Fig. S1. Overexpression of Notch or Fringe bypasses the requirement for spdo function during asymmetric divisions in the CNS. Ventral view of stage 15 embryos stained with anti-Eve (upper panel). (A) In wild-type embryos, five to six Eve⁺ U neurons (arrowheads) have formed by stage 15, each one arising from the asymmetric division of an Eve⁺ precursor that produces an Eve⁺ U neuron (‘A’ daughter cell) and an Eve⁻ U sibling neuron (‘B’ daughter cell). (B) In spdo mutant embryos, no Eve⁺ U neurons develop (arrowheads) as both siblings adopt the Eve⁻ U sibling fate. (C) Overexpression of Spdo in the CNS (via ProsperoGAL4) of otherwise spdo mutant embryos leads to the presence of two to four Eve⁺ U neurons. (D) Overexpression of Delta in the CNS of spdo mutant embryos rarely results in the presence of one or two Eve⁺ U neurons in a hemisegment. (E) Co-overexpression of Neuralized and Delta provides modest restoration of the Eve⁺ U neuron fate. (F,G) ProsGAL4-mediated overexpression of Notch (F) or Fringe (G) in spdo mutant embryos yields a modest to strong restoration of Eve⁺ U neuron development (arrowheads). Note Notch overexpression occasionally results in more Eve⁺ U neurons than observed in wild-type or spdo mutant embryos that overexpress Spdo in the CNS (compare F with A or C), indicating that Notch can promote the Eve⁺ U fate in both siblings in the absence of spdo. Anterior, top.

Fig. S2. The Notch and Fringe transgenes function in a ligand-dependent manner. (Top, A) In the wild type, two rows of cardioblasts develop. (B) Loss of Dl and Ser function leads to a drastic overproduction of cardioblasts owing to defects in lateral inhibition. (C-F) TwistGAL4-mediated overexpression of Delta (C), but neither Notch (D,E) nor Fringe (F), rescues the observed phenotype in Dl and Ser mutant embryos. Cardioblasts visualized by Nmr1 protein expression. Anterior, left. (Middle/Bottom) Eve (middle) and Odd (bottom) expression in the developing Drosophila CNS. (A) A vast excess of Eve⁺ (middle) and Odd⁺ neurons (bottom) form in Delta mutant embryos owing to defects in lateral inhibition, which lead to excess neuroblasts and asymmetric divisions. (B-D) ScabrousGAL4-mediated expression of Delta (B), but neither Notch (C) nor fringe (D), throughout the neuroectoderm and CNS of otherwise Delta mutant embryos rescues the observed phenotypes. Anterior, top.

Fig. S3. Numb localizes asymmetrically upon overexpression of Notch or Fringe in wild-type and spdo mutant embryos. High-magnification images of mitotic Svp⁺ heart precursors labeled for Svp (red) and Numb (green). Numb localizes asymmetrically before precursor division in the wild type. Numb also localizes asymmetrically in wild-type embryos that overexpress Fringe (o/e Fng), spdo mutant embryos, as well as spdo mutant embryos that overexpress Fringe (o/e Fng; spdo⁻) or Notch (o/e N; spdo⁻).