DEV029975 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Additional marker analysis of stage 14 leg primordia. (A) Leg primordia of a stage 14 embryo. elav-gal4 (green) and ct (blue) expression are coincident in the leg primordia and are mutually exclusive to LT activity (red). Combinations of channels are shown below. (B,C) DKO activity is present in a subset of Dll-expressing cells (red; B) that also express ct (red; C). Single channels are shown below. (D) Early second instar larvae carrying LT-Gal4/UAS-GFP transgenes. LT activity is visualized by GFP in the leg imaginal discs (arrows). The inset shows LT activity (red) and the Keilin’s Organ (green) color coded.

• Supplemental Figure S2 -

  Fig. S2. Additional fate mapping experiments. (A) The progeny of cells in which Dll (red) was expressed contribute to the leg and the wing imaginal discs. In the leg, the Dll lineage contributes to the coxopodite and the telopodite. Single channels are shown below. Hth (blue) marks the coxopodite and Dll (green) part of the telopodite. (B) LT lineage visualized in an adult leg, showing that it contributes to the telopodite. The small clone that appears in the coxopodite (arrow) may be due to the imperfection of the LT-Gal4 driver. The LT lineage is marked by GFP (white) and outlined in red. cox, coxa; tro, trochanter; fe, femur; tib, tibia; tar, tarsal segments. (C) Effects on Keilin’s Organ (KO) formation by the expression of the pro-apoptotic gene hid using different Gal4 drivers. For this experiment we counted the number of hairs in each KO in the T3 segment. Driving hid via LT-Gal4 or esg-Gal4 has no effect on KO formation. By contrast, expressing hid via the Dll304-Gal4 and the DKO-Gal4 drivers reduced the number of hairs from 100% to 28% and 50%, respectively.

• Supplemental Figure S3 -

  Fig. S3. Additional experiments addressing the genetic inputs into LT and DKO. (A) Ectopic expression of Dll using the prd-Gal4 driver weakly activates LT. Ventral view of a stage 14 embryo stained with β-gal (LT, red) and Dll (green). The arrows mark the segments where Dll is ectopically expressed and the weak activation of LT. (B) Ectopic expression of btd using prd-Gal4 weakly activates LT. Lateral view of a stage 14 embryo stained with β-gal (LT, red) and Ubx (green). Btd is able to activate LT in the dorsal region of T2 and weakly in the abdomen. (C) In a stage 14 btd^XG81 mutant embryo, the activity of LT (red) and ct (green) is highly reduced (arrow). Compare it with a wild-type pattern in Fig. 4A. (D,D′) Ectopic expression of the Dpp pathway repressor Dad using the prd-Gal4 driver represses DKO activity in the second thoracic segment (arrow). Compare DKO activity in the second thoracic segment (arrow) with that in the first and the third thoracic segments (asterisks). Stage 14 embryo stained for β-gal (DKO, red) and Dll (green). Note that the few remaining DKO-lacZ expressing cells are outside of the leg primordia and are therefore not part of the normal Dll expression pattern (arrowhead). In this experiment, we also included a UAS-Dll transgene, to rule out that the absence of DKO activity is not due to the absence of Dll. (E,E′) Ectopic expression of the dominant form of TCF (TCF^DN) using the prd-Gal4 driver represses DKO activity in the second thoracic segment, where the driver is active (arrow). Compare DKO activity in the second thoracic segment (arrow) with that in the first and the third thoracic segments (asterisks). Stage 14 embryo stained for β-gal (DKO, red) and Dll (green). Note that the few remaining DKO-lacZ expressing cells are outside of the leg primordia and are therefore not part of the normal Dll expression pattern (arrowhead). In this experiment, we also included a UAS-Dll transgene to resupply Dll protein, because Dll304 is repressed by TCF^DN, and DKO requires Dll for activity.

• Supplemental Figure S4 -

  Fig. S4. Examples of neutral clones generated in the telopodite and coxopodite. (A-B′′) Examples of randomly generated, positively marked neutral clones induced at 12-24 hours and dissected at 48-60 hours (A) and 60-72 hours AEL (B). Single channels or combinations of channels are shown. The leg discs are stained for Tsh (red), GFP (green) and β-gal (blue; A) or DAPI (red), GFP (green) and β-gal (blue; B). (A′) A single cell clone originated in a telopodite precursor cell (arrow). (A′′) A four-cell clone originated in the coxopodite (arrow). (A′′′) A single cell clone originated in the KO progenitor domain (arrow). These cells also express ct (data not shown). (B′) A six-cell clone originated in the telopodite progenitor domain (LT, arrow). (B′′) A five-cell clone originated in the coxopodite progenitor domain (arrow). Note that clones originating in the LT domain never cross into the coxopodite domain and vice versa. (C) A neutral clone induced at approx 6 hours of development (prior to LT activation) and dissected 96 hours later (early third instar). Random clones originated at this time can cross the boundary separating the coxopodite and the telopodite (six out of 32 clones). The disc is stained with Hth (red), Dll (green) and β-gal (blue).

• Supplemental Figure S5 -

  Fig. S5. Additional experiments on the telopodite-coxopodite lineages. (A,B) Third instar leg discs containing neutral clones stained for Hth (red), Dll (green) and β-gal (blue, the clone marker). Randomly marked neutral clones induced 12-24 hours of development cross the border between the telopodite and the trochanter (A) or the coxopodite and the trochanter (B). The arrow marks the trochanter domain where Dll and Hth are coexpressed. (C) Forced expression of Hth in the telopodite truncates the leg from the proximal femur to the tarsus. For this experiment we used Dll-Gal4/UAS-flp (line em 212, see Material and methods) in combination with act>stop>Gal4 UAS-GFP and UAS-hth to drive Hth in all the cells of the telopodite. To prevent death we also expressed the caspase inhibitor P35 in the same domain. Carrying out the same experiment with UAS-tsh instead of UAS-hth caused larval lethality, preventing us from analyzing the affects on the adult legs (not shown).