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

Suppressor of Fused (SUFU) is an essential negative regulator of the Hedgehog (HH) pathway and involved in GLI transcription factor regulation. Due to early embryonic lethality of Sufu-/- mice, investigations of SUFU's role later in development are limited to conditional, tissue-specific knockout models. In this study we developed a mouse model (SufuEx456(fl)/Ex456(fl)) with hypomorphic features where embryos were viable up to E18.5, although with a spectrum of developmental defects of varying severity, including polydactyly, exencephaly and omphalocele. Development of certain tissues, like the skeleton, was more affected than that of others such as skin, which remained largely normal. Interestingly, no apparent changes in the dorso-ventral patterning of the neural tube at E9.0 could be seen. Thus, this model provides an opportunity to globally study SUFU's molecular function in organogenesis beyond E9.5. Molecularly, SufuEx456(fl)/Ex456(fl) embryos displayed aberrant mRNA splicing and drastically reduced levels of Sufu wild-type mRNA and SUFU protein in all tissues. As a consequence, at E9.5 the levels of all three different GLI proteins were reduced. Interestingly, despite the reduction of GLI3 protein levels, the critical ratio of the GLI3 full-length transcriptional activator versus GLI3 truncated repressor remained unchanged compared to wild-type embryos. This suggests that the limited amount of SUFU protein present is sufficient for GLI processing but not for stabilization. Our data demonstrate that tissue development is differentially affected in response to the reduced SUFU levels, providing novel insight regarding the requirements of different levels of SUFU for proper organogenesis.

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

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.

Loss of Trp53 promotes medulloblastoma development but not skin tumorigenesis in Sufu heterozygous mutant mice.

Basal cell carcinoma of the skin typically carries genetic alterations in components of the hedgehog (HH) signaling pathway. Previously, we generated a knockout mouse with a loss-of-function mutation in suppressor of fused (Sufu), an essential repressor of the pathway downstream of Hh ligand cell surface reception. Mice heterozygous for the mutated Sufu allele develop a skin phenotype that includes lesions similar to basaloid follicular hamartomas. The purpose of the current study was to test the possibility that the simultaneous loss of the tumor suppressor gene, transformation related protein 53 (Trp53), would aggravate the Sufu skin phenotype since Trp53 loss is known to enhance the growth of other Hh-driven tumors. Consistent with previous reports, medulloblastomas and rhabdomyosarcomas developed in Sufu(+/-) ;Trp53(-/-) mice. However, the characteristic Sufu(+/-) skin phenotype was not altered in the absence of Trp53, and showed no changes in latency, multiplicity, cellular phenotype, or proliferative capacity of the basaloid lesions. This finding was both novel and intriguing and demonstrated a differential, tissue-specific sensitivity to Sufu and Trp53 tumor suppressor gene loss, which may be linked to developmental stage and the degree of proliferative activity in specific cell types.

Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway.

The Hedgehog (Hh) pathway plays important roles during embryogenesis and carcinogenesis. Here, we show that ablation of the mouse Suppressor of fused (Sufu), an intracellular pathway component, leads to embryonic lethality at approximately E9.5 with cephalic and neural tube defects. Fibroblasts derived from Sufu(-/-) embryos showed high Gli-mediated Hh pathway activity that could not be modulated at the level of Smoothened and could only partially be blocked by PKA activation. Despite the robust constitutive pathway activation in the Sufu(-/-) fibroblasts, the GLI1 steady-state localization remained largely cytoplasmic, implying the presence of an effective nuclear export mechanism. Sufu(+/-) mice develop a skin phenotype with basaloid changes and jaw keratocysts, characteristic features of Gorlin syndrome, a human genetic disease linked to enhanced Hh signaling. Our data demonstrate that, in striking contrast to Drosophila, in mammals, Sufu has a central role, and its loss of function leads to potent ligand-independent activation of the Hh pathway.