Transdetermination study

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. We analyzed the expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate our methods for expression profiling as a way to analyze developmental programs, and show that modifications to chromatin structure are key to changes in cell fate. Our findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells. Keywords: cell type comparison, response to genetic alteration (Wingless over-expression) or mechanical injury (regeneration) Seeking to identify genes expressed in TD cells, we overexpressed Wg just after the molt to the third instar, and 3 days later isolated leg discs prior to pupariation. Probes generated from mRNA by linear amplification were applied to DNA microarrays. In order to distill the genes involved in transdetermination from the expression profiles of more than 14,000 genes of transdetermining (TD) cells, we generated nine different types of samples for control and comparative purposes: (1) wild-type wing (W); (2) leg (L) discs of 3rd instar larvae; (3-6) dorsal and ventral cells from both wild-type and Wg-expressing discs (D(wt), V(wt), D(wg) and V(wg)); (7) intact Wg-induced 1st leg discs; (8) micro-dissected TD cells from these discs (TD); and (9) 3/4 fragments of 2nd leg discs that were cultivated to obtain a population of regenerating cells (Sustar and Schubiger, 2005). Last, we prepared a ‘reference sample’ of wild-type leg discs by pooling amplified RNA of 1st, 2nd and 3rd leg discs of third instar male and female wildtype larvae. DNA arrays were hybridized to pairs of probes that had been generated from the same larva or, when this was impractical, to the common reference sample. Strong overexpression of Wg produces ectopic growth in 1st leg discs. Previously, we have shown that this response to Wg overexpression is region specific (Maves and Schubiger, 1998; Sustar and Schubiger, 2005). Cells in the ventral part of the disc (VWg cells) produce only structures characteristic of this region – ‘fate map structures’. Cells in the dorsal part respond in two ways. Dorsal cells in the ‘weak point’ (TD cells) transdetermine to wing; dorsal cells surrounding the transdetermined region (DWg cells) regenerate a new posterior compartment. These two dorsal populations can be distinguished in the disc with the use of a reporter transgene that expresses GFP under the control of the vg boundary enhancer (vgBE) (Kim et al., 1996; Williams et al., 1991). Leg-to- wing transdetermination depends upon vg expression driven by the vgBE and expression of the vgBE-GFP reporter clearly marks transdetermining leg cells (Maves and Schubiger, 1998). The 9 categories of experiments are: category #1: prothoracic leg disc vs. reference (4 independent replicates, 1 dye-swap); category #2: wing vs leg imaginal discs (9 independent replicates, 4 dye-swaps); category #3: wg-induced 1st leg disc vs. reference (6 independent replicates, 3 dye-swaps); category #4: TD cells vs. D(wg) cells (3 independent replicates, 1 dye-swap); category #5: TD cells vs. V(wg) cells (5 independent replicates, 2 dye-swaps); category #6: D(wg) cells vs V(wg) cells (3 independent replicates, 2 dye-swaps); category #7: TD cells vs. D(wt) cells (2 independent replicates, 2 dye-swaps); category #8: D(wt) cells vs V(wt) cells (3 independent replicates, 1 dye-swap); category #9:regenerating discs vs reference (4 independent replicates, 2 dye-swaps);