Identification of pax6-dependent gene regulatory networks in the mouse lens.
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.
Wolf
Louise V
LV
Department of Ophthalmology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
Yang
Ying
Y
Wang
Jinhua
J
Xie
Qing
Q
Braunger
Barbara
B
Tamm
Ernst R
ER
Zavadil
Jiri
J
Cvekl
Ales
A
D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D053263:Gene Regulatory Networks / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D007908:Lens, Crystalline / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013687:Telencephalon / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000235:genetics; D053675:Versicans / Q000235:genetics
D018797:Cell Cycle Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; C493284:Mab21l2 protein, mouse; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; C435983:Spag5 protein, mouse; C509042:Tgfb2 protein, mouse; D053781:Transforming Growth Factor beta2; C508323:Vcan protein, mouse; D053675:Versicans