Phenotypic Convergence: Distinct Transcription Factors Regulate Common Terminal Features.

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.

A complete temporal transcription factor series in the fly visual system.

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.

Neuronal diversity and convergence in a visual system developmental atlas.

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.

Asynchronous transcription and translation of neurotransmitter-related genes characterize the initial stages of neuronal maturation in Drosophila.

Neuron specification and maturation are essential for proper central nervous system development. However, the precise mechanisms that govern neuronal maturation, essential to shape and maintain neuronal circuitry, remain poorly understood. Here, we analyse early-born secondary neurons in the Drosophila larval brain, revealing that the early maturation of secondary neurons goes through 3 consecutive phases: (1) Immediately after birth, neurons express pan-neuronal markers but do not transcribe terminal differentiation genes; (2) Transcription of terminal differentiation genes, such as neurotransmitter-related genes VGlut, ChAT, or Gad1, starts shortly after neuron birth, but these transcripts are, however, not translated; (3) Translation of neurotransmitter-related genes only begins several hours later in mid-pupa stages in a coordinated manner with animal developmental stage, albeit in an ecdysone-independent manner. These results support a model where temporal regulation of transcription and translation of neurotransmitter-related genes is an important mechanism to coordinate neuron maturation with brain development.

Neuronal differentiation strategies: insights from single-cell sequencing and machine learning.

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.

Single-cell transcriptomics in the Drosophila visual system: Advances and perspectives on cell identity regulation, connectivity, and neuronal diversity evolution.

The Drosophila visual system supports complex behaviors and shares many of its anatomical and molecular features with the vertebrate brain. Yet, it contains a much more manageable number of neurons and neuronal types. In addition to the extensive Drosophila genetic toolbox, this relative simplicity has allowed decades of work to yield a detailed account of its neuronal type diversity, morphology, connectivity and specification mechanisms. In the past three years, numerous studies have applied large scale single-cell transcriptomic approaches to the Drosophila visual system and have provided access to the complete gene expression profile of most neuronal types throughout development. This makes the fly visual system particularly well suited to perform detailed studies of the genetic mechanisms underlying the evolution and development of neuronal systems. Here, we highlight how these transcriptomic resources allow exploring long-standing biological questions under a new light. We first present the efforts made to characterize neuronal diversity in the Drosophila visual system and suggest ways to further improve this description. We then discuss current advances allowed by the single-cell datasets, and envisage how these datasets can be further leveraged to address fundamental questions regarding the regulation of neuronal identity, neuronal circuit development and the evolution of neuronal diversity.

A versatile strategy for gene trapping and trap conversion in emerging model organisms.

Genetic model organisms such as Drosophila, C. elegans and the mouse provide formidable tools for studying mechanisms of development, physiology and behaviour. Established models alone, however, allow us to survey only a tiny fraction of the morphological and functional diversity present in the animal kingdom. Here, we present iTRAC, a versatile gene-trapping approach that combines the implementation of unbiased genetic screens with the generation of sophisticated genetic tools both in established and emerging model organisms. The approach utilises an exon-trapping transposon vector that carries an integrase docking site, allowing the targeted integration of new constructs into trapped loci. We provide proof of principle for iTRAC in the emerging model crustacean Parhyale hawaiensis: we generate traps that allow specific developmental and physiological processes to be visualised in unparalleled detail, we show that trapped genes can be easily cloned from an unsequenced genome, and we demonstrate targeting of new constructs into a trapped locus. Using this approach, gene traps can serve as platforms for generating diverse reporters, drivers for tissue-specific expression, gene knockdown and other genetic tools not yet imagined.

Evolution of patterning.

Developing tissues are patterned in space and time; this enables them to differentiate their cell types and form complex structures to support different body plans. Although space and time are two independent entities, there are many examples of spatial patterns that originate from temporal ones. The most prominent example is the expression of the genes hunchback, Krüppel, pdm, and castor, which are expressed temporally in the neural stem cells of the Drosophila ventral nerve cord and spatially along the anteroposterior axis of the blastoderm stage embryo. In this Viewpoint, we investigate the relationship between space and time in specific examples of spatial and temporal patterns with the aim of gaining insight into the evolutionary history of patterning.

[A temporal mechanism for the generation of neuronal diversity]. / Un mécanisme temporel pour la génération de la diversité neuronale.

One of the greatest challenges in neuroscience is to understand how a complex structure, such as the brain, is built. Spatial and temporal patternings of neuronal progenitors are responsible for the generation of most of the neuronal diversity observed in the brain. This review focuses on the temporal patterning of neuronal progenitors, i.e. the sequential expression of transcription factors that changes the capacity of stem cells to generate different neuronal types, and which is conserved in animals. Recent papers have offered a near complete understanding of the mechanism of temporal patterning in the developing visual system of Drosophila, and of how this contributes to the specification of diverse neuronal identities, which are then maintained by the sustained expression of downstream transcription factors. The insect visual system provides a unique model to study the evolution of neuronal cell types, as well as the evolution of neurodevelopmental mechanisms that generate them.

Title: Un mécanisme temporel pour la génération de la diversité neuronale. Abstract: L'un des plus grands défis des neurosciences est de comprendre comment une structure complexe, telle que le cerveau, se construit. L'encodage spatial et temporel des progéniteurs neuronaux permet la génération de l'essentiel de la diversité neuronale. Cette revue se concentre sur l'expression séquentielle de facteurs de transcription temporels, qui modifie la capacité des cellules souches à générer différents types de neurones et qui est conservée chez plusieurs espèces animales. Des publications récentes ont permis, en particulier, une compréhension fine de ce processus au cours du développement du système visuel de la drosophile, en éclairant la manière dont il contribue à la spécification de diverses identités neuronales. Le système visuel des insectes constitue un modèle unique pour étudier l'évolution des mécanismes neurodéveloppementaux qui génèrent la diversité neuronale.

Neuronal Circuit Evolution: From Development to Structure and Adaptive Significance.

Neuronal circuits represent the functional units of the brain. Understanding how the circuits are generated to perform computations will help us understand how the brain functions. Nevertheless, neuronal circuits are not engineered, but have formed through millions of years of animal evolution. We posit that it is necessary to study neuronal circuit evolution to comprehensively understand circuit function. Here, we review our current knowledge regarding the mechanisms that underlie circuit evolution. First, we describe the possible genetic and developmental mechanisms that have contributed to circuit evolution. Then, we discuss the structural changes of circuits during evolution and how these changes affected circuit function. Finally, we try to put circuit evolution in an ecological context and assess the adaptive significance of specific examples. We argue that, thanks to the advent of new tools and technologies, evolutionary neurobiology now allows us to address questions regarding the evolution of circuitry and behavior that were unimaginable until very recently.

Integration of Spatial and Temporal Patterning in the Invertebrate and Vertebrate Nervous System.

The nervous system is one of the most sophisticated animal tissues, consisting of thousands of interconnected cell types. How the nervous system develops its diversity from a few neural stem cells remains a challenging question. Spatial and temporal patterning mechanisms provide an efficient model through which diversity can be generated. The molecular mechanism of spatiotemporal patterning has been studied extensively in Drosophila melanogaster, where distinct sets of transcription factors define the spatial domains and temporal windows that give rise to different cell types. Similarly, in vertebrates, spatial domains defined by transcription factors produce different types of neurons in the brain and neural tube. At the same time, different cortical neuronal types are generated within the same cell lineage with a specific birth order. However, we still do not understand how the orthogonal information of spatial and temporal patterning is integrated into the progenitor and post-mitotic cells to combinatorially give rise to different neurons. In this review, after introducing spatial and temporal patterning in Drosophila and mice, we discuss possible mechanisms that neural progenitors may use to integrate spatial and temporal information. We finally review the functional implications of spatial and temporal patterning and conclude envisaging how small alterations of these mechanisms can lead to the evolution of new neuronal cell types.

Old questions, new models: unraveling complex organ regeneration with new experimental approaches.

How do some animals like crabs, flatworms and salamanders regenerate entire body parts after a severe injury? Which are the mechanisms and how did that regenerative ability evolve over time? The ability to regenerate complex organs is widespread in the animal kingdom, but fundamental, centuries-old questions remain unanswered. Forward genetics approaches that were so successful in probing embryonic development are lacking in most regenerative models, and candidate gene approaches can be biased and misleading. We summarize recent progress in establishing new genetic tools and approaches to study regeneration and provide a personal perspective on the feasibility and value of establishing such tools, based on our experience with a new experimental model, the crustacean Parhyale hawaiensis.

Programmed cell death acts at different stages of Drosophila neurodevelopment to shape the central nervous system.

Nervous system development is a process that integrates cell proliferation, differentiation, and programmed cell death (PCD). PCD is an evolutionary conserved mechanism and a fundamental developmental process by which the final cell number in a nervous system is established. In vertebrates and invertebrates, PCD can be determined intrinsically by cell lineage and age, as well as extrinsically by nutritional, metabolic, and hormonal states. Drosophila has been an instrumental model for understanding how this mechanism is regulated. We review the role of PCD in Drosophila central nervous system development from neural progenitors to neurons, its molecular mechanism and function, how it is regulated and implemented, and how it ultimately shapes the fly central nervous system from the embryo to the adult. Finally, we discuss ideas that emerged while integrating this information.

The genome of the crustacean Parhyale hawaiensis, a model for animal development, regeneration, immunity and lignocellulose digestion.

The amphipod crustacean Parhyale hawaiensis is a blossoming model system for studies of developmental mechanisms and more recently regeneration. We have sequenced the genome allowing annotation of all key signaling pathways, transcription factors, and non-coding RNAs that will enhance ongoing functional studies. Parhyale is a member of the Malacostraca clade, which includes crustacean food crop species. We analysed the immunity related genes of Parhyale as an important comparative system for these species, where immunity related aquaculture problems have increased as farming has intensified. We also find that Parhyale and other species within Multicrustacea contain the enzyme sets necessary to perform lignocellulose digestion ('wood eating'), suggesting this ability may predate the diversification of this lineage. Our data provide an essential resource for further development of Parhyale as an experimental model. The first malacostracan genome will underpin ongoing comparative work in food crop species and research investigating lignocellulose as an energy source.

Cell competition: dying for communal interest.

Viable but slower growing cells are eliminated during embryonic development through the process of cell competition. Two new studies highlight a role for cell competition during adulthood as a surveillance mechanism that ensures tissue integrity during homeostasis, regeneration, and aging.

Common temporal identity factors regulate neuronal diversity in fly ventral nerve cord and mouse retina.

Temporal sequences of transcription factors (tTFs) in Drosophila neural progenitors generate neuronal diversity. Mattar et al. (2015) identify Casz1/Castor as a late temporal identity factor in mouse retinal progenitors that is regulated by the early factor Ikzf1/Hunchback, thus generalizing the notion of tTFs.

A common cellular basis for muscle regeneration in arthropods and vertebrates.

Many animals are able to regenerate amputated or damaged body parts, but it is unclear whether different taxa rely on similar strategies. Planarians and vertebrates use different strategies, based on pluripotent versus committed progenitor cells, respectively, to replace missing tissues. In most animals, however, we lack the experimental tools needed to determine the origin of regenerated tissues. Here, we present a genetically tractable model for limb regeneration, the crustacean Parhyale hawaiensis. We demonstrate that regeneration in Parhyale involves lineage-committed progenitors, as in vertebrates. We discover Pax3/7-expressing muscle satellite cells, previously identified only in chordates, and show that these cells are a source of regenerating muscle in Parhyale. These similarities point to a common cellular basis of regeneration, dating back to the common ancestors of bilaterians.

Reconfiguring gene traps for new tasks using iTRAC.

We recently developed integrase-mediated trap conversion (iTRAC) as a means of exploiting gene traps to create new genetic tools, such as markers for imaging, drivers for gene expression and landing sites for gene and chromosome engineering. The principle of iTRAC is simple: primary gene traps are generated with transposon vectors carrying φC31 integrase docking sites, which are subsequently utilized to integrate different constructs into the selected trapped loci. Thus, iTRAC allows us to reconfigure selected traps for new purposes. Two features make iTRAC an attractive approach for Drosophila research. First, its versatility permits the exploitation of gene traps in an open-ended way, for applications that were not envisaged during the primary trapping screen. Second, iTRAC is readily transferable to new species and provides a means for developing complex genetic tools in drosophilids that lack the facility of Drosophila melanogaster genetics.