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First published online January 27, 2005
doi: 10.1242/10.1242/dev.01653

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bHLH-dependent and -independent modes of Ath5 gene regulation during retinal development

¹ Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132, USA
² Divisions of Developmental Biology and Ophthalmology, Children's Hospital Research Foundation, Cincinnati, OH 45229, USA

View larger version (43K): [in a new window]   Fig. 1. Identification of a Xath5 regulatory fragment that functions in vivo. (A) The pG1X5 construct contains 3.3 kb of 5' Xath5a genomic DNA cloned into the promoterless vector pG1 with a GFP reporter. The TATAA element is at –226 bp, the putative transcription start site is at –205 bp and the translation start site is at +1 bp. (B) In situ hybridization showing that endogenous Xath5 mRNA is expressed in the retina, the pineal gland (arrowhead), and the olfactory placodes (arrows) of a stage 28 embryo (frontal view). (C,D) Frontal and lateral views of a stage 28 transgenic pG1X5 embryo showing expression of the transgene in the same tissues as the endogenous Xath5 mRNA (compare with B).

View larger version (45K): [in a new window]   Fig. 2. Deletion analysis isolates a proximal Xath5 cis-regulatory region. (A) HindIII, DraII and PstI sites were used to delete regions of Xath5 genomic sequence in the pG1X5 construct. All three constructs drove strong transgene expression in the retina. (B) The pG1X5-proximal construct containing sequences from –427 to –1 drove robust retinal expression of the transgene. The pG1X5-TATAA construct containing sequences from –226 to –1 showed no expression. Xath5 sequence from –427 to –226, when fused to the Fos heterologous basal promoter (pG1-cfos-201bp-X5), drove retinal expression, but more weakly. The basal Fos promoter alone (pG1-cfos) did not confer retinal GFP expression. (C) A pG1X5-proximal transgenic embryo (stage 33) shows retinal GFP expression. (D) In situ hybridization of pG1-cfos-201bp-X5 (stage 32) transgenic embryo shows weak GFP expression in the retina.

View larger version (56K): [in a new window]   Fig. 3. The proximal regulatory region mimics endogenous Xath5 retinal expression in the ciliary marginal zone. (A-C) Double in situ hybridization on retinal sections from stage 42 embryos comparing expression of the GFP transgene mRNA (red; A,B) as driven by the pG1X5-proximal construct to endogenous Xath5 mRNA (purple; B,C). (D) Unlike the RNA, GFP protein is found throughout the central retina and in many cell types at stage 42. Bracket marks the CMZ, which lacks GFP fluorescence. (E-G) Higher magnification images of the ciliary marginal zone (CMZ) from A-C showing cell-by-cell correlation of GFP (red) and Xath5 (purple) mRNAs.

View larger version (53K): [in a new window]   Fig. 4. Interspecies Ath5 sequence analysis identifies a highly conserved proximal non-coding region. (A) Pairwise VISTA analysis of X. laevis and mouse Ath5 5' noncoding sequences identifies a highly conserved region (black arrowhead) within the proximal region of Xath5 5' sequence, as well as a weaker region of conservation more distally (–1597 to –1669). Basepair position relative to the Xath5 translation start site (+1 bp) indicated at bottom of the VISTA alignment. Approximate location of Ath5 exon indicated by double arrowed line. (B) Alignment of the conserved region from X. laevis, X. tropicalis, mouse, human, chick, zebrafish and fugu shows blocks of highly conserved nucleotides (gray). Bars mark two conserved E-box binding sites (CANNTG) found in all species.

View larger version (44K): [in a new window]   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.

View larger version (87K): [in a new window]   Fig. 6. 3.3 kb Xath5 transgene expression can be activated by bHLH factors, but is not dependent upon conserved E-boxes or bHLH activity. (A-C) Injection of RNA encoding RFP alone did not alter transgene expression on the injected side. (D-E) Injection of RNA for Xath5 ectopically activated transgene expression on the injected side (bracket in E) compared with the uninjected side (D). RFP marks the region targeted on the injected side (bracket in F). (G) Mutation of E1 and E2 (E1,2) in the pG1X5-3.3 kb transgene did not eliminate retinal transgene expression. Mutation of two additional conserved E-boxes, E3 and E4, also did not abolish transgene activity. (H-J) Injection of RNA for the dominant-negative Xath5-EnR did not suppress expression of the pG1X5-3.3 kb transgene on the injected side (I) compared with the uninjected side (H). RNA encoding RFP (red) was co-injected to mark the injected side (J).

View larger version (21K): [in a new window]   Fig. 7. The distal cis-regulatory region of Xath5 alone is sufficient to promote transgene expression in the developing retina. (A) A PstI fragment that lacks the conserved proximal region promotes retinal transgene expression when coupled to either the pG1X5-TATAA basal promoter (pG1X5 distal + TATAA) or to the Fos basal promoter (pG1X5 distal + Fos). (B) Fluorescent image of a stage 30 pG1X5distal+TATAA transgenic embryo showing expression of the transgene in the retina.

View larger version (96K): [in a new window]   Fig. 8. Cross-species analysis of Ath5 transgene expression. (A) The proximal 600 bp of the Math5 cis-regulatory region drives weak GFP expression in the retina of transgenic frog embryos, as shown by in situ hybridization on a pG1M5-0.6 kb transgenic Xenopus embryo. (B) GFP in situ hybridization in a pG1M5-2.3 kb transgenic embryo. The 2.3 kb Math5 fragment drives strong transgene expression in the retina, cranial ganglia, midbrain and hindbrain regions in transgenic Xenopus embryos. (C) In situ hybridization on retinal sections from stage 41 pG1M5-2.3 kb transgenic embryos shows that the domain of GFP mRNA is restricted to the CMZ (bracket). (D) E13.5 whole embryo image demonstrating retinal expression of the pG1M5-2.3 kb transgene in mouse embryos at E13.5 (arrow). (E) The Xenopus 3.3 kb Xath5 transgene (pG1X5-3.3 kb) also shows retinal expression at E13.5. (F,G) Math5 is not required for expression of the pG1M5-2.3 kb transgene as equivalent fluorescence from the transgene was observed between wild-type (F) and Math5–/– (G) embryos. Scale bar: 500 µm in D.