Changes in core-promoter binding proteins may affect wing differentiation. (A) Expression data from the wing developmental time course for genes that encode core promoter binding proteins, general transcription components, and TBP-associated factors (TAFs). Each row corresponds to a single gene and each column represents an individual time point. Expression values (log2) are color coded according to the legend at the bottom. Specific groups of genes are indicated. The complete clustering of all 72 genes is provided in Additional file7. (B) RT-PCR and western blot analyses were used to examine RNA or protein levels for selected factors in pupal wing tissue. By microarray analysis sa, which is a tTAF, did not increase expression at the time points shown. This was verified by western blot. Levels of the tTAF Mia increased in the wing, both by RT-PCR (at 24 h APF) and western blot (at 36 h APF). Mia-specific bands at 35 and 70 kD (indicated by asterisks) are observed with anti-Mia antibody in select tissues (N. Haugen and D. Wassarman, personal communication). BEAF decreased in the wing over time. Acetylated histone H4 served as a loading control. (C-F) Using UAS-RNAi lines, engrailed-Gal4 was used to inhibit either white (w), or nht in the posterior wing. Tubulin-Gal80TS was used to limit RNAi expression from the second larval instar until eclosion. Compared to controls (C), nht reduction affected posterior wing growth and cuticle integrity (D). Staining for F-actin reveals developing wing hairs at 34 h APF (E). Compared to the anterior control, expression of nht RNAi led to delay in wing hair formation in the posterior (F). nhtz5946 hemizygotes, exhibit ectopic vein and multiple wing hair phenotypes (G, inset). Two nht alleles in trans (nhtz5347/nhtz5946) at 25°C exhibit ectopic vein (indicated by arrowheads in H, with 20X magnification in I). Approximately 10% of nhtz5347/nhtz5946 females exhibit patches of thin, small wing hairs (outlined by dashed line in J).