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Int J Dev Biol
2012 Jan 01;565:357-62. doi: 10.1387/ijdb.120009nd.
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Involvement of the eukaryotic initiation factor 6 and kermit2/gipc2 in Xenopus laevis pronephros formation.
Tussellino M
,
De Marco N
,
Campanella C
,
Carotenuto R
.
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The translation initiation factor Eif6 has been implicated as a regulator of ribosome assembly, selective mRNA translation and apoptosis. Many of these activities depend upon the phosphorylation of eif6 Serine 235 by protein kinase C (PKC). Eif6-60S is probably part of the RNA-induced silencing complex (RISC). eif6 over-expression in Xenopus embryos causes aberrant eye development. kermit2/gipc2 morphants have an eye phenotype similar to that of the eif6 overexpressors. Eye formation is regulated by insulin growth factor (IGF) signalling. eif6 interacts with the IGF receptor (IGFR) and kermit2/gipc2, which also binds to igfr. eif6 over-expression in Xenopus causes also the formation of antero-ventral oedema, suggesting a malfunction of the excretory system. Here we evaluated the pronephros phenotype. The oedema grows into the nephrocoel, expanding its boundary and is accompanied by a strong reduction of the pronephros. The three main components of the pronephros are severely impaired in eif6 over-expressors, while are not affected in eif6 morphants. Conversely, gipc2 depletion induces the oedema phenotype and reduction of the pronephros, while gipc2 overexpression does not. p110*, a constitutively active p110 subunit of the PI3 kinase partially recovers the oedema phenotype. We also determined that PKC-dependent phosphorylation of Ser235 in eif6 is not required to produce defective pronephroi. These results indicate that the levels of eif6 are highly regulated during development and instrumental for proper morphogenesis of the pronephros. Moreover, it appears that for proper pronephros development the gipc2 level should be kept within or over the physiological range and that the oedema phenotype is partly due to the inhibition of IGF signalling.
Fig. 1. The pronephros phenotype in eif6 overexpressors. (A-E) eif6 mRNA and protein are expressed in the pronephros (arrows) of stages 24, 32 and 38 Xenopus laevis embryos. (A,B) Immunofluorescence using anti-Eif6 antibody of section (A) or whole mount (B) of respectively stage 24 and stage 32 embryos indicating that eif6 is present in the pronephros as well as in most tissues; the arrows indicate the pronephros anlagen. (C) Whole mount in situ hybridisation using eif6 antisense probe (stage 38). (D,E) Immunofluorescence with anti-Eif6 antibody of whole mounts (D) or sections (E) of stage 38 embryo. (D Whole mount immunofluorescence using only secondary antibody BODIPY-conjugates as negative control. Staining is absent. (F) Oedema formation (arrowhead) is present in embryos injected with 400 pg of eif6 and 300 pg of GFP into one blastomere at the two-cell stage and harvested at stage 38. (G) Histology with haema- toxylin and eosin on the embryonic sections showed in (F). A reduction of pronephric tubules is evident. The arrow indicates that the glomus is not surrounded by the splanchnic mesoderm, as the oedema has grown in the nephrocoel, expanding the splanchnic mesoderm boundary (see also K). (H) A stage 38 embryo injected with eif6 morpholino: there is no oedema. (I) A section of the same embryo stained with haematoxylin and eosin. eif6 depletion did not produce oedema. (J,K) Embryos injected with 400 pg of S235A and 300 pg of GFP show the oedema (arrowhead). (J) The pronephric reduction is evident in the histological section of embryo shown in (K). The arrow indicates that the glomus is not surrounded by the splanchnic mesoderm. pn, pronephros.
Fig. 2. Oedema phenotype in eif6 overexpressors and in gipc2 loss-of unction embryos. (A-D) Stage 32 and stage 38 embryos (E-H) injected into one blastomere at the two-cell stage with 400 pg of GFP alone (A,E), 400 pg of eif6 and 300 pg of GFP (B,F) 40 ng of gipc2 morpholino and 300 pg of GFP (C,G) or 40 ng of gipc2 mispaired morpholino and 300 pg of GFP (D,H). In the eif6 over-expressors (B,F) and gipc2 morphants (C,G), the oedema phenotype (arrow) appears at stage 32 and increases at stage 38.
Fig. 3. eif6 over-expression and gipc depletion affect lhx1 expression. Embryos injected into one blastomere at the two-cell stage with GFP (A,D), eif6 (B,E) and gipc2 morpholino (C,F). bgal mRNA was co-injected for tracing lineage (stained red). (A-C) Embryos were cultured until stage 22 and whole mount in situ hybridised for expression of lhx1, an early marker of the pronephric anlagen. Injection of the GFP alone had no effect on lhx1 expression (A). eif6 over-expression (B) and gipc2 depletion (C) inhibited most pronephric anlagen formation (arrow). The white lines in the inset of (B) surround the reduced pronephric anlagen. (D-F) Embryos were cultured until stage 25-26 and whole mount in situ hybridised for expression of myod1, a marker of differentiating muscle. No difference in the myod1 expression pattern was found in the eif6 over-expressors (E) and gipc2 morphants (F).
Fig. 4. Whole mount in situ hybridisa- tion for markers of terminal proneph- ros differentiation. Stage 38 embryos injected with GFP alone (A,D,G,J), eif6 (B,E,H,K) or gipc2 morpholino (C,F,I,L). Micrographs of the two sides of the same embryos are shown (A,A B,B etc.). Co-injection of (D-F) GFP mRNA as tracer, and (G-I) bgal mRNA (stained red). It should be observed that the red stained tissue distributed in the most su- perficial tissues is often partially removed during manipulation. The glomus, (arrow) marked by nephrin; proximal tubule (aster- isk), marked by slc5a9; intermediate and distal tubule domain (arrowhead), marked by clcnkb and cdh16, respectively, were reduced in eif6 over-expressors and in gipc2 morphants.
Fig. 5. p110* partially recov- ers the phenotype in eif6 overexpressors. (A-F) Stage 38 embryos injected into one blastomere at the two-cell stage with 400 pg of GFP only (A,D), 400 pg of eif6 and 300 pg of GFP (B,E), or 400 pg of eif6, 300 pg of GFP and 1 ng of p110* (C,F). (D-F) Whole mount in situ hybridisation for clcnkb, an intermediate and distal tubule marker (arrow). p110* partially recovers the oedema phenotype (C) and clcnkb expression (F).
