Developmental Genetic and Molecular Analysis of Drosophila Central Complex Lineages.

Complex behaviors are mediated by a diverse class of neurons and glia produced during development. Both neural stem cell-intrinsic and -extrinsic temporal cues regulate the appropriate number, molecular identity, and circuit assembly of neurons. The Drosophila central complex (CX) is a higher-order brain structure regulating various behaviors, including sensory-motor integration, celestial navigation, and sleep. Most neurons and glia in the adult CX are formed during larval development by 16 Type II neural stem cells (NSCs). Unlike Type I NSCs, which directly give rise to the ganglion mother cells (GMCs), Type II NSCs give rise to multiple intermediate neural progenitors (INPs), and each INP in turn generates multiple GMCs, hence fostering the generation of longer and more diverse lineages. This makes Type II NSCs a suitable model to unravel the molecular mechanisms regulating neural diversity in more complex lineages. In this review, we elaborate on the classification and identification of NSCs based on the types of division adopted and the molecular markers expressed in each type. In the end, we discuss genetic methods for lineage analysis and birthdating. We also explain the temporal expression of stem cell factors and genetic techniques to study how stem cell factors may regulate neural fate specification.

Lineage Analysis and Birthdating of Drosophila Central Complex Lineages.

From insects to humans, the nervous system generates complex behaviors mediated by distinct neural circuits that are composed of diverse cell types. During development, the spatiotemporal gene expression of the neural progenitors expands the diversity of neuronal and glial subtypes. Various neural stem cell-intrinsic and -extrinsic gene programs have been identified that are thought to play a major role in generating diverse neuronal and glial cell types. Drosophila has served as an excellent model system for discovering the fundamental principles of nervous system development and function. The sophisticated genetic tools allow us to link the origin and birth timing (the time when a particular neuron is born during development) of neuron types to unique neural stem cells (NSCs) and to a developmental time. In Drosophila, a special class of NSCs called Type II NSCs has adopted a more advanced division mode to generate lineages for the higher-order brain center, the central complex, which is an evolutionarily conserved brain region found in all insects. Type II NSCs, similar to the human outer radial glia, generate intermediate neural progenitors (INPs), which divide many times to produce about eight to 10 progeny. Both Type II NSCs and INPs express distinct transcription factors and RNA-binding proteins that have been proposed to regulate the specification of cell types populating the adult central complex. Here, we describe the recently invented lineage filtering system, called cell class-lineage intersection (CLIn), which enables the tracking and birthdating of the Type II NSC lineages. Using CLIn, one can easily generate clones of different Type II NSCs and identify not only the origins of neurons of interest but also their birth time.

The conserved RNA-binding protein Imp is required for the specification and function of olfactory navigation circuitry in Drosophila.

Complex behaviors depend on the precise developmental specification of neuronal circuits, but the relationship between genetic programs for neural development, circuit structure, and behavioral output is often unclear. The central complex (CX) is a conserved sensory-motor integration center in insects, which governs many higher-order behaviors and largely derives from a small number of type II neural stem cells (NSCs). Here, we show that Imp, a conserved IGF-II mRNA-binding protein expressed in type II NSCs, plays a role in specifying essential components of CX olfactory navigation circuitry. We show the following: (1) that multiple components of olfactory navigation circuitry arise from type II NSCs. (2) Manipulating Imp expression in type II NSCs alters the number and morphology of many of these circuit elements, with the most potent effects on neurons targeting the ventral layers of the fan-shaped body (FB). (3) Imp regulates the specification of Tachykinin-expressing ventral FB input neurons. (4) Imp is required in type II NSCs for establishing proper morphology of the CX neuropil structures. (5) Loss of Imp in type II NSCs abolishes upwind orientation to attractive odor while leaving locomotion and odor-evoked regulation of movement intact. Taken together, our findings establish that a temporally expressed gene can regulate the expression of a complex behavior by developmentally regulating the specification of multiple circuit components and provides a first step toward a developmental dissection of the CX and its roles in behavior.

Stem cell-specific ecdysone signaling regulates the development and function of a Drosophila sleep homeostat.

Complex behaviors arise from neural circuits that are assembled from diverse cell types. Sleep is a conserved and essential behavior, yet little is known regarding how the nervous system generates neuron types of the sleep-wake circuit. Here, we focus on the specification of Drosophila sleep-promoting neurons-long-field tangential input neurons that project to the dorsal layers of the fan-shaped body neuropil in the central complex (CX). We use lineage analysis and genetic birth dating to identify two bilateral Type II neural stem cells that generate these dorsal fan-shaped body (dFB) neurons. We show that adult dFB neurons express Ecdysone-induced protein E93, and loss of Ecdysone signaling or E93 in Type II NSCs results in the misspecification of the adult dFB neurons. Finally, we show that E93 knockdown in Type II NSCs affects adult sleep behavior. Our results provide insight into how extrinsic hormonal signaling acts on NSCs to generate neuronal diversity required for adult sleep behavior. These findings suggest that some adult sleep disorders might derive from defects in stem cell-specific temporal neurodevelopmental programs.

The RNA-binding protein, Imp specifies olfactory navigation circuitry and behavior in Drosophila.

Complex behaviors depend on the precise developmental specification of neuronal circuits, but the relationship between genetic prograssms for neural development, circuit structure, and behavioral output is often unclear. The central complex (CX) is a conserved sensory-motor integration center in insects that governs many higher order behaviors and largely derives from a small number of Type II neural stem cells. Here, we show that Imp, a conserved IGF-II mRNA-binding protein expressed in Type II neural stem cells, specifies components of CX olfactory navigation circuitry. We show: (1) that multiple components of olfactory navigation circuitry arise from Type II neural stem cells and manipulating Imp expression in Type II neural stem cells alters the number and morphology of many of these circuit elements, with the most potent effects on neurons targeting the ventral layers of the fan-shaped body. (2) Imp regulates the specification of Tachykinin expressing ventral fan-shaped body input neurons. (3) Imp in Type II neural stem cells alters the morphology of the CX neuropil structures. (4) Loss of Imp in Type II neural stem cells abolishes upwind orientation to attractive odor while leaving locomotion and odor-evoked regulation of movement intact. Taken together, our work establishes that a single temporally expressed gene can regulate the expression of a complex behavior through the developmental specification of multiple circuit components and provides a first step towards a developmental dissection of the CX and its roles in behavior.

The Drivers of Diversity: Integrated genetic and hormonal cues regulate neural diversity.

Proper functioning of the nervous system relies not only on the generation of a vast repertoire of distinct neural cell types but also on the precise neural circuitry within them. How the generation of highly diverse neural populations is regulated during development remains a topic of interest. Landmark studies in Drosophila have identified the genetic and temporal cues regulating neural diversity and thus have provided valuable insights into our understanding of temporal patterning of the central nervous system. The development of the Drosophila central complex, which is mostly derived from type II neural stem cell (NSC) lineages, showcases how a small pool of NSCs can give rise to vast and distinct progeny. Similar to the human outer subventricular zone (OSVZ) neural progenitors, type II NSCs generate intermediate neural progenitors (INPs) to expand and diversify lineages that populate higher brain centers. Each type II NSC has a distinct spatial identity and timely regulated expression of many transcription factors and mRNA binding proteins. Additionally, INPs derived from them show differential expression of genes depending on their birth order. Together type II NSCs and INPs display a combinatorial temporal patterning that expands neural diversity of the central brain lineages. We cover advances in current understanding of type II NSC temporal patterning and discuss similarities and differences in temporal patterning mechanisms of various NSCs with a focus on how cell-intrinsic and extrinsic hormonal cues regulate temporal transitions in NSCs during larval development. Cell extrinsic ligands activate conserved signaling pathways and extrinsic hormonal cues act as a temporal switch that regulate temporal progression of the NSCs. We conclude by elaborating on how a progenitor's temporal code regulates the fate specification and identity of distinct neural types. At the end, we also discuss open questions in linking developmental cues to neural identity, circuits, and underlying behaviors in the adult fly.

Identification of a molecular basis for the juvenile sleep state.

Across species, sleep in young animals is critical for normal brain maturation. The molecular determinants of early life sleep remain unknown. Through an RNAi-based screen, we identified a gene, pdm3, required for sleep maturation in Drosophila. Pdm3, a transcription factor, coordinates an early developmental program that prepares the brain to later execute high levels of juvenile adult sleep. PDM3 controls the wiring of wake-promoting dopaminergic (DA) neurites to a sleep-promoting region, and loss of PDM3 prematurely increases DA inhibition of the sleep center, abolishing the juvenile sleep state. RNA-Seq/ChIP-Seq and a subsequent modifier screen reveal that pdm3 represses expression of the synaptogenesis gene Msp300 to establish the appropriate window for DA innervation. These studies define the molecular cues governing sleep behavioral and circuit development, and suggest sleep disorders may be of neurodevelopmental origin.

Non-Cell-Autonomous Function of the GPI-Anchored Protein Undicht during Septate Junction Assembly.

Occluding cell-cell junctions are pivotal during the development of many organs. One example is septate junction (SJ) strands, which are found in vertebrates and invertebrates. Although several proteins have been identified that are responsible for septate junction formation in Drosophila, it is presently unclear how these structures are formed or how they are positioned in a coordinated manner between two neighboring cells and within the tissue. Here, we identified a GPI-anchored protein called Undicht required for septate junction formation. Clonal analysis and rescue experiments show that Undicht acts in a non-cell-autonomous manner. It can be released from the plasma membrane by the proteolytic activity of two related ADAM10-like proteases, Kuzbanian and Kuzbanian-like. We propose that juxtacrine function of Undicht coordinates the formation of septate junction strands on two directly neighboring cells, whereas paracrine activity of Undicht controls the formation of occluding junctions within a tissue.

Playing Well with Others: Extrinsic Cues Regulate Neural Progenitor Temporal Identity to Generate Neuronal Diversity.

During neurogenesis, vertebrate and Drosophila progenitors change over time as they generate a diverse population of neurons and glia. Vertebrate neural progenitors have long been known to use both progenitor-intrinsic and progenitor-extrinsic cues to regulate temporal patterning. In contrast, virtually all temporal patterning mechanisms discovered in Drosophila neural progenitors (neuroblasts) involve progenitor-intrinsic temporal transcription factor cascades. Recent results, however, have revealed several extrinsic pathways that regulate Drosophila neuroblast temporal patterning: nutritional cues regulate the timing of neuroblast proliferation/quiescence and a steroid hormone cue that is required for temporal transcription factor expression. Here, we discuss newly discovered extrinsic cues regulating neural progenitor temporal identity in Drosophila, highlight conserved mechanisms, and raise open questions for the future.

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity.

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. Here we show that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. We show that Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors.

The CD59 family member Leaky/Coiled is required for the establishment of the blood-brain barrier in Drosophila.

The blood-brain barrier of Drosophila is established by the subperineurial glial cells that encase the CNS and PNS. The subperineurial glial cells are thin, highly interdigitated cells with epithelial character. The establishment of extensive septate junctions between these cells is crucial for the prevention of uncontrolled paracellular leakage of ions and solutes from the hemolymph into the nervous system. In the absence of septate junctions, macromolecules such as fluorescently labeled dextran can easily cross the blood-brain barrier. To identify additional components of the blood-brain barrier, we followed a genetic approach and injected Texas-Red-conjugated dextran into the hemolymph of embryos homozygous for chromosomal deficiencies. In this way, we identified the 153-aa-large protein Coiled, a new member of the Ly6 (leukocyte antigen 6) family, as being crucially required for septate junction formation and blood-brain barrier integrity. In coiled mutants, the normal distribution of septate junction markers such as NeurexinIV, Coracle, or Discs large is disturbed. EM analyses demonstrated that Coiled is required for the formation of septate junctions. We further show that Coiled is expressed by the subsperineurial glial cells in which it is anchored to the cell membrane via a glycosylphosphatidylinositol anchor and mediates adhesive properties. Clonal rescue studies indicate that the presence of Coiled is required symmetrically on both cells engaged in septate junction formation.