Larval Central Nervous System Research Articles

Functional analysis by RNAi of an glutaredoxin gene in Helicoverpa armigera

Glutaredoxins play crucial roles in maintaining intracellular redox homeostasis via scavenging of excess reactive oxygen species. In this study, a glutaredoxin domain-containing cysteine-rich gene from Helicoverpa armigera, named HaGdccr, was characterized. Sequence analysis revealed that it contains a glutaredoxin domain and a conserved cysteine and shares high sequence identity with other insect genes. HaGdccr mRNA expression was highest in molting larvae of the 3rd instar and was mainly detected in the central nervous system of larvae and the wings of adults. Quantitative real-time PCR results revealed that the expression of HaGdccr was suppressed at 1 and 6 h and increased at 24 h after the larvae were treated with 4 °C and hydrogen peroxide. When the larvae were exposed to 20 °C, HaGdccr decreased at 1 h and was induced at 12 and 24 h. HaGdccr transcription level was downregulated at 2 and 12 h and upregulated at 24 h after the adults were exposed to 0 °C. However, transcript levels were increased by high temperature in both larvae and adults. After knockdown of HaGdccr by RNA interference, the expression of antioxidant genes, including thioredoxin-like (Trx-like), catalase (CAT), glutathione-S-transferase (GST), thioredoxin reductase (TrxR), and thioredoxin (Trx), was increased, whereas that of thioredoxin peroxidase (Tpx) was decreased. In addition, we found that HaGdccr knockdown enhanced the enzymatic activity of superoxide dismutase and the contents of hydrogen peroxide and ascorbate. Taken together, these results indicate that HaGdccr may play significant roles in protecting organisms against oxidative damage.

Combinatorial action of Grainyhead, Extradenticle and Notch in regulating Hox mediated apoptosis in Drosophila larval CNS.

Hox mediated neuroblast apoptosis is a prevalent way to pattern larval central nervous system (CNS) by different Hox genes, but the mechanism of this apoptosis is not understood. Our studies with Abdominal-A (Abd-A) mediated larval neuroblast (pNB) apoptosis suggests that AbdA, its cofactor Extradenticle (Exd), a helix-loop-helix transcription factor Grainyhead (Grh), and Notch signaling transcriptionally contribute to expression of RHG family of apoptotic genes. We find that Grh, AbdA, and Exd function together at multiple motifs on the apoptotic enhancer. In vivo mutagenesis of these motifs suggest that they are important for the maintenance of the activity of the enhancer rather than its initiation. We also find that Exd function is independent of its known partner homothorax in this apoptosis. We extend some of our findings to Deformed expressing region of sub-esophageal ganglia where pNBs undergo a similar Hox dependent apoptosis. We propose a mechanism where common players like Exd-Grh-Notch work with different Hox genes through region specific enhancers to pattern respective segments of larval central nervous system.

Serotonergic network in the subesophageal zone modulates the motor pattern for food intake in Drosophila

The functional organization of central motor circuits underlying feeding behaviors is not well understood. We have combined electrophysiological and genetic approaches to investigate the regulatory networks upstream of the motor program underlying food intake in the Drosophila larval central nervous system. We discovered that the serotonergic network of the CNS is able to set the motor rhythm frequency of pharyngeal pumping. Pharmacological experiments verified that modulation of the feeding motor pattern is based on the release of serotonin. Classical lesion and laser based cell ablation indicated that the serotonergic neurons in the subesophageal zone represent a redundant network for motor control of larval food intake.

Genetic tools to study juvenile hormone action in Drosophila

The insect juvenile hormone receptor is a basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) domain protein, a novel type of hormone receptor. In higher flies like Drosophila, the ancestral receptor germ cell-expressed (gce) gene has duplicated to yield the paralog Methoprene-tolerant (Met). These paralogous receptors share redundant function during development but play unique roles in adults. Some aspects of JH function apparently require one receptor or the other. To provide a foundation for studying JH receptor function, we have recapitulated endogenous JH receptor expression with single cell resolution. Using Bacteria Artificial Chromosome (BAC) recombineering and a transgenic knock-in, we have generated a spatiotemporal expressional atlas of Met and gce throughout development. We demonstrate JH receptor expression in known JH target tissues, in which temporal expression corresponds with periods of hormone sensitivity. Larval expression largely supports the notion of functional redundancy. Furthermore, we provide the neuroanatomical distribution of JH receptors in both the larval and adult central nervous system, which will serve as a platform for future studies regarding JH action on insect behavior.

Characterization of microfluidic clamps for immobilizing and imaging of Drosophila melanogaster larva's central nervous system.

Drosophila melanogaster is a well-established model organism to understand biological processes and study human diseases at the molecular-genetic level. The central nervous system (CNS) of Drosophila larvae is widely used as a model to study neuron development and network formation. This has been achieved by using various genetic manipulation tools such as microinjection to knock down certain genes or over-express proteins for visualizing the cellular activities. However, visualization of an intact-live neuronal response in larva's Central Nervous System (CNS) is challenging due to robust digging/burrowing behaviour that impedes neuroimaging. To address this problem, dissection is used to isolate and immobilize the CNS from the rest of the body. In order to obtain a true physiological response from the Drosophila CNS, it is important to avoid dissection, while the larva should be kept immobilized. In this paper, a series of microfluidic clamps were investigated for intact immobilization of the larva. As a result, an optimized structure for rapid mechanical immobilization of Drosophila larvae for CNS imaging was determined. The clamping and immobilization processes were characterized by imaging and movement measurement of the CNS through the expression of genetically encoded Calcium sensor GCaMP5 in all sensory and cholinergic interneurons. The optimal structure that included two 3D constrictions inside a narrowed channel considerably reduced the internal CNS capsule movements. It restricts the CNS movement to 10% of the motion from a glued larva and allows motion of only 10 ± 30 μm over 350 s immobilization which was sufficient for CNS imaging. These larva-on-a-chip platforms can be useful for studying CNS responses to sensory cues such as sound, light, chemosensory, tactile, and electric/magnetic fields.

Mosquitocidal Activity and Mode of Action of the Isoxazoline Fluralaner.

Mosquitoes, such as Aedes aegypti and Anopheles gambiae, are important vectors of human diseases. Fluralaner, a recently introduced parasiticide, was evaluated as a mosquitocide in this study. On Ae. aegypti and An. gambiae fourth-instar larvae, fluralaner had 24-h LC50 (lethal concentration for 50% mortality) values of 1.8 ppb and 0.4 ppb, respectively. Following topical application to adult Ae. aegypti, fluralaner toxicity reached a plateau in about 3 days, with 1- and 3-day LD50 (lethal dose for 50% mortality) values of 1.3 ng/mg and 0.26 ng/mg, suggesting a slowly developing toxicity. Fipronil outperformed fluralaner by up to 100-fold in adult topical, glass contact, and feeding assays on Ae. aegypti. These data show that fluralaner does not have exceptional toxicity to mosquitoes in typical exposure paradigms. In electrophysiological recordings on Drosophila melanogaster larval central nervous system, the effectiveness of fluralaner for restoring nerve firing after gamma-aminobutyric acid (GABA) treatment, a measure of GABA antagonism, was similar in susceptible Oregon-R and cyclodiene-resistant rdl-1675 strains, with EC50 (half maximal effective concentration) values of 0.34 µM and 0.29 µM. Although this finding suggests low cross resistance in the presence of rdl, the moderate potency, low contact activity, and slow action of fluralaner argue against its use as an adult mosquitocide for vector control.

Pathogen-induced food evasion behavior in Drosophila larvae.

Recognizing a deadly pathogen and generating an appropriate immune reaction is essential for any organism to survive in its natural habitat. Unlike vertebrates and higher primates, invertebrates depend solely on the innate immune system to defend themselves from an attacking pathogen. In this study, we report a behavioral defense strategy observed in Drosophila larvae that helps them escape and limit an otherwise lethal infection. A bacterial infection in the gut is sensed by the larval central nervous system, which generates an alteration in the larva's food preference, leading it to stop feeding and move away from the infectious food source. We have also found that this behavioral response is dependent on the internal nutritive state of the larvae. Using this novel behavioral assay as a read-out, we further identified hugin neuropeptide to be involved in the evasion response and detection of bacterial signals.

Prevention of medulla neuron dedifferentiation by Nerfin-1 requires inhibition of Notch activity.

The Drosophila larval central nervous system comprises the central brain, ventral nerve cord and optic lobe. In these regions, neuroblasts (NBs) divide asymmetrically to self-renew and generate differentiated neurons or glia. To date, mechanisms of preventing neuron dedifferentiation are still unclear, especially in the optic lobe. Here, we show that the zinc-finger transcription factor Nerfin-1 is expressed in early-stage medulla neurons and is essential for maintaining their differentiation. Loss of Nerfin-1 activates Notch signaling, which promotes neuron-to-NB reversion. Repressing Notch signaling largely rescues dedifferentiation in nerfin-1 mutant clones. Thus, we conclude that Nerfin-1 represses Notch activity in medulla neurons and prevents them from dedifferentiation.

Transcriptome Profiling Identifies Multiplexin as a Target of SAGA Deubiquitinase Activity in Glia Required for Precise Axon Guidance During Drosophila Visual Development.

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for the deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2, and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration observed in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop, and sgf11 larval optic lobes using affinity-purification of KASH-GFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia.

Lineage mapping identifies molecular and architectural similarities between the larval and adult Drosophila central nervous system.

Neurogenesis in Drosophila occurs in two phases, embryonic and post-embryonic, in which the same set of neuroblasts give rise to the distinct larval and adult nervous systems, respectively. Here, we identified the embryonic neuroblast origin of the adult neuronal lineages in the ventral nervous system via lineage-specific GAL4 lines and molecular markers. Our lineage mapping revealed that neurons born late in the embryonic phase show axonal morphology and transcription factor profiles that are similar to the neurons born post-embryonically from the same neuroblast. Moreover, we identified three thorax-specific neuroblasts not previously characterized and show that HOX genes confine them to the thoracic segments. Two of these, NB2-3 and NB3-4, generate leg motor neurons. The other neuroblast is novel and appears to have arisen recently during insect evolution. Our findings provide a comprehensive view of neurogenesis and show how proliferation of individual neuroblasts is dictated by temporal and spatial cues.

Expression of RYamide in the nervous and endocrine system of Bombyx mori

RYamides are neuropeptides encoded by a gene whose precise expression and function have not yet been determined. We identified the RYamide gene transcript (fmgV1g15f, SilkBase database) and predicted two candidates for G-protein coupled RYamide receptors (A19-BAG68418 and A22-BAG68421) in the silkworm Bombyx mori. We cloned the RYamide transcript and described its spatial expression using in situ hybridisation. In the larval central nervous system (CNS) expression of RYamide was restricted to 12–14 small neurons in the brain and two posterior neurons in the terminal abdominal ganglion. During metamorphosis their number decreased to eight protocerebral neurons in the adults. Multiple staining, using various insect neuropeptide antibodies, revealed that neurons expressing RYamide are different from other peptidergic cells in the CNS. We also found RYamide expression in the enteroendocrine cells (EC) of the anterior midgut of larvae, pupae and adults. Two minor subpopulations of these EC were also immunoreactive to antibodies against tachykinin and myosupressin. This expression pattern suggests RYamides may play a role in the regulation of feeding and digestion.

Patterning of brain precursors in ascidian embryos.

In terms of their embryonic origins, the anterior and posterior parts of the ascidian central nervous system (CNS) are associated with distinct germ layers. The anterior part of the sensory vesicle, or brain, originates from ectoderm lineages following a neuro-epidermal binary fate decision. In contrast, a large part of the remaining posterior CNS is generated following neuro-mesodermal binary fate decisions. Here, we address the mechanisms that pattern the anterior brain precursors along the medial-lateral axis (future ventral-dorsal) at neural plate stages. Our functional studies show that Nodal signals are required for induction of lateral genes, including Delta-like, Snail, Msxb and Trp Delta-like/Notch signalling induces intermediate (Gsx) over medial (Meis) gene expression in intermediate cells, whereas the combinatorial action of Snail and Msxb prevents the expression of Gsx in lateral cells. We conclude that despite the distinct embryonic lineage origins within the larval CNS, the mechanisms that pattern neural precursors are remarkably similar.

Expression of a sugar clade gustatory receptor, BmGr6, in the oral sensory organs, midgut, and central nervous system of larvae of the silkworm Bombyx mori

Insects taste nonvolatile chemicals through gustatory receptors (Grs) and make choices for feeding, mating, and oviposition. To date, genome projects have identified 69 Gr genes in the silkworm, Bombyx mori; however, the expression sites of these Grs remain to be explored. In this study, we used reverse transcription (RT)-PCR to investigate expression of the B. mori Gr-6 (BmGr6) gene, a member of the putative sugar clade gene family in various tissues. BmGr6 is expressed in the midgut, central nervous system (CNS), and oral sensory organs. Moreover, immunohistochemistry using an anti-BmGr6 antiserum demonstrated that BmGr6 is expressed in cells by oral sensory organs, midgut and nervous system. Furthermore, double-immunohistochemistry indicated that BmGr6 is expressed in midgut enteroendocrine cells, also in CNS neurosecretory cells. In particular, a portion of BmGr6-expressing cells, in both midgut and CNS, secretes FMRFamide-related peptides (FaRPs). These results suggest that BmGr6 functions not only as a taste receptor, but also as a chemical sensor such as for the regulation of gut movement, physiological conditions, and feeding behavior of larvae.

Expression of the Drosophila homeobox gene, Distal-less, supports an ancestral role in neural development.

Distal-less (Dll) encodes a homeodomain transcription factor expressed in developing appendages of organisms throughout metazoan phylogeny. Based on earlier observations in the limbless nematode Caenorhabditis elegans and the primitive chordate amphioxus, it was proposed that Dll had an ancestral function in nervous system development. Consistent with this hypothesis, Dll is necessary for the development of both peripheral and central components of the Drosophila olfactory system. Furthermore, vertebrate homologs of Dll, the Dlx genes, play critical roles in mammalian brain development. Using fluorescent immunohistochemistry of fixed samples and multiphoton microscopy of living Drosophila embryos, we show that Dll is expressed in the embryonic, larval and adult central nervous system and peripheral nervous system (PNS) in embryonic and larval neurons, brain and ventral nerve cord glia, as well as in PNS structures associated with chemosensation. In adult flies, Dll expression is expressed in the optic lobes, central brain regions and the antennal lobes. Characterization of Dll expression in the developing nervous system supports a role of Dll in neural development and function and establishes an important basis for determining the specific functional roles of Dll in Drosophila development and for comparative studies of Drosophila Dll functions with those of its vertebrate counterparts.

Differential control of Yorkie activity by LKB1/AMPK and the Hippo/Warts cascade in the central nervous system

The Hippo (Hpo) pathway is a highly conserved tumor suppressor network that restricts developmental tissue growth and regulates stem cell proliferation and differentiation. At the heart of the Hpo pathway is the progrowth transcriptional coactivator Yorkie [Yki-Yes-activated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) in mammals]. Yki activity is restricted through phosphorylation by the Hpo/Warts core kinase cascade, but increasing evidence indicates that core kinase-independent modes of regulation also play an important role. Here, we examine Yki regulation in the Drosophila larval central nervous system and uncover a Hpo/Warts-independent function for the tumor suppressor kinase liver kinase B1 (LKB1) and its downstream effector, the energy sensor AMP-activated protein kinase (AMPK), in repressing Yki activity in the central brain/ventral nerve cord. Although the Hpo/Warts core cascade restrains Yki in the optic lobe, it is dispensable for Yki target gene repression in the late larval central brain/ventral nerve cord. Thus, we demonstrate a dramatically different wiring of Hpo signaling in neighboring cell populations of distinct developmental origins in the central nervous system.

Neuronal map reveals the highly regionalized pattern of the juvenile central nervous system of the ascidian Ciona intestinalis.

The dorsally located central nervous system (CNS) is an important hallmark of chordates. Among chordates, tunicate ascidians change their CNS remarkably by means of a metamorphosis from a highly regionalized larval CNS to an oval-shaped juvenile CNS without prominent morphological features. The neuronal organization of the CNS of ascidian tadpole larvae has been well described, but that in the CNS of postmetamorphosis juveniles has not been characterized well. We investigated the number of neural cells, the number and position of differentiated neurons, and their axonal trajectories in the juvenile CNS of the ascidian Ciona intestinalis. The cell bodies of cholinergic, glutamatergic, and GABAergic/glycinergic neurons exhibited different localization patterns along the anterior-posterior axis in the juvenile CNS. Cholinergic neurons extended their axons toward the oral, atrial and body wall muscles and pharyngeal gill to regulate muscle contraction and ciliary movement. Unlike its featureless shape, the juvenile CNS is highly patterned along the anterior-posterior axis. This patterning may be necessary for exerting multiple roles in the regulation of adult tissues distributed throughout the body. This basic information of the juvenile CNS of Ciona will allow in-depth studies of molecular mechanisms underlying the reconstruction of the CNS during ascidian metamorphosis.

Whole-central nervous system functional imaging in larval Drosophila.

Understanding how the brain works in tight concert with the rest of the central nervous system (CNS) hinges upon knowledge of coordinated activity patterns across the whole CNS. We present a method for measuring activity in an entire, non-transparent CNS with high spatiotemporal resolution. We combine a light-sheet microscope capable of simultaneous multi-view imaging at volumetric speeds 25-fold faster than the state-of-the-art, a whole-CNS imaging assay for the isolated Drosophila larval CNS and a computational framework for analysing multi-view, whole-CNS calcium imaging data. We image both brain and ventral nerve cord, covering the entire CNS at 2 or 5 Hz with two- or one-photon excitation, respectively. By mapping network activity during fictive behaviours and quantitatively comparing high-resolution whole-CNS activity maps across individuals, we predict functional connections between CNS regions and reveal neurons in the brain that identify type and temporal state of motor programs executed in the ventral nerve cord.

AdamTS‐A Developmental Function in Neuronal Development and Migration in Drosophila Melanogaster

The Drosophila central nervous system (CNS) is an excellent model system in which to dissect the genetic and molecular processes that control the development of the nervous system. Through a forward genetic screen, we previously identified, a larval phenotype that leads to the formation of ectopic neural masses. These masses are composed of dozens to thousands of neuronal cell bodies at the junctures between the brain and the eyes, the ventral nerve cord and the leg imaginial discs, as well as within nerves. We determined that this mutant phenotype arises due to a transposable element insert within the 5' untranslated region of a disintegrin and metalloprotease with thrombospondin type 1 motif A (AdamTS‐A) gene. The AdamTS family of proteases are secreted proteins thought to play critical roles in extracullular matrix (ECM) formation and function, and their regulation has been linked to cancer progression. We have recently generated RNA and antibody probes to the AdamTS‐A and will report the RNA and protein expression of Adam TS‐A during larval CNS development.Support from the NINDS/NIH grant number NS0365570 and the Opportunities in Genomics Research through the Genome Institute at Washington University in St. Louis, grant number R25H600668703.

Bridging the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster.

The clarification of complete cell lineages, which are produced by specific stem cells, is fundamental for understanding mechanisms, controlling the generation of cell diversity and patterning in an emerging tissue. In the developing Central Nervous System (CNS) of Drosophila, neural stem cells (neuroblasts) exhibit two periods of proliferation: During embryogenesis they produce primary lineages, which form the larval CNS. After a phase of mitotic quiescence, a subpopulation of them resumes proliferation in the larva to give rise to secondary lineages that build up the CNS of the adult fly. Within the ventral nerve cord (VNC) detailed descriptions exist for both primary and secondary lineages. However, while primary lineages have been linked to identified neuroblasts, the assignment of secondary lineages has so far been hampered by technical limitations. Therefore, primary and secondary neural lineages co-existed as isolated model systems. Here we provide the missing link between the two systems for all lineages in the thoracic and abdominal neuromeres. Using the Flybow technique, embryonic neuroblasts were identified by their characteristic and unique lineages in the living embryo and their further development was traced into the late larval stage. This comprehensive analysis provides the first complete view of which embryonic neuroblasts are postembryonically reactivated along the anterior/posterior-axis of the VNC, and reveals the relationship between projection patterns of primary and secondary sublineages.

Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster

Orcokinins are neuropeptides that have been identified in diverse arthropods. In some species, an orcokinin gene encodes two isoforms of mature orcokinin peptide through alternative mRNA splicing. The existence of two orcokinin isoforms was predicted in Drosophila melanogaster as well, but the expression pattern of both isoforms has not been characterized. Here, we use in situ hybridization, antibody staining, and enhancer fusion GAL4 transgenic flies to examine the expression patterns of the A and B forms of orcokinin, and provide evidence that they are expressed differentially in the central nervous system (CNS) and the intestinal enteroendocrine system. The orcokinin A isoform is mainly expressed in the CNS of both larvae and adults. The A form is expressed in 5 pairs of neurons in abdominal neuromeres 1–5 of the larval CNS. In the adult brain, the A form is expressed in one pair of neurons in the posteriorlateral protocerebrum, and an additional four pairs of neurons located near the basement of the accessory medulla. Orcokinin A expression is also observed in two pairs of neurons in the ventral nerve cord (VNC). The orcokinin B form is mainly expressed in intestinal enteroendocrine cells in the larva and adult, with additional expression in one unpaired neuron in the adult abdominal ganglion. Together, our results provide elucidation of the existence and differential expression of the two orcokinin isoforms in the Drosophila brain and gut, setting the stage for future functional studies of orcokinins utilizing the genetically amenable fly model.