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

Abstract

Hedgehog signaling is essential for vertebrate development, however, less is known about the negative regulators that influence this pathway during the differentiation of cell fates. 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 neural differentiation but was inactive during terminal differentiation of neurons and astrocytes. This 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 the differentiation process, but its loss through CRISPIR-Cas9 gene editing resulted in ectopic expression of Hedgehog target genes. Interestingly, these SUFU-deficient cells were unable to differentiate in the absence of retinoic acid, but when differentiated using retinoic acid, 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 phenotype was accompanied by the loss of GLI3, despite the presence of the Gli3 message. Thus, the study indicates the proper timing and proportion of glial cell differentiation requires SUFU, and its normal regulation of GLI3 to maintain Hh signaling in an inactive state.

Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

“…Despite the reports showing that Nek2 regulates Hh and Wnt signalling [20–23] and both pathways are activated during P19 neural differentiation [5,37,40], few publications have explored the role of Nek2 in embryonic development [25,26,44] and to our knowledge, few limited work exists on its involvement in neurogenesis. To first address this shortfall, Nek2 gene expression (Supplemental Figure 1) and protein levels (Fig.…”

“…Wildtype and mutant cells were passaged every 4 days or when they reached a confluency of 70%, whichever occurred first. Following previously published protocols [37,41], differentiation was induced in wildtype and mutant cells by plating 1.05 × 10 6 cells on bacterial grade Petri dishes (Falcon) in the presence of 0.5 μM retinoic acid (RA) for 4 days to form embryoid bodies (EB). These aggregates were collected and re-plated on adherent tissue culture dishes in the presence of 0.5 μM RA, which in wildtype cells, develop into neurons after 10 days and form astrocytes by day 17.…”

“…Wildtype and Nek2 knockout cells were treated with 0.5 μM RA as described [37] for 17 days on poly-L-lysine hydrobromide (Sigma, P5899) coated coverslips. Cells on coverslips were washed with PBS for 1 minute and then fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature.…”

“…Every 24 hours for 96 hours, cells were trypsinized and a 1:1 cell suspension to trypan blue ratio was added and total number of cells counted using a CellDrop BF Brightfield Cell Counter (DeNovix). For RA treatment, cells were processed as described previously [37] and EBs were collected after 4 days. EBs were vigorously resuspended in complete media, before being counted in a 1:1 cell suspension to trypan blue ratio.…”

“…Given that Hh and Wnt signalling are essential in neural differentiation, and that Nek2 acts as a regulator of both pathways, we sought to explore the role of Nek2 in neural differentiation using P19 embryonal carcinoma cells. These cells can be differentiated towards neurons and astrocytes in the presence of retinoic acid (RA) [33–37], which affects Wnt [3,5,38,39] and Hh [37,40] signalling. Herein, we report the presence of Nek2 throughout RA-induced differentiation and through overexpression studies and CRISPR-Cas9 ablation, demonstrate an essential role of Nek2 in neuron and astrocyte differentiation.…”