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During central nervous system development the timing of progenitor differentiation must be precisely controlled to generate the proper number and complement of neuronal cell types. Proneural basic helix-loop-helix (bHLH) transcription factors play a central role in regulating neurogenesis, and thus the timing of their expression must be regulated to ensure that they act at the appropriate developmental time. In the developing retina, the expression of the bHLH factor Ath5 is controlled by multiple signals in early retinal progenitors, although less is known about how these signals are coordinated to ensure correct spatial and temporal pattern of gene expression. Here we identify a key distalXath5 enhancer and show that this enhancer regulates the early phase of Xath5 expression, while the proximal enhancer we previously identified acts later. The distal enhancer responds to Pax6, a key patterning factor in the optic vesicle, while FGF signaling regulates Xath5 expression through sequences outside of this region. In addition, we have identified an inhibitory element adjacent to the conserved distal enhancer region that is required to prevent premature initiation of expression in the retina. This temporal regulation of Xath5 gene expression is comparable to proneural gene regulation in Drosophila, whereby separate enhancers regulate different temporal phases of expression.
Fig. 1. Identification of a conserved minimal Xath5distal enhancer that promotes retinal expression in vivo. (A) Pairwise mVISTA (http://genome.lbl.gov/vista/index.shtml) analysis of X. laevis and X. tropicalis Xath5 5′ distal noncoding sequences identifies a 1 kb highly conserved region. (B) A series of nested deletion constructs were generated to identify a minimal 152 bp enhancer that is sufficient to promote retinal specific expression. + strong expression, −/+ very weak expression, − no expression (C) A stage 33 pG1X5 distal 1 kb transgenic embryo shows retinal GFP expression. (D) The 152 bp distal enhancer also drives GFP in the retina. (E) A pG1X5 distal 73 bp transgenic embryo does not express GFP in the retina but only expresses in the pineal gland and olfactory placodes. (F–H) The zc38 transgenic zebrafish line exhibits specific expression in the retina as shown in confocal z-projections at 18 hpf (F), 24 hpf (G), 52 hpf (H) Anterior is to the right in all panels.
Fig. 2. Expression of the 152 bp distal enhancer is premature compared to endogenous Xath5 expression and an additional 100 bp prevents premature transgene expression. (A–B) pG1 X5 3.3 kb and (C–D) X5 distal 1 kb embryos do not begin expressing GFP in the retina until after stage 22. (E–F) The X5 distal 152 bp mult transgenic embryos express GFP in the retina at both stage 22 and stage 32/33. Addition of 100 bp of sequence 5′ to the distal enhancer prevents premature expression (G–H). Each embryo was imaged at stage 22 and again at stage 32/33. Marks gut autofluorescence. (I) Deletion constructs were assayed for retinal GFP expression at stage 22 identifying a 100 bp region that inhibits precocious expression (marked by bracket). + strong expression, −/+ very weak expression, − no expression.
Fig. 3. The 152 bp Xath5distal enhancer does not depend upon conserved E-boxes. (A) ClustalW alignment of the 152 bp distal enhancer sequence between human, mouse, chick and X. laevis shows blocks of highly conserved sequence (gray). Black lines mark the 4 conserved E-boxes in this region (CANNTG). + strong expression. Mutation of E-boxes E3 and E4 (B) or all 4 E-boxes E3–E6 (B′) does not reduce retinal GFP expression in transgenic embryos. (C) A stage 33 pG1X5 distal 152 bp E3,4 mutembryo shows GFP expression in the retina. (D) A stage 33 pG1X5 distal 152 bp E3,4,5,6 mutembryo show GFP expression in the retina as well as ectopic expression in the neural tube and cranial ganglia. Dashed lines mark the 2 overlapping Pax6 sites in the distal enhancer.
Fig. 4. Pax6 is required for Xath5 expression but is not sufficient to induce Xath5 expression. Overexpression of dnPax6 mRNA decreases Xath5 expression on the injected side (A–B) but does not affect expression of the progenitor markers Vsx1 (C–D) or Rx (E–F). Overexpression of Pax6 mRNA is not sufficient to induce ectopic Xath5 expression (G–H) or ectopic pG1X5 3.3 kb transgene expression (I–J). Gut autofluorescence.
Fig. 5. Expression of the 152 bp distal enhancer is dependent upon conserved Pax6 sites and Pax6 binds to this region in vivo. (A) Sequence from the Xenopus laevis 152 bp enhancer region showing the 2 overlapping Pax6 sites identified using Transfac MATCH version 10.3 (http://www.gene-regulation.com/pub/databases.html#transfac). P1 aligns with the Pax6_01 matrix, while P2 aligns with the Pax6_Q2 matrix. (B) Mutation of one Pax6 site dramatically reduces (−/+) while mutation of both Pax6 sites in the 152 bp distal enhancer results in retinal GFP expression in only a few cells (−/(+)) in transgenic embryos. (C) A stage 33 pG1X5 distal 152 bp transgenic embryo shows strong GFP expression in the retina. (D) A stage 33 pG1X5 distal 152 bp P1P2 mut transgenic embryo does not have retinal GFP expression. (E) ChIP showing Pax6 binds to the distal enhancer region in vivo (group student t test P = 0.002 for enhancer region (IgG vs. Pax6 antibody), and 0.056 for the GFP coding region (IgG vs. Pax6 antibody)). Error bars represent standard deviation. Gut autofluorescence.
Fig. 6. Endogenous Xath5 expression is regulated by FGF signaling. Stage 28 Fzd5-XFD transgenic embryos show decreased expression of bHLH transcription factors Xath5 (B), NeuroD (D) vs. non-transgenic controls (A, C). The expression of retinal progenitor genes Rx (E–F), and Sox2 (G–H) is not affected. Retinal expression of Xer81 is inhibited in Fz5-XFD transgenic embryos (J) vs. non-transgenic controls (I).
Fig. 7. FGF signaling acts to regulate Xath5 expression through the 3.3 kb enhancer sequence but this effect is not mediated via the 152 bp distal enhancer. Overexpression of XFD mRNA in pG1 X5 3.3 kb transgenic embryos inhibits transgene expression on the injected side (A–B). Ath5 expression is blocked in zebrafish embryos treated with SU5402 vs. DMSO treated embryos (C–D). zc38 transgenic embryos treated with SU5402 express GFP throughout the retina, similar to DMSO treated controls (E–F).
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