Abstract
The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. We developed new genetic tools that target neuroblasts and their diverse descendants, increasing our ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. We successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, we expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. Our transgenic tools can be combined to map individual neurons in specific lineages of various genotypes.
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Acknowledgements
We thank Janelia Farm fly core, especially M. Mercer, and Janelia Farm Fly Light, including R. Johnston, E. Willis, S. Tae and R. Vorimo, for their essential technical support. We thank S. Murphy, T. Safford, K. Rokicki, E. Trautman, Y. Yu and the rest of the team that developed the Janelia Farm fly brain workstation for streamlining our confocal image analysis. We thank J. Truman and G. Rubin for helpful discussions and sharing imagery of GR GAL4 lines before publication, and C. Sullivan for administrative support. This work was supported by Howard Hughes Medical Institute and the US National Institutes of Health.
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T.A., C.-F.K., Y.-J.L. and C.-P.Y. designed and conducted the experiments, collected and analyzed the data, and prepared the manuscript. Y.H., B.D.P. and H.L. made DNA constructs. X.J., Y.-F.H. and Y.H. produced data. M.D.S. assisted with data analysis and paper writing. A.K., T.B. and W.F.O. provided the dpn enhancer. C.T.Z. assisted with data production and paper writing. T.L. conceived the project, designed the experiments, analyzed the data and wrote the paper.
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Supplementary Figure 1 Twin-spot MARCM transgenes for GAL4 or LexA drivers
(a) Illustration of genetically simplified twin-spot MARCM compatible with GAL4 or LexA drivers. UAS or lexAop transgenes, carrying a gypsy-insulated spacer between reporter1 and silencer2 or reporter2 and silencer1, are placed in trans on the homologous chromosome arms distal to FRTs. Mitotic recombination across FRTs followed by X segregation of the recombinant chromosomes would result in respective expression of reporter1 and reporter2 in the distinct homozygous daughter cells (e.g. mutant versus wild type) due to their loss of silencer1 or silencer2. (b) With twin-spot MARCM, one can label twin neurons (N) made by one GMC (G) in distinct colors or create a multi-cellular NB clone paired with the neuron(s) derived from the preceding GMC for detailed sublineage analysis. (c) The above UAS and lexAop twin-spot transgenes were inserted into the indicated phiC31 integration sites on four major chromosome arms.
Supplementary Figure 2 Early-larval-derived NB clones of the ten R13C01^dpn-hit lineages
NB clones of the indicated lineages frequently hit by R13C01^dpn were obtained following clone induction at the indicated hours after larval hatching (left panels; 165 mosaic brains were examined), and compared to the ‘full-size’ NB clones of the same lineages reported by Yu et al., 2013 (right panels). The ALl1 (a), VLPl1 (b), VLPl4 (c), and VLPl2 (d) NB clones pair with a green GMC clone (green) and appear full in size. Other NB clones, FLAa2 (e), EBa1 (f), VLPa1 (g), VLPa2 (h), LHl1 (i), and LHl4 (j), show missing of some elaborations (arrows) and their paired GMC clones could not be found, indicating late onset of the lineage-restricted driver in these six lineages. Note presence of background clones (VLPa2 & SEG) in the VLPl2 sample (d). Scale bar: 50 μm.
Supplementary Figure 3 Birth timing of VLPl4 NB clones that exist alone or associate with distinct classes of VLPl4 single-neuron clones
Various numbers of VLPl4 NB clones (Y axis) were obtained following clone induction in specific two-hour windows (X axis). Colors indicate their association with distinct classes of single neurons that primarily target AVLP (a), PVLP (B), LO/PVLP (C), SCL/SIP/SLP (D), and LH/SLP/SIP (E), respectively. ~40 adult brains were analyzed in each two-hour window, except 28-30 hours ALH (#). Four NB clones (*) carry significantly more neuronal cell bodies than those induced in the same window, probably due to the birth of such clones prior to the recorded time of clone induction. * at 60ALH: 83 vs. 67.3±4.0; * at 70ALH: 100 vs. 55.1±3.2; * at 78ALH: 60 vs. 44.8±4.3; * at 8BPF: 93 vs. 20.4±2.6.
Supplementary Figure 4 General genetic schemes for targeted MARCM techniques
Multiple transgenic elements are first assembled on one of the three major chromosomes via homologous recombination. Parental strains carrying various recombinant chromosomes are then established by standard fly genetics. Crossing the parental strains of distinct genotypes yields heterozygous organisms with a complete set of transgenic elements required for targeted MARCM labeling.
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Awasaki, T., Kao, CF., Lee, YJ. et al. Making Drosophila lineage–restricted drivers via patterned recombination in neuroblasts. Nat Neurosci 17, 631–637 (2014). https://doi.org/10.1038/nn.3654
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DOI: https://doi.org/10.1038/nn.3654
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