Fig. 5. Proximal transgene expression is dependent upon conserved E-boxes and bHLH activity, but E-boxes alone are not sufficient for retinal expression. (A) Sequential deletions of the Xath5 proximal region: pG1X5-proximal-401 provided robust retinal GFP expression while pG1X5-proximal-389, -349 and -337 drove weaker retinal expression and at reduced frequency. (B) Mutation of either E1 or E2 (E) reduced the percentage of embryos expressing GFP while mutation of both eliminated all transgene expression. (C-E) Injection of RNA for the dominant-negative Xath5-EnR suppressed expression of the pG1X5-proximal GFP transgene on the injected side (D) compared with the uninjected side (C). RNA encoding RFP (red) was co-injected to mark the injected side (E). (F) Multimerized E-boxes (pG1X5-TATAA+2xE1E2) were not sufficient to promote expression, while the E-boxes and adjacent -box (pG1X5-TATAA+33 bp) promoted non-specific GFP expression throughout the CNS and head musculature (GFP+ overall), but did not promote specific retinal expression. The pG1X5-TATAA+48 bp transgenic construct promoted GFP expression in a Xath5-like pattern, but also in the axial somites, with a small percentage only showing expression in axial somites (contributing to the increase in overall GFP+ embryos). The robustness of transgene expression was reduced by mutation of the -box with the pG1X5-TATAA+48 bp transgenic construct. (G) pG1X5-TATAA+33 bp is expressed non-specifically in CNS and muscle (H) pG1X5-TATAA+48 bp transgene is expressed in Xath5-like pattern and in axial somites. Asterisk indicates gut autofluorescence.