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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.
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