1. Cell Class-Lineage Analysis Reveals Sexually Dimorphic Lineage Compositions in the Drosophila Brain 
Qingzhong Ren, Takeshi Awasaki, Yu-Fen Huang, Zhiyong Liu, Tzumin Lee 
Current Biology 
Volume 26, Issue 19, Pages (October 2016)
DOI: /j.cub
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2. Figure 1 The CLIn System for Cell-Class Lineage Intersection
(A) Schematic illustration showing how CLIn allows one to derive a GAL4 sub-pattern based on cell lineage by removing GAL80 from a given subset of NBs. GAL80 is selectively lost in those NBs expressing recombinase KD (orange), through intermediate steps of the KD/KDRT system and the Cre/loxP system. Loss of GAL80 leads to activation of a ubiquitous LexA::p65 driver in all of the descendants and marked with lexAop-controlled reporter-A (magenta), among which those positive for GAL4 are further marked with UAS-controlled reporter-B (green). dpn, pan NB promotor. Actin: the actin driver is transcriptionally active in all cell types.
(B) Expression pattern of R83D12-GAL4 driving membrane marker (UAS-mCD8GFP, green) shown in adult fly brains, counterstained with nc82 mAb (blue). n = 5.
(C–C″) Composite confocal images of an adult fly brain with R44F03-KD-positive NB lineages (magenta) and R83D12-GAL4-positive neurons (green). (C′) is a merged view of (C) and (C″). A small number of R83D12-GAL4-positive neurons, which extend from specific antennal lobe (AL) glomeruli to the mushroom body calyx (MBc) and the lateral horn (LH), are retained after intersecting with the R44F03-KD-targeted NB lineages, including four AL lineages. Arrowhead indicates cell bodies. n = 10.
(D–D″) Composite confocal images of an adult fly brain with stg14-KD-expressing NB lineages (magenta) and the R83D12-GAL4-positive neurons (green). (D′) is a merged view of (D) and (D″), with (D″) showing the boxed regions in (D′). A subset of R83D12-GAL4-positive neurons was retained after intersecting with the stg-KD-targeted, primarily type II, NB lineages. These neurons innervate the protocerebral bridge (PB), lower layers of the fan-shaped body (FB) and the noduli (NO) in the central complex. n = 9.
Scale bar, 50 μm. See also Table S1.
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3. Figure 2
TH-Positive Neurons Made by Specific Type II NBs and Lineage-Temporal Mapping of These Neurons
(A) Expression pattern of TH-GAL4 (green) in nc82-counterstained adult fly brain (blue).
(B) Marking TH-GAL4-positive neurons of all type II NB lineages targeted with stg-KD.
(A′ and B′) Partial projections of the cell body regions of (A) and (B). Individual cell clusters were outlined by circle.
(C) The genetics schemes to mark individual type II lineages by a ubiquitous LexA driver and its cell-class component by a class-specific GAL4 driver. The stg14 promoter is active mostly in type II NBs of the central brain. The KD recombinase will kickout the KDRT cassette in hs-ATG>KOT>FLP (see Figure S1A for its detailed design) and an intact FLP (blue) is reconstituted under the control of heat shock (hs) promoter. Transient hs induction of FLP will stochastically flip-out the flip-out cassette between pan-NB dpn promoter and Cre (yellow), which, in turn, generates individual NB clones that is positive for LexA::p65 (magenta) and negative for GAL80. Thus, only GAL4 positive cells within the GAL80– lineage are selectively relieved from depression by GAL80 and further marked by UAS-controlled reporter (green).
(D–G′) Composite confocal images of adult fly brains with the offspring of a particular type II NB or its first larval-born INP labeled in magenta, in which TH-GAL4-positive neurons (if any) are further marked in green. The magenta/green and green-only views of the same brains, counterstained with nc82 mAb (blue), are shown side by side. Clusters of neuronal cell bodies (arrowheads) and their cell counts are indicated. Judging from cell numbers (Table 1), the known PPM3-cluster TH neurons exclusively arise from the larval DM6 lineage (D and D′) and most of the PPL1-cluster TH neurons originate from the larval DL1 and DL2 lineage (F–G′). Other larval type II lineages yield 0–1 TH neurons (see Figure S2).
(H–K) TH-GAL4-positive neurons labeled since various hours after larval hatching (ALH). This experiment utilizes 2-hr heat shock (hs) aiming to induce CLIn clones in the targeted NB and its recently derived neuronal precursors present during the stage-specific clone induction. Labeling all of the progeny born after various developmental times can allow one to deduce when a given neuronal subset, marked by the neuronal class driver in CLIn, was born over the ∼100 hr of larval development. The major time window for (H and I) was ALH8–20, (J) ALH16–30, and (K) ALH24–30.
(L–O) Images showing the deduced developmental sequence of DL2-TH neurons.
Scale bar, 50 μm. See also Figures S1 and S2 and Tables S1, S2, and S3.
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4. Figure 3
Blocking Apoptosis throughout Lineage Development Reveals Gender-Dependent Neuronal Lineages
Composite confocal images of adult fly brains carrying various CLIn clones of the DM4 lineage. All cells of the CLIn clones are labeled in magenta, and the cells positive for dsxGAL4 (pC1 cluster) or fruGAL4 (pMP-e cluster) are further marked in green. The clone identity (DM4) and the transgene used to block apoptosis throughout lineage development (A″–D″) are indicated. Notably, male versus female lineages show gender-dependent fruGAL4 neuronal numbers and/or trajectories after blocking apoptosis. Scale bar, 50 μm. See also Figures S3–S5 and Tables S1, S2, and S4.
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5. Figure 4
Both Lineage and Apoptosis Contribute to Final Sexual Dimorphism
Schematic illustration of conventional (left) versus revised (right) views on the origin of sexually dimorphic neuronal lineages. Classical views argue for the production of the same numbers of dsx- or fru-expressing neurons that undergo gender-dependent apoptosis to derive sexually dimorphic lineages. Blocking apoptosis throughout lineage development, by contrast, reveals the ability of male versus female NBs to produce distinct numbers of dsx- or fru-expressing neurons. It ascribes the origin of brain sexual dimorphism to both gender-dependent neurogenesis and differential progeny apoptosis.
Current Biology  , DOI: ( /j.cub )
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