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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). |
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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. |
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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. |
