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.

Phenomenon of reproductive plasticity in ants.

Ant colonies are organized in castes with distinct behaviors that together allow the colony to strive. Reproduction relies on one or a few queens that stay in the nest producing eggs, while females of the worker caste do not reproduce and instead engage in colony maintenance and brood caretaking. Yet, in spite of this clear separation of functions, workers can become reproductive under defined circumstances. Here, we review the context in which workers become reproductive, exhibiting asexual or sexual reproduction depending on the species. Remarkably, the activation of reproduction in these workers can be quite stable, with changes that include behavior and a dramatic extension of lifespan. We compare these changes between species that do or do not have a queen caste. We discuss how the mechanisms underlying reproductive plasticity include changes in hormonal functions and in epigenetic configurations. Further studies are warranted to elucidate not only how reproductive functions have been gradually restricted to the queen caste during evolution but also how reproductive plasticity remains possible in workers of some species.

Spatial patterning controls neuron numbers in the Drosophila visual system.

Neurons must be made in the correct proportions to communicate with the appropriate synaptic partners and form functional circuits. In the Drosophila visual system, multiple subtypes of distal medulla (Dm) inhibitory interneurons are made in distinct, reproducible numbers-from 5 to 800 per optic lobe. These neurons are born from a crescent-shaped neuroepithelium called the outer proliferation center (OPC), which can be subdivided into specific domains based on transcription factor and growth factor expression. We fate mapped Dm neurons and found that more abundant neural types are born from larger neuroepithelial subdomains, while less abundant subtypes are born from smaller ones. Additionally, morphogenetic Dpp/BMP signaling provides a second layer of patterning that subdivides the neuroepithelium into smaller domains to provide more granular control of cell proportions. Apoptosis appears to play a minor role in regulating Dm neuron abundance. This work describes an underappreciated mechanism for the regulation of neuronal stoichiometry.

High-throughput identification of the spatial origins of Drosophila optic lobe neurons using single-cell mRNA-sequencing.

The medulla is the largest neuropil of the Drosophila optic lobe. It contains about 100 neuronal types that have been comprehensively characterized morphologically and molecularly. These neuronal types are specified from a larval neuroepithelium called the Outer Proliferation Center (OPC) via the integration of temporal, spatial, and Notch-driven mechanisms. Although we recently characterized the temporal windows of origin of all medulla neurons, as well as their Notch status, their spatial origins remained unknown. Here, we isolated cells from different OPC spatial domains and performed single-cell mRNA-sequencing to identify the neuronal types produced in these domains. This allowed us to characterize in a high-throughput manner the spatial origins of all medulla neurons and to identify two new spatial subdivisions of the OPC. Moreover, our work shows that the most abundant neuronal types are produced from epithelial domains of different sizes despite being present in a similar number of copies. Combined with our previously published scRNA-seq developmental atlas of the optic lobe, our work opens the door for further studies on how specification factor expression in progenitors impacts gene expression in developing and adult neurons.

Protocol for replacing coding intronic MiMIC and CRIMIC lines with T2A-split-GAL4 lines in Drosophila using genetic crosses.

Here, we present a protocol for generating gene-specific split-GAL4 drivers from coding intronic Minos-mediated integration cassette/CRISPR-mediated integration cassette (MiMIC/CRIMIC) lines in Drosophila. We describe steps for four rounds of in vivo genetic crosses, PCR genotyping, and fluorescence imaging to ensure correct orientation of split-GAL4 integration before establishing stable fly stocks. This protocol offers a cost-effective alternative to traditional microinjection techniques for converting coding intronic MiMIC/CRIMIC lines into gene-specific split-GAL4 lines that are adaptable for fly researchers working on different tissues. For complete details on the use and execution of this protocol, please refer to Chen et al.1.

An Atlas of the Developing Drosophila Visual System Glia and Subcellular mRNA Localization of Transcripts in Single Cells.

Glial cells are essential for proper nervous system development and function. To understand glial development and function, we comprehensively annotated glial cells in a single-cell mRNA-sequencing (scRNAseq) atlas of the developing Drosophila visual system. This allowed us to study their developmental trajectories, from larval to adult stages, and to understand how specific types of glia diversify during development. For example, neuropil glia that are initially transcriptionally similar in larvae, split into ensheathing and astrocyte-like glia during pupal stages. Other glial types, such as chiasm glia change gradually during development without splitting into two cell types. The analysis of scRNA-seq allowed us to discover that the transcriptome of glial cell bodies can be distinguished from that of their broken processes. The processes contain distinct enriched mRNAs that were validated in vivo. Therefore, we have identified most glial types in the developing optic lobe and devised a computational approach to identify mRNA species that are localized to cell bodies or cellular processes.

Using single-cell RNA sequencing to generate predictive cell-type-specific split-GAL4 reagents throughout development.

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.

Using single-cell RNA sequencing to generate cell-type-specific split-GAL4 reagents throughout development.

Cell-type-specific tools facilitate the identification and functional characterization of distinct cell types, which underly the complexity of neuronal circuits. A large collection of existing genetic tools in Drosophila relies on enhancer activity to label different subsets of cells. These enhancer-based GAL4 lines often fail to show a predicable expression pattern to reflect the expression of nearby gene(s), partly due to an incomplete capture of the full gene regulatory elements. While genetic intersectional technique such as the split-GAL4 system further improve cell-type-specificity, it requires significant time and resource to generate and screen through combinations of enhancer expression patterns. In addition, since existing enhancer-based split-GAL4 lines that show cell-type-specific labeling in adult are not necessarily active nor specific in early development, there is a relative lack of tools for the study of neural development. Here, we use an existing single-cell RNA sequencing (scRNAseq) dataset to select gene pairs and provide an efficient pipeline to generate cell-type-specific split-GAL4 lines based on the native genetic regulatory elements. These gene-specific split-GAL4 lines can be generated from a large collection of coding intronic MiMIC/CRIMIC lines either by embryo injection or in vivo cassette swapping crosses and/or CRISPR knock-in at the N or C terminal of the gene. We use the developing Drosophila visual system as a model to demonstrate the high prediction power of scRNAseq-guided gene specific split-GAL4 lines in targeting known cell types. The toolkit allows efficient cluster annotation in scRNAseq datasets but also the identification of novel cell types. Lastly, the gene-specific split-GAL4 lines are broadly applicable to Drosophila tissues. 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 to the fly research community. Significance Statement: Understanding the functional role of individual cell types in the nervous systems has remained a major challenge for neuroscience researchers, partly due to incomplete identification and characterization of underlying cell types. To study the development of individual cell types and their functional roles in health and disease, experimental access to a specific cell type is often a prerequisite. Here, we establish an experimental pipeline to generate gene-specific split-GAL4 guided by single-cell RNA sequencing datasets. These lines show high accuracy for labeling targeted cell types from early developmental stages to adulthood and can be applied to any tissues in Drosophila. The collection of gene-speicifc-split-GAL4 will provide a valuable resource to the entire fly research community.

Temporal regulation of neural diversity in Drosophila and vertebrates.

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.

Coordinated control of neuronal differentiation and wiring by sustained transcription factors.

The large diversity of cell types in nervous systems presents a challenge in identifying the genetic mechanisms that encode it. Here, we report that nearly 200 distinct neurons in the Drosophila visual system can each be defined by unique combinations of on average 10 continuously expressed transcription factors. We show that targeted modifications of this terminal selector code induce predictable conversions of neuronal fates that appear morphologically and transcriptionally complete. Cis-regulatory analysis of open chromatin links one of these genes to an upstream patterning factor that specifies neuronal fates in stem cells. Experimentally validated network models describe the synergistic regulation of downstream effectors by terminal selectors and ecdysone signaling during brain wiring. Our results provide a generalizable framework of how specific fates are implemented in postmitotic neurons.

Insulin signaling in the long-lived reproductive caste of ants.

In most organisms, reproduction is correlated with shorter life span. However, the reproductive queen in eusocial insects exhibits a much longer life span than that of workers. In Harpegnathos ants, when the queen dies, workers can undergo an adult caste switch to reproductive pseudo-queens (gamergates), exhibiting a five-times prolonged life span. To explore the relation between reproduction and longevity, we compared gene expression during caste switching. Insulin expression is increased in the gamergate brain that correlates with increased lipid synthesis and production of vitellogenin in the fat body, both transported to the egg. This results from activation of the mitogen-activated protein kinase (MAPK) branch of the insulin signaling pathway. By contrast, the production in the gamergate developing ovary of anti-insulin Imp-L2 leads to decreased signaling of the AKT/forkhead box O (FOXO) branch in the fat body, which is consistent with their extended longevity.

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.

Neural specification, targeting, and circuit formation during visual system assembly.

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.

A molecular toolkit for superorganisms.

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.

Building a circuit through correlated spontaneous neuronal activity in the developing vertebrate and invertebrate visual systems.

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.

Early behavioral and molecular events leading to caste switching in the ant Harpegnathos.

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.

Integrated Patterning Programs During Drosophila Development Generate the Diversity of Neurons and Control Their Mature Properties.

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.

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.

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.