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.

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