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Insulin-like factor regulates neural induction through an IGF1 receptor-independent mechanism.

Haramoto Y, Takahashi S, Oshima T, Onuma Y, Ito Y, Asashima M - Sci Rep (2015)

Bottom Line: Insulin3 reduced extracellular Wnts and cell surface localised Lrp6.These results suggest that Insulin3 is a novel cell-autonomous inhibitor of Wnt signalling.This study provides the first evidence that an insulin-like factor regulates neural induction through an IGF1R-independent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.

ABSTRACT
Insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) signalling is required for normal embryonic growth and development. Previous reports indicated that the IGF/IGF1R/MAPK pathway contributes to neural induction and the IGF/IGF1R/PI3K/Akt pathway to eye development. Here, we report the isolation of insulin3 encoding a novel insulin-like ligand involved in neural induction. Insulin3 has a similar structure to pro-insulin and mature IGF ligands, but cannot activate the IGF1 receptor. However, similar to IGFs, Insulin3 induced the gene expression of an anterior neural marker, otx2, and enlarged anterior head structures by inhibiting Wnt signalling. Insulin3 are predominantly localised to the endoplasmic reticulum when otx2 is induced by insulin3. Insulin3 reduced extracellular Wnts and cell surface localised Lrp6. These results suggest that Insulin3 is a novel cell-autonomous inhibitor of Wnt signalling. This study provides the first evidence that an insulin-like factor regulates neural induction through an IGF1R-independent mechanism.

No MeSH data available.


Related in: MedlinePlus

Insulin3 inhibits Wnt signalling but cannot activate the IGF1 receptor.mRNAs were injected into the animal pole of both blastomeres at the two-cell stage in X. laevis embryos. (a) One ng of insulin3, IGF1, or IGF1R mRNA was injected per embryo. Embryos were harvested at stage 11 for western blot analysis. (b) One ng of Myc-tagged extracellular domain of IGF1 receptor (IGF1Rex-Myc), IGF1-HA, insulin3-HA, or insulin1-HA mRNA was injected per embryo. Embryos were harvested at stage 10.5 for co-immunoprecipitation assay. (c,d,e) Amounts of mRNA injected per embryos were: (c) FGF4 (10 pg), (d) insulin3 (100 pg), and (e) IGF1 (100 pg). Animal caps were dissected at stage 9 and treated with chemical MEK inhibitors, PD98059 (100 μM) and U0126 (100 μM) until sibling embryos reached stage 10.5. (c) Bra expression induced by FGF4 was inhibited, but otx2 expression induced by (d) insulin3 or (e) IGF1 was not. Full-length blots and gels are presented in Supplementary figure S8.
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f6: Insulin3 inhibits Wnt signalling but cannot activate the IGF1 receptor.mRNAs were injected into the animal pole of both blastomeres at the two-cell stage in X. laevis embryos. (a) One ng of insulin3, IGF1, or IGF1R mRNA was injected per embryo. Embryos were harvested at stage 11 for western blot analysis. (b) One ng of Myc-tagged extracellular domain of IGF1 receptor (IGF1Rex-Myc), IGF1-HA, insulin3-HA, or insulin1-HA mRNA was injected per embryo. Embryos were harvested at stage 10.5 for co-immunoprecipitation assay. (c,d,e) Amounts of mRNA injected per embryos were: (c) FGF4 (10 pg), (d) insulin3 (100 pg), and (e) IGF1 (100 pg). Animal caps were dissected at stage 9 and treated with chemical MEK inhibitors, PD98059 (100 μM) and U0126 (100 μM) until sibling embryos reached stage 10.5. (c) Bra expression induced by FGF4 was inhibited, but otx2 expression induced by (d) insulin3 or (e) IGF1 was not. Full-length blots and gels are presented in Supplementary figure S8.

Mentions: Previous studies reported that IGFs inhibit canonical Wnt signalling67. Here we showed that Insulin3 could also inhibit canonical Wnt signalling (Fig. 4b) indicating it can activate the same signal pathway as IGFs. IGF signals are mainly mediated by means of the IGF1 receptor (IGF1R) and to a lesser extent by the insulin receptor (IR), which are receptor tyrosine kinases (RTKs). We hypothesised that Insulin3 could also activate the IGF1 receptor to induce anterior neural gene expression. Contrary to our hypothesis, our results indicated that Insulin3 cannot activate IGF1R (Fig. 6a), whereas Insulin3 efficiently interacted with the extracellular domain of IGF1R (Fig. 6b). It has been reported that IGFs induce head formation through the IGF/IGF1R/MAPK pathway5. Our results indicate that Insulin3 inhibits Wnt signalling using another mechanism.


Insulin-like factor regulates neural induction through an IGF1 receptor-independent mechanism.

Haramoto Y, Takahashi S, Oshima T, Onuma Y, Ito Y, Asashima M - Sci Rep (2015)

Insulin3 inhibits Wnt signalling but cannot activate the IGF1 receptor.mRNAs were injected into the animal pole of both blastomeres at the two-cell stage in X. laevis embryos. (a) One ng of insulin3, IGF1, or IGF1R mRNA was injected per embryo. Embryos were harvested at stage 11 for western blot analysis. (b) One ng of Myc-tagged extracellular domain of IGF1 receptor (IGF1Rex-Myc), IGF1-HA, insulin3-HA, or insulin1-HA mRNA was injected per embryo. Embryos were harvested at stage 10.5 for co-immunoprecipitation assay. (c,d,e) Amounts of mRNA injected per embryos were: (c) FGF4 (10 pg), (d) insulin3 (100 pg), and (e) IGF1 (100 pg). Animal caps were dissected at stage 9 and treated with chemical MEK inhibitors, PD98059 (100 μM) and U0126 (100 μM) until sibling embryos reached stage 10.5. (c) Bra expression induced by FGF4 was inhibited, but otx2 expression induced by (d) insulin3 or (e) IGF1 was not. Full-length blots and gels are presented in Supplementary figure S8.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481404&req=5

f6: Insulin3 inhibits Wnt signalling but cannot activate the IGF1 receptor.mRNAs were injected into the animal pole of both blastomeres at the two-cell stage in X. laevis embryos. (a) One ng of insulin3, IGF1, or IGF1R mRNA was injected per embryo. Embryos were harvested at stage 11 for western blot analysis. (b) One ng of Myc-tagged extracellular domain of IGF1 receptor (IGF1Rex-Myc), IGF1-HA, insulin3-HA, or insulin1-HA mRNA was injected per embryo. Embryos were harvested at stage 10.5 for co-immunoprecipitation assay. (c,d,e) Amounts of mRNA injected per embryos were: (c) FGF4 (10 pg), (d) insulin3 (100 pg), and (e) IGF1 (100 pg). Animal caps were dissected at stage 9 and treated with chemical MEK inhibitors, PD98059 (100 μM) and U0126 (100 μM) until sibling embryos reached stage 10.5. (c) Bra expression induced by FGF4 was inhibited, but otx2 expression induced by (d) insulin3 or (e) IGF1 was not. Full-length blots and gels are presented in Supplementary figure S8.
Mentions: Previous studies reported that IGFs inhibit canonical Wnt signalling67. Here we showed that Insulin3 could also inhibit canonical Wnt signalling (Fig. 4b) indicating it can activate the same signal pathway as IGFs. IGF signals are mainly mediated by means of the IGF1 receptor (IGF1R) and to a lesser extent by the insulin receptor (IR), which are receptor tyrosine kinases (RTKs). We hypothesised that Insulin3 could also activate the IGF1 receptor to induce anterior neural gene expression. Contrary to our hypothesis, our results indicated that Insulin3 cannot activate IGF1R (Fig. 6a), whereas Insulin3 efficiently interacted with the extracellular domain of IGF1R (Fig. 6b). It has been reported that IGFs induce head formation through the IGF/IGF1R/MAPK pathway5. Our results indicate that Insulin3 inhibits Wnt signalling using another mechanism.

Bottom Line: Insulin3 reduced extracellular Wnts and cell surface localised Lrp6.These results suggest that Insulin3 is a novel cell-autonomous inhibitor of Wnt signalling.This study provides the first evidence that an insulin-like factor regulates neural induction through an IGF1R-independent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.

ABSTRACT
Insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) signalling is required for normal embryonic growth and development. Previous reports indicated that the IGF/IGF1R/MAPK pathway contributes to neural induction and the IGF/IGF1R/PI3K/Akt pathway to eye development. Here, we report the isolation of insulin3 encoding a novel insulin-like ligand involved in neural induction. Insulin3 has a similar structure to pro-insulin and mature IGF ligands, but cannot activate the IGF1 receptor. However, similar to IGFs, Insulin3 induced the gene expression of an anterior neural marker, otx2, and enlarged anterior head structures by inhibiting Wnt signalling. Insulin3 are predominantly localised to the endoplasmic reticulum when otx2 is induced by insulin3. Insulin3 reduced extracellular Wnts and cell surface localised Lrp6. These results suggest that Insulin3 is a novel cell-autonomous inhibitor of Wnt signalling. This study provides the first evidence that an insulin-like factor regulates neural induction through an IGF1R-independent mechanism.

No MeSH data available.


Related in: MedlinePlus