Sufu regulation of Hedgehog signaling in P19 cells is required for proper astrocyte differentiation

Hedgehog signaling is essential for vertebrate development, however, less is known about the negative regulators that influence this pathway. Using the mouse P19 embryonal carcinoma cell model, Suppressor of Fused (SUFU), a negative regulator of the Hedgehog pathway, was investigated during retinoic acid-induced neural differentiation. We found Hedgehog signaling was activated in the early phase of differentiation but was inactive during terminal differentiation of neurons and astrocytes. Early activation was required for neural differentiation however, it alone was not sufficient to induce neural lineages. SUFU, which regulates signaling at the level of GLI, remained relatively unchanged during differentiation, but its loss through CRISPR-Cas9 gene editing resulted in ectopic expression of Hedgehog target genes. Interestingly, these SUFU-deficient cells were unable to differentiate without retinoic acid, and when used they showed delayed and decreased astrocyte differentiation; neuron differentiation was unaffected. Ectopic activation of Hh target genes in SUFU-deficient cells remained throughout retinoic acid-induced differentiation and this was accompanied by the loss of GLI3, despite the presence of the Gli3 message. Thus, the study indicates the proper timing and proportion of astrocyte differentiation requires SUFU, and its normal regulation of GLI3 to maintain Hh signaling in an inactive state.