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During Drosophila embryogenesis, the beta3 tubulin gene is expressed in the visceral and somatic mesoderm as well as in the dorsal vessel. Transcription of the gene is limited to four pairs of cardioblasts per segment. Here we show that its expression in the dorsal vessel (dv) is mediated by a 333-bp enhancer located upstream of the gene. The homeodomain protein Tinman is also expressed in these cardioblasts, implying that Tinman might be a key regulator of the beta3 tubulin gene. Gel retardation and footprint assays indeed revealed two Tinman binding sites within the dv-specific enhancer. We analyzed the relevance of the Tinman binding sites in a transgenic fly assay and observed distinct functions for both sites. The BS(Tin-1460) site is absolutely required for expression in cardioblasts, while BS(Tin-1425) is needed for high-level expression. Thus, these two Tinman binding sites act in concert to drive beta3 tubulin gene expression during heart development. Tinman initially functions in the specification of visceral mesoderm and heart progenitors, but remains expressed in cardioblasts until dorsal closure. Overall, our data demonstrate a late function for Tinman in the regulation of beta3 tubulin gene expression in the forming heart of Drosophila., (Copyright 1999 Academic Press.)

The beta3 tubulin gene of Drosophila is expressed in the major mesodermal derivatives during their differentiation. The gene is subject to complex stage- and tissue-specific transcriptional control by upstream as well as downstream regions. Analysis of the vm1 enhancer, which is responsible for tissue-specific expression in the visceral mesoderm and is localized in the intron, revealed a complex modular arrangement of regulatory elements. In vitro and in vivo experiments uncovered two binding sites, termed UBX1 and UBX2, for the product of the homeotic gene Ultrabithorax (Ubx), which are required for high-level expression in pPS6 and PS7. Further analysis of the vm1 enhancer revealed that deletion of a specific element, termed element 7 (e7), abolishes transcription of the lacZ reporter gene in all parasegments except pPS6/PS7. Gel-retardation and footprint analysis identified a binding site for the homeodomain protein Tinman, which is essential for the specification of the dorsal mesoderm, within e7. Simultaneous deletion of two further sequence blocks in the vml enhancer, named elements 3 (e3), and 6 (e6), results in a reduction analogous to that caused by removal of e7. The e6 sequence contains conserved motifs also found in the visceral enhancer of the Ubx gene. Therefore we conclude that these elements act in concert with the Tinman binding site to achieve high expression levels. Thus the vm1 enhancer of the beta3 tubulin gene contains a complex array of elements that are involved in transactivation by a combination of tissue- and position-specific factors including Tinman and UBX.