Suppressor of Fused Regulation of Hedgehog Signaling is Required for Proper Astrocyte Differentiation

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Abstract
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 increased activity in the early phase of differentiation but was reduced during terminal differentiation of neurons and astrocytes. This early increase in pathway activity 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 towards neural lineages without retinoic acid, and when directed towards these lineages 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, likely acting through Gli3, to reduce Hh signaling during late-stage differentiation.
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