supplementary materials - genes &...

Peng et. al., revised 08/03/09 GENESDEV/2009/124248R1

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Supplementary Materials

Transcription factor choice in the Hippo signaling pathway: homothorax and yorkie regulation

of the microRNA bantam in the progenitor domain of the Drosophila eye imaginal disc

H. Wayne Peng1,2, Matthew Slattery1, Richard S. Mann1,*

1Department of Biochemistry and Molecular Biophysics,

2Integrated Program of Cellular, Molecular and Biophysical Studies,

Columbia University Medical Center

*Correspondence should be addressed to: [email protected]


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Supplementary Figure 1. Loss-of-function tsh clones grow poorly in the eye progenitor domain of tio null

flies.

tsh is located proximal to the FRT used to generate mitotic clones. In addition, it has a highly

related and partially redundant sister gene, tio, located about 180 kb from tsh. For these reasons, we

examined loss of tsh function by generating clones that express a tsh hairpin to induce RNAi in either a

tio null or tio heterozygous background.

(A,B) show examples of eye imaginal discs containing act>stop>Gal4; UAS-tshRNAi flip out clones in

either a tio–/– (A) or tio+/– (B) background. Clones are positively marked by GFP (green) and the discs

were costained for Dlg (blue, to mark the MF) and Tsh (red, which shows incomplete knockdown).

(C) The number of nuclei present in the GFP+ clones from the experiment shown in A,B were counted

and plotted for these two genotypes. More than 50 discs were quantified for each genotype. The data

show that tsh knockdown clones grow more poorly in a tio null background than in a tio heterozygous

background.


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Supplementary Figure 2. Hth+Tsh induce proliferation independently of Wg, N, and JakStat.

(A) Hth+Tsh clones overgrow

(B) Hth+Tsh; fz fz2 clones also overgrow.

(C) fz fz2 clones grow very poorly.

(D) Hth+Tsh clones overgrow.

(E) Hth+Tsh+TCFDN (a dominant negative form of the Wg transcription factor TCF) also overgrow.

(F) Clones expressing the intracellular domain of Notch (NICD) overgrow.

(G) NICD; hthP2 clones also overgrow.

(H) Hth+Tsh clones that also express a dominant negative form of Delta (DlDN) overgrow.

(I) Hth+Tsh clones overgrow.

(J) Hth+Tsh; stat92E6346 clones also overgrow.


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Supplementary Figure 3. Expression of Hpo partially suppresses the overgrowths induced by

Hth+Tsh.

All clones were induced in parallel, at the same time.

(A) Eye disc containing clones expressing Hpo (stained for Tsh, red, and pH3, blue). These clones grow

poorly.

(B) Eye disc containing clones expressing Hpo+Tsh+Hth stained for Tsh (red) and pH3 (blue). These

clones grow larger than Hpo+ clones.

(C) Eye disc containing clones expressing Hth+Tsh stained for Tsh (red) and pH3 (blue). These clones

grow much larger than Hpo+Tsh+Hth clones.


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Supplementary Figure 4. Yki+ or Ban+ clones do not affect the expression of Hth, Tsh, or

differentiation markers.

(A) Eye disc containing Yki+ clones (green) stained for Hth (blue) and Tsh (red). Hth and Tsh expression

patterns are normal.

(B) Eye disc containing Yki+ clones (green) stained for CycB (purple). Although Yki+ clones overgrow, they

do not induce additional CycB expression.

(C) Eye disc containing Yki+ clones (green) stained for Elav (purple). No affect on Elav expression is

observed.

(D) Eye disc containing clones ectopically expressing bantam (green) stained for Hth (red) and Eya (blue).

No affect on Hth or Eya expression patterns is observed.


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Supplementary Figure 5. sd null clones survive in the eye progenitor domain.

(A,B) sdΔB null clones in the eye disc (A) or wing disc (B) generated in parallel and stained for GFP (green,

clones are marked by the absence of GFP) and Dlg (red, which marks the MF). In eye discs, sdΔB null

clones grow well in the anterior progenitor domain (A, arrows). In wing discs, sdΔB null clones survive in

the notum domain (B, arrows), but not in the wing pouch (B, arrowheads; the bright green clones are

twin spots, showing that clones were generated but failed to survive).

(C,D) Gal80– act-Gal4 MARCM clones generated in parallel that were either sdΔB; act>Hth+Tsh (C) or sdΔB;

act>GFP+ (D) stained for Dlg (red) and GFP (green), which positively marks the clones. These data

show that Hth+Tsh clones can induce overgrowths in the absence of sd (arrow in C).


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Supplementary Figure 6. CycE, diap1, and expanded expression in Hth+Tsh clones.

(A-C) Eye discs containing Hth+Tsh expressing clones (positively marked in green) stained for (A) CycE

(red) and CycB (blue); (B) diap1-lacZ (red); and (C) ex-lacZ (red). Arrows point to clones. In some

examples, diap1-lacZ is weakly upregulated and ex-lacZ is down-regulated in response to Hth+Tsh.


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Supplementary Figure 7. hthP2 eyes are small.

(A) Eye sizes (normal, small, or no eye) obtained using the EGUF method (Stowers and Schwarz 1999).

The cross used to generate these data was:

yw; FRT82B hthP2/TM6B X yw; ey-Gal4 UAS-flp; FRT82B GMR-hid/TM2

TM6B/TM2 and FRT82B hthP2/TM2 flies have normal sized eyes.

FRT82B GMR-hid/TM6B flies have no eyes due to cell death induced throughout the retina by the

expression of the proapoptotic gene hid via the GMR promoter.

ey-Gal4 UAS-flp; FRT82B hthP2/FRT82B GMR-hid flies can only generate eyes made of hthP2/hthP2 tissue

due to cell death induced by GMR-hid. In this case, although there is significant lethality of this

genotype, 100% of the surviving animals that hatched had small eyes. The observed lethality may

reflect a requirement for hth in other cells that express ey-Gal4 (such as in the nervous system).

(B) Four examples of eyes from yw; ey-Gal4 UAS-flp; FRT82B hthP2/FRT82B GMR-hid flies.

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