Extended Data figure 4: Newly identified tTFs are expressed temporally.

(a) Antibody staining (frontal view) against five of the eight new temporal transcription factors, Opa (red), Erm (blue), Hbn (green), Scro (white), and BarH1 (green), shown in different combinations.

(b) Antibody staining against Opa, Hth, Ey, and Arm (which marks all cells). Opa is expressed in two waves, one succeeding and partially overlapping with the Hth window and one later partially overlapping with Ey. The expression of Opa covers the previous “gap” between Hth- and Ey-expressing neuroblasts.

(c) Antibody staining against Erm and Ey. Erm starts being expressed before Ey, partially overlapping with it (magenta arrowheads).

(d) Antibody staining against Hbn and Ey. Hbn expression begins slightly after Ey and overlaps almost completely with it. Ey-positive, Hbn-negative neuroblasts are indicated with arrowheads.

(e) Antibody staining against Oaz and D. Oaz expression precedes the expression of D.

(f) Antibody staining against Dpn, Oaz, BarH1, and Arm (which marks all cells). BarH1 is expressed after Oaz.

(g) Antibody staining against BarH1 and Tll. BarH1 is expressed before and overlaps with Tll.

(h) Fluorescent in situ hybridization against dpn, Oaz, BarH1 and BarH2. BarH2 is expressed in the same neuroblasts as BarH1, after Oaz has stopped being expressed. Hoechst marks all cells.

(i) Hbn is expressed between the two Opa temporal windows in neurons, while BarH1 is expressed after the second Opa temporal window. Hbn is also expressed in later born neurons, after the second Opa wave, and is lost from the first lamina (in between Opa) as neurons mature.

Scale bars: 10 μm