DEV02148 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Pixie is predominantly in the cytoplasm in S2 cells. Confocal images of Drosophila S2 cells stained with rhodamine-conjugated phalloidin to detect the actin cytoskeleton (red), the DNA dye TO-PRO3 (blue) and antiserum against Pixie (green). Lower panels show cells treated with pixie dsRNA.

• Supplemental Figure 2 -

  Fig. S2. The period of intense apoptosis in the wing pouch in M(3)66D¹/+ wing discs correlates maximally with the slow phase of disc growth. (A-F) Similar to pixie mutant discs, apoptosis is seen in uniformly distributed clusters throughout early to mid third instar M(3)66D¹/+ wing discs. Apoptosis is gradually more localized to the wing pouch during mid to late third instar, when disc growth slows (see G). At 110 hours AEL, a decrease in cell death in the dorsal hinge area relative to the rest of the disc is clearly visible (10/10 discs examined, B,C). In a proportion of these discs, intense cell death in the dorsal pouch is also visible (2/10 discs, C). By 120 hours AEL (when growth has begun to slow), intense cell death in the wing pouch is clearly evident in all eight discs examined (D) and persists through most of the slow phase of growth. Stage of the disc in hours after egg lay (AEL) is indicated; pre-wandering indicates a day before wandering. At the wandering larval stage (F), intensity of apoptosis decreases, but is still higher than control. (G) Graph showing increase in cell number on a logarithmic scale plotted against stage of the discs (shown as hours AEL) of control (grey line) and M(3)66D¹/+ (black line) wing discs. The graphs begin at the beginning of the third larval instar. These Minute larvae undergo the moult into third instar at a similar time to control larvae (at 72 hours AEL), albeit at a smaller size with smaller discs. Numbers in brackets indicate number of discs examined for each point. The points on these graphs represent data pooled from three independent experiments. Numbers in bold indicate points where cell number was counted on a haemocytometer, for all other points cell number was counted from confocal images. The two techniques were found to yield comparable results (see Materials and methods). There is a higher intrinsic variability in M(3)66D¹ /+ disc size. However, both in the control discs and in the Minute discs, a rapid increase in size is followed by a distinct slowing down of growth towards the end of larval life. The time at which apoptosis begins to decrease in the hinge (L) (110 hours AEL) is towards the end of the rapid phase of growth.

• Supplemental Figure 3 -

  Fig. S3. Spatial and temporal dynamics of increases in cell number in developing wing imaginal discs. Wing imaginal disc cells grow faster in the early third instar (A) than the late third instar (B), and in the hinge (right) than the pouch (left). Wild-type GFP¯ clones and GFP⁺ twins were generated in GFP^+/¯ discs, and the number of cells per GFP¯ clone quantified. Bar charts depict distribution of clone cell number; x-axes show the size of individual clones and y-axes the number of clones in each size category. (A) Clones were induced mid-third instar, 66.3±1 hours AEL and examined 30 hours later, number of clones examined (n) = 49 in pouch and 61 in hinge. h ACI, hours after clone induction. (B) Clones were induced 96 hours AEL and examined 48 hours later at wandering; n=85 in pouch and 182 in hinge. MDTs in B resemble MDTs in the anterior compartment in Fig. 5D, but are longer than MDTs characterizing slow growth in Fig. 6D. This is because of developmental delay induced by the culture conditions in this experiment and in Fig. 5B-D, where larval wandering occurred at ~144 hours AEL instead of the more normal 125 hours AEL.

• Supplemental Figure 4 -

  Fig. S4. Spatial and temporal dynamics of the effect of pix^L17 and eIF4A¹⁰⁰⁶ on wing disc clonal growth. Bar charts show raw clone (black bars) and twin (grey bars) cell number data. These data were used to generate Fig. 6D-H. (A-C) pix^L17 clones generated during the fast growth phase. (D-F) pix^L17 clones generated during the slow growth phase. pix^L17 clones grow better in the hinge (D-F, bar charts on right) than during the early fast growth phase. However, in the pouch pix^L17 clones grow poorly during the slow growth phase (D,E); this growth is particularly poor during the 44-hour late window (D), but is significantly improved during the 37-hour window (E). As the median number of divisions (2.3) of twin clones during the late 32-hour window (F) is much lower than during the early 29- and 30-hour windows (3.0 in each), a comparison of the strength of phenotypes is not possible. Increase in number of divisions can allow the differences in growth rate between twin and mutant clone to accumulate. (G,H) eIF4A¹⁰⁰⁶ clones generated during the fast (G) and slow (H) growth phases. In contrast to the growth of pixie mutant clones, the growth of eIF4A¹⁰⁰⁶ clones is similarly impaired in the pouch and hinge. In contrast to pix^L17 clones, eIF4A¹⁰⁰⁶ clones are rarely absent, both in the hinge and pouch, despite the strong reduction in clone size. The scale on the y-axis is different in A.

• Supplemental Figure 5 -

  Fig. S5. pix^L17 clones in wing discs do not ectopically express brinker. pix^L17 wing pouch clones examined at ~110 hours AEL, 30 hours after induction, do not express brinker (brk). (A) brk expression (red) was detected using antibodies against b-galactosidase driven by brk upstream sequences. There is no ectopic expression of brk in the wing pouch. (B,C) Merge images of the GFP and b-galactosidase signals show the position of a GFP¯, pix^L17 clone (white arrows) together with its GFP⁺ twin (black arrow). Part of the clone tissue is shown in B and the rest of the clone tissue is shown magnified on a more basal section in C. pix^L17 clones in the pouch are rare. However, 18 mutant clones examined at 104±6 hours AEL did not reveal brk expression. The clones depicted here are from a disc older among the ones examined.