The UBX-regulated network in the haltere imaginal disc of D. melanogaster

Hox proteins have been proposed to act at multiple levels within regulatory hierarchies and to directly control the expression of a plethora of target genes. However, for any specific Hox protein or tissue, very few direct in vivo-regulated target genes have been identified. Here, we have identified target genes of the Hox protein Ultrabithorax (UBX), which modifies the genetic regulatory network of the wing to generate the haltere, a modified hindwing. We used whole-genome microarrays and custom arrays including all predicted transcription factors and signaling molecules in the D. melanogaster genome to identify differentially expressed genes in wing and haltere imaginal discs. To elucidate the regulation of selected genes in more detail, we isolated cis-regulatory elements (CREs) for genes that were specifically expressed in either the wing disc or haltere disc. We demonstrate that UBX binds directly to sites in one element, and these sites are critical for activation in the haltere disc. These results indicate that haltere and metathoracic segment morphology is not achieved merely by turning off the wing and mesothoracic development programs, but rather specific genes must also be activated to form these structures. The evolution of haltere morphology involved changes in UBX-regulated target genes, both positive and negative, throughout the wing genetic regulatory network. Keywords: tissue comparison Two rounds of microaray expression profiling were performed. 1)Three samples each of wildtype, Cbx mutant, and vg mutant wing imaginal disc tissue were generated. Each sample consists of 60-100 pooled discs. Each sample was hybridized to two whole-genome arrays (GPL4573). Three biological replicates for each condition; two technical replicates for each biological sample (hybridization). 2)Three samples each of wildtype wing and haltere imaginal disc tissue; Cbx mutant wing imaginal disc tissue; vg mutant wing imaginal disc tissue. Each sample consists of 60-250 pooled discs. Each sample was hybridized to a single array that contained 4 replicates of each oligonucleotide sequence (GPL4568). Three biological replicates for each condition. Since each array contains 4 replicate blocks, 4 normalized values are generated for each gene for a given array. These replicates are indicated by "Block1, Block2" etc.