XB-ART-7326
Dev Biol
April 15, 2002;
244
(2):
407-17.
The IGF pathway regulates head formation by inhibiting Wnt signaling in Xenopus.
Abstract
The
insulin-like growth factors (IGFs) are well known mitogens, both in vivo and in vitro, while functions in cellular differentiation have also been indicated. Here, we demonstrate a new role for the IGF pathway in regulating
head formation in Xenopus embryos. Both
IGF-1 and
IGF-2, along with their receptor
IGF-1R, are expressed early during embryogenesis, and the
IGF-1R is present particularly in
anterior and dorsal structures. Overexpression of
IGF-1 leads to
anterior expansion of
head neural
tissue as well as formation of ectopic
eyes and
cement gland, while
IGF-1 receptor depletion using antisense morpholino oligonucleotides drastically reduces
head structures. Furthermore, we demonstrate that IGF signaling exerts this effect by antagonizing the activity of the Wnt signal transduction pathway in the early
embryo, at the level of beta-catenin. Thus, the IGF pathway is required for
head formation during embryogenesis.
PubMed ID:
11944947
Article link:
Dev Biol
Genes referenced:
foxg1
igf1
igf1r
igf2
igf3
ins
ncam1
nodal3.1
otx2
pax6
rho
rpe
sia1
tcf7l1
wnt8a
Morpholinos:
igf1r MO1
Article Images:
[+] show captions
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FIG. 1. Expression of IGF signaling components during Xenopus development. (A) RT-PCR analysis of IGF-1 and IGF-2 transcripts throughout development. (B) RT-PCR analysis of IGF-1 expression at early neurula stage (D, dorsal; V, ventral side of the embryo). (C–G) IGF-1R mRNAs detected by in situ hybridization (bl, blastopore; he, head; cgp, cement gland primordium; cg, cement gland). Expression is first detected at the end of the gastrulation (stage 12) in the presumptive dorsal side of the embryo (C) but excluded from the ventral part of the embryo (D). During neurulation, transcripts are present in the head region and in the anterior mesoderm, but are not detectable in the neural tube and the ventral part of the embryo (E, frontal view; F, lateral view). At tailbud stage, transcripts are present in the head and the anterior part of the neural tube (G).
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FIG. 3. Overexpression of IGF-1 inhibits convergent-extension movements. (A) Dorsal injections of IGF-1 mRNA (1–3 ng in one dorsal blastomere at the 4-cell stage) lead to dorsally curved embryos with truncated posterior tissue. (B) In the presence of activin, control ectodermal explants derived from embryos injected with -galactosidase RNA (1.5 ng/blastomere at the 2-cell stage) elongate (left panel), but explants derived from IGF-1-injected embryos (1.5 ng/blastomere) remain round (right panel).
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FIG. 4. IGF-1 overexpression induces anterior neural markers expression. Whole-mount in situ hybridization shows an expansion of expression of Otx-2 (A, front view; B, lateral view; he, head), NCAM (C), and Pax-6 (D) on the injected side (right side). (E) RT-PCR analysis of ectodermal explants injected with IGF-1 mRNA shows that IGF-1 induces anterior markers (Co, uninjected ectodermal explants; WE, whole embryo at tailbud stage; -, water control).
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FIG. 5. IGF signaling is required for head formation. (A) Western blot analysis of IGF-1R expression in embryos at late blastula stage (stage 9) injected with Mo (20 ng per blastomere at the two-cell stage) specific to the receptor (MoIGF-1R) or control Mo (MoC) (Co, noninjected embryo). -Tubulin is shown as a loading control. (B–E) Whole-mount in situ hybridization analysis of embryos injected in one dorsal blastomere at the four-cell stage with 20 ng of MoIGF-1R shows a strong inhibition of XBF-1 expression (B, embryo stage 13) and NCAM expression (D, embryo stage 19) on the injected side (right). The expression patterns of these two genes are normal in MoC-injected embryos (C, XBF-1; E, NCAM). (F–H) Four-cell embryos were injected in the two dorsal blastomeres with 20 ng of the Mo indicated. The embryo injected with MoIGF-1R shows microcephaly and no apparent eyes (F), whereas the embryos injected with MoC are normal (G). The coinjection of 0.3 ng of IGF-1R mRNA with MoIGF-1R rescues the morpholino-induced phenotype (H).
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FIG. 6. IGF-1 inhibits Wnt signaling in ectodermal explants. (A) IGF-1 mRNA (1.5 ng per blastomere) inhibits induction of Siamois and Xnr-3 by Wnt-8 (10 pg per blastomere), DN-GSK (150 pg per blastomere), and -catenin (50 pg per blastomere) in ectodermal explants by stage 10.5, but fails to block activation of Wnt target genes by VP16 Tcf-3 (50 pg per blastomere). (B) IGF-1 mRNA (1.5 ng per blastomere) inhibits the transcriptional activity of the -catenin/Tcf complex. Embryos were injected at the two-cell stage along with the luciferase reporter plasmid (TOPFLASH) and RNAs as indicated (IGF-1 mRNA, 1.5 ng per blastomere; -catenin mRNA, 50 pg per blastomere). Embryos were collected at stage 10.5 in pools of five embryos and assayed for luciferase expression in triplicate.
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FIG. 7. IGF-1 inhibits Wnt-posteriorizing activity after MBT. (A) Injection of IGF-1 mRNAs (1.5 ng per dorsal blastomere, four-cell stage) rescues posteriorization induced by injection of CSKA-X8 plasmid (75 pg per blastomere). (B) Coinjection of IGF-1 mRNA (2 ng) with tBR mRNA (0.5 ng) into one ventral blastomere at the four-cell stage induces an ectopic head with a visible eye (arrow), unlike tBR mRNA alone which induces only a partial axis with no head structures (arrow).
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igf1 (insulin-like growth factor 1 (somatomedin C)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior down.
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igf1 (insulin-like growth factor 1 (somatomedin C)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
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igf1 (insulin-like growth factor 1 (somatomedin C)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, dorsal view, anterior down.
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