Verification of microarray results by qRT-PCR and qChIP.

<p>(A) Relative expression levels of <i>Gaa</i>, <i>Has2</i>, <i>Isl1</i>, <i>Kif1b</i>, <i>Mtmr2</i>, <i>Pax6</i>, <i>Pcsk1n</i>, <i>and Snca</i> in wild type (WT, shown in black) and Pax6^+/− (shown in gray) lenses were determined using qRT-PCR as described in Methods. B2m, Hprt and Ccni transcripts were tested as internal references, and all were found unchanged between the WT and Pax6^+/− lenses. The data are expressed relative to the unchanged expression level of B2m transcripts. For statistical evaluation of the results, p-values were calculated from paired Student t-tests. (B) Validation of Pax6-binding regions in lens chromatin by qChIPs. A and B are distal regions with Pax6 binding identified in ChIP-Chip experiments and P regions are binding regions around the proximal promoters. At each of these gene loci, a non-specific region (negative signals in ChIP-Chip experiments and no candidate Pax6 binding sites predicted) was also included as a negative control. In addition, Cryaa promoter (Cryaa-P) and +6 kb region serve as positive and negative controls respectively. The specific enrichments of Pax6 binding were detected at Isl1-A, Mtmr2-A, Snca-A, Gaa-P, Kif1b-P, Pcsk1n-B and Pcsk1n-A regions. The calculation of the cutoff value (0.100 of 1% input) for background signals and specific binding signals is described in Materials and Methods.</p

Twenty-seven genes relevant to lens placode formation and lens morphogenesis show differential expression in Pax6^−/− E9.5 mutated lens placodes.

<p>(A) A list of 27 genes includes a combination of well-characterized genes in lens biology and selected differentially expressed genes in Pax6 null (<i>Sey</i>) cortex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Holm1" target="_blank">[45]</a>. The differentially expressed genes in Pax6^−/− E9.5 wild type and mutated lens placodes were identified using the Illumina Mouse6 bead microarrays as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Huang1" target="_blank">[96]</a>. Twenty-four of the 27 genes were differentially expressed in at least 50% of experiments. (B) Relative expression levels of <i>Fat4</i>, <i>Trpm3</i>, <i>Pax6</i>, <i>Has2</i>, <i>Efnb2</i>, and <i>Nav1</i> in wild type (WT, black bars) and Pax6^−/− (open bars) lens placode and mutated ectoderm were determined using qRT-PCR as described in Methods.</p

Pax6 regulates expression of Snca.

<p>(A) Identification of Pax6-binding region by ChIP-Chip in lens chromatin and corresponding luciferase reporter constructs for transfection assays. (B) Pax6 regulates <i>Snca</i> promoter/distal region in cultured cells. Transient transfections were performed in P19 embryonic carcinoma and in αTN4-1 lens cell as described in Methods. (C) Prediction of Pax6 binding sites with novel Pax6 DNA binding motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Xie1" target="_blank">[48]</a>. (D) EMSA validation of Pax6 binding to the probes identified by motif 1-1, 1-2 and 3-3. PD/HD, recombinant Pax6 protein containing both Pax6 paired domain (PD) and homeodomain (HD). P6CON, DNA-binding concensus for Pax6 paired domain.</p

Down-regulation of Kif1b and Snca in Sye/Sey forebrains.

<p>In situ hybridization analysis of E14 wild type (WT) and Pax6^Sey/Sey mouse embryos. Ganglionic eminence, GE.</p

Pax6 regulates expression of Kif1b.

<p>(A) Identification of Pax6-binding regions by ChIP-Chip in lens chromatin and a corresponding luciferase reporter construct for transfection assays. (B) A diagrammatic summary of Pax6 mutants, N50K, R128C, R242T and R317X. β, N-terminal β-turn unit; PD, paird domain; PAI, N-termianl subdomian of PD; RED, C-terminal subdomian of PD; L, linker region; HD, homeodomain; PST, proline-serine-threonine rich transactivation domain. (C) Pax6 activates <i>Kif1b</i> promoter in cultured cells. Transient transfections were performed in P19 embryonic carcinoma and in αTN4-1 lens cell as described in Methods. (D) Prediction of Pax6 binding sites with novel Pax6 DNA binding motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Xie1" target="_blank">[48]</a>. (E) EMSA validation of Pax6 binding to two probes identified by motifs 2-2 and 4-1. PD/HD, recombinant Pax6 protein containing both Pax6 paired domain (PD) and homeodomain (HD). P6CON, DNA-binding concensus for Pax6 paired domain.</p

Diagrammatic summary of novel functions of Pax6 during lens development.

<p>The connections between genes regulated by Pax6 in lens placode were identified via Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, Mountain View, CA). Expression of Stat3 is reduced in the Pax6 mutated E9.5 embryonic tissues. The Tnf promoter region is occupied by Pax6 in lens chromatin.</p

Identification of genes regulated by Pax6 in newborn lens.

<p>Venn diagram showing identification of 76 genes both bound and regulated by Pax6 by intersecting two genome-wide data sets: RNA expression profiling (559 transcripts, P1 lens, Pax6^+/− versus wild type) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Wolf1" target="_blank">[26]</a> and present ChIP-chip studies (2,335 peaks) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507.s005" target="_blank">Table S1</a>) in lens chromatin. The table summarized the known function of six important and validated Pax6 direct target genes from this group of 76 genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone-0054507-g004" target="_blank">Figure 4</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Zhao1" target="_blank">[75]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Bijvoet1" target="_blank">[121]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Bolino1" target="_blank">[122]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Fricker1" target="_blank">[123]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Masliah1" target="_blank">[124]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Pan1" target="_blank">[125]</a>.</p