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Identification of pax6-dependent gene regulatory networks in the mouse lens.

Wolf LV, Yang Y, Wang J, Xie Q, Braunger B, Tamm ER, Zavadil J, Cvekl A - PLoS ONE (2009)

Bottom Line: Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis.Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development.This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Lineage-specific DNA-binding transcription factors regulate development by activating and repressing particular set of genes required for the acquisition of a specific cell type. Pax6 is a paired domain and homeodomain-containing transcription factor essential for development of central nervous, olfactory and visual systems, as well as endocrine pancreas. Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis. Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development. Using DNA microarrays, we have identified 559 genes expressed differentially between 1-day old mouse Pax6 heterozygous and wild type lenses. Of these, 178 (31.8%) were similarly increased and decreased in Pax6 homozygous embryonic telencephalon [Holm PC, Mader MT, Haubst N, Wizenmann A, Sigvardsson M, Götz M (2007) Loss- and gain-of-function analyses reveals targets of Pax6 in the developing mouse telencephalon. Mol Cell Neurosci 34: 99-119]. In contrast, 381 (68.2%) genes were differently regulated between the lens and embryonic telencephalon. Differential expression of nine genes implicated in lens development and homeostasis: Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2, was confirmed by quantitative RT-PCR, with five of these genes: Cspg2, Mab21l2, Olfm3, Spag5 and Tgfb2, identified as candidate direct Pax6 target genes by quantitative chromatin immunoprecipitation (qChIP). In Mab21l2 and Tgfb2 promoter regions, twelve putative individual Pax6-binding sites were tested by electrophoretic mobility shift assays (EMSAs) with recombinant Pax6 proteins. This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs. Collectively, the present studies represent an integrative genome-wide approach to identify downstream networks controlled by Pax6 that control mouse lens and forebrain development.

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A schematic diagram of computational, analytic and confirmatory strategies.The predicted Pax6-targets were validated by qRT-PCR, qChIP and EMSAs. The left arm of the diagram illustrates a strategy focusing on genes with established roles in the lens. The right arm shows a parallel strategy based on comparative expression profiling in lens and embryonic telencephalon. (*) represent data from [17], (**) represent data from [18]. In situ hybridization data, ISH; Pavlidis Template Matching, PTM; shared responses, S; suppression-activation, SA.
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pone-0004159-g008: A schematic diagram of computational, analytic and confirmatory strategies.The predicted Pax6-targets were validated by qRT-PCR, qChIP and EMSAs. The left arm of the diagram illustrates a strategy focusing on genes with established roles in the lens. The right arm shows a parallel strategy based on comparative expression profiling in lens and embryonic telencephalon. (*) represent data from [17], (**) represent data from [18]. In situ hybridization data, ISH; Pavlidis Template Matching, PTM; shared responses, S; suppression-activation, SA.

Mentions: A summary of multiple strategies to identify top candidate genes regulated by Pax6 is shown in Fig. 8. The left arm of the diagram illustrates a procedure to identify a relatively small number of genes that deserve further attention to understand their function and regulation during lens development. The right arm of the analysis shows a parallel approach that considers both shared and opposite (suppression-activation) Pax6's functions in embryonic lens and cortical development. We also included data from a recent study in which 82 Pax6-regulated genes emerged from high-throughput in situ hybridization studies using an atlas of ∼1,000 spatial gene expression patterns of the midgestation mouse embryo [18]. Of these 82, eight genes, i.e. Cdh8, Fgf14, Gabrg2, Neurod1, Neurod6, Pde1c, Tmem2 and Wnt2b, were found common between 381 differentially expressed genes identified here with opposite trends responding to the loss of Pax6 function in newborn mouse lens and embryonic cortex. At present, the end-points of the RNA expression profiling/ChIP approach (present study) and in situ hybridization studies [18], are experimental validations of candidate Pax6-binding sites using EMSAs. This is a necessary but not a sufficient step to further proof that Pax6 is indeed a direct regulator of expression of the gene of interest in a specific cell type.


Identification of pax6-dependent gene regulatory networks in the mouse lens.

Wolf LV, Yang Y, Wang J, Xie Q, Braunger B, Tamm ER, Zavadil J, Cvekl A - PLoS ONE (2009)

A schematic diagram of computational, analytic and confirmatory strategies.The predicted Pax6-targets were validated by qRT-PCR, qChIP and EMSAs. The left arm of the diagram illustrates a strategy focusing on genes with established roles in the lens. The right arm shows a parallel strategy based on comparative expression profiling in lens and embryonic telencephalon. (*) represent data from [17], (**) represent data from [18]. In situ hybridization data, ISH; Pavlidis Template Matching, PTM; shared responses, S; suppression-activation, SA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2612750&req=5

pone-0004159-g008: A schematic diagram of computational, analytic and confirmatory strategies.The predicted Pax6-targets were validated by qRT-PCR, qChIP and EMSAs. The left arm of the diagram illustrates a strategy focusing on genes with established roles in the lens. The right arm shows a parallel strategy based on comparative expression profiling in lens and embryonic telencephalon. (*) represent data from [17], (**) represent data from [18]. In situ hybridization data, ISH; Pavlidis Template Matching, PTM; shared responses, S; suppression-activation, SA.
Mentions: A summary of multiple strategies to identify top candidate genes regulated by Pax6 is shown in Fig. 8. The left arm of the diagram illustrates a procedure to identify a relatively small number of genes that deserve further attention to understand their function and regulation during lens development. The right arm of the analysis shows a parallel approach that considers both shared and opposite (suppression-activation) Pax6's functions in embryonic lens and cortical development. We also included data from a recent study in which 82 Pax6-regulated genes emerged from high-throughput in situ hybridization studies using an atlas of ∼1,000 spatial gene expression patterns of the midgestation mouse embryo [18]. Of these 82, eight genes, i.e. Cdh8, Fgf14, Gabrg2, Neurod1, Neurod6, Pde1c, Tmem2 and Wnt2b, were found common between 381 differentially expressed genes identified here with opposite trends responding to the loss of Pax6 function in newborn mouse lens and embryonic cortex. At present, the end-points of the RNA expression profiling/ChIP approach (present study) and in situ hybridization studies [18], are experimental validations of candidate Pax6-binding sites using EMSAs. This is a necessary but not a sufficient step to further proof that Pax6 is indeed a direct regulator of expression of the gene of interest in a specific cell type.

Bottom Line: Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis.Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development.This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Lineage-specific DNA-binding transcription factors regulate development by activating and repressing particular set of genes required for the acquisition of a specific cell type. Pax6 is a paired domain and homeodomain-containing transcription factor essential for development of central nervous, olfactory and visual systems, as well as endocrine pancreas. Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis. Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development. Using DNA microarrays, we have identified 559 genes expressed differentially between 1-day old mouse Pax6 heterozygous and wild type lenses. Of these, 178 (31.8%) were similarly increased and decreased in Pax6 homozygous embryonic telencephalon [Holm PC, Mader MT, Haubst N, Wizenmann A, Sigvardsson M, Götz M (2007) Loss- and gain-of-function analyses reveals targets of Pax6 in the developing mouse telencephalon. Mol Cell Neurosci 34: 99-119]. In contrast, 381 (68.2%) genes were differently regulated between the lens and embryonic telencephalon. Differential expression of nine genes implicated in lens development and homeostasis: Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2, was confirmed by quantitative RT-PCR, with five of these genes: Cspg2, Mab21l2, Olfm3, Spag5 and Tgfb2, identified as candidate direct Pax6 target genes by quantitative chromatin immunoprecipitation (qChIP). In Mab21l2 and Tgfb2 promoter regions, twelve putative individual Pax6-binding sites were tested by electrophoretic mobility shift assays (EMSAs) with recombinant Pax6 proteins. This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs. Collectively, the present studies represent an integrative genome-wide approach to identify downstream networks controlled by Pax6 that control mouse lens and forebrain development.

Show MeSH