Figure 4. Btd likely acts downstream of Trx to maintain a type II neuroblast functional identity.

(A–D) The btd gene is an excellent candidate target of Trx in the type II neuroblast. (A) The btd mRNA is highly enriched in the lysate extracted from larval brain enriched with type II neuroblasts. The elav transcript is highly enriched in differentiated neurons. The quantification represents the average of three biological replicates. (B) Trx directly binds to the type II neuroblast-specific enhancer element as well as the transcription start site (TSS) of the btd gene. The ChIP experiments were performed using the extract isolated from dissected brat mutant brains that are enriched with type II neuroblasts. Quantification of chromatin immunoprecipitated by the indicated antibodies relative to 5% of input. The quantification represents the average of three biological replicates. (C–D) An enhancer element from the btd gene is sufficient to induce type II neuroblast-specific expression of a UAS-mCD8::gfp reporter transgene in wild-type brain, while the enhancer activity of btd-Gal4 was reduced in rbbp5^null brain. Scale bar, 20 μm. (E–H) btd is required for maintaining the functional identity but not the molecular signature of a type II neuroblast. (E–F) In the 72-hr clones, btd mutant type II neuroblasts maintain a type II neuroblast marker expression profile and are surrounded by 1–2 immature INP-like cells. Three-dimensionally reconstructed images of the clones are shown below. Scale bar, 10 μm. (G) The average number of INPs per clone of the indicated genotypes. (H) The average number of GMCs with or without erm-lacZ expression per type II neuroblast clones of the indicated genotypes at 72 hr after clone induction. (I–J) The immature INP-like cells generated by btd mutant type II neuroblasts are insensitive to loss of brat function. Removing brat function does not lead to supernumerary neuroblast formation in the 72-hr btd mutant type II neuroblast clones. Scale bar, 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.03502.010

Figure 4—figure supplement 1. Global H3K4 mono- or tri-methylation is not required for maintenance of a type II neuroblast functional identity.

(A–H) The core component of the SET1/MLL complex is required for the global methylation of H3K4. (A, C, E, G) Knocking down the function of ash2 or trr leads to global loss of the H3K4 mono-methylation while knocking down the function of dSet1 does not. Scale bar, 10 μm. (B, D, F, H) Knocking down the function of ash2 or dSet1 leads to global loss of the H3K4 mono-methylation while knocking down the function of trr does not. (I–J) trr and dSet1 are dispensable for the maintenance of type II neuroblasts. (I–J) The average number of type II neuroblasts or INPs per brain lobe of the indicated genotypes after knocking down the function of trr or dSet1 for 72 hr. (K–N) trx mutant type II neuroblasts do not display appreciable reduction in the global methylation pattern. Scale bar, 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.03502.011

Figure 4—figure supplement 2. Pnt likely functions to specify an INP identity.

(A) Trx directly binds to transcription start site (TSS) of the pntP1 transcript. Quantification of chromatin immunoprecipitated by the indicated antibodies relative to 5% of input. The quantification represents the average of three biological replicates. The black lines indicate three different pnt transcripts. The magenta lines indicate three UAS-RNAi used to target the common exon of pnt transcripts. (1) UAS-pnt_RNAi (7171), (2) UAS-pnt_RNAi (TRiP.JF02227), and (3) UAS-pnt_RNAi (TRiP.HMSO1452). (B–C) Expression of the UAS-pnt_RNAi transgene efficiently reduces PntP1 protein expression throughout the type II neuroblast lineage. (D–E) Knocking down the function of pnt induces supernumerary neuroblast formation. Scale bar, 10 μm. (F–G) The average number of type II neuroblasts per clone of the indicated genotypes.

DOI: http://dx.doi.org/10.7554/eLife.03502.012