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

Adherence to the Revised NICHD Protocol recommendations for conducting repeated supportive interviews is associated with the likelihood that children will allege abuse.

Because intensely reluctant children often fail to report being abused even when they are supportively interviewed, the Revised NICHD (National Institute of Child Health and Human Development) Protocol (RP) guides interviewers to delay discussion of sensitive topics and build rapport before scheduling a follow-up interview in which children might feel more comfortable. We sought to determine whether adherence to these recommendations was associated with the children’s propensity to make allegations. Repeated forensic interviews were conducted with 202 Israeli children aged 3-14 who did not make allegations in the first interview, but of whom 104 made allegations during the second interview. The interviews were coded to identify interviewers’ provision of support and types of substantive questions (invitations vs. closed-ended), as well as children’s signs of reluctance, responsiveness, and informativeness. Interviewer behavior was represented with a latent variable reflecting the interviewers’ expression of support, use of invitations, and the avoidance of closed-ended questions. Structural Equation Modeling (SEM) showed that adherence to the suggested interviewing model was positively associated with children being more likely to allege abuse (total effect: β = .29). This association was mediated by children’s enhanced cooperativeness in the second interview (indirect effect: β = .16). These findings suggest that repeated interviews can be useful despite the additional financial costs.Nuffield Foundation, Jacobs Foundation, Haruv Institut