Lineage-associated tracts defining the anatomy of the Drosophila first instar larval brain

AbstractFixed lineages derived from unique, genetically specified neuroblasts form the anatomical building blocks of the Drosophila brain. Neurons belonging to the same lineage project their axons in a common tract, which is labeled by neuronal markers. In this paper, we present a detailed atlas of the lineage-associated tracts forming the brain of the early Drosophila larva, based on the use of global markers (anti-Neuroglian, anti-Neurotactin, inscuteable-Gal4>UAS-chRFP-Tub) and lineage-specific reporters. We describe 68 discrete fiber bundles that contain axons of one lineage or pairs/small sets of adjacent lineages. Bundles enter the neuropil at invariant locations, the lineage tract entry portals. Within the neuropil, these fiber bundles form larger fascicles that can be classified, by their main orientation, into longitudinal, transverse, and vertical (ascending/descending) fascicles. We present 3D digital models of lineage tract entry portals and neuropil fascicles, set into relationship to commonly used, easily recognizable reference structures such as the mushroom body, the antennal lobe, the optic lobe, and the Fasciclin II-positive fiber bundles that connect the brain and ventral nerve cord. Correspondences and differences between early larval tract anatomy and the previously described late larval and adult lineage patterns are highlighted. Our L1 neuro-anatomical atlas of lineages constitutes an essential step towards following morphologically defined lineages to the neuroblasts of the early embryo, which will ultimately make it possible to link the structure and connectivity of a lineage to the expression of genes in the particular neuroblast that gives rise to that lineage. Furthermore, the L1 atlas will be important for a host of ongoing work that attempts to reconstruct neuronal connectivity at the level of resolution of single neurons and their synapses

G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila.

We combined Gal4-UAS and the FLP recombinase-FRT and fluorescent reporters to generate cell clones that provide spatial, temporal and genetic information about the origins of individual cells in Drosophila melanogaster. We named this combination the Gal4 technique for real-time and clonal expression (G-TRACE). The approach should allow for screening and the identification of real-time and lineage-traced expression patterns on a genomic scale

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Postembryonic neurogenesis in the optic lobe and central brain of Drosophila melanogaster

Neurogenesis or the proper formation of the nervous system requires three distinct phases: (1) early neurulation involving progenitor proliferation and specification; (2) progenitor migration and extension of fibers; and (3) neural differentiation and connectivity establishment. How connectivity is established remains to be poor understood. We investigated neurogenesis of the central brain and visual processing center in Drosophila. In Drosophila central nervous system (or central brain), there are 100 lineages, each derived from a single neuroblast; where neurons of one lineage remain in close proximity to their mother neuroblast. As described in the Appendix, we used cell type specific markers combined with global neuronal markers to serve as local landmark and mapped out how individual neuroblast lineages progress during development where gross anatomical changes are described. The fly optic lobe, the visual system processing center, is also highly modular. We show that early neurogenesis in the optic lobe is remarkably similar to vertebrates, following a “conveyor belt neurogenesis” (Ch. 4) and show that Jak/Stat and Notch negatively regulates epithelium-to-neuroblast conversion, a non-canonical mode of neurogenesis (Ch. 2). To further gain insights on how connectivity is established in the optic lobe, we reconstructed the global architecture and connectivity of the optic lobe at sequential stages of development. Our analysis reveals three major structural/developmental hallmarks by which the optic lobe, compared to other regions of the fly brain, stands out: large scale neuronal movements, correlated temporal gradients in neuron production and differentiation, highly ordered retinotopic projections in between visual neuropils, and the formation of multiple layers within these neuropils (Ch. 3). The works described in Ch. 2-5 and Appendix 1-3 serve an important platform for understanding how the nervous system is formed in Drosophila

Postembryonic neurogenesis in the optic lobe and central brain of Drosophila melanogaster

Neurogenesis or the proper formation of the nervous system requires three distinct phases: (1) early neurulation involving progenitor proliferation and specification; (2) progenitor migration and extension of fibers; and (3) neural differentiation and connectivity establishment. How connectivity is established remains to be poor understood. We investigated neurogenesis of the central brain and visual processing center in Drosophila. In Drosophila central nervous system (or central brain), there are 100 lineages, each derived from a single neuroblast; where neurons of one lineage remain in close proximity to their mother neuroblast. As described in the Appendix, we used cell type specific markers combined with global neuronal markers to serve as local landmark and mapped out how individual neuroblast lineages progress during development where gross anatomical changes are described. The fly optic lobe, the visual system processing center, is also highly modular. We show that early neurogenesis in the optic lobe is remarkably similar to vertebrates, following a “conveyor belt neurogenesis” (Ch. 4) and show that Jak/Stat and Notch negatively regulates epithelium-to-neuroblast conversion, a non-canonical mode of neurogenesis (Ch. 2). To further gain insights on how connectivity is established in the optic lobe, we reconstructed the global architecture and connectivity of the optic lobe at sequential stages of development. Our analysis reveals three major structural/developmental hallmarks by which the optic lobe, compared to other regions of the fly brain, stands out: large scale neuronal movements, correlated temporal gradients in neuron production and differentiation, highly ordered retinotopic projections in between visual neuropils, and the formation of multiple layers within these neuropils (Ch. 3). The works described in Ch. 2-5 and Appendix 1-3 serve an important platform for understanding how the nervous system is formed in Drosophila

Spatio-temporal pattern of neuronal differentiation in the Drosophila visual system: A user’s guide to the dynamic morphology of the developing optic lobe

Visual information processing in animals with large image forming eyes is carried out in highly structured retinotopically ordered neuropils. Visual neuropils in Drosophila form the optic lobe, which consists of four serially arranged major subdivisions; the lamina, medulla, lobula and lobula plate; the latter three of these are further subdivided into multiple layers. The visual neuropils are formed by more than 100 different cell types, distributed and interconnected in an invariant highly regular pattern. This pattern relies on a protracted sequence of developmental steps, whereby different cell types are born at specific time points and nerve connections are formed in a tightly controlled sequence that has to be coordinated among the different visual neuropils. The developing fly visual system has become a highly regarded and widely studied paradigm to investigate the genetic mechanisms that control the formation of neural circuits. However, these studies are often made difficult by the complex and shifting patterns in which different types of neurons and their connections are distributed throughout development. In the present paper we have reconstructed the three-dimensional architecture of the Drosophila optic lobe from the early larva to the adult. Based on specific markers, we were able to distinguish the populations of progenitors of the four optic neuropils and map the neurons and their connections. Our paper presents sets of annotated confocal z-projections and animated 3D digital models of these structures for representative stages. The data reveal the temporally coordinated growth of the optic neuropils, and clarify how the position and orientation of the neuropils and interconnecting tracts (inner and outer optic chiasm) changes over time. Finally, we have analyzed the emergence of the discrete layers of the medulla and lobula complex using the same markers (DN-cadherin, Brp) employed to systematically explore the structure and development of the central brain neuropil. Our work will facilitate experimental studies of the molecular mechanisms regulating neuronal fate and connectivity in the fly visual system, which bears many fundamental similarities with the retina of vertebrates

G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila

We combine Gal4/UAS, FLP/FRT and fluorescent reporters to generate cell clones that provide spatial, temporal, and genetic information about the origins of individual cells in Drosophila. We name this combination the Gal4 Technique for Real-time and Clonal Expression (G-TRACE). The approach should allow for screening and the identification of real-time and lineage-traced expression patterns on a genomic scale