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WIREs Dev Biol
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Generating mosaics for lineage analysis in flies

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By generating and studying mosaic organisms, we are learning how intricate tissues form as cells proliferate and diversify through organism development. FLP/FRT‐mediated site‐specific mitotic recombination permits the generation of mosaic flies with efficiency and control. With heat‐inducible or tissue‐specific FLP transgenes at our disposal, we can engineer mosaics carrying clones of homozygous cells that come from specific pools of heterozygous precursors. This permits detailed cell lineage analysis followed by mosaic analysis of gene functions in the underlying developmental processes. Expression of transgenes (e.g., reporters) only in the homozygous cells enables mosaic analysis in the complex nervous system. Tracing neuronal lineages by using mosaics revolutionized mechanistic studies of neuronal diversification and differentiation, exemplifying the power of genetic mosaics in developmental biology. WIREs Dev Biol 2014, 3:69–81. doi: 10.1002/wdev.122 This article is categorized under: Nervous System Development > Flies Technologies > Generating Chimeras and Lineage Analysis

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Timeline of Drosophila genetic chimeras and cell lineage analysis. Major advances in Drosophila cell lineage analysis through gynandromorphy (within blue box), irradiation‐induced mitotic recombination (within orange box), or FLP/FRT‐mediated site‐specific recombination (within green box) are shown chronologically. All the modern genetic mosaic tools were established via germ line transformation, which was made possible by Rubin and Spradling in 1982.
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Loss‐of‐function mosaic analysis of neuronal temporal identity. (a–d) In the wild‐type adAL lineage, the VA7l and VA2 uniglomerular PNs are born sequentially (a), as twin‐spot MARCM labeling (b) shows pairing of the smallest VA2‐containing NB clone (c) with the VA7l‐targeting PN (d). (e–h) By contrast, the Kr mutant single‐neuron GMC clone (green) paired with the equivalent VA2‐containing wild‐type NB clone (magenta) redundantly innervates the VA2 glomerulus (e–g). This chronologically abnormal phenotype argues for the acquisition of the next VA2 temporal cell fate by the prospective VA7l PN (h). Scale bar: 10 µm.
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High‐resolution neuronal lineage analysis by twin‐spot MARCM. (a) The adult Drosophila brain (gray) carries neurons with different characteristic morphologies that arise in clones from specific NBs. Exemplified in this illustration are representative neurons present in two antennal lobe (AL) NB clones (dashed circumferences). The anterodorsal AL (adAL) lineage yields distinct uniglomerular projection neurons (PNs), which each target one of the 50 or so AL glomeruli and then project through the MB calyx into the LH (e.g., the right‐hemisphere blue neuron). Besides making uniglomerular PNs that target a different set of AL glomeruli (e.g., the left‐hemisphere blue neuron), the lateral AL (lAL) lineage generates diverse AL local interneurons (LNs, e.g., the orange neuron) plus various non‐AL PNs (e.g., the green neuron) that connect other neuropils. To map individual neurons and further elucidate the origins of neuron diversity requires tracking single neurons serially made by a common NB. (b–d) Systematic analysis of GMC clones (red) paired with NB clones (green) of different sizes allows mapping of the serially derived neurons in the adAL lineage. Only one of the sister neurons derived from each adAL GMC survives into the adult stage, as the NB clones consistently associate with a single‐neuron GMC clone. Notably, the uniglomerular adAL neurons that innervate different AL glomeruli are born sequentially in a defined order from the adAL NB. For instance, the VM3‐, 1‐, and DL2v‐targeting neurons are born at the early (b), middle (c), and late (d) stages, respectively. Scale bar: 10 µm. (e) The lAL NB clone (cell bodies marked with black asterisks) exhibits complex morphologies. Differential labeling of twin neurons derived from common GMCs consistently reveals one PN (magenta) paired with one LN (green) in the lAL lineage. Distinct PN/LN pairs are selectively recovered following clone induction (blue asterisks) in different two‐hour windows after larval hatching (ALH), suggesting neuron fate specification based on the GMC birth order and the A/B binary fate decision. Scale bar: 10 µm.
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Twin‐spot unique labeling techniques. (a) Twin‐spot MARCM involves two repressible marker genes (UAS‐RFP vs UAS‐GFP) that are respectively silenced by a corresponding transgenic miRNA (UAS‐RFPi vs UAS‐GFPi) in heterozygous cells. miRNAs (little Pacman eaters) repress the expression of reporters in heterozygous cells. Opposing de‐repression of either marker gene can occur in the sister cells derived from a heterozygous precursor, when the miRNA transgenes residing distal to FRTs (orange triangles) are differentially lost. (b) Twin‐spot generator allows reconstitution of distinct reporter genes (GFP vs RFP) following site‐specific recombination, which can be segregated into distinct daughter cells for differential labeling. Note the heterozygous mother cell is shown in light yellow to indicate the fact that recombination without segregation could result in co‐expression of both reporters in heterozygous cells. (c) Coupled MARCM involves two independent pairs of transcriptional activators and repressors. GAL80 (GAL4 repressor) and QS (QF repressor) reside on the apposing homologous chromosome arms distal to FRTs. Mitotic recombination leads to respective loss of GAL80 and QS from the two distinct homozygous daughter cells that can be differentially labeled by GAL4‐dependent UAS‐GFP versus QF‐dependent QUAS‐RFP.
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MARCM and discrete sizes of neural clones. (a) MARCM allows the derivation of homozygous mutant cells devoid of the GAL80 transgene (plum) following site‐specific mitotic recombination. Depending on the GAL4 (blue) expression pattern, subsets of GAL80‐negative mutant cells can be uniquely labeled by UAS‐reporter (green). (b) A neuronal lineage forms as one neuroblast (NB) repeatedly buds off ganglion mother cells (GMC) that each divide once to produce two post‐mitotic neurons which often acquire distinct binary cell fates (A vs B) due to differential Notch signaling. Given this general pattern of neurogenesis, MARCM allows labeling of either a multicellular NB clone or a two‐cell GMC clone following mitotic recombination in a dividing NB and marking of either A or B neuron as a single‐neuron clone if mitotic recombination occurs in a dividing GMC. Note only post‐mitotic neurons are marked in the panels given the transient nature of precursors. (c) Dual‐expression‐control MARCM allows differential labeling of distinct populations of GAL80‐minus cells via use of two independent GAL80‐repressible binary transgene induction systems (e.g., GAL4/UAS and LexA::GAD/lexAop). Green: GAL4+ and LexA::GAD–; red: GAL4– and LexA::GAD+; yellow: GAL4+ and LexA::GAD+.
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Genetic mosaics by site‐specific mitotic recombination. (a) A heterozygous parental cell can give rise to two distinct daughter cells homozygous for different alleles of genes and transgenes residing distal to the site of mitotic recombination (orange triangles: FRTs; recombination between FRTs requires FLP). The resulting homozygous daughter cells and their offspring can be distinguished from the surrounding heterozygous cells based on the copy number of the marker transgene (green). One can further enrich clones homozygous for the recessive x mutation by incorporating a dominant slow‐growing Y mutation onto the otherwise wild‐type homologous chromosome arm. (b) A schematic heterozygous wing disc (light green) carries a large x/x mutant clone (unmarked) paired with a tiny Y/Y sister clone (dark green). The mutant clone shows growth advantages due to the absence of the dominant slow‐growth Y mutation.
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