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Dev Biol
2017 Jul 01;4271:148-154. doi: 10.1016/j.ydbio.2017.04.017.
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Eukaryotic initiation factor eIF6 modulates the expression of Kermit 2/XGIPC in IGF- regulated eye development.
De Marco N
,
Tussellino M
,
Carotenuto R
,
Ronca R
,
Rizzolio S
,
Biffo S
,
Campanella C
.
Abstract
The eukaryotic initiation translation factor eIF6 is a highly conserved, essential protein implicated in translation. eIF6 is regulated in vivo by extracellular signals, such as IGF signaling (for a review see Miluzio et al., 2009). In Xenopus, eif6 over-expression causes a delay in eye development (De Marco et al., 2011). In this study we showed that eif6 co-immunoprecipitates with the insulin-like growth factor receptor (igfr) and may function downstream of igf in eye formation. The relationship between eif6 and gipc2, a protein partner of a variety of molecules including membrane proteins, was investigated. gipc2 is required for maintaining igf-induced akt activation on eye development (Wu et al., 2006). Significantly eif6 and gipc2 have opposite effects in eye development. While eif6 is required for eye formation below threshold levels, gipc2 knockdown impairs eye development (De Marco et al., 2011; Wu et al., 2006). In this study, it was shown that in eif6 over-expressors, the delay in eye morphogenesis is reversed by gipc2 injection, while the injection of eif6 down-regulates gipc2 expression. Real-time-PCR indicates that eif6 regulates gipc2 expression in a dose-dependent manner. In contrast, gipc2 knockdown has no significant effect on eif6 mRNA levels. These results suggest that eif6 regulation of gipc2 enables correct morphogenesis of Xenopus eye and stimulate questions on the molecular network implicated in this process.
Fig. 1.
eif6 interaction with the igf pathway. a. Co-immunoprecipitation using anti-eIF6 or anti-IGFR. The two isoforms of eif6 are immunoprecipitated by anti-IGFR and igfr is immunoprecipitated by anti-eIF6, in contrast to the control immunoprecipitation using IgG. b. Immunoblots of eif6-injected embryos or of GFP-injected embryos. Anti-AKT or anti-pAKT, as well as anti-pMAPK or anti-MAPK, were used: there is a clear decrease in pakt level in eif6-injected embryos compared to GFP-injected embryos. In contrast, in embryos injected with eif6 or with GFP no difference is observed between pmapk and mapK levels. Normalization was performed with anti-tubulin. c. Animal caps lysates of embryos injected with p110* of the PI3 kinase were analyzed. pAkt level is higher in embryos injected with p110* than in those injected with GFP. d.In situ hybridizations of neurula embryos using rax1 riboprobe. The hybridization by this marker specific for the eye indicates the extension of the eye field in the observed embryos. 1. Control embryos injected with GFP showing that the rax1 hybridization extension is practically identical in both sides of the embryo. 2. The side of the embryo injected with eif6 shows a rax1 hybridization less extended than the un-injected side. 3. In the embryo side co-injected with p110* and eif6 the eye field stained by rax1 is practically identical in both sides of the embryo indicating that a partial rescue of the eye field occurs in these embryos. Rescue means a partial or total recovery of the hybridization extension as found in the eye-field of the embryo un-injected side. βgal mRNA was visualized with Red-Gal.
Fig. 2.
Co-immunoprecipitations of eif6 and gipc2 and of eif6 and gipc2mut. a. We utilized lysates of embryos injected with pCS2gipc2GFP. IP experiments were performed using anti-GFP antibodies for immunoprecipitation and anti-gipc2 and anti-eif6 antibodies for western blots. In western blots of the immunoprecipitations, the eif6 protein was found in the sample immunoprecipitated with anti-GFP (lane 1) and GFP was found in the sample immunoprecipitated with anti-eif6 (lane 3), indicating that eif6 and gipc2 co-immunoprecipitate. Lane 2=embryo lysate. In control IP using unrelated IgG the gipc2GFP and the eif6 bands are absent (lane 4). Lane 5=only an anti-GFP immunopositive band is present in the immunoprecipitate derived from lysates of pCS2GFP-injected embryos. b. We utilized lysates of embryos injected with pCS2gipc2mutHA (gipc2mut = gipc2 with PDZ domain deletion) to perform the IP experiments using anti-HA and anti-eif6 antibodies. The blots indicate that while in the lysates both eif6 and gipc2mutHA are present (lane 2), this does not occur in the reciprocal IPs, i.e., the gipc2mutHA band is absent in anti-eif6 IP (lane 1), and the eif6 band is absent in the anti-HA IP (lane 3). Therefore, gipc2mutHA does not interact with eif6. Lane 4=control IP using unrelated IgG: no bands are present.
Fig. 3.
Relationship between eif6 and gipc2 expression. a. In situ hybridization performed using gipc2 anti-sense RNA on neurula injected at the 2 cell stage in one blastomere with either GFP (1 and 4), eif6 (2 and 5) or S235A (3 and 6). In GFP injected embryos (1 and 4), the hybridization intensity and pattern is identical in the embryo dorsal portion (1) and in the eye field territory (4, arrows) of both sides. In both eif6 (2 and 5) and S235A (3−6) injected samples, gipc2 is expressed at a lower level in the injected side than in the un-injected side, in particular in the eye field territory (arrows). βgal mRNA was visualized with Red-Gal. b. Real-time PCR using embryos injected with increasing concentration of eif6 (100, 200 and 400 pg) shows a highly significant (P<0.001) dose-dependent decrease of gipc2 mRNA. c. Real-time PCR in eif6 morphants showing a highly significant increase of gipc2 mRNA (P<0001) in contrast to the control mis-paired morpholino.
Fig. 4.
gipc2 rescues the eye-field phenotype of eif6-overexpressors, while gipc2mut does not. a. In situ hybridizations with rax1 and pax6, in GFP, eif6 or eif6+gipc2 injected embryos. The arrows delimitate the site of the presumptive eye field in the side of the embryo where injection was performed. 1,4. The eye field in control GFP-injected embryos at neurula stage incubated with rax1 (1) or pax6 (4) riboprobe. 2,3. rax1 hybridization. 2. eif6-injected neurula shows that the eye field is smaller than in the counter-lateraleye, while the two eye fields have similar shape and extension in the eif6+gipc2-injected embryos (3). 5,6. pax hybridization. In eif6-injected neurula the eye field is smaller than in the counter-lateraleye (5), while in the eif6+gipc2-injected embryos the two eye fields have the same shape and extension (6). b. In situ hybridizations with rax1 of GFP and gipc2mut-injected embryos. Embryos injected with gipc2mut (2) indicate that rax1 riboprobe localization is partially reduced in the injected side, compared with the counter-lateral un-injected side, in contrast to the GFP-injected embryos (1). βgal mRNA was visualized with Red-Gal. c. The percentage of embryos with normal or reduced eyes is summarized.
