Jin Z , Schwend T , Fu J , Bao Z , Liang J , Zhao H , Mei W , Yang J .

R01 GM093217 NIGMS NIH HHS , R01 GM111816 NIGMS NIH HHS , F32 EY021708 NEI NIH HHS , R35 GM131810 NIGMS NIH HHS

	Fig. 1. Members of the Rusc protein family interact with Sufu. (A) Co-immunoprecipitation (CoIP) showing the interaction between hSUFU and hRUSC2. hRUSC2-FLAG and myc-hSUFU were expressed in HEK293T cells alone or in combination. CoIP was performed using an anti-myc antibody (upper panel) or an anti-FLAG antibody (lower panel). (B) CoIP showing that endogenous Sufu and Rusc2 form a complex in mouse whole brain lysate. Sufu was immunoprecipitated. (C) Identity between the Rusc proteins. Protein sequences of Rusc1 and Rusc2 from human (h), mouse (m) and Xenopus (x) were aligned using NCBI BLAST. (D) CoIP showing that mRusc1 and hRUSC2 form complexes with hSUFU. (E,F) CoIP showing that myc-xRusc1 (E) and myc-xRusc2 (F) interact with FLAG-hSUFU. IP, immunoprecipitation; WB, western blot.
	Fig. 6. Expression of rusc1 and rusc2 during Xenopus eye development. (A) RT-PCR showing the temporal expression of rusc1 and rusc2 during Xenopus development. The expression level of rusc1 and rusc2 was normalized to that of odc. Data are shown as mean±s.d. (B) Whole-mount in situ hybridization showing the spatial expression pattern of rusc1, rusc2 and gli1. St., stage. Arrowheads point to the eye domains, which express rusc1 but not gli1. Black, red and yellow arrows point to the trigeminal ganglion, middle lateral line placode, and anterodorsal lateral line placode, respectively.
	Fig. 7. Dominant-negative Rusc enhances Hh signaling in Xenopus embryos and impairs eye development. (A) Schematic of hRUSC2 and deletion derivatives. Whether an hRUSC2 construct interacts with hSUFU in the CoIP experiment is indicated by + or −. (B) CoIP results showing that hSUFU interacts with full-length hRUSC2, RUSC608-903 and RUSC1233-C. (C) CoIP showing that overexpression of RUSC1233-C reduces the binding between hSUFU and full-length hRUSC2. (D) Dual-luciferase assay showing that the activities of Gli1 and Gli2 are enhanced by co-overexpression of RUSC1233-C in NIH3T3 cells. Data are shown as mean±s.d. *P<0.05, **P<0.01. (E) In situ hybridization showing the expression of gli1, pax6, rax and six3 in control (left) and RUSC1233-C overexpression (right) Xenopus embryos at stage 20. At the 8-cell stage, one of the dorsal animal blastomeres was injected with a mixture of RUSC1233-C (1 ng) and n-β-gal (250 pg) encoding RNAs. (F) Overexpression of RUSC1233-C (1 ng) reduced the size of the eye (arrowhead).
	Fig. 8. Rusc1 inhibits Hh signaling during Xenopus eye development. (A) Whole embryo morphology of uninjected embryos and those injected with R1-MO, R1-5mis or R2-MO. Morpholinos (20 ng) were injected into both dorsal blastomeres at the 4-cell stage. (B) Overexpression of myc-xRusc1 rescued the phenotypes induced by unilateral injection of R1-MO. (Left) Summary of embryos with eye defects. (Right) Images of representative embryos. A 50% or greater reduction in eye size is considered ‘severe’; a reduction of less than 50% is considered ‘mild’. (C) RT-PCR showing the expression of gli1, ptc1, ptc2 and hhip in animal caps. Chordin (Chd, 25 pg) was injected into the animal pole of control and R1-MO (40 ng) injected embryos at the 1-cell stage. Animal caps were dissected at the late blastula stage and harvested at stage 22. Data are shown as mean±s.d. *P<0.05, **P<0.01. (D) In situ hybridization showing that unilateral injection of R1-MO (20 ng) enhances the expression of gli1, and reduces the expression of pax6, rax and six3. The expression of shh was not altered by R1-MO injection. Embryos were analyzed at stage 20. (E) In situ hybridization showing that unilateral injection of R1-MO enhances the expression of gli1 in the head region and reduces the expression of pax6, rax and six3 at stage 33. Arrowheads point to eyes on the injected side. (F) Morphology of uninjected embryos and those unilaterally injected with R1-MO alone or R1-MO together with Gli1 morpholino (Gli1 MO). Insets show further examples of the illustrated phenotype. Arrows (D,F) point to the developing eyes.
	Supplemental Figure 5. Effects of xRusc morpholinos. A. Western blot showing that injection of Rusc1 morpholino (R1-MO, 20 ng) and Rusc2 morpholinos (R2-MO, 20 ng) into Xenopus embryos blocked translation of C-terminal myc-tagged xRusc1 (left panel) and xRusc2 (right panel), respectively. R1-MO blocked translation of a C-terminal myc-tagged xRusc1, but not a Nterminal myc-tagged xRusc1 (middle panel). In these experiments, morpholinos were injected at the 1-cell stage. At the 2-cell stage, a mixture of Rusc (1 ng) and myc-GFP RNA (50 pg) were injected into embryos. B. Histological analysis of eyes from a control embryo (left) and R1-MO injected embryos with mildly (middle) and severely (right) affected eyes. C. In situ hybridization showing the expression of gli1 in a control embryo (left) and an embryo bilaterally injected with 40 ng of R1-MO (right). Embryos were analyzed at stage 18.
	Supplemental Figure 6. Knocking down xRusc1 by injection of R1-sb enhances Hh signaling and impairs eye development. A. Schematic diagram showing the design of R1-sb, which blocks rusc1 splicing. Arrowheads indicate primers used in RT-PCR to validate the effect of R1-sb on splicing of rusc1. B. RT-PCR result showing the effect of R1-sb on rusc1 splicing. Fertilized eggs were injected with R1-sb (80 ng) and harvested at stage 33 for RT-PCR. C. Sequences of the PCR products (primers Up + D1) amplified from control and R1-sb injected embryos, showing insertion of intron 5 into rusc1 mRNA in R1-sb injected embryos. D. Whole embryo morphology of a control tadpole and a tadpole that was injected with 20 ng of R1-sb bilaterally at the 4-cell stage. Both dorsal blastomeres were injected. E. In situ hybridization showing the expression of gli1, six3, and rax in control and R1-sb injected embryos. A mixture of R1-sb (20ng) and RNA encoding n-ß-gal (500 pg) was injected into one of the dorsal blastomeres at the 4-cell stage. Embryos were harvested at stage 33. Both un-injected and injected sides of injected embryos are shown. In stage 33 control embryos, gli1 is not expressed in the eye, forming a prominent “gli1-free” domain in the head (pointed by arrows). In R1-sb injected embryos, the gli1-free domain disappears. Cells in the head region express gli1 nearly uniformly.
	rusc1 (RUN and SH3 domain containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, animal view.
	rusc1 (RUN and SH3 domain containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	rusc1 (RUN and SH3 domain containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, lateral view, anterior right, dorsal up.
	rusc2 (RUN and SH3 domain containing 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view.
	rusc2 (RUN and SH3 domain containing 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, anterior view, dorsal up.
	rusc2 (RUN and SH3 domain containing 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, lateral view, anterior right, dorsal up.

Aavikko, Loss of SUFU function in familial multiple meningioma. 2012, Pubmed

Aavikko, Loss of SUFU function in familial multiple meningioma. 2012, Pubmed
Amato, Hedgehog signaling in vertebrate eye development: a growing puzzle. 2004, Pubmed
Bayer, Identification and characterization of Iporin as a novel interaction partner for rab1. 2005, Pubmed
Boehlke, Differential role of Rab proteins in ciliary trafficking: Rab23 regulates smoothened levels. 2010, Pubmed
Briscoe, Making a grade: Sonic Hedgehog signalling and the control of neural cell fate. 2009, Pubmed
Briscoe, The mechanisms of Hedgehog signalling and its roles in development and disease. 2013, Pubmed
Briscoe, Morphogen rules: design principles of gradient-mediated embryo patterning. 2015, Pubmed
Brugières, Incomplete penetrance of the predisposition to medulloblastoma associated with germ-line SUFU mutations. 2010, Pubmed
Chen, Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved. 2009, Pubmed
Cheung, The kinesin protein Kif7 is a critical regulator of Gli transcription factors in mammalian hedgehog signaling. 2009, Pubmed
Chi, Rab23 negatively regulates Gli1 transcriptional factor in a Su(Fu)-dependent manner. 2012, Pubmed
Cooper, Cardiac and CNS defects in a mouse with targeted disruption of suppressor of fused. 2005, Pubmed
Ding, Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of Gli1. 1999, Pubmed
Eggenschwiler, Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway. 2001, Pubmed
Eggenschwiler, Mouse Rab23 regulates hedgehog signaling from smoothened to Gli proteins. 2006, Pubmed
Endoh-Yamagami, The mammalian Cos2 homolog Kif7 plays an essential role in modulating Hh signal transduction during development. 2009, Pubmed
Evans, Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway. 2003, Pubmed
Fukuda, Genome-wide investigation of the Rab binding activity of RUN domains: development of a novel tool that specifically traps GTP-Rab35. 2011, Pubmed
Han, Multisite interaction with Sufu regulates Ci/Gli activity through distinct mechanisms in Hh signal transduction. 2015, Pubmed
He, The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the cilium tip compartment. 2014, Pubmed
Huangfu, Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways from Drosophila to vertebrates. 2006, Pubmed
Hui, Gli proteins in development and disease. 2011, Pubmed
Humke, The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins. 2010, Pubmed
Jia, Suppressor of Fused inhibits mammalian Hedgehog signaling in the absence of cilia. 2009, Pubmed
Jia, Decoding the Hedgehog signal in animal development. 2006, Pubmed
Jiang, Hedgehog signaling in development and cancer. 2008, Pubmed
Jin, The antagonistic action of B56-containing protein phosphatase 2As and casein kinase 2 controls the phosphorylation and Gli turnover function of Daz interacting protein 1. 2011, Pubmed , Xenbase
Jin, PP2A:B56{epsilon}, a substrate of caspase-3, regulates p53-dependent and p53-independent apoptosis during development. 2010, Pubmed , Xenbase
Jin, The 48-kDa alternative translation isoform of PP2A:B56epsilon is required for Wnt signaling during midbrain-hindbrain boundary formation. 2009, Pubmed , Xenbase
Kijima, Two cases of nevoid basal cell carcinoma syndrome associated with meningioma caused by a PTCH1 or SUFU germline mutation. 2012, Pubmed
Kogerman, Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of Gli-1. 1999, Pubmed
Koide, Negative regulation of Hedgehog signaling by the cholesterogenic enzyme 7-dehydrocholesterol reductase. 2006, Pubmed , Xenbase
Law, Antagonistic and cooperative actions of Kif7 and Sufu define graded intracellular Gli activities in Hedgehog signaling. 2012, Pubmed
Lee, Gli1 is a target of Sonic hedgehog that induces ventral neural tube development. 1997, Pubmed , Xenbase
Li, Kif7 regulates Gli2 through Sufu-dependent and -independent functions during skin development and tumorigenesis. 2012, Pubmed
Li, Rab23 GTPase is expressed asymmetrically in Hensen's node and plays a role in the dorsoventral patterning of the chick neural tube. 2007, Pubmed
Liem, Mouse Kif7/Costal2 is a cilia-associated protein that regulates Sonic hedgehog signaling. 2009, Pubmed
Lin, Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling. 2014, Pubmed
Liu, Dual function of suppressor of fused in Hh pathway activation and mouse spinal cord patterning. 2012, Pubmed
Lupo, Mechanisms of ventral patterning in the vertebrate nervous system. 2006, Pubmed
MacDonald, Nesca, a novel neuronal adapter protein, links the molecular motor kinesin with the pre-synaptic membrane protein, syntaxin-1, in hippocampal neurons. 2012, Pubmed
Maurya, Positive and negative regulation of Gli activity by Kif7 in the zebrafish embryo. 2013, Pubmed
Min, The dual regulator Sufu integrates Hedgehog and Wnt signals in the early Xenopus embryo. 2011, Pubmed , Xenbase
Moody, Fates of the blastomeres of the 16-cell stage Xenopus embryo. 1987, Pubmed , Xenbase
Moody, Testing retina fate commitment in Xenopus by blastomere deletion, transplantation, and explant culture. 2012, Pubmed , Xenbase
Murcia, The Oak Ridge Polycystic Kidney (orpk) disease gene is required for left-right axis determination. 2000, Pubmed , Xenbase
Murone, Gli regulation by the opposing activities of fused and suppressor of fused. 2000, Pubmed , Xenbase
Mussolino, A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. 2011, Pubmed
Nguyen, Cooperative requirement of the Gli proteins in neurogenesis. 2005, Pubmed , Xenbase
Pastorino, Identification of a SUFU germline mutation in a family with Gorlin syndrome. 2009, Pubmed
Pearse, Vertebrate homologs of Drosophila suppressor of fused interact with the gli family of transcriptional regulators. 1999, Pubmed , Xenbase
Petrova, Roles for Hedgehog signaling in adult organ homeostasis and repair. 2014, Pubmed
Préat, Characterization of Suppressor of fused, a complete suppressor of the fused segment polarity gene of Drosophila melanogaster. 1992, Pubmed
Rorick, PP2A:B56epsilon is required for eye induction and eye field separation. 2007, Pubmed , Xenbase
Sasaki, A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro. 1997, Pubmed , Xenbase
Schulman, Multiple Hereditary Infundibulocystic Basal Cell Carcinoma Syndrome Associated With a Germline SUFU Mutation. 2016, Pubmed
Schwend, Stabilization of speckle-type POZ protein (Spop) by Daz interacting protein 1 (Dzip1) is essential for Gli turnover and the proper output of Hedgehog signaling. 2013, Pubmed , Xenbase
Stone, Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli. 1999, Pubmed
Sun, Crystal structure and functional implication of the RUN domain of human NESCA. 2012, Pubmed
Svärd, Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway. 2006, Pubmed
Tay, A homologue of the Drosophila kinesin-like protein Costal2 regulates Hedgehog signal transduction in the vertebrate embryo. 2005, Pubmed
Taylor, Mutations in SUFU predispose to medulloblastoma. 2002, Pubmed
Tukachinsky, A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu-Gli protein complexes. 2010, Pubmed
Wang, Suppressor of fused and Spop regulate the stability, processing and function of Gli2 and Gli3 full-length activators but not their repressors. 2010, Pubmed
Wilson, Mechanism and evolution of cytosolic Hedgehog signal transduction. 2010, Pubmed
Wolff, Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo. 2003, Pubmed
Wulfkuhle, Domain analysis of supervillin, an F-actin bundling plasma membrane protein with functional nuclear localization signals. 1999, Pubmed
Zeng, Coordinated translocation of mammalian Gli proteins and suppressor of fused to the primary cilium. 2010, Pubmed
Zhang, A hedgehog-induced BTB protein modulates hedgehog signaling by degrading Ci/Gli transcription factor. 2006, Pubmed
Zhang, Structural insight into the mutual recognition and regulation between Suppressor of Fused and Gli/Ci. 2013, Pubmed
Zhang, Multiple Ser/Thr-rich degrons mediate the degradation of Ci/Gli by the Cul3-HIB/SPOP E3 ubiquitin ligase. 2009, Pubmed