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. 2002 Feb 19;99(4):2134–2139. doi: 10.1073/pnas.032426699

Figure 4.

Figure 4

Spatial and temporal expression of clock genes in the reproductive system of D. melanogaster. (A) 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) staining demonstrating strong per–lacZ expression in the SV (s) and the lower testes (lt), weak signal in the ejaculatory duct (e) and in the upper testes (t), and no signal in the paragonial glands (p). (Inset) Expression of tim-driven green fluorescent protein reporter in the lower testes–SV. (B) Immunofluorescence detecting PER and TIM proteins in the nuclei of SV epithelial cells at ZT 20 but not at ZT 8. (C) In situ hybridization with antisense probes detecting per mRNA in the SV and lower testis. Higher magnification of SV showing that epithelial cells are per-negative at ZT 4 and per-positive at ZT 16. (D) In situ hybridization at ZT 4 with antisense Clk mRNA show colocalization with per in the SV and lower testis. Higher magnification of SV showing that Clk is expressed in epithelial cells at ZT 4 but not at ZT 16. Control hybridization with respective sense probes yielded no signal. Images represent the prevailing pattern observed among at least five specimens used to assess staining with each reagent at a given time point.