Seven-up acts in neuroblasts to specify adult central complex neuron identity and initiate neuroblast decommissioning.

An unanswered question in neurobiology is how are diverse neuron cell types generated from a small number of neural stem cells? In the Drosophila larval central brain, there are eight bilateral Type 2 neuroblast (T2NB) lineages that express a suite of early temporal factors followed by a different set of late temporal factors and generate the majority of the central complex (CX) neurons. The early-to-late switch is triggered by the orphan nuclear hormone receptor Seven-up (Svp), yet little is known about how this Svp-dependent switch is involved in specifying CX neuron identities. Here, we: (1) birth date the CX neurons P-EN and P-FN (early and late, respectively); (2) show that Svp is transiently expressed in all early T2NBs; and (3) show that loss of Svp expands the population of early born P-EN neurons at the expense of late born P-FN neurons. Furthermore, in the absence of Svp, T2NBs fail decommissioning and abnormally extend their lineage into week-old adults. We conclude that Svp is required to specify CX neuron identity, as well as to initiate T2NB decommissioning.

Atlas-builder software and the eNeuro atlas: resources for developmental biology and neuroscience.

A major limitation in understanding embryonic development is the lack of cell type-specific markers. Existing gene expression and marker atlases provide valuable tools, but they typically have one or more limitations: a lack of single-cell resolution; an inability to register multiple expression patterns to determine their precise relationship; an inability to be upgraded by users; an inability to compare novel patterns with the database patterns; and a lack of three-dimensional images. Here, we develop new 'atlas-builder' software that overcomes each of these limitations. A newly generated atlas is three-dimensional, allows the precise registration of an infinite number of cell type-specific markers, is searchable and is open-ended. Our software can be used to create an atlas of any tissue in any organism that contains stereotyped cell positions. We used the software to generate an 'eNeuro' atlas of the Drosophila embryonic CNS containing eight transcription factors that mark the major CNS cell types (motor neurons, glia, neurosecretory cells and interneurons). We found neuronal, but not glial, nuclei occupied stereotyped locations. We added 75 new Gal4 markers to the atlas to identify over 50% of all interneurons in the ventral CNS, and these lines allowed functional access to those interneurons for the first time. We expect the atlas-builder software to benefit a large proportion of the developmental biology community, and the eNeuro atlas to serve as a publicly accessible hub for integrating neuronal attributes - cell lineage, gene expression patterns, axon/dendrite projections, neurotransmitters--and linking them to individual neurons.

Formin-mediated actin polymerization cooperates with Mushroom body defect (Mud)-Dynein during Frizzled-Dishevelled spindle orientation.

To position the mitotic spindle, cytoskeletal components must be coordinated to generate cortical forces on astral microtubules. Although the dynein motor is common to many spindle orientation systems, 'accessory pathways' are often also required. In this work, we identified an accessory spindle orientation pathway in Drosophila that functions with Dynein during planar cell polarity, downstream of the Frizzled (Fz) effector Dishevelled (Dsh). Dsh contains a PDZ ligand and a Dynein-recruiting DEP domain that are both required for spindle orientation. The Dsh PDZ ligand recruits Canoe/Afadin and ultimately leads to Rho GTPase signaling mediated through RhoGEF2. The formin Diaphanous (Dia) functions as the Rho effector in this pathway, inducing F-actin enrichment at sites of cortical Dsh. Chimeric protein experiments show that the Dia-actin accessory pathway can be replaced by an independent kinesin (Khc73) accessory pathway for Dsh-mediated spindle orientation. Our results define two 'modular' spindle orientation pathways and show an essential role for actin regulation in Dsh-mediated spindle orientation.

Seven-up acts in neuroblasts to specify adult central complex neuron identity and initiate neuroblast decommissioning.

An open question in neurobiology is how diverse neuron cell types are generated from a small number of neural stem cells. In the Drosophila larval central brain, there are eight bilateral Type 2 neuroblast (T2NB) lineages that express a suite of early temporal factors followed by a different set of late temporal factors and generate the majority of the central complex (CX) neurons. The early-to-late switch is triggered by the orphan nuclear hormone receptor Seven-up (Svp), yet little is known about this Svp-dependent switch in specifying CX neuron identities. Here, we (i) birthdate the CX neurons P-EN and P-FN (early and late, respectively); (ii) show that Svp is transiently expressed in all early T2NBs; and (iii) show that loss of Svp expands the population of early born P-EN neurons at the expense of late born P-FN neurons. Furthermore, in the absence of Svp, T2NBs fail decommissioning and abnormally extend their lineage into week-old adults. We conclude that Svp is required to specify CX neuron identity, as well as to initiate T2NB decommissioning.

A developmental framework linking neurogenesis and circuit formation in the Drosophila CNS.

The mechanisms specifying neuronal diversity are well characterized, yet it remains unclear how or if these mechanisms regulate neural circuit assembly. To address this, we mapped the developmental origin of 160 interneurons from seven bilateral neural progenitors (neuroblasts) and identify them in a synapse-scale TEM reconstruction of the Drosophila larval central nervous system. We find that lineages concurrently build the sensory and motor neuropils by generating sensory and motor hemilineages in a Notch-dependent manner. Neurons in a hemilineage share common synaptic targeting within the neuropil, which is further refined based on neuronal temporal identity. Connectome analysis shows that hemilineage-temporal cohorts share common connectivity. Finally, we show that proximity alone cannot explain the observed connectivity structure, suggesting hemilineage/temporal identity confers an added layer of specificity. Thus, we demonstrate that the mechanisms specifying neuronal diversity also govern circuit formation and function, and that these principles are broadly applicable throughout the nervous system.

MDN brain descending neurons coordinately activate backward and inhibit forward locomotion.

Command-like descending neurons can induce many behaviors, such as backward locomotion, escape, feeding, courtship, egg-laying, or grooming (we define 'command-like neuron' as a neuron whose activation elicits or 'commands' a specific behavior). In most animals, it remains unknown how neural circuits switch between antagonistic behaviors: via top-down activation/inhibition of antagonistic circuits or via reciprocal inhibition between antagonistic circuits. Here, we use genetic screens, intersectional genetics, circuit reconstruction by electron microscopy, and functional optogenetics to identify a bilateral pair of Drosophila larval 'mooncrawler descending neurons' (MDNs) with command-like ability to coordinately induce backward locomotion and block forward locomotion; the former by stimulating a backward-active premotor neuron, and the latter by disynaptic inhibition of a forward-specific premotor neuron. In contrast, direct monosynaptic reciprocal inhibition between forward and backward circuits was not observed. Thus, MDNs coordinate a transition between antagonistic larval locomotor behaviors. Interestingly, larval MDNs persist into adulthood, where they can trigger backward walking. Thus, MDNs induce backward locomotion in both limbless and limbed animals.

Immunofluorescent antibody staining of intact Drosophila larvae.

Antibody staining is a vital technique for studying the development of many model organisms, including Drosophila. Reliable protocols have long been available for antibody staining of Drosophila whole-mount embryos and dissected larvae. By contrast, methods for staining whole larvae have rarely been reported, are unreliable, and fail to work on large third-instar larvae. This has become a major limitation to understanding the role of multitissue interactions such as neural circuit formation and cell metastasis. We have modified existing embryo protocols to develop a reliable method for antibody staining of whole Drosophila larvae of any developmental stage. The procedure consists of a bleach wash, enzymatic digestion, first fixation, 'cracking', second fixation, (optional) Proteinase K (Pro-K) or sonication treatment, antibody staining, clearing, and mounting. The method takes longer than typical antibody stains of dissected larval tissues-12 or 16 d, depending on the size of the larvae, compared with 2-3 d for embryos or dissected tissue stains-but time is saved by eliminating the need for larval dissections and by allowing hundreds of larvae to be batch-processed. The method also works well for staining embryos, even late-stage embryos with cuticles, allowing characterization from early embryogenesis to the end of larval development.

Genetic screens for enhancers of brahma reveal functional interactions between the BRM chromatin-remodeling complex and the delta-notch signal transduction pathway in Drosophila.

The Drosophila trithorax group gene brahma (brm) encodes the ATPase subunit of a 2-MDa chromatin-remodeling complex. brm was identified in a screen for transcriptional activators of homeotic genes and subsequently shown to play a global role in transcription by RNA polymerase II. To gain insight into the targeting, function, and regulation of the BRM complex, we screened for mutations that genetically interact with a dominant-negative allele of brm (brm(K804R)). We first screened for dominant mutations that are lethal in combination with a brm(K804R) transgene under control of the brm promoter. In a distinct but related screen, we identified dominant mutations that modify eye defects resulting from expression of brm(K804R) in the eye-antennal imaginal disc. Mutations in three classes of genes were identified in our screens: genes encoding subunits of the BRM complex (brm, moira, and osa), other proteins directly involved in transcription (zerknullt and RpII140), and signaling molecules (Delta and vein). Expression of brm(K804R) in the adult sense organ precursor lineage causes phenotypes similar to those resulting from impaired Delta-Notch signaling. Our results suggest that signaling pathways may regulate the transcription of target genes by regulating the activity of the BRM complex.

Even-Skipped(+) Interneurons Are Core Components of a Sensorimotor Circuit that Maintains Left-Right Symmetric Muscle Contraction Amplitude.

Bilaterally symmetric motor patterns--those in which left-right pairs of muscles contract synchronously and with equal amplitude (such as breathing, smiling, whisking, and locomotion)--are widespread throughout the animal kingdom. Yet, surprisingly little is known about the underlying neural circuits. We performed a thermogenetic screen to identify neurons required for bilaterally symmetric locomotion in Drosophila larvae and identified the evolutionarily conserved Even-skipped(+) interneurons (Eve/Evx). Activation or ablation of Eve(+) interneurons disrupted bilaterally symmetric muscle contraction amplitude, without affecting the timing of motor output. Eve(+) interneurons are not rhythmically active and thus function independently of the locomotor CPG. GCaMP6 calcium imaging of Eve(+) interneurons in freely moving larvae showed left-right asymmetric activation that correlated with larval behavior. TEM reconstruction of Eve(+) interneuron inputs and outputs showed that the Eve(+) interneurons are at the core of a sensorimotor circuit capable of detecting and modifying body wall muscle contraction.

A resource for manipulating gene expression and analyzing cis-regulatory modules in the Drosophila CNS.

Here, we describe the embryonic central nervous system expression of 5,000 GAL4 lines made using molecularly defined cis-regulatory DNA inserted into a single attP genomic location. We document and annotate the patterns in early embryos when neurogenesis is at its peak, and in older embryos where there is maximal neuronal diversity and the first neural circuits are established. We note expression in other tissues, such as the lateral body wall (muscle, sensory neurons, and trachea) and viscera. Companion papers report on the adult brain and larval imaginal discs, and the integrated data sets are available online (http://www.janelia.org/gal4-gen1). This collection of embryonically expressed GAL4 lines will be valuable for determining neuronal morphology and function. The 1,862 lines expressed in small subsets of neurons (<20/segment) will be especially valuable for characterizing interneuronal diversity and function, because although interneurons comprise the majority of all central nervous system neurons, their gene expression profile and function remain virtually unexplored.

Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation.

Regulation of stem cell self-renewal versus differentiation is critical for embryonic development and adult tissue homeostasis. Drosophila larval neuroblasts divide asymmetrically to self-renew, and are a model system for studying stem cell self-renewal. Here we identify three mutations showing increased brain neuroblast numbers that map to the aurora-A gene, which encodes a conserved kinase implicated in human cancer. Clonal analysis and time-lapse imaging in aurora-A mutants show single neuroblasts generate multiple neuroblasts (ectopic self-renewal). This phenotype is due to two independent neuroblast defects: abnormal atypical protein kinase C (aPKC)/Numb cortical polarity and failure to align the mitotic spindle with the cortical polarity axis. numb mutant clones have ectopic neuroblasts, and Numb overexpression partially suppresses aurora-A neuroblast overgrowth (but not spindle misalignment). Conversely, mutations that disrupt spindle alignment but not cortical polarity have increased neuroblasts. We conclude that Aurora-A and Numb are novel inhibitors of neuroblast self-renewal and that spindle orientation regulates neuroblast self-renewal.