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Fig. 1. Rax2A2.8 directs gene expression in the eye fields, ventral forebrain, and pineal gland. Fluorescent images of Rax2A2.8/GFP transgenic embryos and tadpoles. Stages are indicated in each panel. Views: (A, E) dorsal; (B) anterior; (C, D, F, G) lateral. Expression of GFP is first evident in the anterior neural plate (A), and is present in the developing eye during neural tube and tailbud stages (B–F). (E) Dorsal view demonstrating eye-specific expression. (G) At tadpole stages, GFP fluorescence is obstructed by the RPE but is readily observed through the clear lens. Abbreviations: B — blastopore, E — eye, VF — ventral forebrain, P — pineal gland, L — lens, R — RPE. Arrows indicate nonspecific nasal expression.
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Fig. 2. Conservation and in silico analysis of the Rax2A regulatory region. (A) Percent identity plot (PIP) alignment of the Rax2A promoter with the Rax1A regulatory sequence (Zhang et al., 2003). Segments of DNA with percent identity above 50% are shown. Alignment analysis was performed using the PipMaker program (http://pipmaker.bx.psu.edu/pipmaker), which plots the percent identity of DNA segments between two or more designated sequences (Schwartz et al., 2000). (B) Schematic representation of the Rax2A regulatory region. Scale indicates transcription start to the right. Longest regions of highest identity revealed by PIP analysis (red boxes) and predicted cis-regulatory elements predicted by MatInspector are shown. Yellow: POU Domain Motif. Light Green: OTX Motif. Blue: SOX Motif. Dark green: Forkhead Motif. Regions of greatest divergence are shown in light blue. Arrows indicate relative locations of highly conserved regions or non-conserved regions between A and B. (C) Alignments showing conservation of predicted transcription factor binding sites within Ultra-Conserved Element 3 (UCE3). Completely conserved residues are shown in gray. Colored boxes indicate core transcription factor binding motifs. Numbers indicate relative position from proximal end of isolated Rax2A regulatory region.
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Fig. 3. Deletion constructs produced to investigate transcriptional activity of the Rax2A regulatory region. Deletion name and schematic representation of transgene constructs are shown: black bar, Rax2A sequence; green bar, GFP; gray bar, heat shock protein gene (HSP) promoter. The entire regulatory sequence isolated is shown in schematic form above and below the transgene constructs: red, regions of high conservation with Rax1A sequence, light blue, regions of divergence with Rax1A sequence; blue, predicted inversion event. Vertical dashed lines delineate the edges of UCE3 (ultra-conserved element 3.)
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Fig. 4. The Rax2A promoter contains cis-elements required for proper spatial and temporal gene expression of the GFP transgene. Fluorescent images of tadpoles generated using Rax2A/GFP 5′-deletion transgenes, as indicated on the right side of the figure. (A–C) Rax2A − 2394; (D–F) Rax2A − 2160; (G–H) Rax2A − 1823; (I–J) Rax2A − 1208; (K) Rax2A − 1090; (L) Rax2A − 944; (M) Rax2A − 818 + FBE. Embryos are shown at early tailbud stages (~ st 24; A, D), mid-tailbud stages (~ st 28; B, E, G, I), or tadpole stages (~ st 41; C, F, H, J–M). All panels show lateral views except dorsal view in (E). White arrows — regions of endogenous expression; red arrows — ectopic expression.
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Fig. 5. Internal deletions of the Rax2A promoter demonstrate additional regulatory regions that provide spatial specificity. Fluorescent images of tadpoles generated using Rax2A/GFP internal deletion transgenes, as indicated on the right side of the figure. (A–B) Rax2A 818–1208; (C–E) Rax2A 818–1236; (F–H) Rax2A 818–2400. Embryos are shown at late neural plate/early neural tube stages (~ st 19/20; A, C, F) or tailbud stages (~ st 26–28; B, D, E, G). Embryo orientation: (A, C, F) anterior views; (B, D, E, G) lateral views. (H) Immuno-labeling of Rax2A 818–2400 transgene expression (brown spots) in the tadpole (st 41) heart (bracket). White arrows — regions of endogenous expression; red arrows — ectopic brain expression; red asterisks — ectopic ventral expression.
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Fig. 6. Cooperative activity of UCE and the proximal FBE directs gene expression throughout developmental stages. Fluorescent images of tadpoles generated using Rax2A-HSP/GFP transgenes, as indicated on the right side of the figure. (A) Rax2A 1703-hsp; (B) Rax2A 1823-hsp; (C) Rax2A 2136-hsp; (D–E) Rax2A 2136 + FBE-hsp. Embryos are shown at tailbud (~ st 26–28; A–D) or tadpole (~ st 41; E) stages. All panels show lateral views except anterior view in D.
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Fig. 7. POU, SOX and OTX sites within UCE3 cooperatively regulate Rax expression in the developing eyes of Xenopus laevis embryos. Fluorescent images of tadpoles generated using Rax2A/GFP with mutations in UCE3 conserved transcription factor binding sites. Transgenes represented are detailed on the right. (A–B) Rax2A UCE delPOU; (C–E) Rax2A mutOtx (− 2179/−1703); (F–H) Rax2A UCE mutSOX; (I–K) Rax2AUCE mutOtx/mutSox. Embryos are shown at tailbud (~ st 26–28; A, C, D, F, G) or tadpole (~ st 41; B, E, H) stages. All panels show lateral views. White arrows, regions of endogenous expression. Red arrows, ectopic expression.
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Fig. 8. Expression of Rax2A-GFP transgenes in the mature retina. Transgene expression was analyzed by in situ hybridization using a GFP riboprobe and sections of stage 41 transgenic tadpoles. (A, A′) Rax2A 2823; (B, B′) Rax2A 2160; (C) Rax2A 1764; (D) Rax2A 944; (E) Rax2A UCE3 mutOTX; (F) Rax2A UCE3 mutOtx/mutSox; (G) Rax2A ΔUCE3. A′ and B′ are high magnification views of A and B, respectively. Red brackets indicate the ciliary marginal zone (CMZ). Red arrowheads indicate examples of transgene expression in the inner nuclear layer. Abbreviations: L — lens; G — ganglion cell layer; I — inner nuclear layer; P — photoreceptor layer.
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Fig. 9. Model of Rax2A transcriptional regulation derived from deletion analysis. Abbreviations: ANP — anterior neural plate; CRE — central repressor element; DAE — distal activator element; DB — dorsal brain; PN — pan-neural.
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