Pax6 regulation of Math5 during mouse retinal neurogenesis

Abstract

Activation of the bHLH factor Math5 (Atoh7) is an initiating event for mammalian retinal neurogenesis, as it is critically required for retinal ganglion cell formation. However, the cis-regulatory elements and trans-acting factors that control Math5 expression are largely unknown. Using a combination of transgenic mice and bioinformatics, we identified a phylogenetically conserved regulatory element that is required to activate Math5 transcription during early retinal neurogenesis. This element drives retinal expression in vivo, in a cross-species transgenic assay. Previously, Pax6 was shown to be necessary for the initiation of Math5 mRNA expression. We extend this finding by showing that the Math5 retinal enhancer also requires Pax6 for its activation, via Pax6 binding to a highly conserved binding site. In addition, our data reveal that other retinal factors are required for accurate regulation of Math5 by Pax6.

Figure 1. Retinal expression of Math5 transgenes

A) Diagram of the Math5 locus, including the coding exon (blue box), and various transgenes containing different 5’ and 3’ Math5 noncoding fragments, driving GFP reporter expression. GFP expression was tested in the developing mouse or frog eye. An arrow denotes the Math5 TATA box. The right column shows number of independent mouse transgenic lines with GFP expression versus the number tested (n ≥ 3 litters scored per line). B) GFP expression in the optic cup of a living E12.5 Math5-GFP2.1 mouse embryo. C) Anti-GFP labeling of E12.5 retinal cryosection from same transgenic line as in B. D) Higher magnification images of anti-GFP (green) and anti-Brn3b (red) double labeling. Arrows point to coexpressing cells. Dorsal and scleral are up, rostral left in B–D; L = lens. Bar = 500 microns in B, 25 microns in C; 50 microns in D.

Figure 2. Math5 transgenes are sensitive to Pax6 gene dosage

A) Math5 mRNA (purple) and Pax6 protein (brown) are completely coexpressed at E11.5. B) After the ganglion cell layer (gcl) forms, differentiated RGCs continue to express Pax6 strongly, but shut off Math5 mRNA. C–E) Math5-GFP2.1 expression in wild type (C,C’), Pax6^Sey/+ (D,D’) and Pax6^Sey/Sey (E,E’) E12.5 embryonic eyes. Panels C–E show GFP fluorescence and C’–E’ bright field images living embryonic eyes (white circles indicate eyes). In both E11.5 and E12.5 embryos, the GFP expression domain is smaller in Pax6^Sey/+ eyes and completely lost in Pax6^Sey/Sey eyes. Rostral is up in A, scleral is up in B, Dorsal is up in C–E. Bar in A, B = 100 microns; C–E = 500 microns.

Figure 3. Pax6 activation of Math5 is cell autonomous

Confocal images of E13.5 of triple labeled retinal sections. Comparison of Math5^LacZ or Brn3b expression in E13.5 α-Cre;Pax6^CKO/+ and α-Cre;Pax6^CKO/CKO retinae. A–C) In α-Cre;Pax6^CKO/+ eyes, Pax6 (red) and βgal (blue) proteins are coexpressed within the Cre expression domain (green). E–G, I–K) Where Pax6 is deleted, Math5^LacZ expression is almost completely lost, but expressed at 1:1 correspondence with Pax6 protein in the central retina. Math5 dependence on Pax6 is highlighted in J and K where a single retinal cell lacking Cre-IRES-GFP autonomously retains both nuclear Pax6 and cytoplasmic βgal expression (arrowhead). White arrows in F,G,J,K point to Cre+; LacZ+; Pax6- cells. Yellow arrows in F,G point to Cre+;Pax6+ cells. See text for details. D,H,L) Two examples of E13.5 α-Cre;Pax6^CKO/CKO peripheral retinal sections with complete loss of Brn3b (blue nuclei) in Pax6^CKO/CKOcells (not red). Scleral is up; scale bar F = 400 microns.

Figure 4. Predicted Pax6 binding sites and phylogenetically conserved regions in mouse, human, frog and chick Ath5/Atoh7 5’ regulatory DNA

A) Pictogram of Mulan alignments of upstream DNA (−3000 to –1), between Math5 vs. Xath5 and Math5 vs. HATH5. Underneath is a diagram of 3 Kb of upstream Math5 DNA (0 = A of ATG start codon). Orange boxes represent ≥70% nucleotide identity between Math5-HATH5, identified by NCBI BLAST. The blue box denotes the TATA box. Grey boxes indicate predicted Pax6 paired- domain binding sites determined by Transfac MATCH program (see Suppl. Table 1). EcoRI, HindIII and PstI restriction sites are the same as in Figure 1. B) Clustal W alignment of Ath5 distal ECRs, containing two completely conserved E boxes highlighted in yellow (E3 and E4, see Hutcheson et al., 2005). Transfac MATCH program predicted Pax6 binding sites are highlighted in teal. For Xath5, the two overlapping sites are distinguished by underlining versus bold faced type. Asterisks denote nucleotides completely conserved among the four species. A line, across the top, marks an 81 nucleotide stretch of high identity. Gaps (dashes) were inserted by the program for optimal alignment.

Figure 5. Pax6 interacts with the Ath5 distal ECR in vivo and in vitro

A) GFP expression in pG1-M5-0.2 distal transient transgenic Xenopus embryos at stage 33. Embryos exhibited retinal expression alone (9/20) or retina plus nervous system expression (11/20) (not shown). pG1-Math5-2.1 also drives GFP expression in the same two expression pattern classes (Hutcheson et al., 2005). B) Real-time quantitative PCR using primers (targets) for the HATH5 dECR and a negative control sequence in HATH5 3’UTR. Results are expressed as a percentage of input chromatin isolated from Ad12 HER10 cells, showing averaged “enrichments” and the standard error of the mean. C) EMSAs of Pax6 paired domain-GST fusion protein with binding sites I,J,K and R in Math5 distal or proximal ECRs. For each binding site, the left lane contains free probe, the middle lane probe plus GST protein alone, and the right lane probe plus Pax6 paired domain-GST fusion protein. Only site J shifts in the presence of Pax6. This binding was completely lost by mutating 3 of 5 core nucleotides (J-Mut).

Figure 6. Distinct Math5 5’ regulatory sequences are activated by different concentrations of Pax6 protein in vitro

Relative promoter activities of Math5-luciferase reporter constructs co-transfected with pCS2, pCS2-Pax6 (25ng or 1µg) into Ad12 HER10 cells. Firefly luciferase values were normalized to pTK-Renilla luciferase values, and pGL2 empty vector activity set to 1.0. The mean values are shown, with error bars representing the standard error of the mean. n = 3 trials, each run in triplicate; *** = p<0.001.