RESUMO
Transcription factors regulate the molecular, morphological, and physiological characteristics of neurons and generate their impressive cell-type diversity. To gain insight into the general principles that govern how transcription factors regulate cell-type diversity, we used large-scale single-cell RNA sequencing to characterize the extensive cellular diversity in the Drosophila optic lobes. We sequenced 55,000 single cells and assigned them to 52 clusters. We validated and annotated many clusters using RNA sequencing of FACS-sorted single-cell types and cluster-specific genes. To identify transcription factors responsible for inducing specific terminal differentiation features, we generated a "random forest" model, and we showed that the transcription factors Apterous and Traffic-jam are required in many but not all cholinergic and glutamatergic neurons, respectively. In fact, the same terminal characters often can be regulated by different transcription factors in different cell types, arguing for extensive phenotypic convergence. Our data provide a deep understanding of the developmental and functional specification of a complex brain structure.
Assuntos
Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurogênese/fisiologia , Animais , Diferenciação Celular , Neurônios Colinérgicos/fisiologia , Análise por Conglomerados , Simulação por Computador , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica/métodos , Proteínas de Homeodomínio , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Neuroglia/fisiologia , Neurônios/fisiologia , Neurotransmissores/genética , Neurotransmissores/fisiologia , Lobo Óptico de Animais não Mamíferos/fisiologia , Fenótipo , Proteínas Proto-Oncogênicas/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Understanding how complex brain wiring is produced during development is a daunting challenge. In Drosophila, information from 800 retinal ommatidia is processed in distinct brain neuropiles, each subdivided into 800 matching retinotopic columns. The lobula plate comprises four T4 and four T5 neuronal subtypes. T4 neurons respond to bright edge motion, whereas T5 neurons respond to dark edge motion. Each is tuned to motion in one of the four cardinal directions, effectively establishing eight concurrent retinotopic maps to support wide-field motion. We discovered a mode of neurogenesis where two sequential Notch-dependent divisions of either a horizontal or a vertical progenitor produce matching sets of two T4 and two T5 neurons retinotopically coincident with pairwise opposite direction selectivity. We show that retinotopy is an emergent characteristic of this neurogenic program and derives directly from neuronal birth order. Our work illustrates how simple developmental rules can implement complex neural organization.
Assuntos
Drosophila/fisiologia , Percepção de Movimento/fisiologia , Retina/fisiologia , Animais , Proteínas de Drosophila/metabolismo , Locomoção/fisiologia , Modelos Neurológicos , Neurônios/fisiologia , Lobo Óptico de Animais não Mamíferos/química , Lobo Óptico de Animais não Mamíferos/metabolismo , Receptores Notch/metabolismo , Retina/citologia , Vias VisuaisRESUMO
Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.
Assuntos
Formigas/crescimento & desenvolvimento , Formigas/genética , Proteínas de Insetos/genética , Receptores Odorantes/genética , Comportamento Social , Sequência de Aminoácidos , Animais , Formigas/anatomia & histologia , Formigas/fisiologia , Antenas de Artrópodes/anatomia & histologia , Antenas de Artrópodes/metabolismo , Sequência de Bases , Comportamento Animal , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Técnicas de Inativação de Genes , Proteínas de Insetos/química , Masculino , Mutação , Feromônios/metabolismo , Receptores Odorantes/químicaRESUMO
In this issue of Cell, Langen et al. use time-lapse multiphoton microscopy to show how Drosophila photoreceptor growth cones find their targets. Based on the observed dynamics, they develop a simple developmental algorithm recapitulating the highly complex connectivity pattern of these neurons, suggesting a basic framework for establishing wiring specificity.
Assuntos
Axônios , Olho Composto de Artrópodes/inervação , Simulação por Computador , Drosophila/crescimento & desenvolvimento , Células Fotorreceptoras de Invertebrados/fisiologia , AnimaisRESUMO
During the approximately 5 days of Drosophila neurogenesis (late embryogenesis to the beginning of pupation), a limited number of neural stem cells produce approximately 200,000 neurons comprising hundreds of cell types. To build a functional nervous system, neuronal types need to be produced in the proper places, appropriate numbers, and correct times. We discuss how neural stem cells (neuroblasts) obtain so-called area codes for their positions in the nervous system (spatial patterning) and how they keep time to sequentially produce neurons with unique fates (temporal patterning). We focus on specific examples that demonstrate how a relatively simple patterning system (Notch) can be used reiteratively to generate different neuronal types. We also speculate on how different modes of temporal patterning that operate over short versus long time periods might be linked. We end by discussing how specification programs are integrated and lead to the terminal features of different neuronal types.
Assuntos
Proteínas de Drosophila , Células-Tronco Neurais , Animais , Drosophila , Proteínas de Drosophila/genética , Neurogênese , NeurôniosRESUMO
Temporal patterning of neural progenitors is one of the core mechanisms generating neuronal diversity in the central nervous system. Here, we show that, in the tips of the outer proliferation center (tOPC) of the developing Drosophila optic lobes, a unique temporal series of transcription factors not only governs the sequential production of distinct neuronal subtypes but also controls the mode of progenitor division, as well as the selective apoptosis of Notch(OFF) or Notch(ON) neurons during binary cell fate decisions. Within a single lineage, intermediate precursors initially do not divide and generate only one neuron; subsequently, precursors divide, but their Notch(ON) progeny systematically die through Reaper activity, whereas later, their Notch(OFF) progeny die through Hid activity. These mechanisms dictate how the tOPC produces neurons for three different optic ganglia. We conclude that temporal patterning generates neuronal diversity by specifying both the identity and survival/death of each unique neuronal subtype.
Assuntos
Sobrevivência Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Neurogênese , Neuropeptídeos/metabolismo , Lobo Óptico de Animais não Mamíferos/citologia , Receptores Notch/metabolismo , Animais , Apoptose , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Células-Tronco Neurais , Lobo Óptico de Animais não Mamíferos/metabolismoRESUMO
During the development of the vertebrate nervous systems, genetic programs assemble an immature circuit that is subsequently refined by neuronal activity evoked by external stimuli. However, prior to sensory experience, the intrinsic property of the developing nervous system also triggers correlated network-level neuronal activity, with retinal waves in the developing vertebrate retina being the best documented example. Spontaneous activity has also been found in the visual system of Drosophila Here, we compare the spontaneous activity of the developing visual system between mammalian and Drosophila and suggest that Drosophila is an emerging model for mechanistic and functional studies of correlated spontaneous activity.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Retina/citologia , Retina/embriologia , Células Receptoras Sensoriais/fisiologia , Animais , Drosophila melanogaster/fisiologia , Olho/citologia , Olho/crescimento & desenvolvimento , Humanos , Modelos Animais , Retina/fisiologia , Células Receptoras Sensoriais/citologiaRESUMO
Ant societies show a division of labor in which a queen is in charge of reproduction while nonreproductive workers maintain the colony. In Harpegnathos saltator, workers retain reproductive ability, inhibited by the queen pheromones. Following the queen loss, the colony undergoes social unrest with an antennal dueling tournament. Most workers quickly abandon the tournament while a few workers continue the dueling for months and become gamergates (pseudoqueens). However, the temporal dynamics of the social behavior and molecular mechanisms underlining the caste transition and social dominance remain unclear. By tracking behaviors, we show that the gamergate fate is accurately determined 3 d after initiation of the tournament. To identify genetic factors responsible for this commitment, we compared transcriptomes of different tissues between dueling and nondueling workers. We found that juvenile hormone is globally repressed, whereas ecdysone biosynthesis in the ovary is increased in gamergates. We show that molecular changes in the brain serve as earliest caste predictors compared with other tissues. Thus, behavioral and molecular data indicate that despite the prolonged social upheaval, the gamergate fate is rapidly established, suggesting a robust re-establishment of social structure.
Assuntos
Formigas , Comportamento Animal , Animais , Feminino , Formigas/genética , Comportamento Animal/fisiologia , Ovário/metabolismo , Reprodução/genética , TranscriptomaRESUMO
The brain consists of thousands of neuronal types that are generated by stem cells producing different neuronal types as they age. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs)1-6. Here we used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila optic lobe neurons. We verify that tTFs regulate the progression of the series by activating the next tTF(s) and repressing the previous one(s), and also identify more complex mechanisms of regulation. Moreover, we establish the temporal window of origin and birth order of each neuronal type in the medulla and provide evidence that these tTFs are sufficient to explain the generation of all of the neuronal diversity in this brain region. Finally, we describe the first steps of neuronal differentiation and show that these steps are conserved in humans. We find that terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons. This comprehensive analysis of a temporal series of tTFs in the optic lobe offers mechanistic insights into how tTF series are regulated, and how they can lead to the generation of a complete set of neurons.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Regulação da Expressão Gênica no Desenvolvimento , Lobo Óptico de Animais não Mamíferos , Fatores de Transcrição , Visão Ocular , Percepção Visual , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Lobo Óptico de Animais não Mamíferos/citologia , RNA-Seq , Análise de Célula Única , Fatores de Transcrição/metabolismoRESUMO
Deciphering how neuronal diversity is established and maintained requires a detailed knowledge of neuronal gene expression throughout development. In contrast to mammalian brains1,2, the large neuronal diversity of the Drosophila optic lobe3 and its connectome4-6 are almost completely characterized. However, a molecular characterization of this neuronal diversity, particularly during development, has been lacking. Here we present insights into brain development through a nearly complete description of the transcriptomic diversity of the optic lobes of Drosophila. We acquired the transcriptome of 275,000 single cells at adult and at five pupal stages, and built a machine-learning framework to assign them to almost 200 cell types at all time points during development. We discovered two large neuronal populations that wrap neuropils during development but die just before adulthood, as well as neuronal subtypes that partition dorsal and ventral visual circuits by differential Wnt signalling throughout development. Moreover, we show that the transcriptomes of neurons that are of the same type but are produced days apart become synchronized shortly after their production. During synaptogenesis we also resolved neuronal subtypes that, although differing greatly in morphology and connectivity, converge to indistinguishable transcriptomic profiles in adults. Our datasets almost completely account for the known neuronal diversity of the Drosophila optic lobes, and serve as a paradigm to understand brain development across species.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Neurônios/classificação , Neurônios/metabolismo , Lobo Óptico de Animais não Mamíferos/citologia , Lobo Óptico de Animais não Mamíferos/crescimento & desenvolvimento , Anatomia Artística , Animais , Apoptose , Atlas como Assunto , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Neurônios/citologia , Pupa/citologia , Pupa/crescimento & desenvolvimento , Análise de Célula Única , Sinapses/metabolismo , Transcriptoma/genética , Vias Visuais , Via de Sinalização WntRESUMO
Eusocial insects live in societies in which distinct family members serve specific roles in maintaining the colony and advancing the reproductive ability of a few select individuals. Given the genetic similarity of all colony members, the diversity of morphologies and behaviors is surprising. Social communication relies on pheromones and olfaction, as shown by mutants of orco, the universal odorant receptor coreceptor, and through electrophysiological analysis of neuronal responses to pheromones. Additionally, neurohormonal factors and epigenetic regulators play a key role in caste-specific behavior, such as foraging and caste switching. These studies start to allow an understanding of the molecular mechanisms underlying social behavior and provide a technological foundation for future studies of eusocial insects. In this review, we highlight recent findings in eusocial insects that advance our understanding of genetic and epigenetic regulations of social behavior and provide perspectives on future studies using cutting-edge technologies.
Assuntos
Comportamento Animal/fisiologia , Epigênese Genética/genética , Insetos/genética , Comportamento Social , Animais , Epigênese Genética/fisiologia , Insetos/fisiologia , Neurônios/metabolismo , Feromônios/genética , Receptores Odorantes/genética , Olfato/genéticaRESUMO
How complex networks of activators and repressors lead to exquisitely specific cell-type determination during development is poorly understood. In the Drosophila eye, expression patterns of Rhodopsins define at least eight functionally distinct though related subtypes of photoreceptors. Here, we describe a role for the transcription factor gene defective proventriculus (dve) as a critical node in the network regulating Rhodopsin expression. dve is a shared component of two opposing, interlocked feedforward loops (FFLs). Orthodenticle and Dve interact in an incoherent FFL to repress Rhodopsin expression throughout the eye. In R7 and R8 photoreceptors, a coherent FFL relieves repression by Dve while activating Rhodopsin expression. Therefore, this network uses repression to restrict and combinatorial activation to induce cell-type-specific expression. Furthermore, Dve levels are finely tuned to yield cell-type- and region-specific repression or activation outcomes. This interlocked FFL motif may be a general mechanism to control terminal cell-fate specification.
Assuntos
Proteínas de Drosophila/genética , Drosophila/embriologia , Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/genética , Animais , Drosophila/citologia , Proteínas de Drosophila/metabolismo , Olho/embriologia , Retroalimentação Fisiológica , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Cell-type-specific tools facilitate the identification and functional characterization of the distinct cell types that form the complexity of neuronal circuits. A large collection of existing genetic tools in Drosophila relies on enhancer activity to label different subsets of cells and has been extremely useful in analyzing functional circuits in adults. However, these enhancer-based GAL4 lines often do not reflect the expression of nearby gene(s) as they only represent a small portion of the full gene regulatory elements. While genetic intersectional techniques such as the split-GAL4 system further improve cell-type-specificity, it requires significant time and resources to screen through combinations of enhancer expression patterns. Here, we use existing developmental single-cell RNA sequencing (scRNAseq) datasets to select gene pairs for split-GAL4 and provide a highly efficient and predictive pipeline (scMarco) to generate cell-type-specific split-GAL4 lines at any time during development, based on the native gene regulatory elements. These gene-specific split-GAL4 lines can be generated from a large collection of coding intronic MiMIC/CRIMIC lines or by CRISPR knock-in. We use the developing Drosophila visual system as a model to demonstrate the high predictive power of scRNAseq-guided gene-specific split-GAL4 lines in targeting known cell types, annotating clusters in scRNAseq datasets as well as in identifying novel cell types. Lastly, the gene-specific split-GAL4 lines are broadly applicable to any other Drosophila tissue. Our work opens new avenues for generating cell-type-specific tools for the targeted manipulation of distinct cell types throughout development and represents a valuable resource for the Drosophila community.
Assuntos
Proteínas de Drosophila , Fatores de Transcrição , Animais , Fatores de Transcrição/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Técnicas Genéticas , Análise de Sequência de RNA , Drosophila melanogaster/metabolismoRESUMO
The generation of neuronal diversity involves temporal patterning mechanisms by which a given progenitor sequentially produces multiple cell types. Several parallels are evident between the brain development programs of Drosophila and vertebrates, such as the successive emergence of specific cell types and the use of combinations of transcription factors to specify cell fates. Furthermore, cell-extrinsic cues such as hormones and signaling pathways have also been shown to be regulatory modules of temporal patterning. Recently, transcriptomic and epigenomic studies using large single-cell sequencing datasets have provided insights into the transcriptional dynamics of neurogenesis in the Drosophila and mammalian central nervous systems. We review these commonalities in the specification of neuronal identity and highlight the conserved or convergent strategies of brain development by discussing temporal patterning mechanisms found in flies and vertebrates.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Vertebrados/metabolismo , Neurônios/metabolismo , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mamíferos/metabolismoRESUMO
The Drosophila visual system has become a premier model for probing how neural diversity is generated during development. Recent work has provided deeper insight into the elaborate mechanisms that control the range of types and numbers of neurons produced, which neurons survive, and how they interact. These processes drive visual function and influence behavioral preferences. Other studies are beginning to provide insight into how neuronal diversity evolved in insects by adding new cell types and modifying neural circuits. Some of the most powerful comparisons have been those made to the Drosophila visual system, where a deeper understanding of molecular mechanisms allows for the generation of hypotheses about the evolution of neural anatomy and function. The evolution of new neural types contributes additional complexity to the brain and poses intriguing questions about how new neurons interact with existing circuitry. We explore how such individual changes in a variety of species might play a role over evolutionary timescales. Lessons learned from the fly visual system apply to other neural systems, including the fly central brain, where decisions are made and memories are stored.
Assuntos
Evolução Biológica , Drosophila melanogaster/metabolismo , Rede Nervosa/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Retina/metabolismo , Visão Binocular/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/classificação , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Discos Imaginais/citologia , Discos Imaginais/metabolismo , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Rede Nervosa/citologia , Neurópilo/citologia , Neurópilo/metabolismo , Organogênese/genética , Células Fotorreceptoras de Invertebrados/citologia , Filogenia , Retina/citologiaRESUMO
In recent work published in Nature, Raj et al. (2010) use single mRNA molecule quantification to show that variation in gene expression in Caenorhabditis elegans increases in mutants displaying incomplete penetrance. They find that a bimodal response is triggered when noisy expression of an upstream regulator crosses a critical threshold.
RESUMO
Social insects, such as ants, bees, wasps, and termites, draw biologists' attention due to their distinctive lifestyles. As experimental systems, they provide unique opportunities to study organismal differentiation, division of labor, longevity, and the evolution of development. Ants are particularly attractive because several ant species can be propagated in the laboratory. However, the same lifestyle that makes social insects interesting also hampers the use of molecular genetic techniques. Here, we summarize the efforts of the ant research community to surmount these hurdles and obtain novel mechanistic insight into the biology of social insects. We review current approaches and propose novel ones involving genomics, transcriptomics, chromatin and DNA methylation profiling, RNA interference (RNAi), and genome editing in ants and discuss future experimental strategies.
Assuntos
Genômica/métodos , Insetos/fisiologia , Animais , Formigas , Comportamento Animal , Cromatina/genética , Genoma de Inseto , Insetos/genética , Interferência de RNA , Análise de Célula Única , Comportamento SocialRESUMO
Although cell fate specification is tightly controlled to yield highly reproducible results and avoid extreme variation, developmental programs often incorporate stochastic mechanisms to diversify cell types. Stochastic specification phenomena are observed in a wide range of species and an assorted set of developmental contexts. In bacteria, stochastic mechanisms are utilized to generate transient subpopulations capable of surviving adverse environmental conditions. In vertebrate, insect, and worm nervous systems, stochastic fate choices are used to increase the repertoire of sensory and motor neuron subtypes. Random fate choices are also integrated into developmental programs controlling organogenesis. Although stochastic decisions can be maintained to produce a mosaic of fates within a population of cells, they can also be compensated for or directed to yield robust and reproducible outcomes.
Assuntos
Linhagem da Célula , Modelos Biológicos , Animais , Bactérias/crescimento & desenvolvimento , Diferenciação Celular , HumanosRESUMO
Like other sensory systems, the visual system is topographically organized: Its sensory neurons, the photoreceptors, and their targets maintain point-to-point correspondence in physical space, forming a retinotopic map. The iterative wiring of circuits in the visual system conveniently facilitates the study of its development. Over the past few decades, experiments in Drosophila have shed light on the principles that guide the specification and connectivity of visual system neurons. In this review, we describe the main findings unearthed by the study of the Drosophila visual system and compare them with similar events in mammals. We focus on how temporal and spatial patterning generates diverse cell types, how guidance molecules distribute the axons and dendrites of neurons within the correct target regions, how vertebrates and invertebrates generate their retinotopic map, and the molecules and mechanisms required for neuronal migration. We suggest that basic principles used to wire the fly visual system are broadly applicable to other systems and highlight its importance as a model to study nervous system development.
Assuntos
Padronização Corporal , Drosophila melanogaster/embriologia , Rede Nervosa/embriologia , Neurônios/fisiologia , Vias Visuais/embriologia , Animais , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Mamíferos/embriologia , Neuroglia/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Neuronal replacement therapies rely on the in vitro differentiation of specific cell types from embryonic or induced pluripotent stem cells, or on the direct reprogramming of differentiated adult cells via the expression of transcription factors or signaling molecules. The factors used to induce differentiation or reprogramming are often identified by informed guesses based on differential gene expression or known roles for these factors during development. Moreover, differentiation protocols usually result in partly differentiated cells or the production of a mix of cell types. In this Hypothesis article, we suggest that, to overcome these inefficiencies and improve neuronal differentiation protocols, we need to take into account the developmental history of the desired cell types. Specifically, we present a strategy that uses single-cell sequencing techniques combined with machine learning as a principled method to select a sequence of programming factors that are important not only in adult neurons but also during differentiation.