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Extracellular signal transduction into cells through ligand-activated receptor tyrosine kinases, such as insulin-like growth factor-1 (IGF-1) receptor (IGF-1R) and insulin receptor (IR) is required for normal embryonic growth and development. The major mediators of IR and IGF-1R are adaptor proteins of the insulin receptor substrate family, the best characterized member of which is IRS-1. Insulin receptor substrate IRS-1 has been shown to influence cell and body size and to interfere with differentiation. We have isolated IRS-1 from Xenopus laevis embryos and analyzed for the first time its spatial and temporal expression pattern during embryogenesis. We found that Xenopus IRS-1 is expressed maternally and constantly during embryogenesis. It is predominantly found in neural tissue at different stages. Furthermore, knock down of IRS-1 in neural tissue by specific antisense morpholino oligonucleotides (MO) resulted in abnormal eye formation accompanied by reduction of the eye-specific marker genes Rx1 and Pax6 and a decreased cell proliferation.
Figure 2. Analysis of the spatial expression pattern of X. laevis IRS-1. Whole-mount in situ hybridizations at the indicated stages. adb, animal dorsal blastomere; anp, anterior neural plate; ba, branchial arches; bp, blastopore; e, eye; hb, hindbrain; hm, hypaxial muscles; l, lens; mb, midbrain; opv, optic vesicle; ov, otic vesicle; pb, presumptive brain; pn, pronephros.Download figure to PowerPoint
Figure 3. Horizontal and transversal section analyses of the IRS-1 expression pattern. anp, anterior neural plate; ba, branchial arches; cmz, ciliary marginal zone; di, diencephalon; g, gill; le, lensepithelium; lf, lens fibers; lpl: lens placode; opv, optic vesicle; ov, otic vesicle; pt, proximal tubule of pronephros; sc, spinal cord.Download figure to PowerPoint
Figure 5. Loss of insulin receptor substrate IRS-1 function in neural tissue results in a severe eye phenotype. A: Upper panel: Representative embryos displaying the observed eye phenotypes upon IRS-1 morpholino oligonucleotide (MO) injection (arrows). Lower panel: Cross-sections through the affected eyes. B: Quantitative representation of the dose-dependent effect of IRS-1 MO is given. C: Coinjection of the δ5′ untranslated region (UTR) IRS-1 construct significantly rescued the IRS-1 MO-induced phenotype. *P < 0.05, n, number of independent experiments; N, total number of embryos examined.Download figure to PowerPoint
Figure 6. Marker gene analyses of Insulin receptor substrate IRS-1-depleted embryos. A: IRS-1 knock down in neural tissue reduces the expression of the eye marker genes Rx1 and Pax6, but not the pan-neural marker gene Sox3 at stage 13. B: At stage 23, expression of Rx1, Pax6, and Otx2 in the eye is affected. In contrast, expression of Emx1, Krox20, and En2 in the brain is not affected. C: Coinjection of 0.5 ng of δ5′ untranslated region (UTR) IRS-1 RNA significantly reverted the IRS-1 morpholino oligonucleotide (MO) (20 ng) -induced reduction of Rx1 expression at stage 13. D,E: Quantitative representations are given. *P < 0.05, **P < 0.005, n, number of independent experiments; N, total number of embryos examined.Download figure to PowerPoint
Figure 7. Insulin receptor substrate IRS-1 knock down affects the size of the lens placode. The width (w) and the height (h) of the the lens placodes visualized by FoxE3 staining were measured (represented by dotted lines), and ellipse area was calculated (π*w*h/4). The area of the lens placode on the IRS-1 morpholino oligonucleotide (MO) -injected side is significantly smaller compared with the uninjected side. In cases of Control MO treatment, no difference in the size of the lens placode of the injected versus uninjected side was observed. In total, 20 embryos from two independent experiments were analyzed. *P < 0.05, is, injected side; uis, uninjected side.Download figure to PowerPoint
Figure 8. Insulin receptor substrate IRS-1 plays a role during cell proliferation but not apoptosis at stage 13. Twenty ng of the indicated morpholino oligonucleotide (MO) and 0.5 ng green fluorescent protein (GFP) RNA were injected into one animal dorsal blastomere at eight-cell stage. Only embryos glowing in the left hemisphere were used for the stainings to lateron identify the injected side. A: Injection of neither IRS-1 nor Control MO had an effect on apoptosis at stage 13 shown by means of TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling) staining. B: Depletion of IRS-1 led to an significant decrease in PH3-positive cells on the injected versus uninjected side, whereas Control MO injection did not alter cell proliferation. For each MO, in total 20 embryos from two independent experiments were analyzed. *P < 0.05.Download figure to PowerPoint
