Enhancer analysis of the Drosophila zinc finger transcription factor Earmuff by gene targeting

Abstract

Background Many transcription factors are involved in the formation of the brain during the development of Drosophila melanogaster. The transcription factor Earmuff (Erm), a member of the forebrain embryonic zinc finger family (Fezf), is one of these important factors for brain development. One major function of Earmuff is the regulation of proliferation within type II neuroblast lineages in the brain; here, Earmuff is expressed in intermediate neural progenitor cells (INPs) and balances neuronal differentiation versus stem cell maintenance. Erm expression during development is regulated by several enhancers. Results In this work we show a functional analysis of erm and some of its enhancers. We generated a new erm mutant allele by gene targeting and reintegrated Gal4 to make an erm enhancer trap strain that could also be used on an erm mutant background. The deletion of three of the previously analysed enhancers showing the most prominent expression patterns of erm by gene targeting resulted in specific temporal and spatial defects in defined brain structures. These defects were already known but here could be assigned to specific enhancer regions. Conclusion This analysis is to our knowledge the first systematic analysis of several large enhancer deletions of a Drosophila gene by gene targeting and will enable deeper analysis of erm enhancer functions 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.…”

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

“…In our study, we decided to use gene targeting for the precise deletion of larger enhancer regions. To our knowledge, this analysis and our recently published analyses of Erm [ 85 ] and DRx [ 55 ] are the first in which several larger enhancer regions were deleted alone and in one combination in Drosophila . In mice, a larger enhancer deletion analysis was made by genome editing of 23 enhancers from seven loci required for limb development [ 86 ].…”

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.…”