Abstract
Reporters are widely used in Drosophila genetics to visualize gene expression and cell lineages. However, uncharacterized limitations in specific reporter lines can lead to data misinterpretation. Here, we identify a consistent, driver-independent tdTomato signal in the adult proventriculus from the widely used lexAop-tdTomato.nls reporter line. This signal was observed across multiple lexA driver combinations and was directly detectable in lexAop-tdTomato.nls responder-alone adult proventriculi lacking any lexA driver and without antibody staining. In contrast, no comparable native red fluorescence was detected in larval proventriculi under the same no-antibody imaging condition. Mouse and rabbit anti-RFP immunostaining further supported the presence of proventriculus-associated tdTomato/RFP antigen in adult responder-alone animals. In larval responder-alone proventriculi, antibody-amplified staining was antibody-source-dependent: a detectable signal was observed only with rabbit anti-RFP, whereas mouse and rat anti-RFP produced no reliable detectable signal under the same staining condition. A driver-matched comparison using lexAop-RFP.nls did not reproduce the proventricular signal, arguing against detectable ectopic activity of the tested lexA driver in this tissue. However, because lexAop-tdTomato.nls and lexAop-RFP.nls differ in reporter/transgene architecture and possibly genomic insertion context, the underlying cause cannot be assigned specifically to the lexAop sequence. Our findings highlight the necessity of including driver-negative and no-antibody controls when using this reporter line in adult Drosophila proventriculus and gut studies.
