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 and became inactive during terminal differentiation of neurons and astrocytes. SUFU, which regulates signaling at the level of GLI, remained relatively unchanged during the differentiation process, however SUFU loss through CRISPIR-Cas9 gene editing resulted in decreased cell proliferation and ectopic expression of Hedgehog target genes. Interestingly, SUFU-deficient cells were unable to differentiate in the absence of retinoic acid, but when differentiated in its presence they showed delayed and decreased astrocyte differentiation; neuron differentiation did not appear to be affected. Retinoic acid-induced differentiation also caused ectopic activation of Hh target genes in SUFU-deficient cells and while the absence of the GLI3 transcriptional inhibitor suggested the pathway was active, no full-length GLI3 was detected even though the message encoding Gli3 was present. Thus, the study would indicate 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

“…We have shown that Nek2 is essential in the differentiation of both neurons and astrocytes in the P19 embryonal carcinoma model (Fig. 5) and previous reports have placed Nek2 as a regulator of the Hh and canonical Wnt pathways [20][21][22][23]27], which are required in this process [3,5,[37][38][39][40] (Supplemental Figure 6). Towards that end, we sought to explore neural differentiation using the P19 model through the context of Wnt and Hh signalling, and although Nek2 appears to affect these pathways, the data did not indicate that their deregulation was the causative factor affecting neural differentiation.…”

“…Although canonical Hh and Wnt signalling is required in P19 cells for proper neural differentiation [3,37,40,54], and there are relationships between these pathways and Nek2, our data strongly suggests that the loss of Nek2 influences neural cell lineages linked to pluripotency and cell proliferation. The data supports this early role, rather than being directly linked to regulating the Wnt and Hh signalling pathways.…”

“…Canonical Hh and Wnt signalling both play an integral role in neural differentiation of P19 cells and have been linked to Nek2 [20][21][22][23]. Wang et al [19] and Zhou et al [20] reported Nek2 serves as a negative regulator of Hh signalling and our previous report has shown that overactivation of Hh signalling through the loss of SUFU inhibited astrocyte differentiation in the P19 model [37] (Spice et al, 2022, under revision). Therefore, Hh signalling was first explored as a potential mechanism for Nek2 in regulating neural differentiation.…”

“…Despite the reports showing that Nek2 regulates Hh and Wnt signalling [20][21][22][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 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.…”