Regulation of Hedgehog and Wnt Signaling in Neural Differentiation of P19 Embryonal Carcinoma Cells

The Hedgehog (Hh) and Wnt protein signaling pathways are essential in the differentiation of neurons and astrocytes. As there are many known and new players involved in regulating these pathways, the role of the regulators Suppressor of Fused (SUFU) and Never in Mitosis Kinase 2 (Nek2) have either not been previously reported or have not been thoroughly explored. To address this shortfall CRISPR gene editing was used to target SUFU and Nek2 in the mouse P19 embryonal carcinoma cell model of neural differentiation. Hh and Wnt signaling were explored in normal P19 neural differentiation, which occurs in the presence of retinoic acid. Both pathways are required for the establishment of neural cell fates, however, neither is sufficient to induce both neurons and astrocyte lineages alone. SUFU was found to be required in the differentiation of astrocytes, but not neurons, and its loss resulted in the loss of the Gli3 transcription factor. The loss of Gli3 in SUFU-deficient cells resulted in constitutive expression of Hh target genes. Nek2 was required for the differentiation of both neurons and astrocytes, however, loss of function experiments did not find a clear link between Nek2, and the Hh or Wnt pathways. Instead, a mechanism was discovered where Nek2 is required to destabilize hypoxia inducible factor 1a, allowing for a required metabolic shift from glycolysis to oxidative phosphorylation during the differentiation of neural cell types. Together this work shows novel mechanisms of regulating neural cell fate through the presence or absence of intracellular regulatory proteins