Larval Central Nervous System Research Articles

Drosophila TET acts with PRC1 to activate gene expression independently of its catalytic activity.

Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS.

Development of Serotonergic and Dopaminergic Neuronal Networks of the Central Nervous System in King Crab, Paralithodes camtschaticus.

This article presents recent findings as regards distribution of cells producing serotonin and dopamine in the larval central nervous system at different developmental stages, including four pelagic larval stages (zoea I-IV), a semibenthic postlarval stage glaucothoe (megalopa), benthic juveniles, and adult red king crabs, Paralithodes camtschaticus, made by using immunocytochemistry and confocal laser scanning microscopy. We have shown that the serotonergic and dopaminergic neurons are present long before the onset of metamorphosis. In the red king crab b larval nervous system, the changes become particularly pronounced during the first metamorphosis from zoea IV to glaucothoe, which may be related to the development of the segmental appendages and maturation of motor behaviors in decapods. This work presents the distribution and dynamics of the development of serotonergic and dopaminergic neuronal networks in king crab show, the potential roles of serotonin and dopamine in the modulation of olfactory and visual processing in the early stages of larval development, and also the mechanosensory and chemosensory processing in the glaucothoe stage during settlement and in their transition from a pelagic to benthic lifestyle.

Ectopic expression of matrix metalloproteinases and filopodia extension via JNK activation are involved in the invasion of blood tumor cells in Drosophila mxc mutant.

Drosophila mxcmbn1 mutant exhibits severe hyperplasia in larval hematopoietic tissue called the lymph glands (LGs). However, the malignant nature of these cells remains unknown. We aimed to identify if mxcmbn1 LG cells behave as malignant tumor cells and uncover the mechanism(s) underlying the malignancy of the mutant hemocytes. When mutant LG cells were allografted into normal adult abdomens, they continued to proliferate; however, normal LG cells did not proliferate. Mutant circulating hemocytes also attached to the larval central nervous system (CNS), where the basement membrane was disrupted. The mutant hemocytes displayed higher expression of matrix metalloproteinase (MMP) 1 and MMP2 and higher activation of the c-Jun N-terminal kinase (JNK) pathway than normal hemocytes. Depletion of MMPs or JNK mRNAs in LGs resulted in reduced numbers of hemocytes attached to the CNS, suggesting that the invasive phenotype involved elevated expression of MMPs via hyperactivation of the JNK pathway. Moreover, hemocytes with elongated filopodia and extra lamellipodia were frequently observed in the mutant hemolymph, which also depended on JNK signaling. Thus, the MMP upregulation and overextension of actin-based cell protrusions were also involved in hemocyte invasion in mxcmbn1 larvae. These findings contribute to the understanding of molecular mechanisms underlying mammalian leukemic invasion.

Neurophysiological action of centrally-acting spider toxin polypeptides derived from Hadronyche versuta and Tegenaria agrestis venoms

Established dogma concerning the action of insecticidal arthropod-derived peptides (e.g., scorpion toxins), was that they acted on the peripheral nervous system and were excluded from the central nervous system (CNS) by barrier systems. Initial evidence for a CNS-directed toxicological effect following parenteral administration was for a novel peptide from the Hobo spider, Tegeneria agrestis. This toxin was inactive on peripheral sensory and motor nerves, but had a potent excitatory effect on the CNS of larval Musca domestica. Recently, a commercialized formulation of GS-omega/kappa-Hxtx-Hv1a (HXTX), derived from the venom of the Australian blue mountain funnel web spider (Hadronyche versuta) was introduced for use in agriculture by Vestaron Corp. Its primary mode of action was found to be central neuroexcitation via positive allosteric modulation of nicotinic acetylcholine receptors (nAchR) of cockroach neurons. In the present study, this peptide showed hyperexcitation followed by a decrease in firing of the Drosophila melanogaster larval CNS that was prevented by co-exposure to 100 nM α-bungarotoxin (α-BGTX), a classical nAchR noncompetitive antagonist. This effect was mirrored in isobologram analysis, which showed clear antagonism between the two toxins when injected into adult houseflies. Interestingly, U1-agatoxin-Ta1b-QA derived from Tegeneria agrestis (VST-7304) had a similar biphasic action, but showed increased nerve discharge when co-exposed with 100 nM α-BGTX, and had additive effects when injected together with α-BGTX in isobologram analyses. Binary mixtures of HXTX or VST-7304 with 30 nM nicotine showed clear evidence of synergized nerve block, which was also observed for mixtures of HXTX and VST-7304. Taken together, these data suggest that HXTX and VST-7304 have somewhat different and complementary modes of action.

Issue Information

Current ProtocolsVolume 3, Issue 2 e467 ISSUE INFORMATIONFree Access Issue Information First published: 25 February 2023 https://doi.org/10.1002/cpz1.467AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Cover: Synthetic assembly DNA cloning for Drosophila melanogaster is described in Venken et al. Different DNA parts, obtained from flies directly, other species, or through synthetic means, are put together using synthetic assembly DNA cloning to generate continuously growing plasmids of increasing complexity. The resulting end products can be two transgenes, a first transgene providing G418 sulfate selection (provided by the NeoR marker) and encoding the binary LexA transactivator driven by the R70B04 enhancer (i.e., the G418 sulfate-selectable LexA transactivator plasmid), and a second transgene providing Blasticidin S selection (provided by the BlastR marker) and encoding the binary LexA-Op responder, reporting green fluorescent protein (sfGFP) reporter expression driven by the LexA binary transactivator, whose expression is regulated by the R70B04 enhancer (i.e., the Blastidicin S-selectable LexA-Op responder plasmid). A double transgenic fly can then be obtained directly by injecting both transgenes (i.e., dual transgenesis) followed by co-selection using both G418 sulfate and Blasticidin S. The double transgenic fly can then be analyzed for expression patterns. Expression analysis of dual transgenic larval animals shows green fluorescent protein (sfGFP) reporter expression against the neuropil marker Discs Large (Dlg) in the larval central nervous system. View all latest articles RelatedInformation

Synergistic modes of interaction between the plant essential oils and the respiratory blocker chlorfenapyr

Plant essential oils are widely acknowledged for their insecticidal activities and synergistic interaction with conventional insecticides, but their insecticidal modes of action and the mechanism of synergy remain less understood. In this study, electrophysiological screenings on the larval central nervous system (CNS) of the common fruit fly, Drosophila melanogaster, and the housefly, Musca domestica, were conducted to identify the neurophysiological effects of the oils and their major constituents. Several oils changed the firing rate of the central motor neurons, and four oils were selected to determine their major active compounds. Eugenol and thymol (87.2% and 31.1% in clove bud and thyme oils, respectively) were inhibitory to the nerve firing rates of the CNS, and exhibited synergistic toxicity to the housefly when blended with a respiratory blocking pyrrole insecticide, chlorfenapyr. On the other hand, trans-cinnamaldehyde and terpinen-4-ol (74.6% and 52.0% in cinnamon and teatree oils) seemed excitatory to the nerves, and displayed antagonistic interaction to chlorfenapyr in their insecticidal activity. Chlorfenapyr led to ATP depletion in the insects, and the inhibitory compounds accelerated the process. On the other hand, nerve-excitatory compounds seemed to nullify the depletion. This was further confirmed with the two CNS-excitatory synthetic insecticides, permethrin and chlorpyrifos, that they exhibited antagonistic toxicity when mixed with chlorfenapyr. Meanwhile, the synergy between the inhibitory compounds and chlorfenapyr was diminished when ATP was artificially injected, indicating that the bioenergetic effects of neuroinhibitors are responsible for their synergistic interactions.

A single-cell transcriptomic atlas of complete insect nervous systems across multiple life stages

Molecular profiles of neurons influence neural development and function but bridging the gap between genes, circuits, and behavior has been very difficult. Here we used single cell RNAseq to generate a complete gene expression atlas of the Drosophila larval central nervous system composed of 131,077 single cells across three developmental stages (1 h, 24 h and 48 h after hatching). We identify 67 distinct cell clusters based on the patterns of gene expression. These include 31 functional mature larval neuron clusters, 1 ring gland cluster, 8 glial clusters, 6 neural precursor clusters, and 13 developing immature adult neuron clusters. Some clusters are present across all stages of larval development, while others are stage specific (such as developing adult neurons). We identify genes that are differentially expressed in each cluster, as well as genes that are differentially expressed at distinct stages of larval life. These differentially expressed genes provide promising candidates for regulating the function of specific neuronal and glial types in the larval nervous system, or the specification and differentiation of adult neurons. The cell transcriptome Atlas of the Drosophila larval nervous system is a valuable resource for developmental biology and systems neuroscience and provides a basis for elucidating how genes regulate neural development and function.

Intact Drosophila central nervous system cellular quantitation reveals sexual dimorphism.

Establishing with precision the quantity and identity of the cell types of the brain is a prerequisite for a detailed compendium of gene and protein expression in the central nervous system (CNS). Currently, however, strict quantitation of cell numbers has been achieved only for the nervous system of Caenorhabditis elegans. Here, we describe the development of a synergistic pipeline of molecular genetic, imaging, and computational technologies designed to allow high-throughput, precise quantitation with cellular resolution of reporters of gene expression in intact whole tissues with complex cellular constitutions such as the brain. We have deployed the approach to determine with exactitude the number of functional neurons and glia in the entire intact larval Drosophila CNS, revealing fewer neurons and more glial cells than previously predicted. We also discover an unexpected divergence between the sexes at this juvenile developmental stage, with the female CNS having significantly more neurons than that of males. Topological analysis of our data establishes that this sexual dimorphism extends to deeper features of CNS organisation. We additionally extended our analysis to quantitate the expression of voltage-gated potassium channel family genes throughout the CNS and uncover substantial differences in abundance. Our methodology enables robust and accurate quantification of the number and positioning of cells within intact organs, facilitating sophisticated analysis of cellular identity, diversity, and gene expression characteristics.

Hox genes collaborate with helix-loop-helix factor Grainyhead to promote neuroblast apoptosis along the anterior-posterior axis of the Drosophila larval central nervous system.

Hox genes code for a family of a homeodomain containing transcription factors that use TALE-HD containing factors Pbx/Exd and Meis/Homothorax to specify the development of the anterior-posterior axis of an organism. However, the absence of TALE-HD containing factors from specific tissues emphasizes the need to identify and validate new Hox cofactors. In Drosophila central nervous system, Hox executes segment-specific apoptosis of neural stem cells (neuroblasts) and neurons. In abdominal segments of larval central nervous system, Hox gene Abdominal-A mediates neuroblast apoptosis with the help of Extradenticle and bHLH factor Grainyhead using a 717-bp apoptotic enhancer. In this study, we show that this enhancer is critical for abdominal neuroblast apoptosis and relies on 2 separable set of DNA-binding motifs responsible for its initiation and maintenance. Our results also show that Abdominal-A and Grainyhead interact through their highly conserved DNA-binding domains, and the DNA-binding specificity of Abdominal-A-homeodomain is important for it to interact with Grainyhead and essential for it to execute neuroblast apoptosis in central nervous system. We also establish that Grainyhead is required for Hox-dependent neuroblast apoptosis in Labial and Sex Combs Reduced expressing regions of the central nervous system, and it can physically interact with all the Hox proteins in vitro. Our biochemical and functional data collectively support the idea that Grainyhead can function as a Hox cofactor and help them carry out their in vivo roles during development.

Mode of action and toxicological effects of the sesquiterpenoid, nootkatone, in insects

Nootkatone, a sesquiterpenoid isolated from Alaskan yellow cedar (Cupressus nootkatensis), is known to possess insect repellent and acaricidal properties and has recently been registered for commercial use by the Environmental Protection Agency. Previous studies failed to elucidate the mechanism of action of nootkatone, but we found a molecular overlay of picrotoxinin and nootkatone indicated a high degree of structural and electrostatic similarity. We therefore tested the hypothesis that nootkatone was a GABA-gated chloride channel antagonist, similar to picrotoxinin. The KD50 and LD50 of nootkatone on the insecticide-susceptible strain of Drosophila melanogaster (CSOR) showed resistance ratios of 8 and 11, respectively, compared to the cyclodiene-resistant strain of RDL1675, indicating significant cross-resistance. Nootkatone reversed GABA-mediated block of the larval CSOR central nervous system; nerve firing of 78 ± 17% of baseline in the CSOR strain was significantly different from 24 ± 11% of baseline firing in the RDL1675 strain (p = 0.035). This finding indicated that the resistance was expressed within the nervous system. Patch clamp recordings on D. melanogaster central neurons mirrored extracellular recordings where nootkatone inhibited GABA-stimulated currents by 44 ± 9% at 100 μM, whereas chloride current was inhibited 4.5-fold less at 100 μM in RDL1675. Taken together, these data suggest nootkatone toxicity in D. melanogaster is mediated through GABA receptor antagonism.

Localization of muscarinic acetylcholine receptor-dependent rhythm-generating modules in the Drosophila larval locomotor network.

Mechanisms of rhythm generation have been extensively studied in motor systems that control locomotion over terrain in limbed animals; however, much less is known about rhythm generation in soft-bodied terrestrial animals. Here we explored how muscarinic acetylcholine receptor (mAChR)-modulated rhythm-generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. We measured fictive motor patterns in isolated CNS preparations, using a combination of Ca2+ imaging and electrophysiology while manipulating mAChR signaling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated bilaterally asymmetric activity in anterior thoracic regions and promoted bursting in posterior abdominal regions. Application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions and blocked the effects of oxotremorine. Oxotremorine triggered fictive forward crawling in preparations without brain lobes. Oxotremorine also potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but it did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca2+ levels and abolished rhythmic activity. Overall, these results suggest that mAChR signaling plays a role in enabling rhythm generation at multiple sites in the larval CNS. This work furthers our understanding of motor control in soft-bodied locomotion and provides a foundation for study of rhythm-generating networks in an emerging genetically tractable locomotor system.NEW & NOTEWORTHY Using a combination of pharmacology, electrophysiology, and Ca2+ imaging, we find that signaling through mACh receptors plays a critical role in rhythmogenesis in different regions of the Drosophila larval CNS. mAChR-dependent rhythm generators reside in distal regions of the larval CNS and provide functional substrates for central pattern-generating networks (CPGs) underlying headsweep behavior and forward locomotion. This provides new insights into locomotor CPG operation in soft-bodied animals that navigate over terrain.

Loss-of-function variants in TIAM1 are associated with developmental delay, intellectual disability, and seizures

TIAM Rac1-associated GEF 1 (TIAM1) regulates RAC1 signaling pathways that affect the control of neuronal morphogenesis and neurite outgrowth by modulating the actin cytoskeletal network. To date, TIAM1 has not been associated with a Mendelian disorder. Here, we describe five individuals with bi-allelic TIAM1 missense variants who have developmental delay, intellectual disability, speech delay, and seizures. Bioinformatic analyses demonstrate that these variants are rare and likely pathogenic. We found that the Drosophila ortholog of TIAM1, still life (sif), is expressed in larval and adult central nervous system (CNS) and is mainly expressed in a subset of neurons, but not in glia. Loss of sif reduces the survival rate, and the surviving adults exhibit climbing defects, are prone to severe seizures, and have a short lifespan. The TIAM1 reference (Ref) cDNA partially rescues the sif loss-of-function (LoF) phenotypes. We also assessed the function associated with three TIAM1 variants carried by two of the probands and compared them to the TIAM1 Ref cDNA function invivo. TIAM1 p.Arg23Cys has reduced rescue ability when compared to TIAM1 Ref, suggesting that it is a partial LoF variant. In ectopic expression studies, both wild-type sif and TIAM1 Ref are toxic, whereas the three variants (p.Leu862Phe, p.Arg23Cys, and p.Gly328Val) show reduced toxicity, suggesting that they are partial LoF variants. In summary, we provide evidence that sif is important for appropriate neural function and that TIAM1 variants observed in the probands are disruptive, thus implicating loss of TIAM1 in neurological phenotypes in humans.

Drosophila functional screening of de novo variants in autism uncovers damaging variants and facilitates discovery of rare neurodevelopmental diseases.

SUMMARYIndividuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through “humanization” rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Versatile Cell and Animal Models for Advanced Investigation of Lead Poisoning.

The heavy metal, lead (Pb) can irreversibly damage the human nervous system. To help understand Pb-induced damage, we applied a genetically encoded Förster resonance energy transfer (FRET)-based Pb biosensor Met-lead 1.44 M1 to two living systems to monitor the concentration of Pb: induced pluripotent stem cell (iPSC)-derived cardiomyocytes as a semi-tissue platform and Drosophila melanogaster fruit flies as an in vivo animal model. Different FRET imaging modalities were used to obtain FRET signals, which represented the presence of Pb in the tested samples in different spatial dimensions. Using iPSC-derived cardiomyocytes, the relationship between beating activity (20–24 beats per minute, bpm) determined from the fluctuation of fluorescent signals and the concentrations of Pb represented by the FRET emission ratio values of Met-lead 1.44 M1 was revealed from simultaneous measurements. Pb (50 μM) affected the beating activity of cardiomyocytes, whereas two drugs that stop the entry of Pb differentially affected this beating activity: verapamil (2 μM) did not reverse the cessation of beating, whereas 2-APB (50 μM) partially restored this activity (16 bpm). The results clearly demonstrate the potential of this biosensor system as an anti-Pb drug screening application. In the Drosophila model, Pb was detected within the adult brain or larval central nervous system (Cha-gal4 > UAS-Met-lead 1.44 M1) using fast epifluorescence and high-resolution two-photon 3D FRET ratio image systems. The tissue-specific expression of Pb biosensors provides an excellent opportunity to explore the possible Pb-specific populations within living organisms. We believe that this integrated Pb biosensor system can be applied to the prevention of Pb poisoning and advanced research on Pb neurotoxicology.

Comparative transcriptome analyses reveal changes of gene expression in larvae hatched from fresh and cryopreserved kelp grouper (Epinephelus moara) embryos

For the first time in fish, this study investigated the changes of gene expression in larvae hatched from fresh and cryopreserved embryos using RNA-seq. The transcriptome of kelp grouper from 1-day larvae hatched from fresh and cryopreserved embryos were obtained by high-throughput sequencing and analyzed by KEGG and GO database. The analysis results showed that 1815 unigenes were significantly differentially expressed (1165 up-regulated vs 650 down-regulated). Significantly down-regulated genes were enriched for eye development, cranial nerve development, sensory light stimulation and neurotransmitter transport terms, predicting that dehydration/rehydration or freezing/thawing during cryopreservation may affect development of the central nervous system, including the primordia of the eye. Moreover, many down-regulated genes were involved in calcium ion regulation, which may affect cell structure, as well as cell proliferation and differentiation. Meanwhile, gene expression of some proteins that protect neurons from the toxic effects of high concentrations of extracellular neurotransmitters significantly decreased, further suggesting that cryopreservation may have an effect on the central nervous system of larvae. In addition, huge amounts of primary data provided in present paper contribute to the genome and proteome databases of the grouper. These results will help other researchers better understand the effect of cryopreservation on fish embryos and improve exploration of the molecular mechanism of fish embryo cryoinjury.

Disruption of left-right axis specification in Ciona induces molecular, cellular, and functional defects in asymmetric brain structures

BackgroundLeft-right asymmetries are a common feature of metazoans and can be found in a number of organs including the nervous system. These asymmetries are particularly pronounced in the simple central nervous system (CNS) of the swimming tadpole larva of the tunicate Ciona, which displays a chordate ground plan. While common pathway elements for specifying the left/right axis are found among chordates, particularly a requirement for Nodal signaling, Ciona differs temporally from its vertebrate cousins by specifying its axis at the neurula stage, rather than at gastrula. Additionally, Ciona and other ascidians require an intact chorionic membrane for proper left-right specification. Whether such differences underlie distinct specification mechanisms between tunicates and vertebrates will require broad understanding of their influence on CNS formation. Here, we explore the consequences of disrupting left-right axis specification on Ciona larval CNS cellular anatomy, gene expression, synaptic connectivity, and behavior.ResultsWe show that left-right asymmetry disruptions caused by removal of the chorion (dechorionation) are highly variable and present throughout the Ciona larval nervous system. While previous studies have documented disruptions to the conspicuously asymmetric sensory systems in the anterior brain vesicle, we document asymmetries in seemingly symmetric structures such as the posterior brain vesicle and motor ganglion. Moreover, defects caused by dechorionation include misplaced or absent neuron classes, loss of asymmetric gene expression, aberrant synaptic projections, and abnormal behaviors. In the motor ganglion, a brain structure that has been equated with the vertebrate hindbrain, we find that despite the apparent left-right symmetric distribution of interneurons and motor neurons, AMPA receptors are expressed exclusively on the left side, which equates with asymmetric swimming behaviors. We also find that within a population of dechorionated larvae, there is a small percentage with apparently normal left-right specification and approximately equal population with inverted (mirror-image) asymmetry. We present a method based on a behavioral assay for isolating these larvae. When these two classes of larvae (normal and inverted) are assessed in a light dimming assay, they display mirror-image behaviors, with normal larvae responding with counterclockwise swims, while inverted larvae respond with clockwise swims.ConclusionsOur findings highlight the importance of left-right specification pathways not only for proper CNS anatomy, but also for correct synaptic connectivity and behavior.

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.

Tissue-specific dynamic codon redefinition in Drosophila

Translational stop codon readthrough occurs in organisms ranging from viruses to mammals and is especially prevalent in decoding Drosophila and viral mRNAs. Recoding of UGA, UAG, or UAA to specify an amino acid allows a proportion of the protein encoded by a single gene to be C-terminally extended. The extended product from Drosophila kelch mRNA is 160 kDa, whereas unextended Kelch protein, a subunit of a Cullin3-RING ubiquitin ligase, is 76 kDa. Previously we reported tissue-specific regulation of readthrough of the first kelch stop codon. Here, we characterize major efficiency differences in a variety of cell types. Immunoblotting revealed low levels of readthrough in malpighian tubules, ovary, and testis but abundant readthrough product in lysates of larval and adult central nervous system (CNS) tissue. Reporters of readthrough demonstrated greater than 30% readthrough in adult brains, and imaging in larval and adult brains showed that readthrough occurred in neurons but not glia. The extent of readthrough stimulatory sequences flanking the readthrough stop codon was assessed in transgenic Drosophila and in human tissue culture cells where inefficient readthrough occurs. A 99-nucleotide sequence with potential to form an mRNA stem-loop 3' of the readthrough stop codon stimulated readthrough efficiency. However, even with just six nucleotides of kelch mRNA sequence 3' of the stop codon, readthrough efficiency only dropped to 6% in adult neurons. Finally, we show that high-efficiency readthrough in the Drosophila CNS is common; for many neuronal proteins, C-terminal extended forms of individual proteins are likely relatively abundant.

Heterologous functional expression of ascidian Nav1 channels and close relationship with the evolutionary ancestor of vertebrate Nav channels

Voltage-gated sodium channels (Nav1s) are responsible for the initiation and propagation of action potentials in neurons, muscle, and endocrine cells. Many clinically used drugs such as local anesthetics and antiarrhythmics inhibit Nav1s, and a variety of inherited human disorders are caused by mutations in Nav1 genes. Nav1s consist of the main α subunit and several auxiliary β subunits. Detailed information on the structure–function relationships of Nav1 subunits has been obtained through heterologous expression experiments and analyses of protein structures. The basic properties of Nav1s, including their gating and ion permeation, were classically described in the squid giant axon and other invertebrates. However, heterologous functional expression of Nav1s from marine invertebrates has been unsuccessful. Ascidians belong to the Urochordata, a sister group of vertebrates, and the larval central nervous system of ascidians shows a similar plan to that of vertebrates. Here, we report the biophysical properties of ascidian Ciona Nav1 (CiNav1a) heterologously expressed in Xenopus oocytes. CiNav1a exhibited tetrodotoxin-insensitive sodium currents with rapid gating kinetics of activation and inactivation. Furthermore, consistent with the fact that the Ciona genome lacks orthologous genes to vertebrate β subunits, the human β1 subunit did not influence the gating properties when coexpressed with CiNav1a. Interestingly, CiNav1a contains an ankyrin-binding motif in the II–III linker, which can be targeted to the axon initial segment of mammalian cortical neurons. Our findings provide a platform to gain insight into the evolutionary and biophysical properties of Nav1s, which are important for the development of targeted therapeutics.

Dissecting Hes-centred transcriptional networks in neural stem cell maintenance and tumorigenesis in Drosophila.

Neural stem cells divide during embryogenesis and juvenile life to generate the entire complement of neurons and glia in the nervous system of vertebrates and invertebrates. Studies of the mechanisms controlling the fine balance between neural stem cells and more differentiated progenitors have shown that, in every asymmetric cell division, progenitors send a Delta-Notch signal to their sibling stem cells. Here, we show that excessive activation of Notch or overexpression of its direct targets of the Hes family causes stem-cell hyperplasias in the Drosophila larval central nervous system, which can progress to malignant tumours after allografting to adult hosts. We combined transcriptomic data from these hyperplasias with chromatin occupancy data for Dpn, a Hes transcription factor, to identify genes regulated by Hes factors in this process. We show that the Notch/Hes axis represses a cohort of transcription factor genes. These are excluded from the stem cells and promote early differentiation steps, most likely by preventing the reversion of immature progenitors to a stem-cell fate. We describe the impact of two of these 'anti-stemness' factors, Zfh1 and Gcm, on Notch/Hes-triggered tumorigenesis.