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Current papers in developmental biology and gene function


Wednesday, September 30th, 2020 - Signaling

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Trostnikov, M. V., Veselkina, E. R., Krementsova, A. V., Boldyrev, S. V., Roshina, N. V. and Pasyukova, E. G. (2020). Modulated Expression of the Protein Kinase GSK3 in Motor and Dopaminergic Neurons Increases Female Lifespan in Drosophila melanogaster. Front Genet 11: 668. PubMed ID: 32695143
Most eukaryotic genes express multiple transcripts and proteins, and a sophisticated gene expression strategy plays a crucial role in ensuring the cell-specificity of genetic information and the correctness of phenotypes. The Drosophila melanogaster gene shaggy encodes several isoforms of the conserved glycogen synthase kinase 3 (GSK3), which is vitally important for multiple biological processes. To characterize the phenotypic effects of differential shaggy expression, this study explored how the multidirectional modulation of the expression of the main GSK3 isoform, Shaggy-PB, in different tissues and cells affects lifespan. To this end, lines with transgenic constructs were used that encode mutant variants of the protein. The effect of shaggy misexpression on lifespan depended on the direction of the presumed change in GSK3 activity and the type of tissue/cell. The modulation of GSK3 activity in motor and dopaminergic neurons improved female lifespan but caused seemingly negative changes in the structural (mitochondrial depletion; neuronal loss) and functional (perturbed locomotion) properties of the nervous system, indicating the importance of analyzing the relationship between lifespan and healthspan in invertebrate models. These findings provide new insights into the molecular and cellular bases of lifespan extension, demonstrating that the fine-tuning of transcript-specific shaggy expression in individual groups of neurons is sufficient to provide a sex-specific increase in survival and slow aging.
Xu, L., Li, P., Hao, X., Lu, Y., Liu, M., Song, W., Shan, L., Yu, J., Ding, H., Chen, S., Yang, A., Zeng, Y. A., Zhang, L. and Jiang, H. (2020). SHANK2 is a frequently amplified oncogene with evolutionarily conserved roles in regulating Hippo signaling. Protein Cell. PubMed ID: 32661924
Dysfunction of the Hippo pathway enables cells to evade contact inhibition and provides advantages for cancerous overgrowth. However, for a significant portion of human cancer, how Hippo signaling is perturbed remains unknown. To answer this question, a genome-wide screening was performed for genes that affect the Hippo pathway in Drosophila and cross-referenced the hit genes with human cancer genome. In this screen, Prosap was identified as a novel regulator of the Hippo pathway that potently affects tissue growth. Interestingly, a mammalian homolog of Prosap, SHANK2, is the most frequently amplified gene on 11q13, a major tumor amplicon in human cancer. Gene amplification profile in this 11q13 amplicon clearly indicates selective pressure for SHANK2 amplification. More importantly, across the human cancer genome, SHANK2 is the most frequently amplified gene that is not located within the Myc amplicon. Further studies in multiple human cell lines confirmed that SHANK2 overexpression causes deregulation of Hippo signaling through competitive binding for a LATS1 (see Drosophila Lats) activator, and as a potential oncogene, SHANK2 promotes cellular transformation and tumor formation in vivo. In cancer cell lines with deregulated Hippo pathway, depletion of SHANK2 restores Hippo signaling and ceases cellular proliferation. Taken together, these results suggest that SHANK2 is an evolutionarily conserved Hippo pathway regulator, commonly amplified in human cancer and potently promotes cancer. This study illustrated oncogenic function of SHANK2, one of the most frequently amplified gene in human cancer. Furthermore, given that in normal adult tissues, SHANK2's expression is largely restricted to the nervous system, SHANK2 may represent an interesting target for anticancer therapy.
Socodato, R., Portugal, C. C., Canedo, T., Rodrigues, A., Almeida, T. O., Henriques, J. F., Vaz, S. H., Magalhaes, J., Silva, C. M., Baptista, F. I., Alves, R. L., Coelho-Santos, V., Silva, A. P., Paes-de-Carvalho, R., Magalhaes, A., Brakebusch, C., Sebastiao, A. M., Summavielle, T., Ambrosio, A. F. and Relvas, J. B. (2020). Microglia Dysfunction Caused by the Loss of Rhoa Disrupts Neuronal Physiology and Leads to Neurodegeneration. Cell Rep 31(12): 107796. PubMed ID: 32579923
Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, this study demonstrates that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of beta-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src (see Drosophila Src64) activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. It was also found that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Abeta oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, these results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.
Nagaoka, T., Furuse, M., Ohtsuka, T., Tsuchida, K. and Kishi, M. (2019). Vangl2 interaction plays a role in the proteasomal degradation of Prickle2. Sci Rep 9(1): 2912. PubMed ID: 30814664
The PET and LIM domain-containing protein, Prickle, plays a key role in planar cell polarity (PCP) in Drosophila. It has been reported that mutations in the PRICKLE2 gene, which encodes one of the human orthologues of Prickle, are associated with human diseases such as epilepsy and autism spectrum disorder. To develop preventive and therapeutic strategies for these intractable diseases, the regulation of Prickle2 protein levels was studied in transfected HEK293T cells. Prickle2 levels were negatively regulated by a physical interaction with another PCP protein, Van Gogh-like 2 (Vangl2; see Drosophila Vang). The Vangl2-mediated reduction in Prickle2 levels was, at least in part, relieved by proteasome inhibitors or by functional inhibition of the Cullin-1 E3 ubiquitin ligase (see Drosophila Cullin 1). Furthermore, the expression of Vangl2 enhanced the polyubiquitination of Prickle2. This ubiquitination was partially blocked by co-expression of a ubiquitin mutant, which cannot be polymerised through their Lys48 residue to induce target proteins toward proteasomal degradation. Together, these results suggest that Prickle2 is polyubiquitinated by the Vangl2 interaction in a Cullin-1-dependent manner to limit its expression levels. This regulation may play a role in the local and temporal fine-tuning of Prickle protein levels during PCP signal-dependent cellular behaviours.
Thevenon, D., Seffouh, I., Pillet, C., Crespo-Yanez, X., Fauvarque, M. O. and Taillebourg, E. (2020). A Nucleolar Isoform of the Drosophila Ubiquitin Specific Protease dUSP36 Regulates MYC-Dependent Cell Growth. Front Cell Dev Biol 8: 506. PubMed ID: 32637412
The c-Myc oncogene is a transcription factor that regulates the expression of a very large set of genes mainly involved in cell growth and proliferation. It is overexpressed in more than 70% of human cancers, illustrating the importance of keeping its levels and activity under control. The ubiquitin proteasome system is a major regulator of MYC levels in humans as well as in model organisms such as Drosophila melanogaster. Although the E3 ligases that promote MYC ubiquitination have been largely investigated, the identity and the role of the deubiquitinating enzymes, which counteract their action is only beginning to be unraveled. Using isoform-specific CRISPR-Cas9 mutagenesis, this study shows that the Drosophila homolog of the Ubiquitin Specific Protease USP36 has different isoforms with specific sub-cellular localizations and that the nucleolar dUSP36-D isoform is specifically required for cell and organismal growth. This isoform interacts with dMYC and the E3 ligase AGO and regulates their stability and ubiquitination levels. Furthermore, this study shows that dUSP36 is ubiquitinated by AGO and is able to self-deubiquitinate. Finally, in vivo evidence is provided supporting the functional relevance of these regulatory relationships. Together these results reveal that dMYC, AGO and dUSP36 form a tripartite, evolutionary conserved complex that acts as a regulatory node to control dMYC protein levels.
Ventura, G., Moreira, S., Barros-Carvalho, A., Osswald, M. and Morais-de-Sa, E. (2020). Lgl cortical dynamics are independent of binding to the Scrib-Dlg complex but require Dlg-dependent restriction of aPKC. Development. PubMed ID: 32665243
Apical-basal polarity underpins the formation of epithelial barriers that are critical for metazoan physiology. Although apical-basal polarity is long known to require the basolateral determinants Lethal Giant Larvae (Lgl), Discs Large (Dlg) and Scribble (Scrib), mechanistic understanding of their function is limited. Lgl plays a role as an aPKC inhibitor, but it remains unclear whether Lgl also forms complexes with Dlg or Scrib. Using fluorescence recovery after photobleaching, this study shows that Lgl does not form immobile complexes at the lateral domain of Drosophila follicle cells. Optogenetic depletion of plasma membrane PIP(2) or dlg mutants accelerate Lgl cortical dynamics. However, Dlg and Scrib are only required for Lgl localization and dynamic behavior in the presence of aPKC function. Furthermore, light-induced oligomerization of basolateral proteins indicates that Lgl is not part of the Scrib-Dlg complex in the follicular epithelium. Thus, Scrib-Dlg are necessary to repress aPKC activity in the lateral domain but do not provide cortical binding sites for Lgl. This work therefore highlights that Lgl does not act in a complex but in parallel with Scrib-Dlg to antagonize apical determinants.

Tuesday, September 29th - Evolution

Huang, J., Sun, W., Seong, K. M., Mittapalli, O., Ojo, J., Coates, B., Paige, K. N., Clark, J. M. and Pittendrigh, B. R. (2020). Dietary antioxidant vitamin C influences the evolutionary path of insecticide resistance in Drosophila melanogaster. Pestic Biochem Physiol 168: 104631. PubMed ID: 32711765
Herbivorous insects encounter a variety of toxic environmental substances ranging from ingested plant defensive compounds to human-introduced insecticidal agents. Dietary antioxidants are known to reduce the negative physiological impacts of toxins in mammalian systems through amelioration of reactive oxygen-related cellular damage. The analogous impacts to insects caused by multigenerational exposure to pesticides and the effects on adaptive responses within insect populations, however, are currently unknown. To address these research gaps, this study used Drosophila as a model system to explore adaptive phenotypic responses to acute dichlorodiphenyltrichloroethane (DDT) exposure in the presence of the dietary antioxidant vitamin C and examined the structural genomic consequences of this exposure. DDT resistance increased significantly among four replicates exposed to a low concentration of DDT for 10 generations. In contrast, dietary intake of vitamin C significantly reduced DDT resistance after mutigenerational exposure to the same concentration of DDT. As to the genomic consequences, no significant differences were predicted in overall nucleotide substitution rates across the genome between any of the treatments. Despite this, replicates exposed to a low concentration of DDT without vitamin C showed the highest number of synonymous and non-synonymous variants (3196 in total), followed by the DDT plus vitamin C (1174 in total), and vitamin C alone (728 in total) treatments. This study demonstrates the potential role of diet (specifically, antioxidant intake) on adaptive genome responses, and thus on the evolution of pesticide resistance within insect populations.
Veenstra, J. A. (2020). Arthropod IGF, relaxin and gonadulin, putative orthologs of Drosophila insulin-like peptides 6, 7 and 8, likely originated from an ancient gene triplication. PeerJ 8: e9534. PubMed ID: 32728497
Insects have several genes coding for insulin-like peptides and they have been particularly well studied in Drosophila. Two other Drosophila insulin-like hormones are either known or suspected to act through a G-protein coupled receptor. Although insulin-related peptides are known from other insect species, Drosophila insulin-like peptide 8, one that uses a GPCR, has so far only been identified from Drosophila and other flies. However, its receptor is widespread within arthropods and hence it should have orthologs. Such putative orthologs were recently identified in decapods and have been called gonadulins. An effort was made to identify gonadulins in other arthropods. Gonadulins were detected in a number of arthropods. Insect gonadulins are expressed in the ovaries and at least in some species also in the testes. In some insects differences in gonadulin expression in the ovary between actively reproducing and non-reproducing females differs more than 100-fold. Putative orthologs of Drosophila ilp 6 were also identified.In cockroaches, termites and stick insects genes coding for the arthropod insulin-like growth factors, gonadulin and relaxin, a third insulin-like peptide, are encoded by genes that are next to one another suggesting that they are the result of a local gene triplication. Insulin signaling pathway evolved differently in insects, decapods and chelicerates. The GPCRs that are related to the Drosophila Ilp 8 receptor similarly show significant differences between those groups. A local gene triplication in an early ancestor likely yielded three genes coding gonadulin, arthropod insulin-like growth factor and relaxin. Orthologs of these genes are now commonly present in arthropods and almost certainly include the Drosophila insulin-like peptides 6, 7 and 8.
Khodursky, S., Svetec, N., Durkin, S. M. and Zhao, L. (2020). The evolution of sex-biased gene expression in the Drosophila brain. Genome Res 30(6): 874-884. PubMed ID: 32554780
Genes with sex-biased expression in Drosophila are thought to underlie sexually dimorphic phenotypes and have been shown to possess unique evolutionary properties. However, the forces and constraints governing the evolution of sex-biased genes in the somatic tissues of Drosophila are largely unknown. Using population-scale RNA sequencing data this study shows that sex-biased genes in the Drosophila brain are highly enriched on the X Chromosome and that most are biased in a species-specific manner. X-linked male-biased genes, and to a lesser extent female-biased genes, are enriched for signatures of directional selection at the gene expression level. By examining the evolutionary properties of gene flanking regions on the X Chromosome, this study found evidence that adaptive cis-regulatory changes are more likely to drive the expression evolution of X-linked male-biased genes than other X-linked genes. Finally, whether constraint due to broad expression across multiple tissues and genetic constraint due to the largely shared male and female genomes could be responsible for the observed patterns of gene expression evolution was examined. Expression breadth did not constrain the directional evolution of gene expression in the brain. Additionally, it was found that the shared genome between males and females imposes a substantial constraint on the expression evolution of sex-biased genes. Overall, these results significantly advance understanding of the patterns and forces shaping the evolution of sexual dimorphism in the Drosophila brain.
Reis, M., Wiegleb, G., Claude, J., Lata, R., Horchler, B., Ha, N. T., Reimer, C., Vieira, C. P., Vieira, J. and Posnien, N. (2020). Multiple loci linked to inversions are associated with eye size variation in species of the Drosophila virilis phylad. Sci Rep 10(1): 12832. PubMed ID: 32732947
The size and shape of organs is tightly controlled to achieve optimal function. Natural morphological variations often represent functional adaptations to an ever-changing environment. For instance, variation in head morphology is pervasive in insects and the underlying molecular basis is starting to be revealed in the Drosophila genus for species of the melanogaster group. However, it remains unclear whether similar diversifications are governed by similar or different molecular mechanisms over longer timescales. To address this issue, species of the virilis phylad were used because they have been diverging from D. melanogaster for at least 40 million years. A comprehensive morphological survey revealed remarkable differences in eye size and head shape among these species with D. novamexicana having the smallest eyes and southern D. americana populations having the largest eyes. The genetic architecture underlying eye size variation is complex with multiple associated genetic variants located on most chromosomes. A genome wide association study (GWAS) strongly suggests that some of the putative causative variants are associated with the presence of inversions. Indeed, northern populations of D. americana share derived inversions with D. novamexicana and they show smaller eyes compared to southern ones. Intriguingly, a significant enrichment of genes was observed that involved in eye development on the 4th chromosome after intersecting chromosomal regions associated with phenotypic differences with those showing high differentiation among D. americana populations. It is proposed that variants associated with chromosomal inversions contribute to both intra- and interspecific variation in eye size among species of the virilis phylad.
Keesey, I. W., Grabe, V., Knaden, M. and Hansson, B. S. (2020). Divergent sensory investment mirrors potential speciation via niche partitioning across Drosophila. Elife 9. PubMed ID: 32602834
The examination of phylogenetic and phenotypic characteristics of the nervous system, such as behavior and neuroanatomy, can be utilized as a means to assess speciation. Recent studies have proposed a fundamental tradeoff between two sensory organs, the eye and the antenna. However, the identification of ecological mechanisms for this observed tradeoff have not been firmly established. This study examines several monophyletic species within the obscura group, and asserts that despite their close relatedness and overlapping ecology, they deviate strongly in both visual and olfactory investment. It is contended that both courtship and microhabitat preferences support the observed inverse variation in these sensory traits. This variation in visual and olfactory investment seems to provide relaxed competition, a process by which similar species can use a shared environment differently and in ways that help them coexist. Moreover, that behavioral separation according to light gradients occurs first, and subsequently, courtship deviations arise.
Watada, M., Hayashi, Y., Watanabe, K., Mizutani, S., Mure, A., Hattori, Y. and Uemura, T. (2020). Divergence of Drosophila species: longevity and reproduction under different nutrient balances. Genes Cells. PubMed ID: 32594638
How nutrition impacts growth, reproduction, and longevity is complex because relationships between these life events are difficult to disentangle. As a first step in sorting out these processes, a comparative analysis was performed of related species of Drosophila with distinct feeding habits. In particular, lifespans and egg laying were examined of two generalists and three specialists on diets with distinct protein-to-carbohydrate ratios. In contrast to the generalist D. melanogaster, adult males of two specialists, D. sechellia and D. elegans, lived longer on a protein-rich diet. These results and previous studies collectively demonstrate that the diet to which larvae of each specialist species has adapted ensures a longer lifespan of adult males of that same species. A species-specific sexual dimorphism of lifespan was found in the above two specialists regardless of the diets, in sharp contrast to D. melanogaster. In D. melanogaster, males lived longer than females, whereas females of D. sechellia and D. elegans were longer-lived than males, and those specialist females were exceedingly low in egg production, relative to the other species. These findings are discuss from perspectives of mechanisms, including a possible contribution of egg production to lifespan.

Monday, September 28th - Adult neural development and function

Huang, B., Li, X., Tu, X., Zhao, W., Zhu, D., Feng, Y., Si, X. and Chen, J. G. (2018). OTX1 regulates cell cycle progression of neural progenitors in the developing cerebral cortex. J Biol Chem 293(6): 2137-2148. PubMed ID: 29273633
The progenitor cells in the cerebral cortex coordinate proliferation and mitotic exit to generate the correct number of neurons and glial cells during development. However, mechanisms for regulating the mitotic cycle of cortical progenitors are not fully understood. Otx1 (see Drosophila ocelliless) is one of the homeobox-containing transcription factors frequently implicated in the development of the central nervous system. Mice bearing a targeted deletion of Otx1 exhibit brain hypoplasia and a decrease in the number of cortical neurons. It was hypothesized that Otx1 might be crucial to the proliferation and differentiation of cortical progenitors. Otx1 knockdown by in utero electroporation in the mouse brain reduced the proportion of the G1 phase while increasing the S and M phases of progenitor cells. The knockdown diminished Tbr1+ neurons (see Drosophila bifid) but increased GFAP+ astrocytes in the early postnatal cortex as revealed by lineage tracing study. Tbr2+ basal progenitors lacking Otx1 were held at the transit-amplifying stage. In contrast, overexpression of wildtype Otx1 but not an astrocytoma-related mutant Y320C inhibited proliferation of the progenitor cells in embryonic cortex. This study demonstrates that Otx1 is one of the key elements regulating cortical neurogenesis, and a loss-of-function in Otx1 may contribute to the overproduction of astrocytes in vivo.
Zhang, Y. F., Liu, L. X., Cao, H. T., Ou, L., Qu, J., Wang, Y. and Chen, J. G. (2015). Otx1 promotes basal dendritic growth and regulates intrinsic electrophysiological and synaptic properties of layer V pyramidal neurons in mouse motor cortex. Neuroscience 285: 139-154. PubMed ID: 25446351
The transcription factor Otx1 (see Drosophila ocelliless) is specifically expressed in layer V pyramidal cells (L5PCs) in the cerebral cortex. Otx1 null mutant mice have a defect in the developmental axon pruning of L5PCs and show epileptic seizures. This study examines the influences of Otx1 on neuronal properties of L5PCs by loss- and gain-of-function approaches. Mice with an Otx1-null mutation had decreased structural measurements of basal dendrites in L5PCs. In contrast, the size of basal dendrites was increased in the Otx1-over-expressed pyramidal cells (PCs) in L2/3 where the gene normally does not express. PCs showed burst and non-burst firing patterns of action potentials. The proportion of burst firing neurons was reduced in the Otx1 mutant but increased in the neurons over-expressing Otx1. Although the burst firing population decreased, the proportion of those bursting neurons with a low threshold increased in the Otx1 mutant mice. Moreover, excitatory facilitating synaptic connections formed between L5PCs were predominant in the Otx1 mutant mice, which greatly contrasted with the predominant depressing synaptic connections in the controls. Taken together, it suggests an enhanced activity of neuronal network in the cortex of Otx1 mutant mice. These data indicate that the Otx1 expression is essential for the normal development of dendritic morphology, intrinsic electrophysiology and synaptic dynamics of L5PCs.
Kosillo, P., Doig, N. M., Ahmed, K. M., Agopyan-Miu, A., Wong, C. D., Conyers, L., Threlfell, S., Magill, P. J. and Bateup, H. S. (2019). Tsc1-mTORC1 signaling controls striatal dopamine release and cognitive flexibility. Nat Commun 10(1): 5426. PubMed ID: 31780742
Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in TSC1 (see Drosophila Tsc1) or TSC2 (see Drosophila Gigas), which encode proteins that negatively regulate mTOR complex 1 (mTORC1). TSC is associated with significant cognitive, psychiatric, and behavioral problems, collectively termed TSC-Associated Neuropsychiatric Disorders (TAND), and the cell types responsible for these manifestations are largely unknown. This study used cell type-specific Tsc1 deletion to test whether dopamine neurons, which modulate cognitive, motivational, and affective behaviors, are involved in TAND. Loss of Tsc1 and constitutive activation of mTORC1 in dopamine neurons causes somatodendritic hypertrophy, reduces intrinsic excitability, alters axon terminal structure, and impairs striatal dopamine release. These perturbations lead to a selective deficit in cognitive flexibility, preventable by genetic reduction of the mTOR-binding protein Raptor (see Drosophila Raptor). These results establish a critical role for Tsc1-mTORC1 signaling in setting the functional properties of dopamine neurons, and indicate that dopaminergic dysfunction may contribute to cognitive inflexibility in TSC.
Wang, N., Dhumale, P., Chiang, J. and Puschel, A. W. (2018). The Sema3A receptor Plexin-A1 suppresses supernumerary axons through Rap1 GTPases. Sci Rep 8(1): 15647. PubMed ID: 30353093
The highly conserved Rap1 GTPases (see Drosophila Rap1) perform essential functions during neuronal development. They are required for the polarity of neuronal progenitors and neurons as well as for neuronal migration in the embryonic brain. Neuronal polarization and axon formation depend on the precise temporal and spatial regulation of Rap1 activity by guanine nucleotide exchange factors (GEFs) and GTPases-activating proteins (GAPs). Several Rap1 GEFs have been identified that direct the formation of axons during cortical and hippocampal development in vivo and in cultured neurons. However little is known about the GAPs that limit the activity of Rap1 GTPases during neuronal development. This study investigated the function of Sema3A and Plexin-A1 as a regulator of Rap1 GTPases during the polarization of hippocampal neurons. Sema3A was shown to suppress axon formation when neurons are cultured on a patterned substrate. Plexin-A1 (see Drosophila Plexin) functions as the signal-transducing subunit of receptors for Sema3A and displays GAP activity for Rap1 GTPases. Sema3A and Plexin-A1 suppress the formation of supernumerary axons in cultured neurons, which depends on Rap1 GTPases.
Teotia, P., Van Hook, M. J., Fischer, D. and Ahmad, I. (2019). Human retinal ganglion cell axon regeneration by recapitulating developmental mechanisms: effects of recruitment of the mTOR pathway. Development 146(13). PubMed ID: 31273087
The poor axon regeneration in the central nervous system (CNS) often leads to permanent functional deficit following disease or injury. For example, degeneration of retinal ganglion cell (RGC) axons in glaucoma leads to irreversible loss of vision. This study tested the hypothesis that the mTOR pathway regulates the development of human RGCs and that its recruitment after injury facilitates axon regeneration. The mTOR pathway is active during RGC differentiation, and using the induced pluripotent stem cell model of neurogenesis this study shows that it facilitates the differentiation, function and neuritogenesis of human RGCs. Using a microfluidic model, it was demonstrated that recruitment of the mTOR pathway facilitates human RGC axon regeneration after axotomy, providing evidence that the recapitulation of developmental mechanism(s) might be a viable approach for facilitating axon regeneration in the diseased or injured human CNS, thus helping to reduce and/or recover loss of function.
Robert, B. J. A., Moreau, M. M., Dos Santos Carvalho, S., Barthet, G., Racca, C., Bhouri, M., Quiedeville, A., Garret, M., Atchama, B., Al Abed, A. S., Guette, C., Henderson, D. J., Desmedt, A., Mulle, C., Marighetto, A., Montcouquiol, M. and Sans, N. (2020). Vangl2 in the Dentate Network Modulates Pattern Separation and Pattern Completion. Cell Rep 31(10): 107743. PubMed ID: 32521268
The organization of spatial information, including pattern completion and pattern separation processes, relies on the hippocampal circuits, yet the molecular and cellular mechanisms underlying these two processes are elusive. This study finds that loss of Vangl2 (see Drosophila Van Gogh), a core PCP gene, results in opposite effects on pattern completion and pattern separation processes. Mechanistically, Vangl2 loss maintains young postmitotic granule cells in an immature state, providing increased cellular input for pattern separation. The genetic ablation of Vangl2 disrupts granule cell morpho-functional maturation and further prevents CaMKII and GluA1 phosphorylation, disrupting the stabilization of AMPA receptors. As a functional consequence, LTP at lateral perforant path-GC synapses is impaired, leading to defects in pattern completion behavior. In conclusion, this study shows that Vangl2 exerts a bimodal regulation on young and mature GCs, and its disruption leads to an imbalance in hippocampus-dependent pattern completion and separation processes.

Friday, September 25th - Chromatin

Kolesnikova, T. D., Kolodyazhnaya, A. V., Pokholkova, G. V., Schubert, V., Dovgan, V. V., Romanenko, S. A., Prokopov, D. Y. and Zhimulev, I. F. (2020). Effects of Mutations in the Drosophila melanogaster Rif1 Gene on the Replication and Underreplication of Pericentromeric Heterochromatin in Salivary Gland Polytene Chromosomes. Cells 9(6). PubMed ID: 32575592
In Drosophila salivary gland polytene chromosomes, a substantial portion of heterochromatin is underreplicated. The combination of mutations SuUR(ES) and Su(var)3-9(06) results in the polytenization of a substantial fraction of unique and moderately repeated sequences but has almost no effect on satellite DNA replication. The Rap1 interacting factor 1 (Rif1) protein is a conserved regulator of replication timing, and in Drosophila, it affects underreplication in polytene chromosomes. This study compared the morphology of pericentromeric regions and labeling patterns of in situ hybridization of heterochromatin-specific DNA probes between wild-type salivary gland polytene chromosomes and the chromosomes of Rif1 mutants and SuUR Su(var)3-9(06) double mutants. Despite general similarities, heterochromatin zones exist that are polytenized only in the Rif1 mutants, and there are zones that are under specific control of Su(var)3-9. In the Rif1 mutants, additional polytenization was found of the largest blocks of satellite DNA (in particular, satellite 1.688 of chromosome X and simple satellites in chromosomes X and 4) as well as partial polytenization of chromosome Y. Data on pulsed incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into polytene chromosomes indicated that in the Rif1 mutants, just as in the wild type, most of the heterochromatin becomes replicated during the late S phase. Nevertheless, a significantly increased number of heterochromatin replicons was noted. These results suggest that Rif1 regulates the activation probability of heterochromatic origins in the satellite DNA region.
Funikov, S. Y., Rezvykh, A. P., Kulikova, D. A., Zelentsova, E. S., Protsenko, L. A., Chuvakova, L. N., Tyukmaeva, V. I., Arkhipova, I. R. and Evgen'ev, M. B. (2020). Adaptation of gene loci to heterochromatin in the course of Drosophila evolution is associated with insulator proteins. Sci Rep 10(1): 11893. PubMed ID: 32681087
Pericentromeric heterochromatin is generally composed of repetitive DNA forming a transcriptionally repressive environment. Dozens of genes were embedded into pericentromeric heterochromatin during evolution of Drosophilidae lineage while retaining activity. However, factors that contribute to insusceptibility of gene loci to transcriptional silencing remain unknown. This study finds that the promoter region of genes that can be embedded in both euchromatin and heterochromatin exhibits a conserved structure throughout the Drosophila phylogeny and carries motifs for binding of certain chromatin remodeling factors, including insulator proteins. Using ChIP-seq data, this study demonstrates that evolutionary gene relocation between euchromatin and pericentric heterochromatin occurred with preservation of sites of insulation of BEAF-32 in evolutionarily distant species, i.e. D. melanogaster and D. virilis. Moreover, promoters of virtually all protein-coding genes located in heterochromatin in D. melanogaster are enriched with insulator proteins BEAF-32, GAF and dCTCF. Applying RNA-seq of a BEAF-32 mutant, this study shows that the impairment of BEAF-32 function has a complex effect on gene expression in D. melanogaster, affecting even those genes that lack BEAF-32 association in their promoters. It is proposed that conserved intrinsic properties of genes, such as sites of insulation near the promoter regions, may contribute to adaptation of genes to the heterochromatic environment and, hence, facilitate the evolutionary relocation of genes loci between euchromatin and heterochromatin.
Maharjan, M., McKowen, J. K. and Hart, C. M. (2020). Overlapping but Distinct Sequences Play Roles in the Insulator and Promoter Activities of the Drosophila BEAF-dependent scs' Insulator. Genetics. PubMed ID: 32554599
Chromatin domain insulators are thought to help partition the genome into genetic units called topologically associating domains (TADs). In Drosophila, TADs are often separated by inter-TAD regions containing active housekeeping genes and associated insulator binding proteins. This raises the question of whether insulator binding proteins are primarily involved in chromosomal TAD architecture or gene activation, or if these two activities are linked. The Boundary Element-Associated Factor of 32 kDa (BEAF-32, or BEAF for short) is usually found in inter-TADs. BEAF was discovered based on binding to the scs' insulator, and is important for the insulator activity of scs' and other BEAF binding sites. There are divergent promoters in scs' with a BEAF binding site by each. This study dissected the scs' insulator to identify DNA sequences important for insulator and promoter activity, focusing on the half of scs' with a high affinity BEAF binding site. The BEAF binding site is important for both insulator and promoter activity, as is another sequence referred to as LS4. Aside from that, different sequences play roles in insulator and promoter activity. So while there is overlap and BEAF is important for both, insulator and promoter activity can be separated.
Kang, J. J., Faubert, D., Boulais, J. and Francis, N. J. (2020). DNA Binding Reorganizes the Intrinsically Disordered C-Terminal Region of PSC in Drosophila PRC1. J Mol Biol. PubMed ID: 32628956
Polycomb Group proteins regulate gene expression by modifying chromatin. Polycomb Repressive Complex 1 (PRC1) has two activities: a ubiquitin ligase activity for histone H2A and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase. The intrinsically disordered C-terminal region of PSC compacts chromatin and inhibits chromatin remodeling and transcription in vitro. Both regions of PSC are essential in vivo. Crosslinking identifies interactions between the C-terminal region of PSC and the core of PRC1, including between N and C-terminal regions of PSC. New contacts and overall more compacted PSC C-terminal region conformations are induced by DNA binding. Protein footprinting of accessible lysine residues reveals an extended, bipartite candidate DNA/chromatin binding surface in the C-terminal region of PSC. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible disordered region of PSC. Intramolecular interactions of PSC detected by crosslinking can bring the high-affinity DNA/chromatin binding region close to the core of PRC1 without disrupting the interface between the ubiquitin ligase and the nucleosome. This approach may be applicable to understanding the global organization of other large intrinsically disordered regions that bind nucleic acids.
Pessoa Rodrigues, C., Herman, J. S., Herquel, B., Valsecchi, C. I. K., Stehle, T., Grun, D. and Akhtar, A. (2020). Temporal expression of MOF acetyltransferase primes transcription factor networks for erythroid fate. Sci Adv 6(21): eaaz4815. PubMed ID: 32671208
Self-renewal and differentiation of hematopoietic stem cells (HSCs) are orchestrated by the combinatorial action of transcription factors and epigenetic regulators. This study has explored the mechanism by which histone H4 lysine 16 acetyltransferase MOF regulates erythropoiesis. Single-cell RNA sequencing and chromatin immunoprecipitation sequencing uncovered that MOF influences erythroid trajectory by dynamic recruitment to chromatin and its haploinsufficiency causes accumulation of a transient HSC population. A regulatory network consisting of MOF, RUNX1 (see Drosophila Runt), and GFI1B is critical for erythroid fate commitment. GFI1B acts as a Mof activator which is necessary and sufficient for cell type-specific induction of Mof expression. Plasticity of Mof-depleted HSCs can be rescued by expression of a downstream effector, Gata1, or by rebalancing acetylation via a histone deacetylase inhibitor. Accurate timing and dosage of Mof expression act as a rheostat for the feedforward transcription factor network that safeguards progression along the erythroid fate.
Hur, W., Kemp, J. P., Jr., Tarzia, M., Deneke, V. E., Marzluff, W. F., Duronio, R. J. and Di Talia, S. (2020). CDK-Regulated Phase Separation Seeded by Histone Genes Ensures Precise Growth and Function of Histone Locus Bodies. Dev Cell. PubMed ID: 32579968
Many membraneless organelles form through liquid-liquid phase separation, but how their size is controlled and whether size is linked to function remain poorly understood. The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. This study shows that Drosophila HLBs form through phase separation. During embryogenesis, the size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic histone gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which facilitates phase separation, and the nuclear concentration of the scaffold protein Multi-sex combs (Mxc), which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Thus, these experiments identify a mechanism linking nuclear body growth and size with gene expression.

Thursday, September 24th - Synapse and vesicles

Dai, J., Aoto, J. and Sudhof, T. C. (2019). Alternative Splicing of Presynaptic Neurexins Differentially Controls Postsynaptic NMDA and AMPA Receptor Responses. Neuron 102(5): 993-1008. PubMed ID: 31005376
AMPA- and NMDA-type glutamate receptors mediate distinct postsynaptic signals that differ characteristically among synapses. How postsynaptic AMPA- and NMDA-receptor levels are regulated, however, remains unclear. Using newly generated conditional knockin mice that enable genetic control of neurexin alternative splicing (see Drosophila Neurexin), this study shows that in hippocampal synapses, alternative splicing of presynaptic neurexin-1 at splice site 4 (SS4) dramatically enhanced postsynaptic NMDA-receptor-mediated, but not AMPA-receptor-mediated, synaptic responses without altering synapse density. In contrast, alternative splicing of neurexin-3 at SS4 suppressed AMPA-receptor-mediated, but not NMDA-receptor-mediated, synaptic responses, while alternative splicing of neurexin-2 at SS4 had no effect on NMDA- or AMPA-receptor-mediated responses. Presynaptic overexpression of the neurexin-1beta and neurexin-3beta SS4+ splice variants, but not of their SS4- splice variants, replicated the respective SS4+ knockin phenotypes. Thus, different neurexins perform distinct nonoverlapping functions at hippocampal synapses that are independently regulated by alternative splicing. These functions transsynaptically control NMDA and AMPA receptors, thereby mediating presynaptic control of postsynaptic responses.
Naito, Y., Tanabe, Y., Lee, A. K., Hamel, E. and Takahashi, H. (2017). Amyloid-beta Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization. Sci Rep 7: 42548. PubMed ID: 28211900
Alzheimer's disease (AD) is characterized by excessive production and deposition of amyloid-beta (Abeta) proteins (see Drosophila Appl) as well as synapse dysfunction and loss. While soluble Abeta oligomers (AbetaOs) have deleterious effects on synapse function and reduce synapse number, the underlying molecular mechanisms are not well understood. This study screened synaptic organizer proteins for cell-surface interaction with AbetaOs and identified a novel interaction between neurexins (NRXs; see Drosophila Neurexin) and AbetaOs. AbetaOs bind to NRXs via the N-terminal histidine-rich domain (HRD) of beta-NRX1/2/3 and alternatively-spliced inserts at splicing site 4 of NRX1/2. In artificial synapse-formation assays, AbetaOs diminish excitatory presynaptic differentiation induced by NRX-interacting proteins including neuroligin1/2 (NLG1/2; see Drosophila Neuroligin) and the leucine-rich repeat transmembrane protein LRRTM2. Although AbetaOs do not interfere with the binding of NRX1beta to NLG1 or LRRTM2, time-lapse imaging revealed that AbetaO treatment reduces surface expression of NRX1beta on axons and that this reduction depends on the NRX1beta HRD. In transgenic mice expressing mutated human amyloid precursor protein, synaptic expression of beta-NRXs, but not alpha-NRXs, decreases. Thus these data indicate that AbetaOs interact with NRXs and that this interaction inhibits NRX-mediated presynaptic differentiation by reducing surface expression of axonal beta-NRXs, providing molecular and mechanistic insights into how AbetaOs lead to synaptic pathology in AD.
Matsuda, K., Budisantoso, T., Mitakidis, N., Sugaya, Y., Miura, E., Kakegawa, W., Yamasaki, M., Konno, K., Uchigashima, M., Abe, M., Watanabe, I., Kano, M., Watanabe, M., Sakimura, K., Aricescu, A. R. and Yuzaki, M. (2016). Transsynaptic Modulation of Kainate Receptor Functions by C1q-like Proteins. Neuron 90(4): 752-767. PubMed ID: 27133466
Postsynaptic kainate-type glutamate receptors (KARs; see Drosophila Glu-RIIA and Glu-RIIB) regulate synaptic network activity through their slow channel kinetics, most prominently at mossy fiber (MF)-CA3 synapses in the hippocampus. Nevertheless, how KARs cluster and function at these synapses has been unclear. This study shows that C1q-like proteins C1ql2 and C1ql3, produced by MFs, serve as extracellular organizers to recruit functional postsynaptic KAR complexes to the CA3 pyramidal neurons. C1ql2 and C1ql3 specifically bound the amino-terminal domains of postsynaptic GluK2 and GluK4 KAR subunits and the presynaptic neurexin 3 containing a specific sequence in vitro. In C1ql2/3 double-null mice, CA3 synaptic responses lost the slow, KAR-mediated components. Furthermore, despite induction of MF sprouting in a temporal lobe epilepsy model, KARs were not recruited to postsynaptic sites in C1ql2/3 double-null mice, leading to reduced recurrent circuit activities. C1q family proteins, broadly expressed, are likely to modulate KAR function throughout the brain and represent promising antiepileptic targets.
Kim, K., Shin, W., Kang, M., Lee, S., Kim, D., Kang, R., Jung, Y., Cho, Y., Yang, E., Kim, H., Bae, Y. C. and Kim, E. (2020). Presynaptic PTPsigma regulates postsynaptic NMDA receptor function through direct adhesion-independent mechanisms. Elife 9. PubMed ID: 32142410
Synaptic adhesion molecules regulate synapse development and function. However, whether and how presynaptic adhesion molecules regulate postsynaptic NMDAR function (see Drosophila Nmdar1 and Nmdar2) remains largely unclear. Presynaptic LAR family receptor tyrosine phosphatases (LAR-RPTPs; see Drosophila Lar) regulate synapse development through mechanisms that include trans-synaptic adhesion; however, whether they regulate postsynaptic receptor functions remains unknown. This study reports that presynaptic PTPsigma, a LAR-RPTP, enhances postsynaptic NMDA receptor (NMDAR) currents and NMDAR-dependent synaptic plasticity in the hippocampus. This regulation does not involve trans-synaptic adhesions of PTPsigma, suggesting that the cytoplasmic domains of PTPsigma, known to have tyrosine phosphatase activity and mediate protein-protein interactions, are important. In line with this, phosphotyrosine levels of presynaptic proteins, including neurexin-1 (see Drosophila Neurexin), are strongly increased in PTPsigma-mutant mice. Behaviorally, PTPsigma-dependent NMDAR regulation is important for social and reward-related novelty recognition. These results suggest that presynaptic PTPsigma regulates postsynaptic NMDAR function through trans-synaptic and direct adhesion-independent mechanisms and novelty recognition in social and reward contexts.
Pechstein, A., Tomilin, N., Fredrich, K., Vorontsova, O., Sopova, E., Evergren, E., Haucke, V., Brodin, L. and Shupliakov, O. (2020). Vesicle Clustering in a Living Synapse Depends on a Synapsin Region that Mediates Phase Separation. Cell Rep 30(8): 2594-2602. PubMed ID: 32101738
Liquid-liquid phase separation is an increasingly recognized mechanism for compartmentalization in cells. Recent in vitro studies suggest that this organizational principle may apply to synaptic vesicle clusters. This study tested this possibility by performing microinjections at the living lamprey giant reticulospinal synapse. Axons are maintained at rest to examine whether reagents introduced into the cytosol enter a putative liquid phase to disrupt critical protein-protein interactions. Compounds that perturb the intrinsically disordered region of synapsin (see Drosophila Synapsin), which is critical for liquid phase organization in vitro, cause dispersion of synaptic vesicles from resting clusters. Reagents that perturb SH3 domain interactions with synapsin are ineffective at rest. These results indicate that synaptic vesicles at a living central synapse are organized as a distinct liquid phase maintained by interactions via the intrinsically disordered region of synapsin.
Khalaj, A. J., Sterky, F. H., Sclip, A., Schwenk, J., Brunger, A. T., Fakler, B. and Sudhof, T. C. (2020). Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism. J Cell Biol 219(9). PubMed ID: 32706374
Neurexins (see Drosophila Neurexin) are presynaptic adhesion molecules that organize synapses by binding to diverse trans-synaptic ligands, but how neurexins are regulated is incompletely understood. This study identified FAM19A/TAFA proteins, "orphan" cytokines, as neurexin regulators that interact with all neurexins, except for neurexin-1gamma, via an unusual mechanism. Specifically, this study showed that FAM19A1-A4 bind to the cysteine-loop domain of neurexins by forming intermolecular disulfide bonds during transport through the secretory pathway. FAM19A-binding required both the cysteines of the cysteine-loop domain and an adjacent sequence of neurexins. Genetic deletion of neurexins suppressed FAM19A1 expression, demonstrating that FAM19As physiologically interact with neurexins. In hippocampal cultures, expression of exogenous FAM19A1 decreased neurexin O-glycosylation and suppressed its heparan sulfate modification, suggesting that FAM19As regulate the post-translational modification of neurexins. Given the selective expression of FAM19As in specific subtypes of neurons and their activity-dependent regulation, these results suggest that FAM19As serve as cell type-specific regulators of neurexin modifications.

Wednesday, September 23rd - Adult neural development and function

Shohayeb, B., Ho, U., Yeap, Y. Y., Parton, R. G., Millard, S. S., Xu, Z., Piper, M. and Ng, D. C. H. (2020). The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia formation and neocortical development. Hum Mol Genet 29(2): 248-263. PubMed ID: 31816041
WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. This study utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, deficient recruitment was observed of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.
Neame, S., Safory, H., Radzishevsky, I., Touitou, A., Marchesani, F., Marchetti, M., Kellner, S., Berlin, S., Foltyn, V. N., Engelender, S., Billard, J. M. and Wolosker, H. (2019). The NMDA receptor activation by d-serine and glycine is controlled by an astrocytic Phgdh-dependent serine shuttle. Proc Natl Acad Sci U S A 116(41): 20736-20742. PubMed ID: 31548413
Astrocytes express the 3-phosphoglycerate dehydrogenase (Phgdh) enzyme required for the synthesis of l-serine from glucose. Astrocytic l-serine was proposed to regulate NMDAR activity by shuttling to neurons to sustain d-serine production. This study report that inhibition of astrocytic Phgdh suppressed the de novo synthesis of l-and d-serine and reduced the NMDAR synaptic potentials and long-term potentiation (LTP) at the Schaffer collaterals-CA1 synapse. Likewise, enzymatic removal of extracellular l-serine impaired LTP, supporting an l-serine shuttle mechanism between glia and neurons in generating the NMDAR coagonist d-serine. Moreover, deletion of serine racemase (SR) in glutamatergic neurons abrogated d-serine synthesis to the same extent as Phgdh inhibition, suggesting that neurons are the predominant source of the newly synthesized d-serine. This study also found that the synaptic NMDAR activation in adult SR-knockout (KO) mice requires Phgdh-derived glycine. Unexpectedly, it was also discovered that glycine regulates d-serine metabolism by a dual mechanism. The first consists of tonic inhibition of SR by intracellular glycine observed in vitro, primary cultures, and in vivo microdialysis. The second involves a transient glycine-induce d-serine release through the Asc-1 transporter, an effect abolished in Asc-1 KO mice and diminished by deleting SR in glutamatergic neurons. These observations suggest that glycine is a multifaceted regulator of d-serine metabolism and implicate both d-serine and glycine in mediating NMDAR synaptic activation at the mature hippocampus through a Phgdh-dependent shuttle mechanism.
Moreno-Bravo, J. A., Roig Puiggros, S., Mehlen, P. and Chedotal, A. (2019). Synergistic Activity of Floor-Plate- and Ventricular-Zone-Derived Netrin-1 in Spinal Cord Commissural Axon Guidance. Neuron 101(4): 625-634. PubMed ID: 30661739
In vertebrates, commissural axons extend ventrally toward the floor plate in the spinal cord and hindbrain. Netrin-1 (see Drosophila Netrins), secreted by floor plate cells, was proposed to attract commissural axons at a distance. However, recent genetic studies in mice have shown that netrin-1 is also produced by ventricular zone (VZ) progenitors and that in the hindbrain, it represents the main source of netrin-1 for commissural axons. This study shows that genetically deleting netrin-1 either from the VZ or the floor plate does not prevent midline crossing in the spinal cord, although axon pathfinding and fasciculation are perturbed. Strikingly, the VZ and floor plate act synergistically, as the simultaneous ablation of netrin-1 from these two sources severely impedes crossing. These results suggest that floor-plate-derived netrin-1 has a distinct impact on commissural axons in the spinal cord and hindbrain.
Lim, N. R., Shohayeb, B., Zaytseva, O., Mitchell, N., Millard, S. S., Ng, D. C. H. and Quinn, L. M. (2017). Glial-specific functions of microcephaly protein WDR62 and interaction with the mitotic kinase AURKA are essential for Drosophila brain growth. Stem Cell Reports. PubMed ID: 28625535
The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribut. This study used Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. It was further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA. Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly.
Gruner, H. N., Kim, M. and Mastick, G. S. (2019). Robo1 and 2 Repellent Receptors Cooperate to Guide Facial Neuron Cell Migration and Axon Projections in the Embryonic Mouse Hindbrain. Neuroscience 402: 116-129. PubMed ID: 30685539
The facial nerve is necessary for our ability to eat, speak, and make facial expressions. Both the axons and cell bodies of the facial nerve undergo a complex embryonic developmental pattern involving migration of the cell bodies caudally and tangentially through rhombomeres, and simultaneously the axons projecting to exit the hindbrain to form the facial nerve. The goal of this study was to test the functions of the chemorepulsive receptors Robo1 and Robo2 (see Drosophila Robo) in facial neuron migration and axon projection by analyzing genetically marked motor neurons in double-mutant mouse embryos through the migration time course, E10.0-E13.5. In Robo1/2 double mutants, axon projection and cell body migration errors were more severe than in single mutants. Most axons did not make it to their motor exit point, and instead projected into and longitudinally within the floor plate. Surprisingly, some facial neurons had multiple axons exiting and projecting into the floor plate. At the same time, a subset of mutant facial cell bodies failed to migrate caudally, and instead either streamed dorsally toward the exit point or shifted into the floor plate. It is concluded that Robo1 and Robo2 have redundant functions to guide multiple aspects of the complex cell migration of the facial nucleus, as well as regulating axon trajectories and suppressing formation of ectopic axons.
Hayashi, C., Suzuki, N., Takahashi, R. and Akazawa, C. (2020). Development of type I/II oligodendrocytes regulated by teneurin-4 in the murine spinal cord. Sci Rep 10(1): 8611. PubMed ID: 32451386
In the spinal cord, the axonal tracts with various caliber sizes are myelinated by oligodendrocytes and function as high-velocity ways for motor and sensory nerve signals. In some neurological disorders, such as multiple sclerosis, demyelination of small caliber axons is observed in the spinal cord. While type I/II oligodendrocytes among the four types are known to myelinate small diameter axons, their characteristics including identification of regulating molecules have not been understood yet. This study found that in the wild-type mouse spinal cord, type I/II oligodendrocytes, positive for carbonic anhydrase II (CAII), were located in the corticospinal tract, fasciculus gracilis, and the inside part of ventral funiculus, in which small diameter axons existed. The type I/II oligodendrocytes started to appear between postnatal day (P) 7 and 11. The type I/II oligodendrocytes were further examined in the mutant mice, whose small diameter axons were hypomyelinated due to the deficiency of teneurin-4 (see Drosophila Tenascin major). In the teneurin-4 deficient mice, type I/II oligodendrocytes were significantly reduced, and the onset of the defect was at P11. These results suggest that CAII-positive type I/II oligodendrocytes myelinate small caliber axons in the spinal cord and teneurin-4 is the responsible molecule for the generation of type I/II oligodendrocytes.

Tuesday, September 23 - Synapse

Li, J., Xie, Y., Cornelius, S., Jiang, X., Sando, R., Kordon, S. P., Pan, M., Leon, K., Sudhof, T. C., Zhao, M. and Arac, D. (2020). Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism. Nat Commun 11(1): 2140. PubMed ID: 32358586
The trans-synaptic interaction of the cell-adhesion molecules teneurins (TENs; see Drosophila Ten-m) with latrophilins (LPHNs/ADGRLs; see Drosophila Cirl) promotes excitatory synapse formation when LPHNs simultaneously interact with FLRTs. Insertion of a short alternatively-spliced region within TENs abolishes the TEN-LPHN interaction and switches TEN function to specify inhibitory synapses. How alternative-splicing regulates TEN-LPHN interaction remains unclear. This study reports the 2.9 Å resolution cryo-EM structure of the TEN2-LPHN3 complex and describes the trimeric TEN2-LPHN3-FLRT3 complex. The structure reveals that the N-terminal lectin domain of LPHN3 binds to the TEN2 barrel at a site far away from the alternatively spliced region. Alternative-splicing regulates the TEN2-LPHN3 interaction by hindering access to the LPHN-binding surface rather than altering it. Strikingly, mutagenesis of the LPHN-binding surface of TEN2 abolishes the LPHN3 interaction and impairs excitatory but not inhibitory synapse formation. These results suggest that a multi-level coincident binding mechanism mediated by a cryptic adhesion complex between TENs and LPHNs regulates synapse specificity.
Ushkaryov, Y. A., Lelianova, V. and Vysokov, N. V. (2019). Catching Latrophilin With Lasso: A Universal Mechanism for Axonal Attraction and Synapse Formation. Front Neurosci 13: 257. PubMed ID: 30967757
Latrophilin-1 (LPHN1; see Drosophila Cirl) was isolated as the main high-affinity receptor for alpha-latrotoxin from black widow spider venom, a powerful presynaptic secretagogue. As an adhesion G-protein-coupled receptor, LPHN1 is cleaved into two fragments, which can behave independently on the cell surface, but re-associate upon binding the toxin. This triggers intracellular signaling that involves the Galphaq/phospholipase C/inositol 1,4,5-trisphosphate cascade and an increase in cytosolic Ca(2+), leading to vesicular exocytosis. This study isolated its endogenous ligand, teneurin-2/Lasso (see Drosophila Ten-m). Both LPHN1 and Ten2/Lasso are expressed early in development and are enriched in neurons. LPHN1 primarily resides in axons, growth cones and presynaptic terminals, while Lasso largely localizes on dendrites. LPHN1 and Ten2/Lasso form a trans-synaptic receptor pair that has both structural and signaling functions. However, Lasso is proteolytically cleaved at multiple sites and its extracellular domain is partially released into the intercellular space, especially during neuronal development, suggesting that soluble Lasso has additional functions. This study discovered that the soluble fragment of Lasso can diffuse away and bind to LPHN1 on axonal growth cones, triggering its redistribution on the cell surface and intracellular signaling which leads to local exocytosis. This causes axons to turn in the direction of spatio-temporal Lasso gradients, while LPHN1 knockout blocks this effect. These results suggest that the LPHN1-Ten2/Lasso pair can participate in long- and short-distance axonal guidance and synapse formation.
Toledo, A., Lang, F., Doengi, M., Morrison, H., Stein, V. and Baader, S. L. (2019). Merlin modulates process outgrowth and synaptogenesis in the cerebellum. Brain Struct Funct 224(6): 2121-2142. PubMed ID: 31165301
Neurofibromatosis type 2 (NF2) patients are prone to develop glial-derived tumors in the peripheral and central nervous system (CNS). The Nf2 gene product Merlin (see Drosophila Merlin) is not only expressed in glia, but also in neurons of the CNS, where its function still remains elusive. This study shows that cerebellar Purkinje cells (PCs) of isoform-specific Merlin-deficient mice were innervated by smaller vGluT2-positive clusters (see Drosophila Vesicular glutamate transporter) at presynaptic terminals than those of wild-type mice. This was paralleled by a reduction in frequency and amplitude of miniature excitatory postsynaptic currents (mEPSC). On the contrary, in conditional transgenic mice in which Merlin expression was specifically ablated in PCs (L7Cre;Nf2(fl/fl)), enlarged vGluT2-positive clusters were found in their presynaptic buttons together with increased amplitudes of miniature postsynaptic currents. The presynaptic terminals of these PCs innervating neurons of the deep cerebellar nuclei were also enlarged. When exploring mice with Merlin-deficient granule cells (GCs) (Math1Cre;Nf2(fl/fl)), this study found cerebellar extracts to contain higher amounts of vGluT1 present in parallel fiber terminals. In parallel, mEPSC frequency was increased in Math1Cre;Nf2(fl/fl) mice. On the contrary, VGluT2 clusters in cerebellar glomeruli composed of NF2-deficient presynaptic Mossy fiber terminals and NF2-deficient postsynaptic GC were reduced in size as shown for isoform-specific knockout mice. These changes in Math1Cre;Nf2(fl/fl)-deficient mice were paralleled by an increased activation of Rac1-Cofilin signaling which is known to impact on cytoskeletal reorganization and synapse formation. Consistent with the observed synaptic alterations in these transgenic mice, this study observed altered ultrasonic vocalization, which is known to rely on proper cerebellar function. No gross morphological changes or motor coordination deficits were observed in any of these transgenic mice. It is therefore concluded that Merlin does not regulate overall cerebellar development, but impacts on pre- and post-synaptic terminal organization.
Bodner, O., Radzishevsky, I., Foltyn, V. N., Touitou, A., Valenta, A. C., Rangel, I. F., Panizzutti, R., Kennedy, R. T., Billard, J. M. and Wolosker, H. (2020). D-Serine Signaling and NMDAR-Mediated Synaptic Plasticity Are Regulated by System A-Type of Glutamine/D-Serine Dual Transporters. J Neurosci 40(34): 6489-6502. PubMed ID: 32661027
D-serine is a physiologic coagonist of NMDA receptors (NMDARs; see Drosophila Nmdar1 and Nmdar2) required for synaptic plasticity, but mechanisms that terminate D-serine signaling are unclear. In particular, the identity of unidirectional plasma membrane transporters that mediate D-serine reuptake has remained elusive. This study reports that D-serine and glutamine share the same neuronal transport system, consisting of the classic system A transporters Slc38a1 and Slc38a2 (see Drosophila CG32081). This study shows that these transporters are not saturated with glutamine in vivo and regulate the extracellular levels of D-serine and NMDAR activity. Glutamine increased the NMDAR-dependent long-term potentiation and the isolated NMDAR potentials at the Schaffer collateral-CA1 synapses, but without affecting basal neurotransmission in male mice. Glutamine did not increase the NMDAR potentials in slices from serine racemase knock-out mice, which are devoid of D-serine, indicating that the effect of glutamine is caused by outcompeting D-serine for a dual glutamine-D-serine transport system. Inhibition of the system A reduced the uptake of D-serine in synaptosomes and neuronal cultures of mice of either sex, while increasing the extracellular D-serine concentration in slices and in vivo by microdialysis. When compared with Slc38a2, the Slc38a1 transporter displayed more favorable kinetics toward the D-enantiomer. Biochemical experiments with synaptosomes from Slc38a1 knock-down mice of either sex further support its role as a D-serine reuptake system. This study identifies the first concentrative and electrogenic transporters mediating D-serine reuptake in vivo. In addition to their classical role in the glutamine-glutamate cycle, system A transporters regulate the synaptic turnover of D-serine and its effects on NMDAR synaptic plasticity.
Risher, W. C., Kim, N., Koh, S., Choi, J. E., Mitev, P., Spence, E. F., Pilaz, L. J., Wang, D., Feng, G., Silver, D. L., Soderling, S. H., Yin, H. H. and Eroglu, C. (2018). Thrombospondin receptor alpha2delta-1 promotes synaptogenesis and spinogenesis via postsynaptic Rac1. J Cell Biol 217(10): 3747-3765. PubMed ID: 30054448
Astrocytes control excitatory synaptogenesis by secreting thrombospondins (TSPs), which function via their neuronal receptor, the calcium channel subunit alpha2delta-1 (see Drosophila straightjacket). alpha2delta-1 is a drug target for epilepsy and neuropathic pain; thus the TSP-alpha2delta-1 interaction is implicated in both synaptic development and disease pathogenesis. However, the mechanism by which this interaction promotes synaptogenesis and the requirement for alpha2delta-1 for connectivity of the developing mammalian brain are unknown. This study shows that global or cell-specific loss of alpha2delta-1 yields profound deficits in excitatory synapse numbers, ultrastructure, and activity and severely stunts spinogenesis in the mouse cortex. Postsynaptic but not presynaptic alpha2delta-1 is required and sufficient for TSP-induced synaptogenesis in vitro and spine formation in vivo, but an alpha2delta-1 mutant linked to autism cannot rescue these synaptogenesis defects. Finally, this study reveals that TSP-alpha2delta-1 interactions control synaptogenesis postsynaptically via Rac1, suggesting potential molecular mechanisms that underlie both synaptic development and pathology.
Zhou, X., Gueydan, M., Jospin, M., Ji, T., Valfort, A., Pinan-Lucarre, B. and Bessereau, J. L. (2020). The netrin receptor UNC-40/DCC assembles a postsynaptic scaffold and sets the synaptic content of GABAA receptors. Nat Commun 11(1): 2674. PubMed ID: 32471987
Increasing evidence indicates that guidance molecules used during development for cellular and axonal navigation also play roles in synapse maturation and homeostasis. In C. elegans the netrin receptor UNC-40/DCC (see Drosophila Frazzled) controls the growth of dendritic-like muscle cell extensions towards motoneurons and is required to recruit type A GABA receptors (GABAARs; see Drosophila Rdl) at inhibitory neuromuscular junctions. This study show that activation of UNC-40 assembles an intracellular synaptic scaffold by physically interacting with FRM-3, a FERM protein orthologous to FARP1/2. FRM-3 then recruits LIN-2, the ortholog of CASK (see Drosophila Cask), that binds the synaptic adhesion molecule NLG-1/Neuroligin (see Drosophila Neuroligin) and physically connects GABAARs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4 (a member of the ADAMTS family of proteases), which controls the localization of GABAARs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABAARs through the UNC-40-dependent intracellular scaffold. Since DCC is detected at GABA synapses in mammals, DCC might also tune inhibitory neurotransmission in the mammalian brain.

Monday, October 22nd - Signaling

Chowdhury, M. I. H., Nishioka, T., Mishima, N., Ohtsuka, T., Kaibuchi, K. and Tsuboi, D. (2020). Prickle2 and Igsf9b Coordinately Regulate the Cytoarchitecture of the Axon Initial Segment. Cell Struct Funct 45(2): 143-154. PubMed ID: 32641624
Prickle2 (see Drosophila Prickle) has been identified in genetic studies of subjects with autism spectrum disorder (ASD) and epilepsy, but the pathological mechanism of Prickle2 remains to be fully understood. Proteomic analysis of Prickle2 with mass spectrometry revealed twenty-eight Prickle2 interactors, including immunoglobulin superfamily member 9b (Igsf9b), in the brain. In this study, because Igsf9 family proteins are associated with psychiatric diseases and seizures, the physiological interaction between Prickle2 and Igsf9b was studied. Prickle2 colocalized with Igsf9b in cultured hippocampal neurons. Knockdown of Prickle2 affected the subcellular localization of Igsf9b. Interestingly, Igsf9b localized along axonal processes in a pattern opposite to the ASD-related molecule ANK3/AnkG. AnkG is a major component of the axon initial segment (AIS), where a variety of ASD and epilepsy susceptibility proteins accumulate. Igsf9b-knockdown neurons displayed altered AnkG localization. Prickle2 depletion caused defects in AnkG and voltage-gated Na+ channel localization, resulting in altered network activity. These results support the idea that Prickle2 regulates AnkG distribution by controlling the proper localization of Igsf9b. The novel function of Prickle2 in AIS cytoarchitecture provides new insights into the shared pathology of ASD and epilepsy (Chowdhury, 2020).
Sturm, S., Dowle, A., Audsley, N. and Isaac, R. E. (2020). The structure of the Drosophila melanogaster sex peptide: Identification of hydroxylated isoleucine and a strain variation in the pattern of amino acid hydroxylation. Insect Biochem Mol Biol 124: 103414. PubMed ID: 32589920
In Drosophila melanogaster mating triggers profound changes in the behaviour and reproductive physiology of the female. Many of these post-mating effects are elicited by sex peptide (SP), a 36-mer pheromone made in the male accessory gland and passed to the female in the seminal fluid. The peptide comprises several structurally and functionally distinct domains, one of which consists of five 4-hydroxyprolines and induces a female immune response. The SP gene predicts an isoleucine (Ile(14)) sandwiched between two of the hydroxyprolines of the mature secreted peptide. This study used matrix-assisted laser desorption ionisation mass spectrometry together with Fourier-transform ion cyclotron resonance mass spectrometry to show that Ile(14) is modified by oxidation of the side chain - a very unusual post-translational modification. Mass spectrometric analysis of glands from different geographical populations of male D. melanogaster show that SP with six hydroxylated side chains is the most common form of the peptide, but that a sub-strain of Canton-S flies held at Leeds only has two or three hydroxylated prolines and an unmodified Ile(14). The D. melanogaster genome has remarkably 17 putative hydroxylase genes that are strongly and almost exclusively expressed in the male accessory gland, suggesting that the gland is a powerhouse of protein oxidation. Strain variation in the pattern of sex peptide hydroxylation might be explained by differences in the expression of individual hydroxylase genes (Sturm, 2020).
Chowdhury, M. I. H., Nishioka, T., Mishima, N., Ohtsuka, T., Kaibuchi, K. and Tsuboi, D. (2020). Prickle2 and Igsf9b Coordinately Regulate the Cytoarchitecture of the Axon Initial Segment. Cell Struct Funct 45(2): 143-154. PubMed ID: 32641624
Prickle2 (see Drosophila Prickle) has been identified in genetic studies of subjects with autism spectrum disorder (ASD) and epilepsy, but the pathological mechanism of Prickle2 remains to be fully understood. Proteomic analysis of Prickle2 with mass spectrometry revealed twenty-eight Prickle2 interactors, including immunoglobulin superfamily member 9b (Igsf9b), in the brain. In this study, because Igsf9 family proteins are associated with psychiatric diseases and seizures, the physiological interaction between Prickle2 and Igsf9b was studied. Prickle2 colocalized with Igsf9b in cultured hippocampal neurons. Knockdown of Prickle2 affected the subcellular localization of Igsf9b. Interestingly, Igsf9b localized along axonal processes in a pattern opposite to the ASD-related molecule ANK3/AnkG. AnkG is a major component of the axon initial segment (AIS), where a variety of ASD and epilepsy susceptibility proteins accumulate. Igsf9b-knockdown neurons displayed altered AnkG localization. Prickle2 depletion caused defects in AnkG and voltage-gated Na+ channel localization, resulting in altered network activity. These results support the idea that Prickle2 regulates AnkG distribution by controlling the proper localization of Igsf9b. The novel function of Prickle2 in AIS cytoarchitecture provides new insights into the shared pathology of ASD and epilepsy.
Araki, Y., Hong, I., Gamache, T. R., Ju, S., Collado-Torres, L., Shin, J. H. and Huganir, R. L. (2020). SynGAP isoforms differentially regulate synaptic plasticity and dendritic development. Elife 9. PubMed ID: 32579114
SynGAP, ortholog of Drosophila raskol is a synaptic Ras GTPase-activating protein (GAP) with four C-terminal splice variants: alpha1, alpha2, beta, and gamma. Although studies have implicated SYNGAP1 in several cognitive disorders, it is not clear which SynGAP isoforms contribute to disease. This study demonstrates that SynGAP isoforms exhibit unique spatiotemporal expression patterns and play distinct roles in neuronal and synaptic development in mouse neurons. SynGAP-alpha1, which undergoes liquid-liquid phase separation with PSD-95 (see Drosophila Disks large), is highly enriched in synapses and is required for LTP. In contrast, SynGAP-beta, which does not bind PSD-95 PDZ domains, is less synaptically targeted and promotes dendritic arborization. A mutation in SynGAP-alpha1 that disrupts phase separation and synaptic targeting abolishes its ability to regulate plasticity and instead causes it to drive dendritic development like SynGAP-beta. These results demonstrate that distinct intrinsic biochemical properties of SynGAP isoforms determine their function, and individual isoforms may differentially contribute to the pathogenesis of SYNGAP1-related cognitive disorders (Araki, 2020).
Davis, T. B., Yang, M., Schell, M. J., Wang, H., Ma, L., Pledger, W. J. and Yeatman, T. J. (2018). PTPRS Regulates Colorectal Cancer RAS Pathway Activity by Inactivating Erk and Preventing Its Nuclear Translocation. Sci Rep 8(1): 9296. PubMed ID: 29915291
Colorectal cancer (CRC) growth and progression is frequently driven by RAS pathway activation through upstream growth factor receptor activation or through mutational activation of KRAS or BRAF. This study describes an additional mechanism by which the RAS pathway may be modulated in CRC. PTPRS (see Drosophila Lar), a receptor-type protein tyrosine phosphatase, appears to regulate RAS pathway activation through ERK (see Drosophila Rolled). PTPRS modulates ERK phosphorylation and subsequent translocation to the nucleus. Native mutations in PTPRS, present in ~10% of CRC, may reduce its phosphatase activity while increasing ERK activation and downstream transcriptional signaling.
Ariss, M. M., Terry, A. R., Islam, A., Hay, N. and Frolov, M. V. (2020). Amalgam regulates the receptor tyrosine kinase pathway through Sprouty in glial cell development. J Cell Sci. PubMed ID: 32878945
The receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. This study profiles the Drosophila larval brain by single cell RNA-sequencing and identified Amalgam (Ama), encoding a cell adhesion protein of the immunoglobulin IgLON family, that regulates the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood-brain barrier (BBB) that leads to hemocyte infiltration and neuronal death. Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPOUTY2 in glioblastoma cell lines suggesting that the relationship between Ama and Sty is conserved.

Friday, September 18th - Synapse and Vesicles

Vickers, E., Osypenko, D., Clark, C., Okur, Z., Scheiffele, P. and Schneggenburger, R. (2020). LTP of inhibition at PV interneuron output synapses requires developmental BMP signaling. Sci Rep 10(1): 10047. PubMed ID: 32572071
Parvalbumin (PV)-expressing interneurons (PV-INs) mediate well-timed inhibition of cortical principal neurons, and plasticity of these interneurons is involved in map remodeling of primary sensory cortices during critical periods of development. To assess whether bone morphogenetic protein (BMP) signaling contributes to the developmental acquisition of the synapse- and plasticity properties of PV-INs, this study investigated conditional/conventional double KO mice of BMP-receptor 1a (BMPR1a; targeted to PV-INs) and 1b (BMPR1a/1b (c)DKO mice) (see Drosophila Thickveins). Spike-timing dependent LTP at the synapse between PV-INs and principal neurons of layer 4 in the auditory cortex was absent, concomitant with a decreased paired-pulse ratio (PPR). On the other hand, baseline synaptic transmission at this connection, and action potential (AP) firing rates of PV-INs were unchanged. To explore possible gene expression targets of BMP signaling, the mRNA levels of the BDNF receptor TrkB and of P/Q-type Ca(2+) channel alpha-subunits were measured, but noexpression changes of the corresponding genes were found in PV-INs of BMPR1a/1b (c)DKO mice. This study suggests that BMP-signaling in PV-INs during and shortly after the critical period is necessary for the expression of LTP at PV-IN output synapses, involving gene expression programs that need to be addressed in future work.
Bhouri, M., Morishita, W., Temkin, P., Goswami, D., Kawabe, H., Brose, N., Sudhof, T. C., Craig, A. M., Siddiqui, T. J. and Malenka, R. (2018). Deletion of LRRTM1 and LRRTM2 in adult mice impairs basal AMPA receptor transmission and LTP in hippocampal CA1 pyramidal neurons. Proc Natl Acad Sci U S A 115(23): E5382-E5389. PubMed ID: 29784826
Leucine-rich repeat transmembrane (LRRTM; see Drosophila Toll-6 & Toll-7 and Capricious) proteins are synaptic cell adhesion molecules that influence synapse formation and function. This study took advantage of the generation of a LRRTM1 and LRRTM2 double conditional knockout mouse (LRRTM1,2 cKO) to examine the role of LRRTM1,2 at mature excitatory synapses in hippocampal CA1 pyramidal neurons. Genetic deletion of LRRTM1,2 in vivo in CA1 neurons dramatically impaired long-term potentiation (LTP), an impairment that was rescued by simultaneous expression of LRRTM2, but not LRRTM4. Mutation or deletion of the intracellular tail of LRRTM2 did not affect its ability to rescue LTP, while point mutations designed to impair its binding to presynaptic neurexins (see Drosophila Neurexin) prevented rescue of LTP. These proteins at mature synapses also caused a decrease in AMPA (see Drosophila GluR1A) receptor-mediated, but not NMDA receptor-mediated, synaptic transmission and had no detectable effect on presynaptic function. Imaging of recombinant photoactivatable AMPA receptor subunit GluA1 in the dendritic spines of cultured neurons revealed that it was less stable in the absence of LRRTM1,2. These results illustrate the advantages of conditional genetic deletion experiments for elucidating the function of endogenous synaptic proteins and suggest that LRRTM1,2 proteins help stabilize synaptic AMPA receptors at mature spines during basal synaptic transmission and LTP.
Roppongi, R. T., Dhume, S. H., Padmanabhan, N., Silwal, P., Zahra, N., Karimi, B., Bomkamp, C., Patil, C. S., Champagne-Jorgensen, K., Twilley, R. E., Zhang, P., Jackson, M. F. and Siddiqui, T. J. (2020). LRRTMs Organize Synapses through Differential Engagement of Neurexin and PTPsigma. Neuron 106(1): 108-125 e112. PubMed ID: 31995730
Presynaptic neurexins (Nrxs) and type IIa receptor-type protein tyrosine phosphatases (RPTPs) organize synapses through a network of postsynaptic ligands. This study shows that leucine-rich-repeat transmembrane neuronal proteins (LRRTMs; see Drosophila Capricious) differentially engage the protein domains of Nrx (see Drosophila (see Drosophila Neurexin)) but require its heparan sulfate (HS) modification to induce presynaptic differentiation. Binding to the HS of Nrx is sufficient for LRRTM3 and LRRTM4 to induce synaptogenesis. Mammalian Nrx1gamma was identified as a potent synapse organizer, and LRRTM4 is revealed as its postsynaptic ligand. Mice expressing a mutant form of LRRTM4 that cannot bind to HS show structural and functional deficits at dentate gyrus excitatory synapses. Through the HS of Nrx, LRRTMs also recruit PTPsigma to induce presynaptic differentiation but function to varying degrees in its absence. PTPsigma forms a robust complex with Nrx, revealing an unexpected interaction between the two presynaptic hubs. These findings underscore the complex interplay of synapse organizers in specifying the molecular logic of a neural circuit.
Zhang, P., Lu, H., Peixoto, R. T., Pines, M. K., Ge, Y., Oku, S., Siddiqui, T. J., Xie, Y., Wu, W., Archer-Hartmann, S., Yoshida, K., Tanaka, K. F., Aricescu, A. R., Azadi, P., Gordon, M. D., Sabatini, B. L., Wong, R. O. L. and Craig, A. M. (2018). Heparan Sulfate Organizes Neuronal Synapses through Neurexin Partnerships. Cell 174(6): 1450-1464 e1423. PubMed ID: 30100184
Synapses are fundamental units of communication in the brain. The prototypical synapse-organizing complex neurexin-neuroligin mediates synapse development and function and is central to a shared genetic risk pathway in autism and schizophrenia. Neurexin's role in synapse development is thought to be mediated purely by its protein domains, but this study reveals a requirement for a rare glycan modification. Mice lacking heparan sulfate (HS) on neurexin-1 show reduced survival, as well as structural and functional deficits at central synapses. HS directly binds postsynaptic partners neuroligins and LRRTMs, revealing a dual binding mode involving intrinsic glycan and protein domains for canonical synapse-organizing complexes. Neurexin HS chains also bind novel ligands, potentially expanding the neurexin interactome to hundreds of HS-binding proteins. Because HS structure is heterogeneous, these findings indicate an additional dimension to neurexin diversity, provide a molecular basis for fine-tuning synaptic function, and open therapeutic directions targeting glycan-binding motifs critical for brain development.
Yamagata, A., Goto-Ito, S., Sato, Y., Shiroshima, T., Maeda, A., Watanabe, M., Saitoh, T., Maenaka, K., Terada, T., Yoshida, T., Uemura, T. and Fukai, S. (2018). Structural insights into modulation and selectivity of transsynaptic neurexin-LRRTM interaction. Nat Commun 9(1): 3964. PubMed ID: 30262834
Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) function as postsynaptic organizers that induce excitatory synapses. Neurexins (Nrxns) and heparan sulfate proteoglycans have been identified as presynaptic ligands for LRRTMs. Specifically, LRRTM1 and LRRTM2 bind to the Nrxn splice variant lacking an insert at the splice site 4 (S4). This study reports the crystal structure of the Nrxn1beta-LRRTM2 complex at 3.4 A resolution. The Nrxn1beta-LRRTM2 interface involves Ca(2+)-mediated interactions and overlaps with the Nrxn-neuroligin interface. Together with structure-based mutational analyses at the molecular and cellular levels, the present structural analysis unveils the mechanism of selective binding between Nrxn and LRRTM1/2 and its modulation by the S4 insertion of Nrxn.
Scott, J., Thakar, S., Mao, Y., Qin, H., Hejran, H., Lee, S. Y., Yu, T., Klezovitch, O., Cheng, H., Mu, Y., Ghosh, S., Vasioukhin, V. and Zou, Y. (2019). Apical-Basal Polarity Signaling Components, Lgl1 and aPKCs, Control Glutamatergic Synapse Number and Function. iScience 20: 25-41. PubMed ID: 31546104
Normal synapse formation is fundamental to brain function. An apical-basal polarity (A-BP) protein, Lgl1 (see Drosophila Lgl), is present in the postsynaptic density and negatively regulates glutamatergic synapse numbers by antagonizing the atypical protein kinase Cs (aPKCs). A planar cell polarity protein, Vangl2 (see Drosophila Vang), which inhibits synapse formation, was decreased in synaptosome fractions of cultured cortical neurons from Lgl1 knockout embryos. Conditional knockout of Lgl1 in pyramidal neurons led to reduction of AMPA/NMDA ratio and impaired plasticity. Lgl1 is frequently deleted in Smith-Magenis syndrome (SMS). Lgl1 conditional knockout led to increased locomotion, impaired novel object recognition and social interaction. Lgl1+/- animals also showed increased synapse numbers, defects in open field and social interaction, as well as stereotyped repetitive behavior. Social interaction in Lgl1+/- could be rescued by NMDA antagonists. These findings reveal a role of apical-basal polarity proteins in glutamatergic synapse development and function and also suggest a potential treatment for SMS patients with Lgl1 deletion.

Thursday September 17th - Neural development

Hing, H., Reger, N., Snyder, J. and Fradkin, L. G. (2020). Interplay between axonal Wnt5-Vang and dendritic Wnt5-Drl/Ryk signaling controls glomerular patterning in the Drosophila antennal lobe. PLoS Genet 16(5): e1008767. PubMed ID: 32357156
Despite the importance of dendritic targeting in neural circuit assembly, the mechanisms by which it is controlled still remain incompletely understood. Previously work showed that in the developing Drosophila antennal lobe, the Wnt5 protein forms a gradient that directs the ~45° rotation of a cluster of projection neuron (PN) dendrites, including the adjacent DA1 and VA1d dendrites. The Van Gogh (Vang) transmembrane planar cell polarity (PCP) protein is required for the rotation of the DA1/VA1d dendritic pair. Cell type-specific rescue and mosaic analyses showed that Vang functions in the olfactory receptor neurons (ORNs), suggesting a codependence of ORN axonal and PN dendritic targeting. Loss of Vang suppressed the repulsion of the VA1d dendrites by Wnt5, indicating that Wnt5 signals through Vang to direct the rotation of the DA1 and VA1d glomeruli. The Derailed (Drl)/Ryk atypical receptor tyrosine kinase is also required for the rotation of the DA1/VA1d dendritic pair. Antibody staining showed that Drl/Ryk is much more highly expressed by the DA1 dendrites than the adjacent VA1d dendrites. Mosaic and epistatic analyses showed that Drl/Ryk specifically functions in the DA1 dendrites in which it antagonizes the Wnt5-Vang repulsion and mediates the migration of the DA1 glomerulus towards Wnt5. Thus, the nascent DA1 and VA1d glomeruli appear to exhibit Drl/Ryk-dependent biphasic responses to Wnt5. This work shows that the final patterning of the fly olfactory map is the result of an interplay between ORN axons and PN dendrites, wherein converging pre- and postsynaptic processes contribute key Wnt5 signaling components, allowing Wnt5 to orient the rotation of nascent synapses through a PCP mechanism.
Seroka, A., Yazejian, R. M., Lai, S. L. and Doe, C. Q. (2020). A novel temporal identity window generates alternating Eve(+)/Nkx6(+) motor neuron subtypes in a single progenitor lineage. Neural Dev 15(1): 9. PubMed ID: 32723364
Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. In vertebrates, these mechanisms generate "cardinal classes" of neurons that share a transcription factor identity and common morphology. In Drosophila, two cardinal classes are Even-skipped (Eve)(+) motor neurons projecting to dorsal longitudinal muscles, and Nkx6/HGTX(+) motor neurons projecting to ventral oblique muscles. Cross-repressive interactions prevent stable double-positive motor neurons. The Drosophila neuroblast 7-1 (NB7-1) lineage uses a temporal transcription factor cascade to generate five distinct Eve(+) motor neurons; the origin and development of Nkx6(+) motor neurons remains unclear. Lineage analysis was used to birth-date the VO motor neuron to the Kr(+) Pdm(+) neuroblast temporal identity window. Lineage analysis identifies an Nkx6(+) neuron born from the Kr(+) Pdm(+) temporal identity window in the NB7-1 lineage, resulting in alternation of cardinal motor neuron subtypes within this lineage (Eve>Nkx6 > Eve). Co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage - the first evidence that this TTF combination specifies neuronal identity. Moreover, the Kr/Pdm combination promotes Nkx6 expression, which itself is necessary and sufficient for motor neuron targeting to ventral oblique muscles, thereby revealing a molecular specification pathway from temporal patterning to cardinal transcription factor expression to motor neuron target selection. This study has shown that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm > Nkx6 pathway is necessary and sufficient to promote VO motor neuron targeting to the correct ventral muscle group.
Yoong, L. F., Lim, H. K., Tran, H., Lackner, S., Zheng, Z., Hong, P. and Moore, A. W. (2020). Atypical myosin tunes dendrite arbor subdivision. Neuron. PubMed ID: 32155441
Dendrite arbor pattern determines the functional characteristics of a neuron. It is founded on primary branch structure, defined through cell intrinsic and transcription-factor-encoded mechanisms. Developing arbors have extensive acentrosomal microtubule dynamics, and this study reports an unexpected role for the atypical actin motor Myo6 in creating primary branch structure by specifying the position, polarity, and targeting of these events. In vivo time-lapse imaging was carried out of Drosophila adult sensory neuron differentiation, integrating machine-learning-based quantification of arbor patterning with molecular-level tracking of cytoskeletal remodeling. This revealed that Myo6 and the transcription factor Knot regulate transient surges of microtubule polymerization at dendrite tips; they drive retrograde extension of an actin filament array that specifies anterograde microtubule polymerization and guides these microtubules to subdivide the tip into multiple branches. Primary branches delineate functional compartments; this tunable branching mechanism is key to define and diversify dendrite arbor compartmentalization.
Liu, X., Fang, Z., Wen, J., Tang, F., Liao, B., Jing, N., Lai, D. and Jin, Y. (2020). SOX1 Is Required for the Specification of Rostral Hindbrain Neural Progenitor Cells from Human Embryonic Stem Cells. iScience 23(9): 101475. PubMed ID: 32905879
Region-specific neural progenitor cells (NPCs) can be generated from human embryonic stem cells (hESCs) by modulating signaling pathways. However, how intrinsic transcriptional factors contribute to the neural regionalization is not well characterized. This study generated region-specific NPCs from hESCs and found that SOX1 (see Drosophila Dichaete) is highly expressed in NPCs with the rostral hindbrain identity. Moreover, it was found that OTX2 (see Drosophila Ocelliless) inhibits SOX1 expression, displaying exclusive expression between the two factors. Furthermore, SOX1 knockout (KO) leads to the upregulation of midbrain genes and downregulation of rostral hindbrain genes, indicating that SOX1 is required for specification of rostral hindbrain NPCs. SOX1 chromatin immunoprecipitation sequencing analysis reveals that SOX1 binds to the distal region of GBX2 (see Drosophila unplugged to activate its expression. Overexpression of GBX2 largely abrogates SOX1-KO-induced aberrant gene expression. Taken together, this study uncovers previously unappreciated role of SOX1 in early neural regionalization and provides new information for the precise control of the OTX2/GBX2 interface.
Meng, J. L., Wang, Y., Carrillo, R. A. and Heckscher, E. S. (2020). Temporal transcription factors determine circuit membership by permanently altering motor neuron-to-muscle synaptic partnerships. Elife 9. PubMed ID: 32391795
How circuit wiring is specified is a key question in developmental neurobiology. Previously, using the Drosophila motor system as a model, the classic temporal transcription factor Hunchback was found to act in NB7-1 neuronal stem cells to control the number of NB7-1 neuronal progeny form functional synapses on dorsal muscles (Meng, 2019). However, it is unknown to what extent control of motor neuron-to-muscle synaptic partnerships is a general feature of temporal transcription factors. Additional temporal transcription factor manipulations-prolonging expression of Hunchback in NB3-1-were performed, as well as precociously expressing Pdm and Castor in NB7-1. Confocal microscopy, calcium imaging, and electrophysiology were used to show that in every manipulation there are permanent alterations in neuromuscular synaptic partnerships. These data show temporal transcription factors, as a group of molecules, are potent determinants of synaptic partner choice and therefore ultimately control circuit membership.
Poe, A. R., Xu, Y., Zhang, C., Lei, J., Li, K., Labib, D. and Han, C. (2020). Low FoxO expression in Drosophila somatosensory neurons protects dendrite growth under nutrient restriction. Elife 9. PubMed ID: 32427101
During prolonged nutrient restriction, developing animals redistribute vital nutrients to favor brain growth at the expense of other organs. In Drosophila, such brain sparing relies on a glia-derived growth factor to sustain proliferation of neural stem cells. However, whether other aspects of neural development are also spared under nutrient restriction is unknown. This study shows that dynamically growing somatosensory neurons in the Drosophila peripheral nervous system exhibit organ sparing at the level of arbor growth: Under nutrient stress, sensory dendrites preferentially grow as compared to neighboring non-neural tissues, resulting in dendrite overgrowth. These neurons express lower levels of the stress sensor FoxO than neighboring epidermal cells, and hence exhibit no marked induction of autophagy and a milder suppression of Tor signaling under nutrient stress. Preferential dendrite growth allows for heightened animal responses to sensory stimuli, indicative of a potential survival advantage under environmental challenges.

Wednesday, September 16th - Adult development

Nishimura, T. (2020). Feedforward Regulation of Glucose Metabolism by Steroid Hormones Drives a Developmental Transition in Drosophila. Curr Biol. PubMed ID: 32679096
Metabolism must be coupled with developmental transition to fulfill the energy requirements during an organism's life cycle. In most animals, steroid hormones are crucial regulators of life-stage transitions until adulthood. In the fruit fly Drosophila, ecdysteroid titers drive developmental transitions, such as molting and metamorphosis. Although the timings of molting and larval-pupal transition are dependent on environmental and internal conditions, metamorphosis is a temporally controlled life transition event that solely relies on energetic macromolecules accumulated during the larval period. The ecdysteroid signaling cascade and the energetics of metamorphosis have been determined. However, the molecular mechanisms that regulate metabolic homeostasis during metamorphosis remain largely unknown. This study shows that the programmed regulation of carbohydrate metabolism by steroid hormones directs the prepupal-pupal transition in Drosophila. Pupation is associated with a transient increase in glucose oxidation. Mechanistically, after pupariation, ecdysteroid signaling and the competence factor Ftz-F1 regulates the systematic degradation of circulating trehalose via the transient induction of trehalose transporters and trehalase (Treh) in a timely manner. Trehalose metabolism is crucial for energy homeostasis at the prepupal-pupal transition. Moreover, trehalose catabolism acts upstream of ecdysteroid biosynthesis and signaling. These findings lead to the hypothesis that trehalose breakdown not only defines energy costs by providing a carbon source but also facilitates pupation by inducing water loss in the puparium. This work sheds light on the ways in which a life-stage transition is driven by the preprogrammed coordination between steroid hormones and catabolism of stored nutrients.
Nam, S. and Cho, K. O. (2020). Wingless and Archipelago, a fly E3 ubiquitin ligase and a homolog of human tumor suppressor FBW7, show an antagonistic relationship in wing development. BMC Dev Biol 20(1): 14. PubMed ID: 32594913
Archipelago (Ago) is a Drosophila homolog of mammalian F-box and WD repeat domain-containing 7 (FBW7, also known as FBXW7). In previous studies, FBW7 has been addressed as a tumor suppressor mediating ubiquitin-dependent proteolysis of several oncogenic proteins. Ubiquitination is a type of protein modification that directs protein for degradation as well as sorting. The level of beta-catenin (β-cat), an intracellular signal transducer in Wnt signaling pathway, is reduced upon overexpression of FBW7 in human cancer cell lines. Loss of function mutations in FBW7 and overactive Wnt signaling have been reported to be responsible for human cancers. This study found that Ago is important for the formation of shafts in chemosensory bristles at wing margin. This loss of shaft phenotype by knockdown of ago was rescued by knockdown of wingless (wg) whereas wing notching phenotype by knockdown of wg was rescued by knockdown of ago, establishing an antagonistic relationship between ago and wg. In line with this finding, knockdown of ago increased the level of Armadillo (Arm), a homolog of β-cat, in Drosophila tissue. Furthermore, knockdown of ago increased the level of Distal-less (Dll) and extracellular Wg in wing discs. In S2 cells, the amount of secreted Wg was increased by knockdown of Ago but decreased by Ago overexpression. Therefore, Ago plays a previously unidentified role in the inhibition of Wg secretion. Ago-overexpressing clones in wing discs exhibited accumulation of Wg in endoplasmic reticulum (ER), suggesting that Ago prevents Wg protein from moving to Golgi from ER. It is concluded that Ago plays dual roles in inhibiting Wg signaling. First, Ago decreases the level of Arm, by which Wg signaling is downregulated in Wg-responding cells. Second, Ago decreases the level of extracellular Wg by inhibiting movement of Wg from ER to Golgi in Wg-producing cells.
Vanyai, H. K., Garnham, A., May, R. E., McRae, H. M., Collin, C., Wilcox, S., Smyth, G. K., Thomas, T. and Voss, A. K. (2019). MOZ directs the distal-less homeobox gene expression program during craniofacial development. Development 146(14). PubMed ID: 31340933
Oral clefts are common birth defects. Individuals with oral clefts who have identical genetic mutations regularly present with variable penetrance and severity. Epigenetic or chromatin-mediated mechanisms are commonly invoked to explain variable penetrance. However, specific examples of these are rare. Two functional copies of the MOZ (KAT6A, MYST3; see Drosophila Mof) gene, encoding a MYST family lysine acetyltransferase chromatin regulator, are essential for human craniofacial development, but the molecular role of MOZ in this context is unclear. Using genetic interaction and genomic studies, this study investigated the effects of loss of MOZ on the gene expression program during mouse development. Among the more than 500 genes differentially expressed after loss of MOZ, 19 genes had previously been associated with cleft palates. These included four distal-less homeobox (DLX) transcription factor-encoding genes, Dlx1, Dlx2, Dlx3 and Dlx5 and DLX target genes (including Barx1, Gbx2, Osr2 and Sim2). MOZ occupied the Dlx5 locus and was required for normal levels of histone H3 lysine 9 acetylation. MOZ affected Dlx gene expression cell-autonomously within neural crest cells. This study identifies a specific program by which the chromatin modifier MOZ regulates craniofacial development.
Nandy, N. and Roy, J. K. (2020). Rab11 is essential for lgl mediated JNK-Dpp signaling in dorsal closure and epithelial morphogenesis in Drosophila. Dev Biol 464(2): 188-201. PubMed ID: 32562757
Dorsal closure during Drosophila embryogenesis provides a robust genetic platform to study the basic cellular mechanisms that govern epithelial wound healing and morphogenesis. As dorsal closure proceeds, the lateral epithelial tissue (LE) adjacent to the dorsal opening advance contra-laterally, with a simultaneous retraction of the amnioserosa. The process involves a fair degree of coordinated cell shape changes in the dorsal most epithelial (DME) cells as well as a few penultimate rows of lateral epithelial (LE) cells (collectively referred here as Dorsolateral Epithelial (DLE) cells), lining the periphery of the amnioserosa, which in due course of time extend contra-laterally and ultimately fuse over the dorsal hole, giving rise to a dorsal epithelial continuum. The JNK-Dpp signaling in the dorsolateral epidermis, plays an instrumental role in guiding their fate during this process. A large array of genes have been reported to be involved in the regulation of this core signaling pathway, yet the mechanisms by which they do so is hitherto unclear, which forms the objective of this study. This study shows a probable mechanism via which lgl, a conserved tumour suppressor gene, regulates the JNK-Dpp pathway during dorsal closure and epithelial morphogenesis. A conditional/targeted knock-down of lgl in the dorsolateral epithelium of embryos results in failure of dorsal closure. Interestingly, a similar phenotype was observed in a Rab11 knockdown condition. This experiment suggests Rab11 interacts with lgl as they seem to synergize in order to regulate the core JNK-Dpp signaling pathway during dorsal closure and also during adult thorax closure process.
Papakyrikos, A. M., Kim, M. J. and Wang, X. (2020). Drosophila PTPMT1 Has a Function in Tracheal Air Filling. iScience 23(7): 101285. PubMed ID: 32629421
The fly trachea is the equivalent of the mammalian lung and is a useful model for human respiratory diseases. However, little is known about the molecular mechanisms underlying tracheal air filling during larval development. This study discovered that PTPMT1 has a function in tracheal air filling. PTPMT1 is a widely conserved, ubiquitously expressed mitochondrial phosphatase. To reveal PTPMT1's functions in genetically tractable invertebrates and whether those functions are tissue specific, a Drosophila model of PTPMT1 depletion was generated. Fly PTPMT1 mutants showed impairments in tracheal air filling and subsequent activation of innate immune responses. On a cellular level, these defects are preceded by aggregation of mitochondria within the tracheal epithelial cells. This work demonstrates a cell-type-specific role for PTPMT1 in fly tracheal epithelial cells to support air filling and to prevent immune activation. The establishment of this model will facilitate exploration of PTPMT1's physiological functions in vivo.
Ojha, S. and Tapadia, M. G. (2020). Transcriptome profiling identifies multistep regulation through E93, Forkhead and Ecdysone Oxidase in survival of Malpighian tubules during metamorphosis in Drosophila. Int J Dev Biol 64(4-5-6): 341-351. PubMed ID: 32658993
Drosophila metamorphosis is associated with substantial metabolic activity involving cell death and cell proliferation leading to differentiation of adult tissues and structures. Unlike other larval tissues, Malpighian tubules (MTs) exhibit apoptotic immunity and do not undergo cell death but are carried over to the adult with some cell reorganisation. They persist despite the fact that they express apoptotic proteins and caspases. This study analysed the global transcription changes in MTs and compared with salivary glands, to decipher the biology of MTs. Gene set enrichment analysis indicated reduced expression of many ecdysone induced genes, including the critical regulator of cell death, E93 in MTs. It is hypothesized that reduction of E93 could be because of overexpression of ecdysone oxidase, which is high in MTs and is responsible for regulation of hormone titer by degradation of ecdysone. Ectopic expression of E93 in MTs results in cell death through autophagy. Fork head, which is crucial for survival, is enriched in the MT transcriptome, and its down regulation in MTs could be consequent to overexpression of E93. Together these data suggests that the cascade of events initiated by ecdysone mediates survival of MTs through concerted action of multiple factors.

Tuesday, September 15th - Synapse and vesicles

Nath, V. R., Mishra, S., Basak, B., Trivedi, D. and Raghu, P. (2020). Extended synaptotagmin regulates membrane contact site structure and lipid transfer function in vivo. EMBO Rep: e50264. PubMed ID: 32716137
Inter-organelle communication between closely apposed membranes is proposed at membrane contact sites (MCS). However, the regulation of MCS structure and their functional relevance in vivo remain debated. The extended synaptotagmins (Esyt) are evolutionarily conserved proteins proposed to function at MCS. However, loss of all three Esyts in yeast or mammals shows minimal phenotypes questioning the functional importance of Esyt. This study reports that in Drosophila photoreceptors, MCS number is regulated by PLCβ activity. Photoreceptors of a null allele of Drosophila extended synaptotagmin (dEsyt) show loss of ER-PM MCS. Loss of dEsyt results in mislocalization of RDGB, an MCS localized lipid transfer protein, required for photoreceptor structure and function, ultimately leading to retinal degeneration. dEsyt depletion enhanced the retinal degeneration, reduced light responses and slower rates of plasma membrane PIP(2) resynthesis seen in rdgB mutants. Thus, dEsyt function and PLCβ signaling regulate ER-PM MCS structure and lipid transfer in Drosophila photoreceptors.
Nguyen, T. H., Han, T. H., Newfeld, S. J. and Serpe, M. (2020). Selective Disruption of Synaptic BMP Signaling by a Smad Mutation Adjacent to the Highly Conserved H2 Helix. Genetics. PubMed ID: 32737119
Phosphorylated Smad (pMad in Drosophila) accumulates at synaptic junctions in protein complexes with genetically distinct composition and regulation. This study examined Mad alleles to search for molecular features relevant to pMad accumulation at synaptic junctions. Strong Mad alleles generally disrupt both synaptic and nuclear pMad, whereas moderate Mad alleles have a wider range of phenotypes and can selectively impact different BMP signaling pathways. Interestingly, regulatory Mad mutations reveal that synaptic pMad appear to be more sensitive to a net reduction in Mad levels than nuclear pMad. Importantly, a previously uncharacterized allele, Mad8, showed markedly reduced synaptic pMad but only moderately diminished nuclear pMad. The postsynaptic composition and electrophysiological properties of Mad8 NMJs were similarly altered. Using biochemical approaches, this study examined how a single point mutation in Mad8 could influence the Mad-receptor interface and identified a key motif, the H2 helix. This study highlights the biological relevance of Smad-dependent, synaptic BMP signaling and uncovers a highly conserved structural feature of Smads, critical for normal development and function.
Mathew, R., Rios-Barrera, L. D., Machado, P., Schwab, Y. and Leptin, M. (2020). Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism. Embo j: e105332. PubMed ID: 32657472
Plasma membranes fulfil many physiological functions. In polarized cells, different membrane compartments take on specialized roles, each being allocated correct amounts of membrane. The Drosophila tracheal system, an established tubulogenesis model, contains branched terminal cells with subcellular tubes formed by apical plasma membrane invagination. This study shows that apical endocytosis and late endosome-mediated trafficking are required for membrane allocation to the apical and basal membrane domains. Basal plasma membrane growth stops if endocytosis is blocked, whereas the apical membrane grows excessively. Plasma membrane is initially delivered apically and then continuously endocytosed, together with apical and basal cargo. An organelle is described carrying markers of late endosomes and multivesicular bodies (MVBs) that is abolished by inhibiting endocytosis and which is suggested to act as transit station for membrane destined to be redistributed both apically and basally. This is based on the observation that disrupting MVB formation prevents growth of both compartments.
Olarte, M. J., Kim, S., Sharp, M. E., Swanson, J. M. J., Farese, R. V., Jr. and Walther, T. C. (2020). Determinants of Endoplasmic Reticulum-to-Lipid Droplet Protein Targeting. Dev Cell. PubMed ID: 32730754
Lipid droplet (LD) formation from the endoplasmic reticulum (ER) is accompanied by the targeting and accumulation of specific hydrophobic, membrane-embedded proteins on LDs. The determinants of this process are unknown. The hydrophobic membrane motifs of two Drosophila melanogaster proteins, GPAT4 and ALG14, that utilize this pathway were studied, and crucial sequence features were identified that mediate LD accumulation. Molecular dynamics simulations and studies in cells reveal that LD targeting of these motifs requires deeply inserted tryptophans that have lower free energy in the LD oil phase and positively charged residues near predicted hairpin hinges that become less constrained in the LD environment. Analyzing hydrophobic motifs from similar LD-targeting proteins, it appears that the distribution of tryptophan and positively charged residues distinguishes them from non-LD-targeting membrane motifs. These studies identify specific sequence features and principles of hydrophobic membrane motifs that mediate their accumulation on LDs.
Sando, R., Jiang, X. and Sudhof, T. C. (2019).. Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins. Science 363(6429). PubMed ID: 30792275
Bidirectional signaling by cell adhesion molecules is thought to mediate synapse formation, but the mechanisms involved remain elusive. This study found that the adhesion G protein-coupled receptors latrophilin-2 and latrophilin-3 (see Drosophila Cirl) selectively direct formation of perforant-path and Schaffer-collateral synapses, respectively, to hippocampal CA1-region neurons. Latrophilin-3 binds to two transcellular ligands: fibronectin leucine-rich repeat transmembrane proteins (FLRTs) and teneurins (see Drosophila Tenascin major). In transgenic mice in vivo, both binding activities were required for input-specific synapse formation, which suggests that coincident binding of both ligands is necessary for synapse formation. In cultured neurons in vitro, teneurin or FLRT alone did not induce excitatory synapse formation, whereas together they potently did so. Thus, postsynaptic latrophilins promote excitatory synapse formation by simultaneous binding of two unrelated presynaptic ligands, which is required for formation of synaptic inputs at specific dendritic localizations.
Mathew, R., Rios-Barrera, L. D., Machado, P., Schwab, Y. and Leptin, M. (2020). Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism. Embo J: e105332. PubMed ID: 32657472
Plasma membranes fulfil many physiological functions. In polarized cells, different membrane compartments take on specialized roles, each being allocated correct amounts of membrane. The Drosophila tracheal system, an established tubulogenesis model, contains branched terminal cells with subcellular tubes formed by apical plasma membrane invagination. This study shows that apical endocytosis and late endosome-mediated trafficking are required for membrane allocation to the apical and basal membrane domains. Basal plasma membrane growth stops if endocytosis is blocked, whereas the apical membrane grows excessively. Plasma membrane is initially delivered apically and then continuously endocytosed, together with apical and basal cargo. An organelle is described carrying markers of late endosomes and multivesicular bodies (MVBs) that is abolished by inhibiting endocytosis and which is suggested to act as transit station for membrane destined to be redistributed both apically and basally. This is based on the observation that disrupting MVB formation prevents growth of both compartments.

Monday, September 14th - Cytoskeleton and junctions

Krol, A., Henle, S. J. and Goodrich, L. V. (2016). Fat3 and Ena/VASP proteins influence the emergence of asymmetric cell morphology in the developing retina. Development 143(12): 2172-2182. PubMed ID: 27122175
Neurons exhibit asymmetric morphologies throughout development - from migration to the elaboration of axons and dendrites - that are correctly oriented for the flow of information. For instance, retinal amacrine cells migrate towards the inner plexiform layer (IPL) and then retract their trailing processes, thereby acquiring a unipolar morphology with a single dendritic arbor restricted to the IPL. This study provides evidence that the Fat-like cadherin Fat3 (see Drosophila Fat) acts during multiple stages of amacrine cell development in mice to orient overall changes in cell shape towards the IPL. Using a time-lapse imaging assay, this study found that developing amacrine cells are less directed towards the IPL in the absence of Fat3, during both migration and retraction. Consistent with its predicted role as a cell-surface receptor, Fat3 functions cell-autonomously and is able to influence the cytoskeleton directly through its intracellular domain, which can bind and localize Ena/VASP family (see Drosophila Enabled) actin regulators. Indeed, a change in Ena/VASP protein distribution is sufficient to recapitulate the Fat3 mutant amacrine cell phenotype. Thus, Fat-like proteins might control the polarized development of tissues by sculpting the cytoskeleton of individual cells.
Butler, M. T. and Wallingford, J. B. (2018). Spatial and temporal analysis of PCP protein dynamics during neural tube closure. Elife 7. PubMed ID: 30080139
Planar cell polarity (PCP) controls convergent extension and axis elongation in all vertebrates. Although asymmetric localization of PCP proteins is central to their function, little is understood about PCP protein localization during convergent extension. This study used quantitative live imaging to simultaneously monitor cell intercalation behaviors and PCP protein dynamics in the Xenopus laevis neural plate epithelium. Asymmetric enrichment of PCP proteins was observed, but more interestingly, tight correlation of PCP protein enrichment was observed with actomyosin-driven contractile behavior of cell-cell junctions. In addition to expected patterns of spatial asymmetry, PCP protein enrichment is tightly linked to cell-cell junction behavior: Prickle2 (Pk2) (Drosophila homolog: Prickle) and Vangl2 (Drosophila homolog: Van Gogh) were dynamically enriched specifically at shrinking cell-cell junctions and depleted from elongating junctions during cell intercalation. The turnover rates of junctional PCP proteins also correlated with the contractile behavior of individual junctions. All these dynamic relationships were disrupted when PCP signaling was manipulated. Together, these results provide a dynamic and quantitative view of PCP protein localization during convergent extension and suggest a complex and intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking during cell intercalation in the closing vertebrate neural tube.
Liao, K. A., Gonzalez-Morales, N. and Schock, F. (2020). Characterizing the actin-binding ability of Zasp52 and its contribution to myofibril assembly. PLoS One 15(7): e0232137. PubMed ID: 32614896
In sarcomeres, α-actinin crosslinks thin filaments and anchors them at the Z-disc. Drosophila melanogaster Zasp52 also localizes at Z-discs and interacts with α-actinin via its extended PDZ domain, thereby contributing to myofibril assembly and maintenance, yet the detailed mechanism of Zasp52 function is unknown. This study shows a strong genetic interaction between actin and Zasp52 during indirect flight muscle assembly, indicating that this interaction plays a critical role during myofibril assembly. The results suggest that Zasp52 contains an actin-binding site, which includes the extended PDZ domain and the ZM region. Zasp52 binds with micromolar affinity to monomeric actin. A co-sedimentation assay indicates that Zasp52 can also bind to F-actin. Finally, in vivo rescue assays of myofibril assembly were used to show that the α-actinin-binding domain of Zasp52 is not sufficient for a full rescue of Zasp52 mutants suggesting additional contributions of Zasp52 actin-binding to myofibril assembly.
Mukherjee, A., Brooks, P. S., Bernard, F., Guichet, A. and Conduit, P. T. (2020). Microtubules originate asymmetrically at the somatic golgi and are guided via Kinesin2 to maintain polarity within neurons. Elife 9. PubMed ID: 32657758
Neurons contain polarised microtubule arrays essential for neuronal function. How microtubule nucleation and polarity are regulated within neurons remains unclear. This study shows that γ-tubulin localises asymmetrically to the somatic Golgi within Drosophila neurons. Microtubules originate from the Golgi with an initial growth preference towards the axon. Their growing plus ends also turn towards and into the axon, adding to the plus-end-out microtubule pool. Any plus ends that reach a dendrite, however, do not readily enter, maintaining minus-end-out polarity. Both turning towards the axon and exclusion from dendrites depend on Kinesin-2, a plus-end-associated motor that guides growing plus ends along adjacent microtubules. It is proposed that Kinesin-2 engages with a polarised microtubule network within the soma to guide growing microtubules towards the axon; while at dendrite entry sites engagement with microtubules of opposite polarity generates a backward stalling force that prevents entry into dendrites and thus maintains minus-end-out polarity within proximal dendrites.
Nakamura, M., Verboon, J. M., Prentiss, C. L. and Parkhurst, S. M. (2020). The kinesin-like protein Pavarotti functions noncanonically to regulate actin dynamics. J Cell Biol 219(9). PubMed ID: 32673395
Pavarotti, the Drosophila MKLP1 orthologue, is a kinesin-like protein that works with Tumbleweed (MgcRacGAP) as the centralspindlin complex. This complex is essential for cytokinesis, where it helps to organize the contractile actomyosin ring at the equator of dividing cells by activating the RhoGEF Pebble. Actomyosin rings also function as the driving force during cell wound repair. Previous work has shown that Tumbleweed and Pebble are required for the cell wound repair process. This study shows that Pavarotti also functions during wound repair and confirm that while Pavarotti, Tumbleweed, and Pebble are all used during this cellular repair, each has a unique localization pattern and knockdown phenotype, demonstrating centralspindlin-independent functions. Surprisingly, it was found that the classically microtubule-associated Pavarotti binds directly to actin in vitro and in vivo and has a noncanonical role directly regulating actin dynamics. Finally, this actin regulation by Pavarotti was shown to not be specific to cellular wound repair but is also used in normal development.
Panda, P., Kovacs, L., Dzhindzhev, N., Fatalska, A., Persico, V., Geymonat, M., Riparbelli, M. G., Callaini, G. and Glover, D. M. (2020). Tissue specific requirement of Drosophila Rcd4 for centriole duplication and ciliogenesis. J Cell Biol 219(8). PubMed ID: 32543652
Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to centrosomes. This study shows that rcd4 mutants exhibit fewer centrioles, aberrant mitoses, and reduced basal bodies in sensory organs. Rcd4 interacts with the C-terminal part of Ana1, which is essential for such conversion. Whereas ana3 mutants are male sterile, reflecting a requirement for Ana3 for centriole development in the male germ line, rcd4 mutants are fertile and have male germ line centrioles of normal length. Thus, Rcd4 is essential in somatic cells but is not absolutely required in spermatogenesis, indicating tissue-specific roles in centriole and basal body formation.

Friday, September 11th - Disease Models

Swanson, L. C., Rimkus, S. A., Ganetzky, B. and Wassarman, D. A. (2020). Loss of the Antimicrobial Peptide Mechnikowin Protects Against Traumatic Brain Injury Outcomes in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32631949
Neuroinflammation is a major pathophysiological feature of traumatic brain injury (TBI). Early and persistent activation of innate immune response signaling pathways by primary injuries is associated with secondary cellular injuries that cause TBI outcomes to change over time. A Drosophila melanogaster model was used to investigate the role of antimicrobial peptides (AMPs) in acute and chronic outcomes of closed-head TBI. AMPs are effectors of pathogen and stress defense mechanisms mediated by the evolutionarily conserved Toll Immune-deficiency (Imd) innate immune response pathways that activate Nuclear Factor kappa B (NF-kB) transcription factors. This study analyzed the effect of null mutations in 10 of the 14 known Drosophila AMP genes on TBI outcomes. Mutation of Metchnikowin (Mtk) was unique in protecting flies from mortality within the 24 h following TBI under two diet conditions that produce different levels of mortality. In addition, Mtk mutants had reduced behavioral deficits at 24 h following TBI and increased lifespan either in the absence or presence of TBI. Using a transcriptional reporter of gene expression, it was found that TBI increased Mtk expression in the brain. Quantitative analysis of mRNA in whole flies revealed that expression of other AMPs in the Toll and Imd pathways as well as NF-κB transcription factors were not altered in Mtk mutants. Overall, these results demonstrate that Mtk plays an infection-independent role in the fly nervous system, and TBI-induced expression of Mtk in the brain activates acute and chronic secondary injury pathways that are also activated during normal aging.
Solana-Manrique, C., Sanz, F. J., Ripolles, E., Bano, M. C., Torres, J., Munoz-Soriano, V. and Paricio, N. (2020). Enhanced activity of glycolytic enzymes in Drosophila and human cell models of Parkinson's disease based on DJ-1 deficiency. Free Radic Biol Med. PubMed ID: 32726690
Parkinson's disease (PD) is a neurodegenerative debilitating disorder characterized by progressive disturbances in motor, autonomic and psychiatric functions. One of the genes involved in familial forms of the disease is DJ-1, whose mutations cause early-onset PD. Besides, it has been shown that an over-oxidized and inactive form of the DJ-1 protein is found in brains of sporadic PD patients. Interestingly, the DJ-1 protein plays an important role in cellular defense against oxidative stress and also participates in mitochondrial homeostasis. Flies mutant for the DJ-1β gene, the Drosophila ortholog of human DJ-1, exhibited disease-related phenotypes such as motor defects, increased reactive oxygen species production and high levels of protein carbonylation. The present study demonstrated that DJ-1β mutants also show a significant increase in the activity of several regulatory glycolytic enzymes. Similar results were obtained in DJ-1-deficient SH-SY5Y neuroblastoma cells, thus suggesting that loss of DJ-1 function leads to an increase in the glycolytic rate. In such a scenario, an enhancement of the glycolytic pathway could be a protective mechanism to decrease ROS production by restoring ATP levels, which are decreased due to mitochondrial dysfunction. The results also show that meclizine and dimethyl fumarate, two FDA-approved compounds with different clinical applications, are able to attenuate PD-related phenotypes in both models. Moreover, it was found that they may exert their beneficial effect by increasing glycolysis through the activation of key glycolytic enzymes. Taken together, these results are consistent with the idea that increasing glycolysis could be a potential disease-modifying strategy for PD, as recently suggested. Besides, they also support further evaluation and potential repurposing of meclizine and dimethyl fumarate as modulators of energy metabolism for neuroprotection in PD.
Shorey, M., Stone, M. C., Mandel, J. and Rolls, M. M. (2020). Neurons survive simultaneous injury to axons and dendrites and regrow both types of processes in vivo. Dev Biol. PubMed ID: 32687893
Neurons extend dendrites and axons to receive and send signals. If either type of process is removed, the cell cannot function. Rather than undergoing cell death, some neurons can regrow axons and dendrites. Axon and dendrite regeneration have been examined separately and require sensing the injury and reinitiating the correct growth program. Whether neurons in vivo can sense and respond to simultaneous axon and dendrite injury with polarized regeneration has not been explored. To investigate the outcome of simultaneous axon and dendrite damage, a Drosophila model system was used in which neuronal polarity, axon regeneration, and dendrite regeneration have been characterized. After removal of the axon and all but one dendrite, the remaining dendrite was converted to a process that had a long unbranched region that extended over long distances and a region where shorter branched processes were added. These observations suggested axons and dendrites could regrow at the same time. To further test the capacity of neurons to implement polarized regeneration after axon and dendrite damage, all neurites were removed from mature neurons. In this case a long unbranched neurite and short branched neurites were regrown from the stripped cell body. Moreover, the long neurite had axonal plus-end-out microtubule polarity and the shorter neurites had mixed polarity consistent with dendrite identity. The long process also accumulated endoplasmic reticulum at its tip like regenerating axons. It is concluded that neurons in vivo can respond to simultaneous axon and dendrite injury by initiating growth of a new axon and new dendrites.
Rani, L., Saini, S., Shukla, N., Chowdhuri, D. K. and Gautam, N. K. (2020). High sucrose diet induces morphological, structural and functional impairments in the renal tubules of Drosophila melanogaster: A model for studying type-2 diabetes mediated renal tubular dysfunction. Insect Biochem Mol Biol: 103441. PubMed ID: 32735915
Continuous feeding of high dietary sugar is strongly associated with type 2 diabetes (T2D) and its secondary complications. Diabetic nephropathy (DN) is a major secondary complication that leads to glomerular and renal tubular dysfunction. The present study is aimed to investigate the effects of chronic exposure of high sugar diet (HSD) on renal tubules. Malpighian tubules (MTs), a renal organ of Drosophila, were used as a model in the study. Feeding of HSD develops T2D condition in Drosophila. The MTs showed structural abnormalities in 20 days of HSD fed flies. Impaired insulin signaling, oxidative stress, enhanced levels of AGE-RAGE and induction of apoptosis were observed in the MTs of these flies. Further, altered expression of transporters, enhanced uric acid level and reduced fluid secretion rate confirmed the impaired function of MTs in these flies. RNA-seq and RT-PCR analyses in the MTs of HSD fed-and control-flies revealed the altered expression of candidate genes that regulate several important pathways including ECM, AGE-RAGE, TGF-β, galactose, starch and sucrose metabolism that are well known mediators of renal tubular dysfunction in diabetic nephropathy (DN) patients. Disruption of insulin signaling in the MTs also causes renal tubular dysfunction similar to HSD fed flies. Overall, the study suggests that phenotypes observed in the MTs of HSD fed flies recapitulate several hallmarks of renal tubular dysfunction in DN patients. Therefore, it is concluded that MTs of HSD fed flies may be used for deciphering the underlying mechanisms of T2D mediated renal tubular dysfunction.
Saikumar, J., Byrns, C. N., Hemphill, M., Meaney, D. F. and Bonini, N. M. (2020). Dynamic neural and glial responses of a head-specific model for traumatic brain injury in Drosophila. Proc Natl Acad Sci U S A 117(29): 17269-17277. PubMed ID: 32611818
Traumatic brain injury (TBI) is the strongest environmental risk factor for the accelerated development of neurodegenerative diseases. There are currently no therapeutics to address this due to lack of insight into mechanisms of injury progression, which are challenging to study in mammalian models. This study has developed and extensively characterized a head-specific approach to TBI in Drosophila, a powerful genetic system that shares many conserved genes and pathways with humans. The Drosophila TBI (dTBI) device inflicts mild, moderate, or severe brain trauma by precise compression of the head using a piezoelectric actuator. Head-injured animals display features characteristic of mammalian TBI, including severity-dependent ataxia, life span reduction, and brain degeneration. Severe dTBI is associated with cognitive decline and transient glial dysfunction, and stimulates antioxidant, proteasome, and chaperone activity. Moreover, genetic or environmental augmentation of the stress response protects from severe dTBI-induced brain degeneration and life span deficits. Together, these findings present a tunable, head-specific approach for TBI in Drosophila that recapitulates mammalian injury phenotypes and underscores the ability of the stress response to mitigate TBI-induced brain degeneration.
Shah, E. J., Gurdziel, K. and Ruden, D. M. (2020). Drosophila Exhibit Divergent Sex-Based Responses in Transcription and Motor Function After Traumatic Brain Injury. Front Neurol 11: 511. PubMed ID: 32636795
Every year, millions of people in the US suffer brain damage from mild to severe traumatic brain injuries (TBI) that result from a sudden impact to the head. Despite TBI being a leading cause of death and disability worldwide, sex differences that contribute to varied outcomes post-injury are not extensively studied and therefore, poorly understood. This study aimed to explore biological sex as a variable influencing response to TBI using Drosophila melanogaster as a model, since flies have been shown to exhibit symptoms commonly seen in other mammalian models of TBI. After inflicting TBI using the high-impact trauma device, w (1118) fly brains were isolated and gene transcription changes were assessed in male and female flies at control and 1, 2, and 4 hr after TBI. The results suggest that overall, Drosophila females show more gene transcript changes than males. Females also exhibit upregulated expression changes in immune response and mitochondrial genes across all time-points. In addition, the impact of injury on mitochondrial health and motor function was assessed in both sexes before and after injury. Although both sexes report similar changes in mitochondrial oxidation and negative geotaxis, locomotor activity appears to be more impaired in males than females. These data suggest that sex-differences not only influence the response to TBI but also contribute to varied outcomes post-injury.

Thursday, September 10 - Signaling

Tarczewska, A., Wycisk, K., Orlowski, M., Waligorska, A., Dobrucki, J., Drewniak-Switalska, M., Berlicki, L. and Ozyhar, A. (2020). Nuclear immunophilin FKBP39 from Drosophila melanogaster drives spontaneous liquid-liquid phase separation. Int J Biol Macromol 163: 108-119. PubMed ID: 32615218
The FKBP39 from Drosophila melanogaster is a multifunctional regulatory immunophilin. It contains two globular domains linked by a highly charged disordered region. The N-terminal domain shows homology to the nucleoplasmin core domain, and the C-terminal domain is characteristic for the family of the FKBP immunophilin ligand binding domain. The specific partially disordered structure of the protein inspired an investigation of whether FKBP39 can drive spontaneous liquid-liquid phase separation (LLPS). Preliminary analyses using CatGranule and Pi-Pi contact predictors suggested a propensity for LLPS. Microscopy observations revealed that FKBP39 can self-concentrate to form liquid condensates. It was also found that FKBP39 can lead to LLPS in the presence of RNA and peptides containing Arg-rich linear motifs derived from selected nuclear and nucleolar proteins. These heterotypic interactions have a stronger propensity for driving LLPS when compared to the interactions mediated by self-associating FKBP39 molecules. To investigate whether FKBP39 can drive LLPS in the cellular environment, it was analysed in fusion with YFP in COS-7 cells. The specific distribution and diffusion kinetics of FKBP39 examined by FRAP experiments provided evidence that immunophilin is an important driver of phase separation. The ability of FKBP39 to go into heterotypic interaction may be fundamental for ribosome subunits assembly.
May, C., Ji, S., Syed, Z. A., Revoredo, L., Daniel, E. J. P., Gerken, T. A., Tabak, L. A., Samara, N. L. and Ten Hagen, K. G. (2020). Differential splicing of the lectin domain of an O-glycosyltransferase modulates both peptide and glycopeptide preferences. J Biol Chem. PubMed ID: 32669364
Mucin-type O-glycosylation is an essential post-translational modification required for protein secretion, extracellular matrix formation and organ growth. O-glycosylation is initiated by a large family of enzymes (GALNTs in mammals and PGANTs in Drosophila) that catalyze the addition of N-acetylgalactosamine (GalNAc) onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes contain 2 functional domains; a catalytic domain and a C-terminal ricin-like lectin domain comprised of 3 potential GalNAc recognition repeats termed α, β and γ. The catalytic domain is responsible for binding donor and acceptor substrates and catalyzing transfer of GalNAc, while the lectin domain recognizes more distant extant GalNAc on previously glycosylated substrates. This study interrogate how the differentially spliced α repeat of the PGANT9A and PGANT9B O-glycosyltransferases confers distinct preferences for a variety of endogenous substrates. Through biochemical analyses and in silico modeling using preferred substrates, it was found that a combination of charged residues within the α repeat and charged residues in the flexible gating loop of the catalytic domain distinctively influence the peptide substrate preferences of each splice variant. Moreover, PGANT9A and PGANT9B also display unique glycopeptide preferences. These data illustrate how changes within the non-catalytic lectin domain can alter the recognition of both peptide and glycopeptide substrates. Overall, these results elucidate a novel mechanism for modulating substrate preferences of O-glycosyltransferases via alternative splicing within specific subregions of functional domains.
Menant, A. and Karess, R. E. (2020). Mutations in the Drosophila rough deal gene affecting RZZ kinetochore function. Biol Cell. PubMed ID: 32602944
The RZZ complex, composed of the proteins Rough-Deal (Rod), Zw10, and Zwilch, plays a central role in the spindle assembly checkpoint (SAC), which assures proper sister chromatid segregation during mitosis. RZZ contributes to the regulation of the SAC by helping to recruit Mad1-Mad2 and the microtubule motor dynein to unattached kinetochores. It is an important component of the outer kinetochore and specifically the fibrous corona whose expansion is believed to facilitate microtubule capture. How RZZ carries out its diverse activities is only poorly understood. The C-terminal region of the Rod subunit is relatively well-conserved across metazoan phylogeny, but no function has been attributed to it. To explore the importance of the Rod_C domain in RZZ function in Drosophila, a series of point mutations was generated in a stretch of 200 residues within this domain, and their phenotypes are reported in this study. Several of the mutations profoundly disrupt recruitment of RZZ to kinetochores, including one in a temperature-sensitive manner, while still retaining the capacity to assemble into a complex with Zw10 and Zwilch. Others affect aspects of dynein activity or recruitment at the kinetochore. These results suggest that the Rod_C domain participates in the protein interactions necessary for RZZ recruitment and functionality at kinetochores.
Stepanik, V., Sun, J. and Stathopoulos, A. (2020). FGF Pyramus Has a Transmembrane Domain and Cell-Autonomous Function in Polarity. Curr Biol. PubMed ID: 32619487
Most fibroblast growth factors (FGFs) function as receptor ligands through their conserved FGF domain, but sequences outside this domain vary and are not well studied. This core domain of 120 amino acids (aa) is flanked in all FGFs by highly divergent amino-terminal and carboxy-terminal sequences of variable length. Drosophila has fewer FGF genes, with only three identified to date, pyramus (pyr), thisbe (ths), and branchless (bnl), and all three encoding relatively large FGF proteins (~80 kDa). It was hypothesized that the longer FGF proteins present in Drosophila and other organisms may relate to an ancestral form, in which multiple functions or regulatory properties are present within a single polypeptide. This study focused analysis on Pyr, finding that it harbors a transmembrane domain (TMD) and extended C-terminal intracellular domain containing a degron. The intracellular portion limits Pyr levels, whereas the TMD promotes spatial precision in the paracrine activation of Heartless FGF receptor. Additionally, degron deletion mutants that upregulate Pyr exhibit cell polarity defects that lead to invagination defects at gastrulation, demonstrating a previously uncharacterized cell-autonomous role. In summary, these data show that Pyr is the first demonstrated transmembrane FGF, that it has both extracellular and intracellular functions, and that spatial distribution and levels of this particular FGF protein are tightly regulated. These results suggest that other FGFs may be membrane tethered or multifunctional like Pyr.
Muliyil, S., Levet, C., Dusterhoft, S., Dulloo, I., Cowley, S. A. and Freeman, M. (2020). ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration. Embo j: e104415. PubMed ID: 32715522
Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. This study revealed an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age-related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age-related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. The glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, this study demonstrates the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, this study has discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina.
Sheng, Z. and Du, W. (2020). NatB regulates Rb mutant cell death and tumor growth by modulating EGFR/MAPK signaling through the N-end rule pathways. PLoS Genet 16(6): e1008863. PubMed ID: 32559195
Inactivation of the Rb tumor suppressor causes context-dependent increases in cell proliferation or cell death. In a genetic screen for factors that promoted Rb mutant cell death in Drosophila, this study identified Psid, a regulatory subunit of N-terminal acetyltransferase B (NatB). NatB subunits were required for elevated EGFR/MAPK signaling and Rb mutant cell survival. NatB regulates the posttranscriptional levels of the highly conserved pathway components Grb2/Drk, MAPK, and PP2AC but not that of the less conserved Sprouty. Interestingly, NatB increased the levels of positive pathway components Grb2/Drk and MAPK while decreased the levels of negative pathway component PP2AC, which were mediated by the distinct N-end rule branch E3 ubiquitin ligases Ubr4 and Cnot4, respectively. These results suggest a novel mechanism by which NatB and N-end rule pathways modulate EGFR/MAPK signaling by inversely regulating the levels of multiple conserved positive and negative pathway components. As inactivation of Psid blocked EGFR signaling-dependent tumor growth, this study raises the possibility that NatB is potentially a novel therapeutic target for cancers dependent on deregulated EGFR/Ras signaling.

Wednesday, September 9th - Adult Physiology

Krycer, J. R., Quek, L. E., Francis, D., Zadoorian, A., Weiss, F. C., Cooke, K. C., Nelson, M. E., Diaz-Vegas, A., Humphrey, S. J., Scalzo, R., Hirayama, A., Ikeda, S., Shoji, F., Suzuki, K., Huynh, K., Giles, C., Varney, B., Nagarajan, S. R., Hoy, A. J., Soga, T., Meikle, P. J., Cooney, G. J., Fazakerley, D. J. and James, D. E. (2020). Insulin signalling requires glucose to promote lipid anabolism in adipocytes. J Biol Chem. PubMed ID: 32723868
Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilisation based on nutritional status. This is coordinated by insulin, which triggers kinase signalling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, this study sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3'-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signalling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilises protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo. Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.
Schotthofer, S. K. and Bohrmann, J. (2020). Analysing bioelectrical phenomena in the Drosophila ovary with genetic tools: tissue-specific expression of sensors for membrane potential and intracellular pH, and RNAi-knockdown of mechanisms involved in ion exchange. BMC Dev Biol 20(1): 15. PubMed ID: 32635900
Changes in transcellular bioelectrical patterns are known to play important roles during developmental and regenerative processes. The Drosophila follicular epithelium has proven to be an appropriate model system for studying the mechanisms by which bioelectrical signals emerge and act. Two genetically-encoded fluorescent sensors for V(mem) and pH(i), ArcLight and pHluorin-Moesin, were expressed in the follicular epithelium of Drosophila. In a RNAi-knockdown screen, five genes of ion-transport mechanisms and gap-junction subunits were identified exerting influence on ovary development and/or oogenesis. Loss of ovaries or small ovaries were the results of soma knockdowns of the innexins inx1 and inx3, and of the DEG/ENaC family member ripped pocket (rpk). Germline knockdown of rpk also resulted in smaller ovaries. Soma knockdown of the V-ATPase-subunit vha55 caused size-reduced ovaries with degenerating follicles from stage 10A onward. In addition, soma knockdown of the open rectifier K(+)channel 1 (ork1) resulted in a characteristic round-egg phenotype with altered microfilament and microtubule organisation in the follicular epithelium. The genetic tool box of Drosophila provides means for a refined and extended analysis of bioelectrical phenomena. Tissue-specifically expressed V(mem)- and pH(i)-sensors exhibit some practical advantages compared to fluorescent indicator dyes. Their use confirms that the ion-transport mechanisms targeted by inhibitors play important roles in the generation of bioelectrical signals. Moreover, modulation of bioelectrical signals via RNAi-knockdown of genes coding for ion-transport mechanisms and gap-junction subunits exerts influence on crucial processes during ovary development and results in cytoskeletal changes and altered follicle shape. Thus, further evidence amounts for bioelectrical regulation of developmental processes via the control of both signalling pathways and cytoskeletal organisation.
Krittika, S. and Yadav, P. (2020). Dietary protein restriction deciphers new relationships between lifespan, fecundity and activity levels in fruit flies Drosophila melanogaster. Sci Rep 10(1): 10019. PubMed ID: 32572062
Drosophila melanogaster has been used in Diet Restriction (DR) studies for a few decades now, due to easy diet implementation and its short lifespan. Since the concentration of protein determines the trade-offs between lifespan and fecundity, it is important to understand the level of protein and the extent of its influence on lifespan, fecundity and activity of fruit flies. This study intended to assess the effect of a series of protein restricted diets from age 1 day of the adult fly on these traits to understand the possible variations in trade-off across tested diets. Lifespan under different protein concentrations remains unaltered, even though protein restricted diets exerted an age-specific influence on fecundity. Interestingly, there was no difference in lifetime activity of the flies in most of the tested protein restricted (PR) diets, even though a sex-dependent influence of protein concentrations was observed. Additionally, not all concentrations of PR diet increase activity, thereby suggesting that the correlation between lifespan and the lifetime activity can be challenged under protein-restricted condition. Therefore, the PR does not need to exert its effect on lifespan and fecundity only but can also influence activity levels of the flies, thereby emphasizing the role of nutrient allotment between lifespan, fecundity and activity.
Scanlan, J. L., Gledhill-Smith, R. S., Battlay, P. and Robin, C. (2020). Genomic and transcriptomic analyses in Drosophila suggest that the ecdysteroid kinase-like (EcKL) gene family encodes the 'detoxification-by-phosphorylation' enzymes of insects. Insect Biochem Mol Biol 123: 103429. PubMed ID: 32540344
Phosphorylation is a phase II detoxification reaction that, among animals, occurs near exclusively in insects. It is proposed that members of the arthropod-specific ecdysteroid kinase-like (EcKL) gene family encode detoxicative kinases. To test this hypothesis, the EcKL gene family was annotated in 12 species of Drosophila and explored their evolution within the genus. Many ancestral EcKL clades are evolutionarily unstable, while others are conserved as single-copy orthologs. Relationships were demonstrated between xenobiotic induction, detoxification tissue-enriched expression and evolutionary instability in the EcKLs and the P450s. A systematic method was devised for identifying candidate detoxification genes in large gene families that is concordant with experimentally determined functions of P450 genes in D. melanogaster. Applying this method to the EcKLs suggested a significant proportion of these genes play roles in detoxification, and that the EcKLs may constitute a detoxification gene family in insects. Additionally, it is estimated that between 11 and 16 uncharacterised D. melanogaster P450s are strong detoxification candidates. Lastly, previously unreported genomic and transcriptomic variation was found in a number of EcKLs and P450s associated with toxic stress phenotypes using a targeted phenome-wide association study (PheWAS) approach in D. melanogaster, presenting multiple future avenues of research for detoxification genetics in this species.
Suito, T., Nagao, K., Takeuchi, K., Juni, N., Hara, Y. and Umeda, M. (2020). Functional expression of Delta12 fatty acid desaturase modulates thermoregulatory behaviour in Drosophila. Sci Rep 10(1): 11798. PubMed ID: 32678126
Polyunsaturated fatty acids (PUFAs) play crucial roles in adaptation to cold environments in a wide variety of animals and plants. However, the mechanisms by which PUFAs affect thermoregulatory behaviour remain elusive. This study investigated the roles of PUFAs in thermoregulatory behaviour of Drosophila melanogaster. To this end, transgenic flies expressing Caenorhabditis elegans δ12 fatty acid desaturase (FAT-2), which converts mono-unsaturated fatty acids to PUFAs such as linoleic acid [C18:2 (n-6)] and linolenic acid [C18:3 (n-3)]. Neuron-specific expression of FAT-2 using the GAL4/UAS expression system led to increased contents of C18:2 (n-6)-containing phospholipids in central nerve system (CNS) and caused significant decreases in preferred temperature of third instar larvae. In genetic screening and calcium imaging analyses of thermoreceptor-expressing neurons, it was demonstrated that ectopic expression of FAT-2 in TRPA1-expressing neurons led to decreases in preferred temperature by modulating neuronal activity. It is conclude that functional expression of FAT-2 in a subset of neurons changes the thermoregulatory behaviour of D. melanogaster, likely by modulating quantities of PUFA-containing phospholipids in neuronal cell membranes.
Sujkowski, A., Gretzinger, A., Soave, N., Todi, S. V. and Wessells, R. (2020). α- and β-adrenergic octopamine receptors in muscle and heart are required for Drosophila exercise adaptations. PLoS Genet 16(6): e1008778. PubMed ID: 32579604
Endurance exercise has broadly protective effects across organisms, increasing metabolic fitness and reducing incidence of several age-related diseases. Drosophila has emerged as a useful model for studying changes induced by chronic endurance exercise, as exercising flies experience improvements to various aspects of fitness at the cellular, organ and organismal level. The activity of octopaminergic neurons is sufficient to induce the conserved cellular and physiological changes seen following endurance training. All 4 octopamine receptors are required in at least one target tissue, but only one, Octβ1R, is required for all of them. This study perform tissue- and adult-specific knockdown of α- and β-adrenergic octopamine receptors in several target tissues. Reduced expression of Octβ1R in adult muscles abolishes exercise-induced improvements in endurance, climbing speed, flight, cardiac performance and fat-body catabolism in male Drosophila. Importantly, Octβ1R and OAMB expression in the heart is also required cell-nonautonomously for adaptations in other tissues, such as skeletal muscles in legs and adult fat body. These findings indicate that activation of distinct octopamine receptors in skeletal and cardiac muscle are required for Drosophila exercise adaptations, and suggest that cell non-autonomous factors downstream of octopaminergic activation play a key role.

Tuesday, September 8th - RNA and transposons

Chang, L. W., Tseng, I. C., Wang, L. H. and Sun, Y. H. (2020). Isoform-specific functions of an evolutionarily conserved 3 bp micro-exon alternatively spliced from another exon in Drosophila homothorax gene. Sci Rep 10(1): 12783. PubMed ID: 32732884
Micro-exons are exons of very small size (usually 3-30 nts). Some micro-exons are alternatively spliced. Their functions, regulation and evolution are largely unknown. This study presents an example of an alternatively spliced 3 bp micro-exon (micro-Ex8) in the homothorax (hth) gene in Drosophila. Hth is involved in many developmental processes. It contains a MH domain and a TALE-class homeodomain (HD). It binds to another homeodomain Exd via its MH domain to promote the nuclear import of the Hth-Exd complex and serve as a cofactor for Hox proteins. The MH and HD domains in Hth as well as the HTh-Exd interaction are highly conserved in evolution. The alternatively spliced micro-exon lies between the exons encoding the MH and HD domains. This study provides clear proof that the micro-Ex8 is produced by alternative splicing from a 48 bp full-length exon 8 (FL-Ex8) and the micro-Ex8 is the first three nt is FL-Ex8. The micro-Ex8 is the ancient form and the 3 + 48 organization of alternatively spliced overlapping exons only emerged in the Schizophora group of Diptera and is absolutely conserved in this group. Several strategies were used to test the in vivo function of the two types of isoforms, and the micro-Ex8 and FL-Ex8 isoforms were found to have largely overlapping functions but also have non-redundant functions that are tissue-specific, which supports their strong evolutionary conservation. Since the different combinations of protein interaction of Hth with Exd and/or Hox can have different DNA target specificity, this finding of alternatively spliced isoforms adds to the spectrum of structural and functional diversity under developmental regulation.
Son, W. and Choi, K. W. (2020). The Classic Lobe Eye Phenotype of Drosophila Is Caused by Transposon Insertion-Induced Misexpression of a Zinc-Finger Transcription Factor. Genetics. PubMed ID: 32641295
Drosophila Lobe (L) alleles were first discovered about hundred years ago as spontaneous dominant mutants with characteristic developmental eye defects. However, the molecular basis for L dominant eye phenotypes has not been clearly understood. A previous work reported an identification of CG10109/PRAS40 as the L gene, but subsequent analyses suggested that PRAS40 may not be related to L. This study revisited the L gene to clarify this discrepancy and understand the basis for the dominance of L mutations. Genetic analysis localized the L gene to Oaz, which encodes a homolog of the vertebrate zinc finger protein 423 (Zfp423) family transcriptional regulators. RNAi knockdown of Oaz almost completely restores all L dominant alleles tested. L(rev6-3), a revertant allele of the L(2) dominant eye phenotype, has an in-frame deletion in the Oaz coding sequence. Molecular analysis of L dominant mutants identified allele-specific insertions of natural transposons or alterations of a preexisting transposon L(2) -specific mutations in roo[ ]Mohr) in the Oaz region. In addition, additional L(2) -reversion alleles were obtained by CRISPR targeting at Oaz. These new loss-of-function Oaz mutations suppress the dominant L eye phenotype. L protein is not expressed in wild-type eye disc but is ectopically expressed in L(2)/+ mutant eye disc. Male recombination was induced between Oaz-GAL4 insertions and the L(2) mutation through homologous recombination. By using the L(2) -recombined GAL4 reporters, this study showed that Oaz-GAL4 is ectopically expressed in L(2) eye imaginal disc. Taken together, these data suggest that neomorphic L eye phenotypes are likely due to misregulation of Oaz by spontaneous transposon insertions.
Mohamed, M., Dang, N. T., Ogyama, Y., Burlet, N., Mugat, B., Boulesteix, M., Merel, V., Veber, P., Salces-Ortiz, J., Severac, D., Pelisson, A., Vieira, C., Sabot, F., Fablet, M. and Chambeyron, S. (2020). A Transposon Story: From TE Content to TE Dynamic Invasion of Drosophila Genomes Using the Single-Molecule Sequencing Technology from Oxford Nanopore. Cells 9(8). PubMed ID: 32722451
Transposable elements (TEs) are the main components of genomes. However, due to their repetitive nature, they are very difficult to study using data obtained with short-read sequencing technologies. This paper describes an efficient pipeline to accurately recover TE insertion (TEI) sites and sequences from long reads obtained by Oxford Nanopore Technology (ONT) sequencing. With this pipeline, the landscapes of the most recent TEIs could be precisely describe in wild-type strains of Drosophila melanogaster and Drosophila simulans. Their comparison suggests that this subset of TE sequences is more similar than previously thought in these two species. The chromosome assemblies obtained using this pipeline also allowed recovering piRNA cluster sequences, which was impossible using short-read sequencing. Finally, the pipeline was used to analyze ONT sequencing data from a D. melanogaster unstable line in which LTR transposition was derepressed for 73 successive generations. It was possible to rely on single reads to identify new insertions with intact target site duplications. Moreover, the detailed analysis of TEIs in the wild-type strains and the unstable line did not support the trap model claiming that piRNA clusters are hotspots of TE insertions.
Mishra, R., Kunar, R., Mandal, L., Alone, D. P., Chandrasekharan, S., Tiwari, A. K., Tapadia, M. G., Mukherjee, A. and Roy, J. K. (2020). A Forward Genetic Approach to Mapping a P-Element Second Site Mutation Identifies DCP2 as a Novel Tumour Suppressor in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32591349
The use of transposons to create mutations has been the cornerstone of Drosophila genetics in the past few decades. Second-site mutations caused by transpositions are often devoid of transposons and thereby affect subsequent analyses. In a P-element mutagenesis screen, a second site mutation was identified on chromosome 3, wherein the homozygous mutants exhibit classic hallmarks of tumour suppressor mutants, including brain tumour and lethality; hence the mutant line was initially named as lethal (3) tumorous brain [l(3)tb]. Classical genetic approaches relying on meiotic recombination and subsequent complementation with chromosomal deletions and gene mutations mapped the mutation to CG6169, the mRNA decapping protein 2 (DCP2), on the left arm of the third chromosome (3L). Thus the mutation was renamed as DCP2(l(3)tb) Fine mapping of the mutation further identified the presence of a Gypsy--LTR like sequence in the 5'UTR coding region of DCP2, along with the expansion of the adjacent upstream intergenic AT-rich sequence. The mutant phenotypes are rescued by the introduction of a functional copy of DCP2 in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. With the increasing repertoire of genes being associated with tumour biology, this is the first instance of mRNA decapping protein being implicated in Drosophila tumourigenesis. These findings, therefore, imply a plausible role for the mRNA degradation pathway in tumorigenesis and identify DCP2 as a potential candidate for future explorations of cell cycle regulatory mechanisms.
Malik, S., Jang, W., Kim, J. Y. and Kim, C. (2020). Mechanisms ensuring robust repression of the Drosophila female germline stem cell maintenance factor Nanos via posttranscriptional regulation. Faseb J. PubMed ID: 32654316
During oogenesis in the Drosophila ovary, numerous translational regulators promote the self-renewal or differentiation of stem cells. An intriguing question is how these regulators combine to execute translational regulation. This study examined mechanisms for the posttranscriptional regulation of nos, a critical stem cell self-renewal factor in the Drosophila ovary; specifically, regulators that promote differentiation of the stem cell daughter. Previous studies showed that Bam, Bgcn, Mei-P26, and Sxl form a complex and repress nos expression through the nos 3'UTR. To further elucidate mechanistic processes of Nos translational regulation, nos repression was reconstituted in cultured Drosophila cells. Ago1 and Brat were identified as new members, and it was shown that Ago1 acts through miRNA binding sites in the proximal region of the nos 3'UTR, whereas Sxl acts via an Sxl binding sequence in the distal region. Combining these findings with published reports, it is proposed that additional factors Bam, Bgcn, Mei-P26, and Brat are recruited to nos mRNAs through interaction with Ago1 and Sxl. These findings elucidate mechanisms of nos regulation by diverse translational repressors.
Gonzalez de Cozar, J. M., Carretero-Junquera, M., Ciesielski, G. L., Miettinen, S. M., Varjosalo, M., Kaguni, L. S., Dufour, E. and Jacobs, H. T. (2020). A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1. J Biochem. PubMed ID: 32589740
In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modeling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, whilst loss of the novel HBD led to a ∼90% drop in K[cat] with a decreased K[M] and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo crosslinking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, it was not possible to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor.

Monday, September 7th - Methods

Sethi, A., Gu, M., Gumusgoz, E., Chan, L., Yan, K. K., Rozowsky, J., Barozzi, I., Afzal, V., Akiyama, J. A., Plajzer-Frick, I., Yan, C., Novak, C. S., Kato, M., Garvin, T. H., Pham, Q., Harrington, A., Mannion, B. J., Lee, E. A., Fukuda-Yuzawa, Y., Visel, A., Dickel, D. E., Yip, K. Y., Sutton, R., Pennacchio, L. A. and Gerstein, M. (2020). Supervised enhancer prediction with epigenetic pattern recognition and targeted validation. Nat Methods 17(8): 807-814. PubMed ID: 32737473
Enhancers are important non-coding elements, but they have traditionally been hard to characterize experimentally. The development of massively parallel assays allows the characterization of large numbers of enhancers for the first time. This study developed a framework using Drosophila STARR-seq to create shape-matching filters based on meta-profiles of epigenetic features. These features were integrated with supervised machine-learning algorithms to predict enhancers. It was further demonstrated that this model could be transferred to predict enhancers in mammals. The predictions were comprehensively validated using a combination of in vivo and in vitro approaches, involving transgenic assays in mice and transduction-based reporter assays in human cell lines (153 enhancers in total). The results confirmed that this model can accurately predict enhancers in different species without re-parameterization. Finally, the transcription factor binding patterns at predicted enhancers versus promoters were examined. These patterns enable the construction of a secondary model that effectively distinguishes enhancers and promoters.
Mangione, F. and Martin-Blanco, E. (2020). Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages. J Vis Exp(160). PubMed ID: 32568222
Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described in this study is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.
Men, J., Li, A., Jerwick, J., Li, Z., Tanzi, R. E. and Zhou, C. (2020). Non-invasive red-light optogenetic control of Drosophila cardiac function. Commun Biol 3(1): 336. PubMed ID: 32601302
Drosophila is a powerful genetic model system for cardiovascular studies. Recently, optogenetic pacing tools have been developed to control Drosophila heart rhythm noninvasively with blue light, which has a limited penetration depth. This study developed both a red-light sensitive opsin expressing Drosophila system and an integrated red-light stimulation and optical coherence microscopy (OCM) imaging system. Noninvasive control of Drosophila cardiac rhythms is demonstrated using a single light source, including simulated tachycardia in ReaChR-expressing flies and bradycardia and cardiac arrest in halorhodopsin (NpHR)-expressing flies at multiple developmental stages. By using red excitation light, it was possible to pace flies at higher efficiency and with lower power than with equivalent blue light excitation systems. The recovery dynamics after red-light stimulation of NpHR flies were observed and quantified. The combination of red-light stimulation, OCM imaging, and transgenic Drosophila systems provides a promising and easily manipulated research platform for noninvasive cardiac optogenetic studies.
de Mena, L. and Rincon-Limas, D. E. (2020). PhotoGal4: A Versatile Light-Dependent Switch for Spatiotemporal Control of Gene Expression in Drosophila Explants. iScience 23(7): 101308. PubMed ID: 32652492
This paper presents PhotoGal4, a phytochrome B-based optogenetic switch for fine-tuned spatiotemporal control of gene expression in Drosophila explants. This switch integrates the light-dependent interaction between phytochrome B and PIF6 from plants with regulatory elements from the yeast Gal4/UAS system. PhotoGal4 efficiently activates and deactivates gene expression upon red- or far-red-light irradiation, respectively. In addition, this optogenetic tool reacts to different illumination conditions, allowing for fine modulation of the light-dependent response. Importantly, by simply focusing a laser beam, PhotoGal4 induces intricate patterns of expression in a customized manner. For instance, personalized patterns of GFP fluorescence such as emoji-like shapes or letterform logos were successfully sketched in Drosophila explants, illustrating the exquisite precision and versatility of this tool. Hence, it is anticipated that PhotoGal4 will expand the powerful Drosophila toolbox and will provide a new avenue to investigate intricate and complex problems in biomedical research.
Raghuraman, B. K., Hebbar, S., Kumar, M., Moon, H., Henry, I., Knust, E. and Shevchenko, A. (2020). Absolute Quantification of Proteins in the Eye of Drosophila melanogaster. Proteomics: e1900049. PubMed ID: 32663363
Absolute (molar) quantification of proteins determines their molar ratios in complexes, networks and metabolic pathways. This study employed MS Western workflow to determine molar abundances of proteins potentially critical for morphogenesis and phototransduction (PT) in eyes of Drosophila melanogaster. A single chimeric 264 kDa protein standard was used that covers, in total, 197 peptides from 43 proteins. The majority of proteins were independently quantified with 2 to 4 proteotypic peptides with the coefficient of variation of less than 15%, better than 1000-fold dynamic range and sub-femtomole sensitivity. Molar abundances were determined of the components of the PT machinery and the rhabdomere, the photosensitive organelle of the fly eye, and how they changed when rhabdomere morphogenesis is perturbed by genetic manipulation of the evolutionary conserved gene crumbs (crb). Data are available via ProteomeXchange with identifier PXD018001.
Stergachis, A. B., Debo, B. M., Haugen, E., Churchman, L. S. and Stamatoyannopoulos, J. A. (2020). Single-molecule regulatory architectures captured by chromatin fiber sequencing. Science 368(6498): 1449-1454. PubMed ID: 32587015
Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. An approach was developed for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N(6)-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. This approach and results open new vistas on the primary architecture of gene regulation.

Friday, September 4th - Behavior

Riddle, N. C. (2020). Variation in the response to exercise stimulation in Drosophila: marathon runner versus sprinter genotypes. J Exp Biol. PubMed ID: 32737212
Animals' behaviors vary in response to their environment, both biotic and abiotic. These behavioral responses have significant impacts on animal survival and fitness, and thus, many behavioral responses are at least partially under genetic control. In Drosophila for example, genes impacting aggression, courtship behavior, circadian rhythms, and sleep have been identified. Animal activity also is influenced strongly by genetics. Previous work used the Drosophila melanogaster Genetics Reference Panel (DGRP) to investigate activity levels and identified over 100 genes linked to activity. This study re-examine these data to determine if Drosophila strains differ in their response to rotational exercise stimulation, not simply in the amount of activity, but in activity patterns and timing of activity. Specifically, it was asked if there are fly strains exhibiting either a "marathoner" pattern of activity, i.e. remaining active throughout the two-hour exercise period, or a "sprinter" pattern, i.e. carrying out most of the activity early in the exercise period. The DGRP strains examined differ significantly in how much activity is carried out at the beginning of the exercise period, and this pattern is influenced by both sex and genotype. Interestingly, there is no clear link between the activity response pattern and lifespan of the animals. Using GWASs, ten high confidence candidate genes were identified that control to which degree Drosophila exercise behaviors fit a marathoner or sprinter activity pattern. This finding suggests that, similar to other aspects of locomotor behavior, timing of activity patterns in response to exercise stimulation is under genetic control.
Schleyer, M., Weiglein, A., Thoener, J., Strauch, M., Hartenstein, V., Kantar Weigelt, M., Schuller, S., Saumweber, T., Eichler, K., Rohwedder, A., Merhof, D., Zlatic, M., Thum, A. S. and Gerber, B. (2020). Identification of Dopaminergic Neurons That Can Both Establish Associative Memory and Acutely Terminate Its Behavioral Expression. J Neurosci 40(31): 5990-6006. PubMed ID: 32586949
An adaptive transition from exploring the environment in search of vital resources to exploiting these resources once the search was successful is important to all animals. The neuronal circuitry that allows larval Drosophila melanogaster of either sex to negotiate this exploration-exploitation transition was examined. This was done by combining Pavlovian conditioning with high-resolution behavioral tracking, optogenetic manipulation of individually identified neurons, and EM data-based analyses of synaptic organization. Optogenetic activation of the dopaminergic neuron DAN-i1 was found to both establish memory during training and acutely terminate learned search behavior in a subsequent recall test. Its activation leaves innate behavior unaffected, however. Specifically, DAN-i1 activation can establish associative memories of opposite valence after paired and unpaired training with odor, and its activation during the recall test can terminate the search behavior resulting from either of these memories. These results further suggest that in its behavioral significance DAN-i1 activation resembles, but does not equal, sugar reward. Dendrogram analyses of all the synaptic connections between DAN-i1 and its two main targets, the Kenyon cells and the mushroom body output neuron MBON-i1, further suggest that the DAN-i1 signals during training and during the recall test could be delivered to the Kenyon cells and to MBON-i1, respectively, within previously unrecognized, locally confined branching structures. This would provide an elegant circuit motif to terminate search on its successful completion.
Khallaf, M. A., Auer, T. O., Grabe, V., Depetris-Chauvin, A., Ammagarahalli, B., Zhang, D. D., Lavista-Llanos, S., Kaftan, F., Weissflog, J., Matzkin, L. M., Rollmann, S. M., Lofstedt, C., Svatos, A., Dweck, H. K. M., Sachse, S., Benton, R., Hansson, B. S. and Knaden, M. (2020). Mate discrimination among subspecies through a conserved olfactory pathway. Sci Adv 6(25): eaba5279. PubMed ID: 32704542
Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, this study identified two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. These results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner.
Tadres, D. and Louis, M. (2020). PiVR: An affordable and versatile closed-loop platform to study unrestrained sensorimotor behavior. PLoS Biol 18(7): e3000712. PubMed ID: 32663220
Tools enabling closed-loop experiments are crucial to delineate causal relationships between the activity of genetically labeled neurons and specific behaviors. The Raspberry Pi Virtual Reality (PiVR) system was developed to conduct closed-loop optogenetic stimulation of neural functions in unrestrained animals. PiVR is an experimental platform that operates at high temporal resolution (70 Hz) with low latencies (<30 milliseconds), while being affordable (<US$500) and easy to build (<6 hours). Through extensive documentation, this tool was designed to be accessible to a wide public, from high school students to professional researchers studying systems neuroscience. The functionality of PiVR was illustrated by focusing on sensory navigation in response to gradients of chemicals (chemotaxis) and light (phototaxis). How Drosophila adult flies perform negative chemotaxis was tested by modulating their locomotor speed to avoid locations associated with optogenetically evoked bitter taste. In Drosophila larvae, innate positive chemotaxis was used to compare behavior elicited by real- and virtual-odor gradients. Finally, how positive phototaxis emerges in zebrafish larvae was examined from the modulation of turning maneuvers to orient in virtual white-light gradients. Besides its application to study chemotaxis and phototaxis, PiVR is a versatile tool designed to bolster efforts to map and to functionally characterize neural circuits.
Misra, S. and Wolfner, M. F. (2020). Drosophila seminal Sex Peptide associates with rival as well as own sperm, providing SP function in polyandrous females. Elife 9. PubMed ID: 32672537
When females mate with more than one male, the males' paternity share is affected by biases in sperm use. These competitive interactions occur while female and male molecules and cells work interdependently to optimize fertility, including modifying the female's physiology through interactions with male seminal fluid proteins (SFPs). Some modifications last long-term, indirectly benefiting later males. Indeed, rival males tailor their ejaculates accordingly. This study shows that SFPs from one male can directly benefit a rival's sperm. Sex Peptide (SP) that a female Drosophila receives from a male can bind sperm that she had stored from a previous male, and rescue the sperm utilization and fertility defects of an SP-deficient first-male. Other seminal proteins received in the first mating 'primed' the sperm (or the female) for this binding. Thus, SP from one male can directly benefit another, making SP a key molecule in inter-ejaculate interaction.
Melnattur, K., Zhang, B. and Shaw, P. J. (2020). Disrupting flight increases sleep and identifies a novel sleep-promoting pathway in Drosophila. Sci Adv 6(19): eaaz2166. PubMed ID: 32494708
Sleep is plastic and is influenced by ecological factors and environmental changes. The mechanisms underlying sleep plasticity are not well understood. This study shows that manipulations that impair flight in Drosophila increase sleep as a form of sleep plasticity. Flight was disrupted by blocking the wing-expansion program, genetically disrupting flight, and by mechanical wing perturbations. A new sleep regulatory circuit is defined starting with specific wing sensory neurons, their target projection neurons in the ventral nerve cord, and the neurons they connect to in the central brain. In addition, a critical neuropeptide (Burs) and its receptor (Rickets) were identified that link wing expansion and sleep. Disrupting flight activates these sleep-promoting projection neurons, as indicated by increased cytosolic calcium levels, and stably increases the number of synapses in their axonal projections. These data reveal an unexpected role for flight in regulating sleep and provide new insight into how sensory processing controls sleep need.

Thursday, September 3rd - Adult Neural Development and Function

Marin, E. C., Buld, L., Theiss, M., Sarkissian, T., Roberts, R. J. V., Turnbull, R., Tamimi, I. F. M., Pleijzier, M. W., Laursen, W. J., Drummond, N., Schlegel, P., Bates, A. S., Li, F., Landgraf, M., Costa, M., Bock, D. D., Garrity, P. A. and Jefferis, G. (2020). Connectomics Analysis Reveals First-, Second-, and Third-Order Thermosensory and Hygrosensory Neurons in the Adult Drosophila Brain. Curr Biol. PubMed ID: 32619476
Animals exhibit innate and learned preferences for temperature and humidity-conditions critical for their survival and reproduction. Leveraging a whole-brain electron microscopy volume, this study examined the adult Drosophila melanogaster circuitry associated with antennal thermo- and hygrosensory neurons. Two new target glomeruli were identified in the antennal lobe, in addition to the five known ones, and the ventroposterior projection neurons (VP PNs) that relay thermo- and hygrosensory information to higher brain centers, including the mushroom body and lateral horn, seats of learned and innate behavior. This paper presents the first connectome of a thermo- and hygrosensory neuropil, the lateral accessory calyx (lACA), by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. A few mushroom body-intrinsic neurons solely receive thermosensory input from the lACA, while most receive additional olfactory and thermo- and/or hygrosensory PN inputs. Furthermore, several classes of lACA-associated neurons form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a hub for thermo- and hygrosensory circuitry. For example, DN1a neurons link thermosensory PNs in the lACA to the circadian clock via the accessory medulla. Finally, this study surveyed strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron targeted by dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor circuits. These data provide a comprehensive first- and second-order layer analysis of Drosophila thermo- and hygrosensory systems and an initial survey of third-order neurons that could directly modulate behavior.
Otto, N., Pleijzier, M. W., Morgan, I. C., Edmondson-Stait, A. J., Heinz, K. J., Stark, I., Dempsey, G., Ito, M., Kapoor, I., Hsu, J., Schlegel, P. M., Bates, A. S., Feng, L., Costa, M., Ito, K., Bock, D. D., Rubin, G. M., Jefferis, G. and Waddell, S. (2020). Input Connectivity Reveals Additional Heterogeneity of Dopaminergic Reinforcement in Drosophila. Curr Biol. PubMed ID: 32619479
Different types of Drosophila dopaminergic neurons (DANs) reinforce memories of unique valence and provide state-dependent motivational control. Prior studies suggest that the compartment architecture of the mushroom body (MB) is the relevant resolution for distinct DAN functions. This study used a recent electron microscope volume of the fly brain to reconstruct the fine anatomy of individual DANs within three MB compartments. The 20 DANs of the γ5 compartment, at least some of which provide reward teaching signals, can be clustered into 5 anatomical subtypes that innervate different regions within γ5. Reconstructing 821 upstream neurons reveals input selectivity, supporting the functional relevance of DAN sub-classification. Only one PAM-γ5 DAN subtype γ5(fb) receives direct recurrent feedback from γ5β'2a mushroom body output neurons (MBONs) and behavioral experiments distinguish a role for these DANs in memory revaluation from those reinforcing sugar memory. Other DAN subtypes receive major, and potentially reinforcing, inputs from putative gustatory interneurons or lateral horn neurons, which can also relay indirect feedback from MBONs. The single aversively reinforcing PPL1-γ1pedc DAN was similarly reconstructed. The γ1pedc DAN inputs mostly differ from those of γ5 DANs and they cluster onto distinct dendritic branches, presumably separating its established roles in aversive reinforcement and appetitive motivation. Tracing also identified neurons that provide broad input to γ5, β'2a, and γ1pedc DANs, suggesting that distributed DAN populations can be coordinately regulated. These connectomic and behavioral analyses therefore reveal further complexity of dopaminergic reinforcement circuits between and within MB compartments.
Tamberg, L., Jaago, M., Saalik, K. L., Sirp, A., Tuvikene, J., Shubina, A., Kiir, C. S., Nurm, K., Sepp, M., Timmusk, T. and Palgi, M. (2020). Daughterless, the Drosophila orthologue of TCF4, is required for associative learning and maintenance of synaptic proteome. Dis Model Mech. PubMed ID: 32641419
Mammalian Transcription Factor 4 (TCF4) has been linked to schizophrenia and intellectual disabilities like Pitt-Hopkins syndrome (PTHS). This study shows that similarly to mammalian TCF4, fruit fly orthologue Daughterless (Da) is expressed widely in the Drosophila brain. Furthermore, silencing of da, using several central nervous system-specific Gal4 driver lines, impairs appetitive associative learning of the larvae and leads to decreased levels of the synaptic proteins Synapsin (Syn) and Discs large 1 (Dlg1) suggesting the involvement of Da in memory formation. This study demonstrates that Syn and dlg1 are direct target genes of Da in adult Drosophila heads, since Da binds to the regulatory regions of these genes and the modulation of Da levels alter the levels of Syn and dlg1 mRNA. Silencing of da also affects negative geotaxis of the adult flies suggesting the impairment of locomotor function. Overall, these findings suggest that Da regulates Drosophila larval memory and adult negative geotaxis possibly via its synaptic target genes Syn and dlg1 These behavioural phenotypes can be further used as a PTHS model to screen for therapeutics.
Lee, J., Yoon, K. J., Park, P., Lee, C., Kim, M. J., Han, D. H., Kim, J. I., Kim, S., Lee, H. R., Lee, Y., Jang, E. H., Ko, H. G., Kong, Y. Y. and Kaang, B. K. (2020). Neur1 and Neur2 are required for hippocampus-dependent spatial memory and synaptic plasticity. Hippocampus. PubMed ID: 32644222
Neur1 and Neur2, mouse homologs of the Drosophila neur gene, consist of two neuralized homology repeat domains and a RING domain. Both Neur1 and Neur2 are expressed in the whole adult brain and encode E3 ubiquitin ligases, which play a crucial role in the Notch signaling pathways. A previous study reported that overexpression of Neur1 enhances hippocampus-dependent memory, whereas the role of Neur2 remains largely unknown. This study aimed to elucidate the respective roles of Neur1 and Neur2 in hippocampus-dependent memory using three lines of genetically modified mice: Neur1 knock-out, Neur2 knock-out, and Neur1 and Neur2 double knock-out (D-KO). The results showed that spatial memory was impaired when both Neur1 and Neur2 were deleted, but not in the individual knock-out of either Neur1 or Neur2. In addition, basal synaptic properties estimated by input-output relationships and paired-pulse facilitation did not change, but a form of long-term potentiation that requires protein synthesis was specifically impaired in the D-KO mice. These results collectively suggest that Neur1 and Neur2 are crucially involved in hippocampus-dependent spatial memory and synaptic plasticity.
McKellar, C. E., Siwanowicz, I., Dickson, B. J. and Simpson, J. H. (2020). Controlling motor neurons of every muscle for fly proboscis reaching. Elife 9. PubMed ID: 32584254
This study describes the anatomy of all the primary motor neurons in the fly proboscis and characterize their contributions to its diverse reaching movements. Pairing this behavior with the wealth of Drosophila's genetic tools offers the possibility to study motor control at single-neuron resolution, and soon throughout entire circuits. As an entry to these circuits, detailed anatomy is provided of proboscis motor neurons, muscles, and joints. A collection of fly strains was created to individually manipulate every proboscis muscle through control of its motor neurons, the first such collection for an appendage. A model is generated of the action of each proboscis joint and found that only a small number of motor neurons are needed to produce proboscis reaching. Comprehensive control of each motor element in this numerically simple system paves the way for future study of both reflexive and flexible movements of this appendage.
Okubo, T. S., Patella, P., D'Alessandro, I. and Wilson, R. I. (2020). A Neural Network for Wind-Guided Compass Navigation. Neuron. PubMed ID: 32681825
Spatial maps in the brain are most accurate when they are linked to external sensory cues. This study shows that the compass in the Drosophila brain is linked to the direction of the wind. Shifting the wind rightward rotates the compass as if the fly were turning leftward, and vice versa. The mechanisms are described of several computations that integrate wind information into the compass. First, an intensity-invariant representation of wind direction is computed by comparing left-right mechanosensory signals. Then, signals are reformatted to reduce the coding biases inherent in peripheral mechanics, and wind cues are brought into the same circular coordinate system that represents visual cues and self-motion signals. Because the compass incorporates both mechanosensory and visual cues, it should enable navigation under conditions where no single cue is consistently reliable. These results show how local sensory signals can be transformed into a global, multimodal, abstract representation of space.

Wednesday, September 2nd - Enhancers and gene regulation

Immarigeon, C., Bernat-Fabre, S., Guillou, E., Verger, A., Prince, E., Benmedjahed, M. A., Payet, A., Couralet, M., Monte, D., Villeret, V., Bourbon, H. M. and Boube, M. (2020). Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo. J Biol Chem. PubMed ID: 32737196
The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? Previous work established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. This study observed mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. This study further shows that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp- dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, a direct interaction between Med19 and Med1 was shown by GST-pull-down experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit - TF partnerships are probably oversimplified models. Several mechanisms are proposed to account for the transcriptional regulation of GATAs-targeted genes.
Myasnikova, E. and Spirov, A. (2020). Gene regulatory networks in Drosophila early embryonic development as a model for the study of the temporal identity of neuroblasts. Biosystems: 104192. PubMed ID: 32619531
Genes belonging to the "gap" and "gap-like" family constitute the best-studied gene regulatory networks (GRNs) in Drosophila embryogenesis. Gap genes are a core of two subnetworks controlling embryonic segmentation: (hunchback, hb; Krüppel, Kr; giant, gt; and knirps, kni) and (hb; Kr; pou-domain, pdm; and, probably, castor, cas). Of particular interest is that (hb, Kr, pdm, cas) also specifies the temporal identity of stem cells, neuroblasts, in Drosophila neurogenesis. This GRN controls the sequential differentiation of neuroblasts during the asymmetric cell division. In the last decades, modeling of the patterning of gene ensemble (hb, Kr, gt, kni) in segmentation was in the center of attention. This study now shows that the previously published and extensively studied model at a certain level of external factors is able to reproduce temporal patterns of (hb, Kr, pdm, cas) in neurogenesis with minor evolutionary explicable modifications. This result testifies in favor of a hypothesis that the similarity of two gene ensembles active in segmentation and neurogenesis is a result of co-option of the network architecture in evolution from the common ancestral form. By means of the model dynamical analysis, it is shown that the establishment of the robust patterns in both systems could be explained in terms of the action of attractors in the gap gene dynamical system. This study formulates the common principles underlying the robustness of both GRNs in segmentation and neurogenesis due to the similar functional organization of the gene ensembles as having the same evolutionary origin.
Nagarkar, S., Wasnik, R., Govada, P., Cohen, S. and Shashidhara, L. S. (2020). Promoter Proximal Pausing Limits Tumorous Growth Induced by the Yki Transcription Factor in Drosophila. Genetics. PubMed ID: 32737120
Promoter proximal pausing (PPP) of RNA Polymerase II has emerged as a crucial rate-limiting-step in the regulation of gene expression. Regulation of PPP is brought about by complexes 7SK snRNP, P-TEFb (Cdk9/cycT) and the Negative Elongation Factor (NELF) which are highly conserved from Drosophila to humans. This study shows that RNAi-mediated depletion of bin3 or Hexim of the 7SK snRNP complex or depletion of individual components of the NELF complex enhance Yki-driven growth leading to neoplastic transformation of Drosophila wing imaginal discs. Increased CDK9 expression cooperates with Yki, in driving neoplastic growth. Interestingly, over-expression of CDK9 on its own or in the background of depletion of one of the components of 7SK snRNP or the NELF complex necessarily and specifically needed Yki over-expression to cause tumorous growth. Genome-wide gene expression analyses suggested that deregulation of protein homeostasis is associated with tumorous growth of wing imaginal discs. As both Fat/Hippo/Yki pathway and PPP are highly conserved, these observations may provide insights into mechanisms of oncogenic function of YAP, the orthologue of Yki in human.
Johnson, D. M., Wells, M. B., Fox, R., Lee, J. S., Loganathan, R., Levings, D., Bastien, A., Slattery, M. and Andrew, D. J. (2020). CrebA increases secretory capacity through direct transcriptional regulation of the secretory machinery, a subset of secretory cargo, and other key regulators. Traffic. PubMed ID: 32613751
Specialization of many cells, including the acinar cells of the salivary glands and pancreas, milk-producing cells of mammary glands, mucus-secreting goblet cells, antibody-producing plasma cells, and cells that generate the dense extracellular matrices of bone and cartilage, requires scaling up both secretory machinery and cell-type specific secretory cargo. Using tissue-specific genome-scale analyses, this study determined how increases in secretory capacity are coordinated with increases in secretory load in the Drosophila salivary gland (SG), an ideal model for gaining mechanistic insight into the functional specialization of secretory organs. The findings show that CrebA, a bZIP transcription factor, directly binds genes encoding the core secretory machinery, including protein components of the signal recognition particle and receptor, ER cargo translocators, Cop I and Cop II vesicles, as well as the structural proteins and enzymes of these organelles. CrebA directly binds a subset of SG cargo genes and CrebA binds and boosts expression of Sage, a SG-specific transcription factor essential for cargo expression. To further enhance secretory output, CrebA binds and activates Xbp1 and Tudor-SN. Thus, CrebA directly upregulates the machinery of secretion and additional factors to increase overall secretory capacity in professional secretory cells; concomitant increases in cargo are achieved both directly and indirectly.
Koromila, T., Gao, F., Iwasaki, Y., He, P., Pachter, L., Gergen, J. P. and Stathopoulos, A. (2020). Odd-paired is a pioneer-like factor that coordinates with Zelda to control gene expression in embryos. Elife 9. PubMed ID: 32701060
Pioneer factors such as Zelda (Zld) help initiate zygotic transcription in Drosophila early embryos, but whether other factors support this dynamic process is unclear. Odd-paired (Opa), a zinc-finger transcription factor expressed at cellularization, controls the transition of genes from pair-rule to segmental patterns along the anterior-posterior axis. Finding that Opa also regulates expression through enhancer sog_Distal along the dorso-ventral axis, it was hypothesized Opa's role is more general. Chromatin-immunoprecipitation (ChIP-seq) confirmed its in vivo binding to sog_Distal but also identified widespread binding throughout the genome, comparable to Zld. Furthermore, chromatin assays (ATAC-seq) demonstrate that Opa, like Zld, influences chromatin accessibility genome-wide at cellularization, suggesting both are pioneer factors with common as well as distinct targets. Lastly, embryos lacking opa exhibit widespread, late patterning defects spanning both axes. Collectively, these data suggest Opa is a general timing factor and likely late-acting pioneer factor that drives a secondary wave of zygotic gene expression.
Liu, Y., Barr, K. and Reinitz, J. (2020). Fully interpretable deep learning model of transcriptional control. Bioinformatics 36(Supplement_1): i499-i507. PubMed ID: 32657418
The universal expressibility assumption of Deep Neural Networks (DNNs) is the key motivation behind recent works in the systems biology community to employ DNNs to solve important problems in functional genomics and molecular genetics. Typically, such investigations have taken a 'black box' approach in which the internal structure of the model used is set purely by machine learning considerations with little consideration of representing the internal structure of the biological system by the mathematical structure of the DNN. DNNs have not yet been applied to the detailed modeling of transcriptional control in which mRNA production is controlled by the binding of specific transcription factors to DNA, in part because such models are in part formulated in terms of specific chemical equationsthat appear different in form from those used in neural networks. This paper gives an example of a DNN which can model the detailed control of transcription in a precise and predictive manner. Its internal structure is fully interpretable and is faithful to underlying chemistry of transcription factor binding to DNA. A DNN was derived from a systems biology model that was not previously recognized as having a DNN structure. Although the DNN was applied to data from the early embryo of the fruit fly Drosophila, this system serves as a test bed for analysis of much larger datasets obtained by systems biology studies on a genomic scale.

Tuesday, September 1st - Disease models

Jin, K., Wilson, K. A., Beck, J. N., Nelson, C. S., Brownridge, G. W., 3rd, Harrison, B. R., Djukovic, D., Raftery, D., Brem, R. B., Yu, S., Drton, M., Shojaie, A., Kapahi, P. and Promislow, D. (2020). Genetic and metabolomic architecture of variation in diet restriction-mediated lifespan extension in Drosophila. PLoS Genet 16(7): e1008835. PubMed ID: 32644988
In most organisms, dietary restriction (DR) increases lifespan. However, several studies have found that genotypes within the same species vary widely in how they respond to DR. To explore the mechanisms underlying this variation, 178 inbred Drosophila melanogaster lines were exposed to a DR or ad libitum (AL) diet, and a panel of 105 metabolites was measured under both diets. Twenty four out of 105 metabolites were associated with the magnitude of the lifespan response. These included proteinogenic amino acids and metabolites involved in α-ketoglutarate (α-KG)/glutamine metabolism. The role of &alpha-KG/glutamine synthesis pathways in the DR response through genetic manipulations. Covariance network analysis was used to investigate diet-dependent interactions between metabolites, identifying the essential amino acids threonine and arginine as "hub" metabolites in the DR response. Finally, a novel metabolic and genetic bipartite network analysis was employed to reveal multiple genes that influence DR lifespan response, some of which have not previously been implicated in DR regulation. One of these is CCHa2R, a gene that encodes a neuropeptide receptor that influences satiety response and insulin signaling. Across the lines, variation in an intronic single nucleotide variant of CCHa2R correlated with variation in levels of five metabolites, all of which in turn were correlated with DR lifespan response. Inhibition of adult CCHa2R expression extended DR lifespan of flies, confirming the role of CCHa2R in lifespan response. These results provide support for the power of combined genomic and metabolomic analysis to identify key pathways underlying variation in this complex quantitative trait.
Morton, D. J., Jalloh, B., Kim, L., Kremsky, I., Nair, R. J., Nguyen, K. B., Rounds, J. C., Sterrett, M. C., Brown, B., Le, T., Karkare, M. C., McGaughey, K. D., Sheng, S., Leung, S. W., Fasken, M. B., Moberg, K. H. and Corbett, A. H. (2020). A Drosophila model of Pontocerebellar Hypoplasia reveals a critical role for the RNA exosome in neurons. PLoS Genet 16(7): e1008901. PubMed ID: 32645003
The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, tissue-specific RNAi drivers were used. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, CRISPR/Cas9 gene editing technology was used to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.
Molon, M., Dampc, J., Kula-Maximenko, M., Zebrowski, J., Molon, A., Dobler, R., Durak, R. and Skoczowski, A. (2020). Effects of Temperature on Lifespan of Drosophila melanogaster from Different Genetic Backgrounds: Links between Metabolic Rate and Longevity. Insects 11(8):E470. PubMed ID: 32722420
Despite many studies of the aging process, questions about key factors ensuring longevity have not yet found clear answers. Temperature seems to be one of the most important factors regulating lifespan. However, the genetic background may also play a key role in determining longevity. The aim of this study was to investigate the relationship between the temperature, genetic background (fruit fly origin), and metabolic rate on lifespan. Experiments were performed with the use of the wild type Drosophila melanogaster fruit flies originating from Australia, Canada, and Benin and the reference OregonR strain. The metabolic rate of D. melanogaster was measured at 20 °C, 25 °C, and 28 °C in an isothermal calorimeter. A strong negative relationship was found between the total heat flow and longevity. A high metabolic rate leads to increased aging in males and females in all strains. Furthermore, the results showed that temperature has a significant effect on fecundity and body weight. The usefulness of the isothermal calorimetry method to study the effect of environmental stress conditions on the metabolic activity of insects was also shiown. This may be particularly important for the forecasting of impact of global warming on metabolic activity and lifespan of various insects.
Haghi, M., Masoudi, R. and Najibi, S. M. (2020). Distinctive alteration in the expression of autophagy genes in Drosophila models of amyloidopathy and tauopathy. Ups J Med Sci: 1-9. PubMed ID: 32657227
Alzheimer's disease (AD) is one the most common types of dementia. Plaques of amyloid beta and neurofibrillary tangles of tau are two major hallmarks of AD. Metabolism of these two proteins, in part, depends on autophagy pathways. Autophagy dysfunction and protein aggregation in AD may be involved in a vicious circle. The aim of this study was to investigate whether tau or amyloid beta 42 (Aβ42) could affect expression of autophagy genes, and whether they exert their effects in the same way or not. Misxpression levels of some autophagy genes, Hook, Atg6, Atg8, and Cathepsin D, were measured using quantitative PCR in transgenic Drosophila melanogaster expressing either Aβ42 or Tau R406W. Hook mRNA levels were downregulated in Aβ42-expressing flies both 5 and 25 days old, while they were increased in 25-day-old flies expressing Tau R406W. Both Atg6 and Atg8 were upregulated at day 5 and then downregulated in 25-day-old flies expressing either Aβ42 or Tau R406W. Cathepsin D expression levels were significantly increased in 5-day-old flies expressing Tau R406W, while there was no significant change in the expression levels of this gene in 5-day-old flies expressing Aβ42. Expression levels of Cathepsin D were significantly decreased in 25-day-old transgenic flies expressing Tau R406W or Aβ42. CONCLUSION: It is concluded that both Aβ42 and Tau R406W may affect autophagy through dysregulation of autophagy genes. Interestingly, it seems that these pathological proteins exert their toxic effects on autophagy through different pathways and independently.
King, L. B., Boto, T., Botero, V., Aviles, A. M., Jomsky, B. M., Joseph, C., Walker, J. A. and Tomchik, S. M. (2020). Developmental loss of neurofibromin across distributed neuronal circuits drives excessive grooming in Drosophila. PLoS Genet 16(7): e1008920. PubMed ID: 32697780
Neurofibromatosis type 1 is a monogenetic disorder that predisposes individuals to tumor formation and cognitive and behavioral symptoms. The neuronal circuitry and developmental events underlying these neurological symptoms are unknown. To better understand how mutations of the underlying gene (NF1) drive behavioral alterations, grooming was examined in the Drosophila neurofibromatosis 1 model. Mutations of the fly NF1 ortholog drive excessive grooming, and increased grooming was observed in adults when Nf1 was knocked down during development. Furthermore, intact Nf1 Ras GAP-related domain signaling was required to maintain normal grooming. The requirement for Nf1 was distributed across neuronal circuits, which were additive when targeted in parallel, rather than mapping to discrete microcircuits. Overall, these data suggest that broadly-distributed alterations in neuronal function during development, requiring intact Ras signaling, drive key Nf1-mediated behavioral alterations. Thus, global developmental alterations in brain circuits/systems function may contribute to behavioral phenotypes in neurofibromatosis type 1.
Mahoney, R., Ochoa Thomas, E., Ramirez, P., Miller, H. E., Beckmann, A., Zuniga, G., Dobrowolski, R. and Frost, B. (2020).. Pathogenic Tau Causes a Toxic Depletion of Nuclear Calcium. Cell Rep 32(2): 107900. PubMed ID: 32668249
Synaptic activity-induced calcium (Ca(2+)) influx and subsequent propagation into the nucleus is a major way in which synapses communicate with the nucleus to regulate transcriptional programs important for activity-dependent survival and memory formation. Nuclear Ca(2+) shapes the transcriptome by regulating cyclic AMP (cAMP) response element-binding protein (CREB). This study utilized a Drosophila model of tauopathy and induced pluripotent stem cell (iPSC)-derived neurons from humans with Alzheimer's disease to study the effects of pathogenic tau, a pathological hallmark of Alzheimer's disease and related tauopathies, on nuclear Ca(2+). Pathogenic tau was found to deplete nuclear Ca(2+) and CREB to drive neuronal death, that CREB-regulated genes are over-represented among differentially expressed genes in tau transgenic Drosophila, and it was found that activation of big potassium (BK) channels elevates nuclear Ca(2+) and suppresses tau-induced neurotoxicity. These studies identify nuclear Ca(2+) depletion as a mechanism contributing to tau-induced neurotoxicity, adding an important dimension to the calcium hypothesis of Alzheimer's disease.
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