Figure S1. Pasha protein is absent in pasha−/− PNs at 18h APF.
(A and B) Pasha localizes to the nucleus of WT adPNs and DL1 single neurons and all surrounding cells in 18h APF pupal brains at equal levels. Pasha staining is shown separately in A1 for the adPN clone and the inset in B for a single neuron, both outlined in yellow.
(C and D) Pasha protein is absent from pasha−/− adPNs (C and C1) and DL1 single neurons (D and inset) at 18h APF while heterozygous neighboring cells express Pasha in the nucleus. The MARCM clones are outlined in yellow based on GFP-staining; the actual clone may also contain some Gal4-GH146 negative, and hence GFP-negative cells (e.g., in C1).
Green is mCD8-GFP labeled MARCM clones, red labels anti-Pasha and blue is DAPI. Scale bars represent 20μm. All images are single confocal sections.
Figure S2. Dicer-2 cannot compensate for Dicer-1 function during PN targeting.
(A1) In Dicer-1−/− DL1 single neurons dendrites mistarget to other glomueruli besides DL1 (arrowheads). In Dicer-1−/− adPNs (A2) and lPNs (A3) the dendritic mass is significantly reduced and dendrites spill non-specifically into inappropriate glomeruli. All these dendritic phenotypes cannot be rescued or altered by overexpressing Dicer-2 in Dicer-1−/− DL1 single neurons (arrowheads in B1, compare to A1), adPNs (B2, compare to A2), and lPNs (B3, compare to A3).
Green is mCD8-GFP labeled MARCM clones, red labels the presynaptic marker nc82. Scale bar represents 20μm. All images are z-projections of confocal stacks.
Figure S3. bantam sensor is de-repressed in AGO1k08121 mutant clones of larval wing discs.
(A–C) pasha−/− clones are marked by the absence of a LacZ reporter gene (red, A). An example is outlined by dashed line. GFP levels are elevated compared to heterozygous tissue (asterisk), presumably due to the absence of mature miRNA within the clone (B). n=11.
(D–F) In Dicer-1−/− clones the effect of bantam sensor de-repression is similar. n=12.
(G–K) FRT42D control (G) or FRT42D AGO1k08121 mutant clones (J), respectively, are marked by the lack of -Gal staining (examples outlined by white dashed line) and contain two copies of the same bantam sensor transgene as in B and E, which is located also on chromosome 2R (H, K). Twin spot clones contain two copies of LacZ (examples marked by yellow dashed line) but lack any bantam sensor transgene (H, K). Heterozygous tissue bears one copy of LacZ and bantam sensor transgene and is marked by an asterisk (H, K).
(L) The extent of bantam sensor de-repression in AGO1k08121 mutant clones was determined as follows: we subtracted the mean fluorescence intensity of twin spot clones (no bantam sensor) from the homozygous (two copies of bantam sensor) and heterozygous clones (one copy of bantam sensor) separately, and then calculated the ratio of the homozygous and the heterozygous values. In control clones, bantam sensor expression is increased by ~2.5 fold compared to heterozygous tissue. In AGO1k08121 mutant clones bantam sensor expression is up-regulated ~3.7 fold, showing a significant increase in GFP levels compared to the control (p=1.7×10−6; n= 13 or 16 independent clones for control or AGO1k08121 mutants, respectively). Error bars indicate SEM.
Larval genotypes: (A–C) Hs-FLP1; bantam sensor/+; arm-LacZ FRT82B/FRT2A FRT82B pashaLL03660 y+; (D–F) Hs-FLP1; bantam sensor/+; arm-LacZ FRT82B/FRT2A FRT82B Dicer-1LL06357 y+; (G–I) Hs-FLP1; FRT42D bantam sensor/FRT42D arm-LacZ; (J–K) Hs-FLP1; FRT42D AGO1k08121 bantam sensor/FRT42D arm-LacZ
Green represents bantam sensor GFP expression, red anti-β-Galactosidase. Scale bar represents 20μm. All images are single confocal sections of 3rd instar wing imaginal discs.