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

Abstract

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.

Generation of Mutants from the 57B Region of Drosophila melanogaster

“…Another advantage is the integration of an attP site in the locus instead of the deleted sequence, which could be used afterward to reintegrate various DNA sequences like a Gal4 sequence or a reporter gene with the help of specific reintegration vectors [73]. In our hands, this worked pretty well and we generated new mutant alleles for the genes otp, DRx, hbn, and earmuff (erm) [74] and reintegrated Gal4 into the locus to make Gal4 strains of the corresponding genes [44][45][46]75]. Our targeting efficiencies were in the same range as those reported by [73] between 1/1000 and 1/2000.…”

“…Summarized our previous work, we isolated three homeobox genes from the 57B region, the gene orthopedia (otp) [38,39], Drosophila Retinal homeobox (DRx) [40,41], and homeobrain (hbn) [42,43]. All genes are located in a 77 kb interval and build a cluster of homeobox genes showing very interesting expression patterns during development mainly in the nervous system [43][44][45][46]. To analyse the function of these genes we isolated mutants from the 57B region performing a classical EMS mutagenesis experiment using a deficiency uncovering the 57B region and succeeded in the isolation of lethal mutant alleles for all three genes [39,43,47].…”

Orthopedia expression during Drosophila melanogaster nervous system development and its regulation by microRNA-252

“…Our targeting efficiency of 1/775 was even better than those reported by Baena-Lopez et al which were between 1/1000 and 1/2000. In similar experiments we made mutants using comparably small deletions for the genes DRx and homeobrain with efficiencies ranging between 1/600 and 1/700 (Kl€ oppel et al, 2021;Hildebrandt et al, 2022), whereas for the earmuff gene a deletion of 1.5 kb resulted in a drop in the efficiency to 1/1894 (Hildebrandt et al, 2021). Using an attP site integrated in the otp locus instead of the deleted sequences it was then possible to reintegrate Gal4 into the locus to generate an otp Gal4 strain.…”