nervioso , Vías Visuales/fisiología , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila , Drosophila melanogaster , Vuelo Animal/fisiología , Interneuronas/clasificación , Interneuronas/fisiología , Movimiento (Física) , Sistema Nervioso/citología , Estimulación Luminosa/métodos , Transducción de Señal/fisiologíaRESUMEN
Insufficient dietary intake of vitamin A causes various human diseases. For instance, chronic vitamin A deprivation causes blindness, slow growth, impaired immunity, and an increased risk of mortality in children. In contrast to these diverse effects of vitamin A deficiency (VAD) in mammals, chronic VAD in flies neither causes obvious developmental defects nor lethality. As in mammals, VAD in flies severely affects the visual system: it impairs the synthesis of the retinal chromophore, disrupts the formation of the visual pigments (Rhodopsins), and damages the photoreceptors. However, the molecular mechanisms that respond to VAD remain poorly understood. To identify genes and signaling pathways that are affected by VAD, we performed RNA-sequencing and differential gene expression analysis in Drosophila melanogaster. We found an upregulation of genes that are essential for the synthesis of the retinal chromophore, specific aminoacyl-tRNA synthetases, and major nutrient reservoir proteins. We also discovered that VAD affects several genes that are required for the termination of the light response: for instance, we found a downregulation of both arrestin genes that are essential for the inactivation of Rhodopsin. A comparison of the VAD-responsive genes with previously identified blue light stress-responsive genes revealed that the two types of environmental stress trigger largely nonoverlapping transcriptome responses. Yet, both stresses increase the expression of seven genes with poorly understood functions. Taken together, our transcriptome analysis offers insights into the molecular mechanisms that respond to environmental stresses.
Asunto(s)
Proteínas de Drosophila , Deficiencia de Vitamina A , Animales , Drosophila melanogaster/genética , Expresión Génica , Rodopsina/genética , Vitamina ARESUMEN
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 (EC(50) = 25 microM). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states ( approximately 25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors were more sensitive to HA (EC(50) = 14 and 1.2 microM, respectively), with much smaller single-channel conductances ( approximately 4 pS). Null mutations of hclA (ort(US6096)) 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.
Asunto(s)
Canales de Cloruro/fisiología , Proteínas de Drosophila/fisiología , Proteínas del Tejido Nervioso/fisiología , Células Fotorreceptoras de Invertebrados/fisiología , Receptores Histamínicos/fisiología , Sinapsis/fisiología , Animales , Línea Celular , Canales de Cloruro/biosíntesis , Canales de Cloruro/genética , Drosophila , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Mutación , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuroglía/fisiología , Lóbulo Óptico de Animales no Mamíferos/citología , Lóbulo Óptico de Animales no Mamíferos/metabolismo , Lóbulo Óptico de Animales no Mamíferos/fisiología , Técnicas de Placa-Clamp , Estimulación Luminosa/métodos , Células Fotorreceptoras de Invertebrados/citología , Transmisión Sináptica/fisiologíaRESUMEN
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 shibire(ts1), 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 shibire(ts1), 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.
Asunto(s)
Conducta Animal/fisiología , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Animales , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Femenino , Masculino , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/metabolismo , Parálisis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Sinapsis/metabolismo , Temperatura , Toxina Tetánica/genética , Toxina Tetánica/metabolismoRESUMEN
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.
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Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Células Fotorreceptoras de Invertebrados/fisiología , Regiones Promotoras Genéticas/genética , Rodopsina/genética , Visión Ocular/genética , Animales , Emparejamiento Base , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Mutación , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neurogénesis/genética , Proteínas Nucleares/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Retroalimentación Fisiológica , Redes Reguladoras de Genes , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Mitosis , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/metabolismo , Proteínas Quinasas/genética , Proteínas Señalizadoras YAPRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Células Fotorreceptoras de Vertebrados/fisiología , Receptores de Hidrocarburo de Aril/metabolismo , Retina/fisiología , Alelos , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Embrión no Mamífero/metabolismo , Embrión no Mamífero/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Mutación , Fenotipo , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/metabolismo , Regiones Promotoras Genéticas , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de Hidrocarburo de Aril/genética , Retina/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Especificidad de la Especie , Transcripción GenéticaRESUMEN
The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s. Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc and initiates the assembly of undifferentiated cells into ommatidia. The 'founder cell' R8 is specified first and recruits R1-6 and then R7. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes, including rhabdomere formation, synaptogenesis and eventually rh expression. In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).