Fig. 6
PI3K downregulation contributes to the Mycn cKO phenotype. a Hindlimbs of mice with indicated genotypes at postnatal day 16. Wildtype control (Ctrl), heterozygous Pten mutant (Prx1-Cre:Ptenfl/+, Pten Het), Mycn Knockout (Prx1-Cre:Mycnfl/fl, Mycn cKO), Mycn cKO in which one allele of Pten is deleted (Prx1-Cre:Mycnfl/fl:Ptenfl/+, Mycn cKO:Pten Het), and Mycn Knockout overexpressing Mir17-92 (Prx1-Cre:Mycnfl/fl:Mir17-92Tg, Mycn cKO:17 Tg). Pten heterozygous deletion partially rescues the skeletal abnormalities of Mycn cKO mutants. Cutaneous syndactyly and brachydactyly are partially rescued, while the fused metatarsals and shortening of mid phalanxes remain. The fifth metacarpal bones, subjected to measurement in b, are indicated by brackets. Scale bars, 0.5 cm. b Quantification of the length (µm) of fifth metacarpal bones of mice with indicated genotypes (n = 6 for Ctrl and Mycn cKO, n = 5 for Pten Het and Mycn cKO:Pten Het, n = 4 for Mycn cKO:17 Tg; *p < 0.01 vs. Ctrl, **p < 0.01 vs. Mycn cKO). c PI3K signaling was assessed by p-Akt (Thr308) in limb bud cells isolated from E 10.5 mice with indicated genotype. d Immunoblot analysis for phospho-Pten (p-Pten) and total Pten (t-Pten) isolated from Mycn cKO and wildtype littermate limb bud cells. The p-Pten level is decreased in Mycn-deficient skeletal progenitor cells whereas the t-Pten level is unchanged. e Immunoblot analysis for casein kinase 2 beta phosphorylation on S209 (p-CK2) in limb bud mesenchymal cells isolated from E10.5 Mycn cKO and wildtype littermates. p-CK2 is decreased in Mycn-deficient cells; *non-specific band. f Proposed model. Upregulation of TGF-β signaling plays a causal role in the skeletal defect of Feingold syndrome type 2 (Mir17-92 mutation), whereas downregulation of the PI3K signaling plays a major pathophysiologic role in Feingold syndrome type 1 (Mycn mutation). Mycn partially regulates miR-17-92 miRNA levels, but the contribution of this regulation in limb development is limited (dotted arrow). Overexpression of miR-17-92 miRNAs can suppress Pten, although miR-17-92 miRNAs have limited regulatory effects on Pten expression at the physiological level (dotted inhibitory line)
