DEV01927 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 (Adobe PDF) - Table S1. Nine categories of microarray experiments. All replicate experiments within a category were performed with independently isolated samples. D_Wg and V_Wg samples in category 6 were the same as in categories 4 and 5, respectively. Pair-wise comparisons were performed with samples from the same discs. Samples in category 7 were the same as in category 4 (TD cells) or in 8 (wild-type D cells). The reference sample was produced by pooling equal proportions of 1st, 2nd and 3rd wild-type leg imaginal discs isolated from male and female 3rd instar larvae.

• Supplemental Table 10 (Adobe PDF) - Table S10. Computational search for regulatory elements.

  Supervised search for vgBE-like elements

  In the non-overlapping wing, leg and transdetermination-induced gene sets, we searched for clusters of the three motifs, which we define as vgBE-like enhancers: binding sites for dTCF, Su(H) and Scalloped (Sd) (Berman et al., 2002; Halder et al., 1998; van de Wetering et al., 1997). We queried 10 kb of upstream sequence for the TD genes using the Cister software (default parameters), which applies a hidden Markov model to locate clusters of binding sites for the three transcription factors (Frith et al., 2001). Each position in the sequence was assigned a probability of containing a cluster of binding sites. Using a cutoff of 0.75, 45 genes were identified with at least one predicted enhancer and are listed in the table. Positions separated by more than 500 bp were defined to be separate clusters. The predicted sequences for these genes were analyzed for each of the three binding sites; there were a total of 55 vgBE-like predicted enhancers. To compare this observation with what would be expected by chance, the CISTER software was used to search for vgBE-like enhancers in the upstream sequences (10 kb) of all genes (n=13324). One-hundred and forty-three genes were then sampled at random (10,000 times) from the entire set of genes and the number of enhancers was tallied. Within the randomly sampled gene sets, the average number of predicted enhancers was 45 and the frequency of observing 55 or more predicted enhancers was 0.067.

  Unsupervised search for common regulatory elements in the D. melanogaster genome

  We searched for novel cis-regulatory motifs in an unbiased manner using the software FReduce (H.L., unpublished), an updated version of the motif analysis software Reduce (Bussemaker et al., 2001). Reduce enumerates all possible motifs (oligonucleotide sequence) up to a specified length and finds motifs that show significant correlation with expression values from a single microarray experiment. FReduce is an updated version that allows for motifs with degenerate sequence symbols (e.g. W=A or T). To search for motifs associated with TD, we ran FReduce on the TD versus D experiments (taking the median across the replicates) and searched within the 5 kb upstream sequence for each gene. The most significant motif correlated with expression in TD was TATCGAYW (P=5.248075e-09). This motif closely resembles the DRE site TATCGATA. The table lists the genes that are upregulated in TD (median expression value>1, log2) and have at least one occurrence of DRE within their 5 kb upstream sequence. Genes that overlap with the TD set are highlighted in yellow and those referred to in the text are in blue.

• Supplemental Table 11 (Adobe PDF) - Table S11. PcG and lama genes function in transdetermination. After expression of Wg in control and mutant animals, transdetermination was monitored by measuring the fraction of 1st leg discs that express vgBE-lacZ, the relative proportion of the discs that lacZ-expressing cells represent, and the fraction of 1st legs in which wing cuticle was present in adults. The genotype of the assayed leg discs is listed in column 1. Columns 2 and 3 list the relative frequency (% of the total number of cases) of vgBE-lacZ expression in leg discs and wing-cuticle, respectively. Anti-b-galactosidase staining was monitored by expression in wing discs of the same animals, since the vgBE-lacZ expression in wing discs is independent of transdetermination. Column 4 lists the median area of transdetermination as the ratio of vgBE-lacZ expressing cells to total disc area. P-values are listed.

• Supplemental Table 2 (Adobe PDF) - Table S2. Genes preferentially expressed in wild-type wing and leg discs. To identify genes with elevated expression in wing and leg discs, nine comparisons of a single wing disc to a pair of leg imaginal discs isolated from the same animal were performed as detailed in Table S1. The median of the expression ratios was determined and the genes were ranked according to their median ratios. For this and in all tables, yellow background indicates genes that encode transcription factors and genes referred to in the text are highlighted in blue.

• Supplemental Table 3 (Adobe PDF) - Table S3. Genes preferentially expressed in dorsal or ventral cell populations of wild-type and Wg overexpressing leg discs. For wild-type discs, single 1st leg discs were cut approximately along the DV boundary. Probes prepared from these fragments were used to measure the expression ratios of three replicate D_WT-to-V_WT comparisons. Median expression ratios of genes with transcripts enriched in the dorsal and ventral fragments. A similar procedure was followed for Wg-expressing discs, except that the D_Wg cells exclude GFP-marked cells of the ‘weak point’ that transdetermine. The table summarizes three replicate direct comparisons for each genotype.
• Supplemental Table 4 (Adobe PDF) - Table S4. Genes preferentially expressed in regenerating cells. Disc fragments consisting of the three-quarter piece of a 2nd leg disc were cultivated in the abdomen of a female fly; after 3-5 days, RNA was extracted and probes were compared with the common leg reference sample. This three-quarter piece, which excludes the upper median quarter of the disc, regenerates but does not transdetermine (G.S., unpublished). Four replicate experiments were performed and the results were summarized by calculating the median values.

• Supplemental Table 5 (Adobe PDF) - Table S5. Genes preferentially expressed in TD cells. GFP-positive cells were dissected from Wg overexpressing 1st leg discs (TD cells), and the remaining disc was cut approximately along the DV boundary (Fig. 2). This procedure generated TD, D_Wg and V_Wg populations. For each preparation, four to eight discs were fragmented. Three replicate experiments of TD-to-D_Wg and five independent TD-to-V_Wg comparisons were performed. The fold induction in TD cells was calculated as the median of these eight experiments. Genes in the TD list that were also identified in other comparisons are listed. Background colors identify genes identified in three comparisons (gold), genes encoding transcription factors (yellow) and genes referred to in the text (blue).

• Supplemental Table 6 (Adobe PDF) - Table S6. Evolutionary conservation of the lama gene. Alignment of the D. melanogaster (DRO-Q94898), mouse (MUS-Q8BHG8), human (HOM-Q8NHP8), C. elegans (CEL-O62146) and Dictyostelium discoideum (DIC-Q8MN53) sequences reveals a high degree of conservation (the numbers indicate accession numbers). Residues are in the one-letter code. Identical residues or conservative exchanges in all five sequences are highlighted in red and matches in at least two sequences are highlighted in blue. !, either I or V; $, either L or M; %, either F or Y; #, N, D, Q, E, B or Z. Multiple sequence alignment with hierarchical clustering was performed with Multalin version 5.4.1 (Corpet, 1988) using the default settings.

• Supplemental Table 7 (Adobe PDF) - Table S7. Cluster analysis identifies genes enriched in transdetermining cells. This table lists the identification numbers and annotated function for genes enriched in TD cells (yellow) of sub-clusters I-IV. For details of the cluster analysis see legend to Fig. 3.

• Supplemental Table 8 (Adobe PDF) - Table S8. Cluster analysis identifies genes depleted in transdetermining cells. This table lists the identification numbers and annotated function for the genes depleted in TD cells (blue) of sub-clusters V-VI. For details of the cluster analysis see legend to Fig. 3.

• Supplemental Table 9 (Adobe PDF) - Table S9. Alignment of D. melanogaster, D. yakuba and D. pseudoobscura vgBE sequences. Sequences were obtained from the UCSC Genome Browser webpage. The Su(H)-binding site (Kim et al., 1996) is labeled in green, the two putative Pan sites (Klein and Arias, 1999) in red and the site matching the Sd consensus (Halder et al., 1998) in blue.