Molecular dissection of Pax6 DNA-binding domains and their roles in mouse cerebral cortex development

Generation of the brain depends on proper regulation of progenitor proliferation and differentiation during development. Many such factors known to affect proliferation and differentiation are transcription factors. In particular, the transcription factor Pax6 has received much attention because of its potency to control various aspects of brain development. During development of the telencephalon Pax6 regulates patterning, cell proliferation and neurogenesis, but how Pax6 mediates and coordinates these diverse functions at the molecular level is not well understood. It has previously been demonstrated that the homeodomain of Pax6 plays a role in establishing the pallial-subpallial boundary. However it is not involved in other processes during telencephalic development as shown by the analysis of Pax64NEU mice, which are characterized by a point-mutation in the DNA-binding helix of the homeodomain. In order to gain more insights into the molecular network underlying the mild homeodomain function in the developing telencephalon, transcriptome analysis with Pax64NEU mice was performed. Almost no transcriptional changes were detected, suggesting that transcriptional regulation by the homeodomain of Pax6 has no major impact on forebrain development. Additionally, these results implied that the majority of effects exerted through Pax6 during telencephalic development are mediated by the bipartite paired-domain (PD). Therefore the main focus of this thesis was to examine the specific roles of the Pax6 paired-domain and its individual DNA-binding subdomains (PAI and RED) during forebrain development. The role of these DNA-binding domains was examined using mice with point-mutations in the PAI (Pax6Leca4, N50K) and RED (Pax6Leca2 R128C) subdomains and showed that the mutations in these subdomains exert opposing roles regulating proliferation in the developing cortex. While the mutated PAI domain resulted in reduced proliferation of both apical and basal progenitors, the mutated RED domain provoked increased proliferation. However, the PAI domain largely mediates the neurogenic function of Pax6. Additionally, genome-wide transcriptome analysis was able to unravel the key signatures mediated by the distinct domains. In summary, Pax6 exerts its key roles during forebrain development by use of distinct subdomains to regulate proliferation and differentiation. Thus Pax6 is able to coordinate and fine tune patterning, neurogenesis and proliferation in a simultaneous manner in different radial glial subpopulations. The transcriptional regulation through Pax6 may not only be restricted to protein coding genes, but may also include control of microRNA (miRNA) expression. Such small RNA molecules have recently been implicated in proliferation and differentiation during development, however expression and the role of single microRNAs is still poorly understood. Towards this end, miRNA expression profiling was performed using an embryonic stem cell differentiation system at different stages of neuronal differentiation in order to identify new miRNAs involved in radial glia specification and differentiation. This analysis revealed a number of microRNAs induced during differentiation from neural progenitors to neurons. Most strikingly only four miRNA candidates were found with exclusively high expression in progenitor cells. These data suggest that also Pax6 may play a role in transcriptional regulation beyond mRNAs

Pax6 interactions with chromatin and identification of its novel direct target genes in lens and forebrain.

Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and β-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6⁺/⁻ lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6⁻/⁻ lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues

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

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

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

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

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