Suppressor of Fused Plays an Important Role in Regulating Mesodermal Differentiation of Murine Embryonic Stem Cells In Vivo

Abstract

The hedgehog (Hh) signaling pathway plays fundamental roles during embryonic development and tumorigenesis. Previously, we have shown that ablation of the tumor suppressor and negative regulator, Suppressor of fused (Sufu), within this pathway causes embryonic lethality around E9.5 in the mouse. In this study, we examine how lack of Sufu influences early cell fate determination processes. We established embryonic stem cell (ESC) lines from preimplantation Sufu(-/-) and wild-type mouse embryos and show that these ESCs express the typical pluripotency markers, alkaline phosphatase, SSEA-1, Oct4, Sox2, and Nanog. We demonstrate that these ESCs express all core Hh pathway components and that glioma-associated protein (Gli)1 mRNA levels are increased in Sufu(-/-) ESCs. Upon spontaneous differentiation of Sufu(-/-) ESCs into embryoid bodies (EBs) in vitro, the Hh pathway is strongly upregulated as indicated by an increase in both Gli1 and patched1 (Ptch1) gene expression. Interestingly, developing Sufu(-/-) EBs were smaller than their wild-type counterparts and showed decreased expression of the ectodermal markers, Fgf5 and Sox1. In vivo teratoma formation revealed that Sufu(-/-) ESCs have a limited capacity for differentiation as the resulting tumors lacked the mesodermal derivatives, cartilage and bone. However, Sufu(-/-) ESCs were able to develop into chondrocytes and osteocytes in vitro, which suggests a differential response of ESCs compared with in vivo conditions. Our findings suggest a regulatory function of the Hh signaling pathway in early mesodermal cell fate determination and emphasize the role of Sufu as a key molecule in this process.

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

“…Although it is difficult to explain these differences, our work showed the knockout of Sufu was accompanied by the consistent over-activation of Hh target gene expression (Figure 5 and Figure 8), although Gli2 was not examined. There was, however no detectable change in neuron differentiation in SUFU-deficient cells (Figure 6), which is in support of the work by Hoelzl et al [43], who found neuronal tissue derived from mouse embryonic stem cells depleted of SUFU. Furthermore, our results that showed a delay and decrease in glial cell differentiation with the loss of SUFU (Figure 6) may explain the disorganization of glial cells observed in the conditional Sufu knockouts described in mouse embryos [15,47].…”

“…The mechanism of SUFU depletion was not determined, nor were SUFU post-translational modifications examined, but it should be noted that differential phosphorylation and ubiquitination can both promote or inhibit SUFU stability and/or GLI interaction [46][47][48][49]. CRISPR-Cas9, used to deplete SUFU from P19 cells, show the loss of SUFU induces the ectopic transcription of Hh target genes (Figure 4 and Figure 5) and while supported by studies using mice, mESCs and hESCs [15,36,[50][51][52], this is in contrast with a study showing the reduction in expression in adult neural stem cells [27]. Interestingly, SUFU-deficient P19 cells did not differentiate in the absence of RA (Figure 6A) despite a previous report that Gli2 overexpression in P19 cells was sufficient to induce neuron differentiation under similar conditions [20].…”

“…Conditional knockouts of Sufu at various stages of brain development also results in decreased neuron differentiation and disorganized glial cell arrangements [15,47], and in human embryonic stem cells there is an increase to neuroectodermal gene expression following SHH treatment [33]. In contrast, however, no change is seen in ectoderm differentiation following Sufu knockout in mouse ESCs [43]. Discrepancies also exist within the same cell type as evident in P19 cells where Gli2 overexpression induces neuron differentiation in the absence of RA [20], while Gli2 knockdown produces the opposite effect albeit under different differentiation conditions [21].…”

Differential requirement of SUFU in tissue development discovered in a hypomorphic mouse model

“…Complementary DNA (cDNA) synthesis, qRT-PCR as well as analysis were performed as described previously (Hoelzl et al, 2015). At least three independent experiments were performed where each reaction was measured in triplicates.…”

“…Immunohistochemical staining on formalin-fixed, paraffin-embedded (FFPE) tissue sections was performed as described previously (Hoelzl et al, 2015). Briefly, tissue was harvested and fixed in 4% PFA overnight, followed by embedding and sectioning.…”