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Supplementary Methods
Fly stocks. btlLG18, spry5, pnt88, UAS-NACT, and Nl1N-ts1 have been described1-5. pnt88 was
recombined onto an FRT82B6 cu sr e ca chromosome. spry5 was recombined onto an FRT2A7
chromosome (S. Toering and M.A.K., unpublished). A third chromosome insertion of btl-Gal4
was generated by mobilization of the original second chromosome insertion8.
Generation and labeling of homozygous mutant cell clones. To generate the mutant clones,
embryos of genotype y, w, hs-FLP1.22; btl-GAL4, UAS-DsRED (or DsRED2nls), UAS-GFP;
btlX, FRT2A (or FRT82B, pntX)/UASi-GFPhp, th, FRT2A (or FRT82B, cu, UASi-GFPhp) were
collected for 1 hr at 25o C, aged for 2 hrs, subjected to a heat shock at 38o C for 45–60 min, and
then returned to 25o C and allowed to develop. Control wild-type cell clones were generated in
the same way in y, w, hs-FLP1.22; btl-GAL4, UAS-DsRED2nls, UAS-GFP; FRT2A,FRT82B/
UASi-GFPhp, th, FRT2A embryos. Wild-type clones in a heterozygous btl background were
generated in y, w, hs-FLP1.22; btl-GAL4, UAS-DsRED2nls, UAS-GFP; btlX, UASi-GFPhp, th,
FRT2A/FRT2A, FRT82B embryos.
Mutant clones were identified and characterized primarily in early third instar larvae. Older
larvae are difficult to score because tracheal histoblasts begin populating anterior dorsal
branches. Younger larvae are hard to score accurately because they contain residual GFP in
tracheal fusion cells. The numbers and positions of all cells in each mosaic dorsal branch were
recorded and cells marked with GFP were noted along with any unusual structural features of the
cells and branches. When multiple cells with terminal cell or fusion cell morphology were
present in a branch, each terminal cell was considered a DB1 cell and each fusion cell was
considered a DB2 cell. The average number of GFP-marked cells per mosaic dorsal branch
varied from ~1.2 to ~1.7 in different experiments, presumably due to variability in heat induction
of the hsFLP1.22 transgene.
Because FLP expression was induced at 2-3 hrs of development and btl expression initiates at
~4.5-5.5 hrs of development9,10, it is possible that recombination to generate clones occasionally
occurred after initiation of btl expression but before the final embryonic tracheal cell division
that occurs at ~5.5 hrs of development11. If so, the resultant homozygous mutant daughter cells
would inherit a limited amount of wild-type btl mRNA and/or protein that could ameliorate the
mutant phenotype of these clones.
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