Figure 2.

Knockdown of pico by RNAi Reduces Tissue Size

Moderate ubiquitous expression of pico^IR results in a reduction in overall body size.

(A and B) A tub-GAL4 fly and a tub-GAL4 UAS-pico^IR (tub > pico^IR) fly are shown for comparison. Quantitative measurements of female flies (n = 50 per genotype) show a 23% reduction in body weight in tub > pico^IR flies (p < 0.01).

(C) Representative RT-PCR analysis showing reduction in pico and pico-L expression in tub-GAL4 and UAS-pico^IR flies using primers recognizing both transcripts. A range of cDNA concentrations normalized against GAPDH was used to assess transcript levels.

(D and E) Expression of pico^IR knocks down levels of Myc-tagged pico in wing imaginal discs. (D) High levels of ectopic pico in the posterior compartment marked by GFP (in green) were detected by Myc staining (in red) in en-GAL4, UAS-Myc-pico discs. (E) No Myc staining was detected in discs coexpressing pico^IR.

(F and G) Compared with control (F), expression of two copies of pico^IR (G) results in a significant reduction in adult wing size (p < 0.01). Male wings are shown. Wing area is expressed as a percentage of control (±SD). The reduction in wing size is due to fewer and smaller cells, as revealed by relative bristle density measurements (see insets).

(H–J) Consequences of pico^IR overexpression in the posterior compartment of the wing under the control of en-GAL4. (H) Compared to control, expression of pico^IR with en-GAL4 results in a reduction in size of the posterior compartment of the adult wing. A, anterior; P, posterior. Numerical scale is in arbitrary units. Error bars indicate 1 SD. (I and J) pico^IR-induced growth retardation is not due to aberrant cell cycle phasing. Flow cytometry was performed on dissociated wing disc cells overexpressing GFP alone (I) or pico^IR and GFP (J). GFP-positive experimental populations are marked in green; GFP-negative internal controls are marked in gray. Comparison of representative cell cycle profiles shows that pico^IR does not alter cell cycle phasing. Percentage of cells in G1, S, and G2 phases of the anterior and posterior compartments is shown in insets. Graphs of forward scatter (FSC; bottom panels) show that pico^IR results in a modest reduction in cell size at the third instar larval stage. The ratio of the mean FSC value of GFP-positive verses GFP-negative cells is shown in the top right corner of the bottom panels.

(K and L) The distribution of clonal cell number for control (K) and pico^IR-expressing clones (L). Cell-doubling time (DT) is markedly increased by pico^IR. Insets show representative clones of each genotype as visualized by nuclear GFP.