Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper.

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.

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.

Integration of temporal and spatial patterning generates neural diversity.

In the Drosophila optic lobes, 800 retinotopically organized columns in the medulla act as functional units for processing visual information. The medulla contains over 80 types of neuron, which belong to two classes: uni-columnar neurons have a stoichiometry of one per column, while multi-columnar neurons contact multiple columns. Here we show that combinatorial inputs from temporal and spatial axes generate this neuronal diversity: all neuroblasts switch fates over time to produce different neurons; the neuroepithelium that generates neuroblasts is also subdivided into six compartments by the expression of specific factors. Uni-columnar neurons are produced in all spatial compartments independently of spatial input; they innervate the neuropil where they are generated. Multi-columnar neurons are generated in smaller numbers in restricted compartments and require spatial input; the majority of their cell bodies subsequently move to cover the entire medulla. The selective integration of spatial inputs by a fixed temporal neuroblast cascade thus acts as a powerful mechanism for generating neural diversity, regulating stoichiometry and the formation of retinotopy.

Identification of enhancers that drive the spatially restricted expression of Vsx1 and Rx in the outer proliferation center of the developing Drosophila optic lobe.

Combinatorial spatial and temporal patterning of stem cells is a powerful mechanism for the generation of neural diversity in insect and vertebrate nervous systems. In the developing Drosophila medulla, the neural stem cells of the outer proliferation center (OPC) are spatially patterned by the mutually exclusive expression of three homeobox transcription factors: Vsx1 in the center of the OPC crescent (cOPC), Optix in the main arms (mOPC), and Rx in the posterior tips (pOPC). These spatial factors act together with a temporal cascade of transcription factors in OPC neuroblasts to specify the greater than 80 medulla cell types. Here, we identify the enhancers that are sufficient to drive the spatially restricted expression of the Vsx1 and Rx genes in the OPC. We show that removal of the cOPC enhancer in the Muddled inversion mutant leads to the loss of Vsx1 expression in the cOPC. Analysis of the evolutionarily conserved sequences within these enhancers suggests that direct repression by Optix may restrict the expression of Vsx1 and Rx to the cOPC and pOPC, respectively.

Temporal patterning of Drosophila medulla neuroblasts controls neural fates.

In the Drosophila optic lobes, the medulla processes visual information coming from inner photoreceptors R7 and R8 and from lamina neurons. It contains approximately 40,000 neurons belonging to more than 70 different types. Here we describe how precise temporal patterning of neural progenitors generates these different neural types. Five transcription factors-Homothorax, Eyeless, Sloppy paired, Dichaete and Tailless-are sequentially expressed in a temporal cascade in each of the medulla neuroblasts as they age. Loss of Eyeless, Sloppy paired or Dichaete blocks further progression of the temporal sequence. We provide evidence that this temporal sequence in neuroblasts, together with Notch-dependent binary fate choice, controls the diversification of the neuronal progeny. Although a temporal sequence of transcription factors had been identified in Drosophila embryonic neuroblasts, our work illustrates the generality of this strategy, with different sequences of transcription factors being used in different contexts.

Cell migration in Drosophila optic lobe neurons is controlled by eyeless/Pax6.

In the developing Drosophila optic lobe, eyeless, apterous and distal-less, three genes that encode transcription factors with important functions during development, are expressed in broad subsets of medulla neurons. Medulla cortex cells follow two patterns of cell movements to acquire their final position: first, neurons are arranged in columns below each neuroblast. Then, during pupation, they migrate laterally, intermingling with each other to reach their retinotopic position in the adult optic lobe. eyeless, which encodes a Pax6 transcription factor, is expressed early in progenitors and controls aspects of this cell migration. Its loss in medulla neurons leads to overgrowth and a failure of lateral migration during pupation. These defects in cell migration among medulla cortex cells can be rescued by removing DE-Cadherin. Thus, eyeless links neurogenesis and neuronal migration.

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.

Mint/X11 PDZ domains from non-bilaterian animals recognize and bind Ca V 2 calcium channel C-termini in vitro .

PDZ domain mediated interactions with voltage-gated calcium (Ca V ) channel C-termini play important roles in localizing membrane Ca 2+ signaling. The first such interaction was described between the scaffolding protein Mint-1 and Ca V 2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. In addition to Ca V 2 channels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast and bacterial 2-hybrid experiments, we show that Mint and Ca V 2 channels from cnidarians and placozoans interact in vitro , and in situ hybridization revealed co-expression in dissociated neurons from the cnidarian Nematostella vectensis . Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly binds the divergent C-terminal ligands of cnidarian and placozoan Ca V 2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore Ca V 2 channel C-terminus. Altogether, our analyses suggest that the capacity of Mint to bind CaV2 channels predates pre-bilaterian animals, and that evolutionary changes in Ca V 2 channel C-terminal sequences resulted in altered binding modalities with Mint.

Mint/X11 PDZ domains from non-bilaterian animals recognize and bind CaV2 calcium channel C-termini in vitro.

PDZ domain mediated interactions with voltage-gated calcium (CaV) channel C-termini play important roles in localizing membrane Ca2+ signaling. The first such interaction was described between the scaffolding protein Mint-1 and CaV2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. In addition to CaV2 chanels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast and bacterial 2-hybrid experiments, we show that Mint and CaV2 channels from cnidarians and placozoans interact in vitro, and in situ hybridization revealed co-expression in dissociated neurons from the cnidarian Nematostella vectensis. Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly bound the divergent C-terminal ligands of cnidarian and placozoan CaV2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore CaV2 channel C-terminus. Altogether, our analyses suggest that the capacity of Mint to bind CaV2 channels predates bilaterian animals, and that evolutionary changes in CaV2 channel C-terminal sequences resulted in altered binding modalities with Mint.

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.

Spalt and disco define the dorsal-ventral neuroepithelial compartments of the developing Drosophila medulla.

Spatial patterning of neural stem cell populations is a powerful mechanism by which to generate neuronal diversity. In the developing Drosophila medulla, the symmetrically dividing neuroepithelial cells of the outer proliferation center crescent are spatially patterned by the nonoverlapping expression of 3 transcription factors: Vsx1 in the center, Optix in the adjacent arms, and Rx in the tips. These spatial genes compartmentalize the outer proliferation center and, together with the temporal patterning of neuroblasts, act to diversify medulla neuronal fates. The observation that the dorsal and ventral halves of the outer proliferation center also grow as distinct compartments, together with the fact that a subset of neuronal types is generated from only one half of the crescent, suggests that additional transcription factors spatially pattern the outer proliferation center along the dorsal-ventral axis. Here, we identify the spalt (salm and salr) and disco (disco and disco-r) genes as the dorsal-ventral patterning transcription factors of the outer proliferation center. Spalt and Disco are differentially expressed in the dorsal and ventral outer proliferation center from the embryo through to the third instar larva, where they cross-repress each other to form a sharp dorsal-ventral boundary. We show that hedgehog is necessary for Disco expression in the embryonic optic placode and that disco is subsequently required for the development of the ventral outer proliferation center and its neuronal progeny. We further demonstrate that this dorsal-ventral patterning axis acts independently of Vsx1-Optix-Rx and thus propose that Spalt and Disco represent a third outer proliferation center patterning axis that may act to further diversify medulla fates.

Imp and Syp mediated temporal patterning of neural stem cells in the developing Drosophila CNS.

The assembly of complex neural circuits requires that stem cells generate diverse types of neurons in the correct temporal order. Pioneering work in the Drosophila embryonic ventral nerve cord has shown that neural stem cells are temporally patterned by the sequential expression of rapidly changing transcription factors to generate diversity in their progeny. In recent years, a second temporal patterning mechanism, driven by the opposing gradients of the Imp and Syp RNA-binding proteins, has emerged as a powerful way to generate neural diversity. This long-range temporal patterning mechanism is utilized in the extended neural stem cell lineages of the postembryonic fly brain. Here, we review the role played by Imp and Syp gradients in several neural stem cell lineages, focusing on how they specify sequential neural fates through the post-transcriptional regulation of target genes, including the Chinmo and Mamo transcription factors. We further discuss how upstream inputs, including hormonal signals, modify the output of these gradients to couple neurogenesis with the development of the organism. Finally, we review the roles that the Imp and Syp gradients play beyond the generation of diversity, including the regulation of stem cell proliferation, the timing of neural stem cell lineage termination, and the coupling of neuronal birth order to circuit assembly.

Conserved role of the Vsx genes supports a monophyletic origin for bilaterian visual systems.

BACKGROUND: Components of the genetic network specifying eye development are conserved from flies to humans, but homologies between individual neuronal cell types have been difficult to identify. In the vertebrate retina, the homeodomain-containing transcription factor Chx10 is required for both progenitor cell proliferation and the development of the bipolar interneurons, which transmit visual signals from photoreceptors to ganglion cells. RESULTS: We show that dVsx1 and dVsx2, the two Drosophila homologs of Chx10, play a conserved role in visual-system development. DVSX1 is expressed in optic-lobe progenitor cells, and, in dVsx1 mutants, progenitor cell proliferation is defective, leading to hypocellularity. Subsequently, DVSX1 and DVSX2 are coexpressed in a subset of neurons in the medulla, including the transmedullary neurons that transmit visual information from photoreceptors to deeper layers of the visual system. In dVsx mutant adults, the optic lobe is reduced in size, and the medulla is small or absent. These results suggest that the progenitor cells and photoreceptor target neurons of the vertebrate retina and fly optic lobe are ancestrally related. Genetic and functional homology may extend to the neurons directly downstream of the bipolar and transmedullary neurons, the vertebrate ganglion cells and fly lobula projection neurons. Both cell types project to visual-processing centers in the brain, and both sequentially express the Math5/ATO and Brn3b/ACJ6 transcription factors during their development. CONCLUSIONS: Our findings support a monophyletic origin for the bilaterian visual system in which the last common ancestor of flies and vertebrates already contained a primordial visual system with photoreceptors, interneurons, and projection neurons.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization.

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Eye evolution at high resolution: the neuron as a unit of homology.

Based on differences in morphology, photoreceptor-type usage and lens composition it has been proposed that complex eyes have evolved independently many times. The remarkable observation that different eye types rely on a conserved network of genes (including Pax6/eyeless) for their formation has led to the revised proposal that disparate complex eye types have evolved from a shared and simpler prototype. Did this ancestral eye already contain the neural circuitry required for image processing? And what were the evolutionary events that led to the formation of complex visual systems, such as those found in vertebrates and insects? The recent identification of unexpected cell-type homologies between neurons in the vertebrate and Drosophila visual systems has led to two proposed models for the evolution of complex visual systems from a simple prototype. The first, as an extension of the finding that the neurons of the vertebrate retina share homologies with both insect (rhabdomeric) and vertebrate (ciliary) photoreceptor cell types, suggests that the vertebrate retina is a composite structure, made up of neurons that have evolved from two spatially separate ancestral photoreceptor populations. The second model, based largely on the conserved role for the Vsx homeobox genes in photoreceptor-target neuron development, suggests that the last common ancestor of vertebrates and flies already possessed a relatively sophisticated visual system that contained a mixture of rhabdomeric and ciliary photoreceptors as well as their first- and second-order target neurons. The vertebrate retina and fly visual system would have subsequently evolved by elaborating on this ancestral neural circuit. Here we present evidence for these two cell-type homology-based models and discuss their implications.

A Unique Class of Neural Progenitors in the Drosophila Optic Lobe Generates Both Migrating Neurons and Glia.

How neuronal and glial fates are specified from neural precursor cells is an important question for developmental neurobiologists. We address this question in the Drosophila optic lobe, composed of the lamina, medulla, and lobula complex. We show that two gliogenic regions posterior to the prospective lamina also produce lamina wide-field (Lawf) neurons, which share common progenitors with lamina glia. These progenitors express neither canonical neuroblast nor lamina precursor cell markers. They bifurcate into two sub-lineages in response to Notch signaling, generating lamina glia or Lawf neurons, respectively. The newly born glia and Lawfs then migrate tangentially over substantial distances to reach their target tissue. Thus, Lawf neurogenesis, which includes a common origin with glia, as well as neuronal migration, resembles several aspects of vertebrate neurogenesis.

Specification and development of the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain.

The central neuroendocrine system in the Drosophila brain includes two centers, the pars intercerebralis (PI) and pars lateralis (PL). The PI and PL contain neurosecretory cells (NSCs) which project their axons to the ring gland, a complex of peripheral endocrine glands flanking the aorta. We present here a developmental and genetic study of the PI and PL. The PI and PL are derived from adjacent neurectodermal placodes in the dorso-medial head. The placodes invaginate during late embryogenesis and become attached to the brain primordium. The PI placode and its derivatives express the homeobox gene Dchx1 and can be followed until the late pupal stage. NSCs labeled by the expression of Drosophila insulin-like peptide (Dilp), FMRF, and myomodulin form part of the Dchx1 expressing PI domain. NSCs of the PL can be followed throughout development by their expression of the adhesion molecule FasII. Decapentaplegic (Dpp), secreted along the dorsal midline of the early embryo, inhibits the formation of the PI and PL placodes; loss of the signal results in an unpaired, enlarged placodeal ectoderm. The other early activated signaling pathway, EGFR, is positively required for the maintenance of the PI placode. Of the dorso-medially expressed head gap genes, only tailless (tll) is required for the specification of the PI. Absence of the corpora cardiaca, the endocrine gland innervated by neurosecretory cells of the PI and PL, does not affect the formation of the PI/PL, indicating that inductive stimuli from their target tissue are not essential for early PI/PL development.