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. 2019 Sep 23;8:e48056. doi: 10.7554/eLife.48056

Figure 6. Mamo maintains α’/β’ MB neuron markers.

(A) Scheme for temperature shift assay. Temperature-sensitive GAL80 was inactivated for 21 days after adult eclosion. E = embryo, L = larva, p=pupa, A = adult (A’) MB cell body marker expression of the three MB neuron types. (B–D) Adult MB cell bodies (OK107 > GFP+tub-GAL80ts) immunostained for GFP, Abrupt, and Trio. Colored arrows identify MB neuron types based on marker expression: red=γ (TrioPM/Abrupt+), orange=α’/β’ (TrioPM,Cyto /Abrupt-), blue=α/β (Trio-/Abrupt-). Images are representative of n > 6. A single focal plane is shown. Scale bar = 20 μm. Notice loss of cytoplasmic Trio staining after depleting expressing mamo-RNAi (C and C’, green arrows = TrioPM/Abrupt- neurons). Overexpressing Mamo (mamo-GOF) after adult eclosion results in increased numbers of TrioPM,Cyto/Abrupt- neurons (green filled area indicates MB calyx) (D and D’). (E) Percent of MB neurons expressing both cytoplasmic and plasma membrane Trio in orange (TrioPM,Cyto/Abrupt-) and percent of neurons expressing only plasma membrane Trio (TrioPM/Abrupt-) in green (mean ± SEM, n = 3 brains). Time course of Mamo depletion with adult induced RNAi is shown in Figure 6—figure supplement 1.

Figure 6—source data 1. Quantification of Trio expression (PM, Cyto).
elife-48056-fig6-data1.xlsx (9.7KB, xlsx)
DOI: 10.7554/eLife.48056.027

Figure 6.

Figure 6—figure supplement 1. Temperature shift assay for effectively repressing Mamo.

Figure 6—figure supplement 1.

(A and B) Scheme for temperature shift assay. Heat shock inactivated the temperature-sensitive GAL80 for 7 days after adult eclosion (A) and for 21 days after adult eclosion (B). (C–E) Adult MB cell bodies (OK107 > GFP+tub-GAL80ts) immunostained for GFP (green) and Mamo (magenta) and Trio (cyan). Colored arrows identify MB neuron types red=γ (Trio+PM/Mamo+low ), orange=α’/β’ (Trio+PM,Cyto/ Mamo+high), blue=α/β (Trio-/Mamo-) based on marker expression. Images are representative of n > 6. A single focal plane is shown. Scale bar = 20 μm. Depleting mamo (mamo-RNAi) for 7 days after adult eclosion does not eliminate Trio+PM,Cyto staining (E’). Most Mamo staining is still visible (E). Depleting mamo (mamo-RNAi) for 21 days after adult eclosion shows effectively diminish Mamo expression and shows qualitative reduction of Trio+PM,Cyto staining (D’ and Figure 6C and C’).
Figure 6—figure supplement 2. Mamo stimulates α’/β’ specific gene expression.

Figure 6—figure supplement 2.

(A) Scheme for temperature shift assay. Heat shock inactivated the temperature-sensitive GAL80 for 7 days at 2 days after pupal formation. (B–C) Adult MB cell bodies (OK107 > GFP+tub-GAL80ts) immunostained for GFP (green) and Abrupt (magenta) and Trio (cyan). Colored arrows identify MB neuron types red=γ ( Trio+PM/Mamo+low ), orange=α’/β’ (Trio+PM,Cyto/ Mamo+high), blue=α/β (Trio-/Mamo-) based on marker expression. Images are representative of n > 6. A single focal plane is shown. Scale bar = 20 μm. Transgene induction at pupal development was more effective at increasing TrioPM,Cyto/Abrupt- cells (C and C’).