Inst Erm and Ey. Erm starts becoming expressed just before Ey, partially overlapping with it (magenta arrowheads). (d) Antibody staining against Hbn and Ey. Hbn expression begins slightly soon after Ey and overlaps just about fully 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 soon 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 within the identical neuroblasts as BarH1, after Oaz has stopped getting expressed. Hoechst marks all cells.Author Manuscript Author ManuscriptNature. Author manuscript; offered in PMC 2022 October 06.Konstantinides et al.Page(i) Hbn is expressed between the two Opa temporal windows in neurons, when BarH1 is expressed just after the second Opa temporal window. Hbn is also expressed in later born neurons, just after the second Opa wave, and is lost from the first lamina (in among Opa) as neurons mature. Scale bars: ten mAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptExtended Data figure 5: Negative genetic interactions among tTFs(a) Diagram of genetic interactions involving tTFs in medulla neuroblasts. Red “X”`s inside the diagram indicate no genetic interaction. Inside the early unit (green box), we identified 3 new tTFs: Opa, Erm and Oaz. Hth will not activate Erm or Opa. Furthermore, Opa does not repress Hth or activate Erm. Inside the middle unit (red box), we identified three new temporal things: Homeobrain (Hbn), Scarecrow (Scro) and Opa. Opa isn’t inhibited by D or BarH1. Oaz can also be not inhibited by D or BarH1. Inside the late unit (blue box) we identified one particular new temporal aspect: BarH1. Tailless (Tll) will not be essential to inhibit BarH1. (b) In cells expressing hth RNAi driven by Vsx-Gal4 (GFP: green), Opa expression isn’t impacted, indicating that Hth does not activate Opa. (c) Erm is unaffected in cells expressing hth RNAi driven by Vsx-Gal4 (GFP: green). Therefore, Hth doesn’t activate Erm.Nature. Author manuscript; readily available in PMC 2022 October 06.Konstantinides et al.Web page(d) In opa mutant clones (GFP: green), Hth and Erm expression are unaffected, indicating that Opa doesn’t inhibit Hth and does not activate Erm. (e) In opa mutant clones (GFP: green), Ey expression is delayed, indicating that Opa aids to time the expression of Ey.Resibufogenin In Vivo (f-j) In cells expressing Oaz RNAi driven by Vsx-Gal4 (GFP: green), (f) Hth, Ey, Slp, (g) Opa, (h) Erm, Hbn, Tll, (i) Scro, D, and (j) BarH1 are unaffected, indicating that Oaz does not regulate their expression.Cephapirin In Vivo (k) Scro knock-down upon expression of scro RNAi (GFP: green) does not affect the expression of Hbn or Slp, as was anticipated offered that Scro is coexpressed with each (see Figure two).PMID:27108903 (l) In negatively marked D mutant clones, the expression of Opa is unaffected, indicating that D will not inhibit Opa. (m) Similarly, in negatively marked D mutant clones, the expression of Oaz is unaffected, indicating that D doesn’t inhibit of Oaz. (n) In Tll mutant clones (GFP: green), neither D nor BarH1 expression are affected, indicating that Tll will not be necessary to inhibit either factor. (o-q) In cells expressing RNAi against BarH1, (o) Opa, (p) Oaz, and (q) D are unaffected, indicating that BarH1 doesn’t regulate their expr.