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Fig. S1. Heterozygosity of klu suppresses supernumerary type II neuroblasts in brat11/DG19310 mutant brains. (A-B) Ase serves as a marker for an intermediate stage of maturation. (A-A′′′′) Larvae carrying GFP-marked wild-type type II neuroblast lineage clones (outlined by the yellow dotted line) were aged for 16 hours after clone induction, and brains were stained for the markers indicated. Scale bar: 10 µm. (B) Summary of the cell fate marker expression pattern in the type II neuroblast lineage. (C-I) brat functions in immature INPs to suppress reversion into type II neuroblasts and to initiate the specification of INP identity. (C-G′′) Larvae carrying GFP-marked wild-type or brat mutant type II neuroblast mosaic clones (outlined by the yellow dotted line) were aged for 24 or 72 hours after clone induction, and brains were stained for the markers indicated. (H) Quantification of various cell types in the wild-type or brat mutant type II neuroblast clone. (I) Summary of the identity of cells in the brat mutant type II neuroblast clone. supernum neurob, supernumerary neuroblast. (J-L) Heterozygosity of klu suppresses supernumerary type II neuroblasts in sensitized brat mutant brains. (J-K′′′) brat DG19310/11; klu+/+ or brat DG19310/11; klu−/+ mutant larvae were aged for 96 hours ALH, and brains were stained for the markers indicated. The white dotted line separates the central brain (left) from the optic lobe (right). (L) Average type II neuroblasts per brain lobe in larvae of the genotype indicated. Scale bar: 20 µm. Type II neuroblast (Dpn+ Ase−, white arrow); Ase− immature INP (Dpn− Ase−, white arrowhead); Ase+ immature INP (Dpn− Ase+, yellow arrow); INP (Dpn+ Ase+, yellow arrowhead).
Fig. S2. klu mutant neuroblasts show asymmetric localization of apical and basal proteins and do not display aberrant activation of caspases. (A,B) Telophase klu mutant neuroblasts show asymmetric localization of aPKC, Miranda and Numb. Scale bar: 5 µm. (C-D′′′) Removal of the Df(3L)H99 locus does not block premature loss of neuroblasts in klu mutant type II neuroblast clones. Larvae carrying GFP-marked klu single-mutant or klu,H99 double-mutant type II neuroblast mosaic clones (outlined by the yellow dotted line) were aged for 72 hours after clone induction, and brains were stained for the markers indicated. Scale bar: 10 µm. (E-F′′) klu mutant neuroblasts do not show aberrant activation of caspases. Wild-type or klu mutant brains overexpressing the UAS-apoliner transgene were stained for the markers indicated. Scale bar: 10 µm. (G-I) Overexpression of the caspase inhibitor protein p35 does not suppress premature loss of neuroblasts in klu mutant brains. klu mutant brains alone or overexpressing the UAS-p35 transgene were stained for the markers indicated. The yellow line separates the central brain (left) from the optic lobe (right). Scale bar: 20 µm. Type I neuroblast (Dpn+ Ase+, green arrow); GMC (Dpn− Ase+, green arrowhead); type II neuroblast (Dpn+ Ase−, white arrow); Ase− immature INP (Dpn− Ase−, white arrowhead); Ase+ immature INP (Dpn− Ase+, yellow arrow); INP (Dpn+ Ase+, yellow arrowhead).
Fig. S3. klu-lacZ is detectable in both type I and II neuroblasts and their progenitor progeny in larval brains. (A-A′′′) Larvae carrying a klu-lacZ enhancer trap transgene were aged for 96 hours after larval hatching, and larval brains were stained for the markers indicated. The white line separates the central brain (left) from the optic lobe (right). Type I neuroblast (Dpn+ Ase+, green arrow); type II neuroblast (Dpn+ Ase−, white arrow).
Fig. S4. Erm-GAL4 is not expressed in type II neuroblasts. (A-A′′) Larvae carrying an Erm-GAL4 and an UAS-prospero transgene were raised at 31.5°C to induce the expression of Prospero for 96 hours after larval hatching, and larval brains were stained for the markers indicated. The white line separates the central brain (left) from the optic lobe (right). Type I neuroblast (Dpn+ Ase+, green arrow); GMC (Dpn− Ase+, green arrowhead); type II neuroblast (Dpn+ Ase−, white arrow).
Fig. S5. Overexpression of various truncated Klu transgenic proteins in larval brains. (A-C′′′′) Larvae carrying Wor-GAL4 in combination with one of several UAS-klu transgenes were raised at 31.5°C to induce the expression of Klu for 96 hours after larval hatching, and larval brains were stained for the markers indicated. The white line separates the central brain (left) from the optic lobe (right). Type I neuroblast (Dpn+ Ase); type II neuroblast (Dpn+ Ase−).
Fig. S6. numb functions in immature INPs to suppress reversion into type II neuroblasts and to initiate specification of INP identity. (A-C′′) Larvae carrying GFP-marked wild-type or numb mutant type II neuroblast mosaic clones (outlined by the yellow dotted line) were aged for 24 or 72 hours after clone induction, and brains were stained for the markers indicated. (D) Summary of the identity of cells in the numb mutant type II neuroblast clone. supernum neurob, supernumerary neuroblast. Type II neuroblast (Dpn+ Ase−, white arrow); Ase− immature INP (Dpn− Ase−, white arrowhead).
Fig. S7. Overexpression of klu enhances the reversion of GMCs into neuroblasts in numb mutant type I neuroblast clones. (A-C′′′) Larvae carrying GFP-marked wild-type or numb mutant type I neuroblast mosaic clones (outlined by the yellow dotted line) alone or overexpressing klu were aged for 48 hours after clone induction, and brains were stained for the markers indicated. Scale bar: 10 µm. (D) Average type I neuroblasts per brain lobe in larvae of the genotype indicated. Type I neuroblast (Dpn+ Ase+, green arrow); GMC (Dpn− Ase+, green arrowhead).
