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First published online 16 August 2006
doi: 10.1242/dev.02547

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Kermit 2/XGIPC, an IGF1 receptor interacting protein, is required for IGF signaling in Xenopus eye development

¹ Cell and Molecular Biology Graduate Group, University of Pennsylvania School of Medicine, 364 Clinical Research Building, 415 Curie Blvd, Philadelphia, PA 19104, USA.
² Department of Medicine (Hematology-Oncology), University of Pennsylvania School of Medicine, 364 Clinical Research Building, 415 Curie Blvd, Philadelphia, PA 19104, USA.

View larger version (56K): [in a new window]   Fig. 1. Temporal and spatial pattern of kermit2 expression. (A) RT-PCR analysis of kermit2 temporal expression. Total RNA was isolated from embryos at different stages and analyzed by RT-PCR. -RT, negative control without reverse transcriptase. ODC was used as a loading control. (B) RT-PCR analysis of kermit2 spatial expression. Stage 16 embryos were dissected into dorsal, ventral, anterior and posterior pieces, as shown in the diagram. Stage 20 embryos were dissected into anterior and posterior pieces. EF1 was used as a loading control. kermit2 is equally expressed in anterior, posterior, dorsal and ventral regions at mid-neurula stage (stage16), but is localized to the anterior region by stage 20. Anterior marker Otx2, posterior dorsal marker HoxD1, and posterior ventral marker Vent-1 were used as positive controls. (C-F) In situ hybridization of neurula and tadpole stage embryos. (C,D) Stage 20, anterior view, dorsal towards the top. (C) Sense control; (D) antisense probe. At stage 20, kermit2 is expressed in the cement gland and neural plate border, which is adjacent to and/or overlapping the presumptive eye region. (E,F) Antisense probe. By tailbud stages, kermit2 expression becomes further restricted, strongly expressed in the cement gland and otic vesicles (ov: arrow) at stage 26 (E), and is also expressed in the pronephros and branchial arches in stage 33 tadpoles (F).

View larger version (63K): [in a new window]   Fig. 2. Kermit 2 loss-of-function phenotype. (A) A kermit 2-directed antisense morpholino blocks translation of kermit 2-GFP mRNA (5'UTR-kermit 2-GFP, which contains the morpholino target sequence in the 5'UTR), but not kermit 2-GFP mRNA lacking the 5'UTR. Kermit 2-GFP mRNA (1 ng) with or without the 5'UTR was injected into one-cell embryos together with kermit 2 morpholino (K2M; 20 ng). Embryos were harvested at stage 10 and analyzed by western blot with GFP antibodies. ß-Tubulin was used as a loading control. (B) Unilateral injection of kermit 2 morpholino (40 ng) into one dorsal blastomere at the four-cell stage completely blocked eye development in 60% of embryos (lower left panel, n=38) and reduced eye formation in an additional 32% (not shown). Co-expression of kermit2-GFP mRNA, which lacked the morpholino target sequence, restored eye formation in 72% of kermit2-depleted embryos (lower right panel, n=43). kermit2-GFP mRNA alone did not cause apparent embryonic defects (upper right panel).

View larger version (83K): [in a new window]   Fig. 3. Depletion of kermit 2/XGIPC specifically inhibits Xenopus eye development. (A-D) Depletion of kermit 2 does not affect the expression of dorsal mesoderm markers chordin and goosecoid. Embryos are viewed from the vegetal side and dorsal is towards the top. Control morpholino or kermit 2 morpholino (40 ng) was injected into two dorsal animal blastomeres of four-cell embryos. Embryos were fixed at the gastrula stage (stage 10) and whole-mount in situ hybridization was performed for chordin (A,B) and goosecoid (C,D). (E-L) Depletion of kermit 2 specifically reduces marker gene expression within the presumptive eye field in stage 20 embryos. Embryos are viewed from the anterior side with dorsal towards the top. Control or kermit 2 morpholino was injected into one dorsal animal blastomere of four-cell/eight-cell embryos with 500 pg of mRNA for nuclear ß-galactosidase. Embryos were fixed at stage 20 and ß-galactosidase activity was measured in situ (red) followed by whole-mount in situ hybridization for Bf1 (E,F), Otx2 (G,H), Pax6 (I,J) and Xrx (K,L). Bf1 expression was not affected by depletion of kermit 2 (F). Expression of Otx2 (H) and Pax6 (J) were reduced only within the presumptive eyeforming region (red arrowheads) in embryos injected with kermit 2 morpholino. The expression of the eye marker Xrx was also inhibited (L red arrowhead). (M-P) Depletion of kermit 2 does not reduce Xrx (N) or Pax6 (P) expression in early neurula stage embryos (stage 14). (Q-S) Kermit 2 mRNA restored Xrx expression in kermit 2-depleted embryos. Red arrowhead in R indicates strongly reduced Xrx expression within presumptive eye domain in kermit 2-depleted embryo; red arrow in S indicates recovered Xrx expression in embryo co-injected with kermit 2 morpholino and kermit 2 mRNA.

View larger version (57K): [in a new window]   Fig. 4. Interaction between kermit2/XGIPC and XIGF1R in eye development. (A) Coimmunoprecipitation of kermit 2 and full-length XIGF1R. Embryos were injected at the one-cell stage with mRNAs encoding XIGF1R (2 ng) and GFP-tagged kermit 2 (1 ng), and cultured until the gastrula stage. XIGF1R/kermit 2 complexes were immunoprecipitated from embryo lysates with anti-GFP antibody and XIGF1R was visualized by western blotting. Mouse IgG was used as a negative control. (B) Kermit 2 morpholino and DN-IGFR synergistically inhibit eye formation in embryos. Kermit 2 morpholino (20 ng), DN-IGFR mRNA (500 pg), or both, were injected into two dorsal animal blastomeres of four-cell/eight-cell embryos. Embryos were cultured until tadpole stages to score phenotypes. The percentage of embryos with either strong or mild reduction in eyes is tabulated in the panel on the right side. (C) Kermit 2 morpholino and DN-IGFR synergistically reduce expression of Pax6 in presumptive eye domain (arrowhead). Microinjections were performed as in Fig. 2B. Nuclear ß-galactosidase mRNA was co-injected as a lineage tracer. Embryos were fixed at stage 20 and ß-galactosidase activity was measured in situ (red) followed by whole-mount in situ hybridization for Pax6.

View larger version (47K): [in a new window]   Fig. 5. Kermit 2/XGIPC is required for IGF1 induced eye formation in whole embryos and in animal cap explants. (A) Kermit 2 is required for IGF1 induced eye formation in Xenopus embryos. XIGF1 mRNA (1 ng), kermit 2 morpholino (40 ng), or both, were injected into one dorsal animal blastomere of four-cell/eight-cell embryos. IGF1 injection alone leads to embryos with expanded eyes and cement glands and co-injection of kermit 2 morpholino specifically inhibits the IGF1-induced eye phenotype. (B) Kermit 2 is required for IGF1-induced eye marker expression in animal cap explants. XIGF1 mRNA alone (2 ng), or with control morpholino (CM) or kermit 2 morpholino (K2M) was injected into four animal blastomeres of four-cell/eight-cell embryos. Animal caps were explanted from stage 9 blastulae, cultured until stage 20, and then harvested for RT-PCR. St. 20 WE indicates the whole embryo control; uninjected represents animal caps from uninjected embryos; -RT, without reverse transcriptase; EF1 is the loading control. Kermit 2 morpholino specifically reduces XIGF1-induced expression of the presumptive eye markers Xrx and Pax6.

View larger version (39K): [in a new window]   Fig. 6. Kermit 2/XGIPC is required to maintain IGF1 induced phosphorylation of AKT in oocytes and animal cap explants. (A) Kermit 2 is required for maintaining IGF1-induced AKT phosphorylation in oocytes. Oocytes were injected with 40 ng of control morpholino (CM) or kermit 2 morpholino (K2M), cultured for 48 hours and then treated with recombinant human IGF1 protein (18 ng/ml) for 30 minutes or overnight. Kermit 2 is required for IGF1-induced AKT phosphorylation/activation after overnight treatment, but not for short-term phosphorylation of AKT or MAPK. Total AKT or total MAPK was used as the loading control. (B) Effects of kermit 2 morpholino on the endogenous activation of AKT and MAPK. Control morpholino or kermit 2 morpholino was injected into four animal blastomeres of four-cell/eight-cell embryos. Animal caps were explanted at stage 9 and harvested after 2 hours or overnight. Kermit 2 morpholino reduces endogenous AKT phosphorylation after overnight incubation but not after 2 hours incubation and does not affect MAPK phosphorylation at either time point. (C) Kermit 2 is required for IGF1-induced AKT phosphorylation in stage 20 animal cap explants. 500 pg of XIGF1 mRNA and 400 pg of GFP mRNA were injected into one-cell embryos and 40 ng of kermit 2 morpholino was injected into four animal blastomeres of four-cell/eight-cell embryos. Animal caps were dissected at stage 9 and harvested at stage 20. GFP was used as the injection control.

View larger version (57K): [in a new window]   Fig. 7. Activation of PI3K partially rescues eye development in kermit 2/XGIPC-depleted embryos. (A) p110*, a constitutively active subunit of PI3 kinase, induces AKT phosphorylation/activation in stage 10 animal cap explants. mRNA encoding p110* was injected into fertilized eggs and animal caps were explanted at the gastrula stage and analyzed by western blot with antibodies recognizing AKT phosphorylated at serine-473 (upper panel) or general anti-AKT antibodies (lower panel). (B) p110* partially rescues eye development in kermit 2-depleted embryos. Kermit 2 morpholino (40 ng), 3 ng of p110* mRNA, or both, were injected into two dorsal animal blastomeres of four-cell/eight-cell embryos. No apparent eyes are present in embryos injected with the kermit 2 morpholino, while small eyes are present in over 70% of embryos co-injected with p110* mRNA and morpholino. p110* injection alone does not affect embryo development. (C) The percentage of embryos with normal, small or absent/miniscule eyes is summarized. (D) p110* partially recovers the expression of Xrx in kermit 2-depleted embryos. One dorsal-animal blastomere of four-cell/eight-cell embryos was injected with kermit 2 morpholino with or without p110* mRNA (right side, indicated by GFP lineage tracer) and harvested at stage 20. Whole-mount in situ hybridization was performed for Xrx. Embryos are viewed from the anterior side with dorsal towards the top.

View larger version (72K): [in a new window]   Fig. 8. Kermit 2/XGIPC is required for cell survival, but not for cell proliferation in Xenopus. (A-F) Control morpholino (A), kermit 2 morpholino alone (B,E,F), kermit 2 morpholino with 3 ng of kermit 2 mRNA (C) or 1 ng of DN-IGFR (D) was injected into the right side dorsal animal blastomere of four-cell/eight-cell embryos. The left side serves as a control. TUNEL staining and phosphorylated histone H3 staining were performed on neurula stage embryos. Embryos are viewed from the dorsal side, anterior towards the top. Compared with the control side (left side), the side injected with kermit 2 morpholino (right side) shows a substantial increase in the number of TUNEL-positive nuclei (B), without apparent change in the number of mitotic cells (E,F). The increased apoptosis in kermit 2-depleted embryos can be rescued by co-expression of kermit 2 mRNA lacking the 5'UTR (C). DN-IGFR also leads to elevated cell death on the injected side, as shown in D. (G-I) Co-injection of Bcl2 mRNA recovers Pax6 eye expression in kermit 2-depleted embryos. Microinjections were performed as above and nß-gal was used as the lineage tracer. Embryos were fixed at stage 20 and ß-galactosidase activity was measured in situ (red) followed by whole-mount in situ hybridization for Pax6. Red arrowhead in H indicates strongly reduced eye region in kermit 2 morpholino-injected embryo and red arrow in I indicates recovered eye expression domain by Bcl2 mRNA.

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