Regulatory modules mediating the complex neural expression patterns of the homeobrain gene during Drosophila brain development.

BACKGROUND: The homeobox gene homeobrain (hbn) is located in the 57B region together with two other homeobox genes, Drosophila Retinal homeobox (DRx) and orthopedia (otp). All three genes encode transcription factors with important functions in brain development. Hbn mutants are embryonic lethal and characterized by a reduction in the anterior protocerebrum, including the mushroom bodies, and a loss of the supraoesophageal brain commissure. RESULTS: In this study we conducted a detailed expression analysis of Hbn in later developmental stages. In the larval brain, Hbn is expressed in all type II lineages and the optic lobes, including the medulla and lobula plug. The gene is expressed in the cortex of the medulla and the lobula rim in the adult brain. We generated a new hbnKOGal4 enhancer trap strain by reintegrating Gal4 in the hbn locus through gene targeting, which reflects the complete hbn expression during development. Eight different enhancer-Gal4 strains covering 12 kb upstream of hbn, the two large introns and 5 kb downstream of the gene, were established and hbn expression was investigated. We characterized several enhancers that drive expression in specific areas of the brain throughout development, from embryo to the adulthood. Finally, we generated deletions of four of these enhancer regions through gene targeting and analysed their effects on the expression and function of hbn. CONCLUSION: The complex expression of Hbn in the developing brain is regulated by several specific enhancers within the hbn locus. Each enhancer fragment drives hbn expression in several specific cell lineages, and with largely overlapping patterns, suggesting the presence of shadow enhancers and enhancer redundancy. Specific enhancer deletion strains generated by gene targeting display developmental defects in the brain. This analysis opens an avenue for a deeper analysis of hbn regulatory elements in the future.

The Drosophila homeodomain transcription factor Homeobrain is involved in the formation of the embryonic protocerebrum and the supraesophageal brain commissure.

During the embryonic development of Drosophila melanogaster many transcriptional activators are involved in the formation of the embryonic brain. In our study we show that the transcription factor Homeobrain (Hbn), a member of the 57B homeobox gene cluster, is an additional factor involved in the formation of the embryonic Drosophila brain. Using a Hbn antibody and specific cell type markers a detailed expression analysis during embryonic brain development was conducted. We show that Hbn is expressed in several regions in the protocerebrum, including fibre tract founder cells closely associated with the supraesophageal brain commissure and also in the mushroom bodies. During the formation of the supraesophageal commissure, Hbn and FasII-positive founder cells build an interhemispheric bridge priming the commissure and thereby linking both brain hemispheres. The Hbn expression is restricted to neural but not glial cells in the embryonic brain. In a mutagenesis screen we generated two mutant hbn alleles that both show embryonic lethality. The phenotype of the hbn mutant alleles is characterized by a reduction of the protocerebrum, a loss of the supraesophageal commissure and mushroom body progenitors and also by a dislocation of the optic lobes. Extensive apoptosis correlates with the impaired formation of the embryonic protocerebrum and the supraesophageal commissure. Our results show that Hbn is another important factor for embryonic brain development in Drosophila melanogaster.

The cis-regulatory code of Hox function in Drosophila.

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, our understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, we have identified binding regions for the Hox TF Deformed (Dfd) in the Drosophila genome. Our analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. We also find that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, our results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo.

Vectors for efficient and high-throughput construction of fluorescent drosophila reporters using the PhiC31 site-specific integration system.

The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements or enhancers. Here, we present a rapid and highly efficient system for the large-scale analysis of enhancer elements, site-specifically integrated into the Drosophila genome. This system, which is scalable for either small projects or high-throughput approaches, makes use of the Gateway cloning technology and the PhiC31 site-specific integration system, which allows the insertion of constructs at predetermined genomic locations. Thus, this system allows not only a fast and easy analysis of reporter gene expression in live animals, but also the simultaneous analysis of different regulatory outputs on a cellular resolution by recombining in the same animal distinct enhancer elements fused to different fluorescent proteins.