Fig. 2 Deletion mapping of the retinal enhancer (RetE). A series of deletion constructs were generated (A) to identify the region required for shh expression in the retina. Transgenes carrying the region from + 549 to + 2381 and + 549 to + 2020 drive expression in the GCL and INL (B, C). The transgene carrying only the conserved region as ar-A from + 2021 to + 2381 failed to show any GFP expression in the retina (D). Transient expression of deletion constructs (E–J). The constructs carrying shh sequence from + 729 to + 2020 and + 549 to + 1035 mediate expression in the retina (E, F). The construct containing shh sequences from + 549 to + 829 did not give retina expression (G). Embryos injected with the -2.4shh:gfpRetE plasmid with the minimal region + 729 to + 1035 have GFP expression in the retina (H), while embryos injected with the -2.4shh:gfp + 549/2020 plasmid with an internal deletion from + 729 to + 1035 failed to drive expression in the retina (I). Embryos injected with the RetE (+ 549 to + 2020) in the context of a heterologous thymidine kinase (-37Tk) promoter -37tk:gfpRetE show retina expression (J). Anterior is to the top in all images and embryos were photographed at 72 hpf. (K–O) In vivo time lapse imaging of -2.4shh:gfpRetE expression in the retina. Single frames taken from a film recording of -2.4shh:gfpRetE expression in the retina, starting at 35 hpf and ending at 54 hpf. The spread of GFP expression is marked by arrowheads. R, retinal expression; L, lens; GCL, ganglion cell layer; INL, inner nuclear layer. Scale bar, 50 μm, panels B–J; 25 μm, panels K–O. +/- Indicates the presence or absence of GFP expression in the retina.
Reprinted from Developmental Biology, 314(1), Vinothkumar, S., Rastegar, S., Takamiya, M., Ertzer, R., and Strähle, U., Sequential and cooperative action of Fgfs and Shh in the zebrafish retina, 200-214, Copyright (2008) with permission from Elsevier. Full text @ Dev. Biol.