SUMMARYAlthough most metazoan genes undergo alternative splicing, the functional relevance of the majority of alternative splicing products is still unknown. Here we explore this problem in the Drosophila Hox gene Ultrabithorax (Ubx). Ubx produces a family of six protein isoforms through alternative splicing. To investigate the functional specificity of the Ubx isoforms, we studied their role during the formation of the Drosophila halteres, small dorsal appendages that are essential for normal flight. Our work shows that isoform Ia, which is encoded by all Ubx exons, is more efficient than isoform IVa, which lacks the amino acids coded by two small exons, in controlling haltere development and regulating Ubx downstream targets. However, our experiments also demonstrate that the functional differences among the Ubx isoforms can be compensated for by increasing the expression levels of the less efficient form. The analysis of the DNA-binding profiles of Ubx isoforms to a natural Ubx target, spalt, shows no major differences in isoform DNA-binding activities, suggesting that alternative splicing might primarily affect the regulatory capacity of the isoforms rather than their DNA-binding patterns. Our results suggest that to obtain distinct functional outputs during normal development genes must integrate the generation of qualitative differences by alternative splicing to quantitative processes affecting isoform protein expression levels. (IV). Each Ubx protein variant (i.e. I, II or IV) is named as 'a' or 'b' depending on: (a) the lack of, or (b) the inclusion of a small nine amino acid segment encoded between the two donor splicing sites at the 3Đ end of the first exon (O'Connor et al., 1988;Kornfeld et al., 1989) (Fig. 1A). Previous studies have revealed that each isoform has distinct expression in the embryo, that class 'b' isoforms are much less abundant than class 'a' proteins, and that isoform Ia is the predominant one in most stages (O'Connor et al., 1988;Kornfeld et al., 1989;LĂłpez and Hogness, 1991;Artero et al., 1992; Bomze and LĂłpez, 1994;LĂłpez et al., 1996). The extent to which the different isoforms possess different functions remains unclear. A pioneer analysis was done with the Ubx MX17 mutation, an inversion with breakpoints to the left and right of the second microexon, and which forms only isoforms IVa and IVb (Busturia et al., 1990;Subramaniam et al., 1994). Homozygous Ubx MX17 adults present small changes in phenotype, most obviously a partial transformation of haltere to wing. This effect, however, was attributed to a reduction in Ubx protein expression in the haltere disc and not to changes in the distribution of the different Ubx proteins, thus suggesting all Ubx isoforms are functionally equivalent (Busturia et al., 1990).Other experiments, however, showed that changes in the activities of the Ia and IVa isoforms affected the peripheral nervous system (Mann and Hogness, 1990;Subramaniam et al., 1994) or the Keilin's organs, particular sensory structures (Gebelein et al., 2002), differently...