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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.


Rescue experiments of insulin3 over-expressed or knockdown phenotypes by modulation of Wnt signalling.(a–d) Rescue of insulin3 over-expressed phenotype by treatment of Wnt signal activator, LiCl. (a) Uninjected control embryos. (b,c) Microinjection of 500 pg of insulin3 mRNA at the two-cell stage induces giant cement gland (b). This phenotype is inhibited by treatment of 0.3M LiCl at the gastrulae stage for 5 min. (c). (d) LiCl treatment can rescue enlarged cement gland induced by insulin3 over-expression. Phenotypic index: Small, small-sized cement gland and head structure. Large, enlarged cement gland. Phenotypes were counted at St. 25. (e–h) Rescue of insulin3 MO phenotype by mouse dkk1 protein. (e) Uninjected control embryos. (f,g) Microinjection of 30 ng insulin3 MO at the two-cell stage induces anterior defects with small eyes (f). This phenotype is rescued by the injection of mouse dkk1 protein (1 ng) into blastocoel at St. 9 (g). (h) Dkk1 protein injection into the blastocoel can rescue small-sized eye phenotypes in insulin3 morphant. Phenotypic index: small, small-sized eye. Large, enlarged eye.
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f5: Rescue experiments of insulin3 over-expressed or knockdown phenotypes by modulation of Wnt signalling.(a–d) Rescue of insulin3 over-expressed phenotype by treatment of Wnt signal activator, LiCl. (a) Uninjected control embryos. (b,c) Microinjection of 500 pg of insulin3 mRNA at the two-cell stage induces giant cement gland (b). This phenotype is inhibited by treatment of 0.3M LiCl at the gastrulae stage for 5 min. (c). (d) LiCl treatment can rescue enlarged cement gland induced by insulin3 over-expression. Phenotypic index: Small, small-sized cement gland and head structure. Large, enlarged cement gland. Phenotypes were counted at St. 25. (e–h) Rescue of insulin3 MO phenotype by mouse dkk1 protein. (e) Uninjected control embryos. (f,g) Microinjection of 30 ng insulin3 MO at the two-cell stage induces anterior defects with small eyes (f). This phenotype is rescued by the injection of mouse dkk1 protein (1 ng) into blastocoel at St. 9 (g). (h) Dkk1 protein injection into the blastocoel can rescue small-sized eye phenotypes in insulin3 morphant. Phenotypic index: small, small-sized eye. Large, enlarged eye.

Mentions: Wnt and BMP inhibition is important to induce anterior neural tissues. To address a question that insulin3 act as a Wnt inhibitor or BMP inhibitor, we verified these activities. Insulin3 over-expressed embryos are more similar to dkk1, a Wnt inhibitor, over-expressed embryos than chd, a BMP inhibitor, over-expressed embryos (Fig. 4a). Insulin3 can inhibit Wnt signalling (Fig. 4b). Insulin3 and dkk1 also can inhibit BMP signalling but their activities are obviously weak compared with chordin (Fig. 4c). Previous study showed that neural induction in Xenopus requires inhibition of Wnt/beta-catenin signalling21 and BMP signalling is sensitive to Wnt inhibition in animal caps of early gastrulae embryos22. These reports and our results indicate that BMP signal inhibition by insulin3 and dkk1 is the result of Wnt inhibition. Indeed, insulin3 over-expressed phenotype was rescued by treatment of LiCl, an activator of Wnt signalling at the gastrulae stage, (Fig. 5a–d) and insulin3 knockdown phenotype by MO was rescued by treatment of mouse dkk1 protein, an inhibitor of Wnt signalling (Fig. 5e–h). These results suggest that Insulin3 mainly acts as a Wnt modulator in vivo.


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)

Rescue experiments of insulin3 over-expressed or knockdown phenotypes by modulation of Wnt signalling.(a–d) Rescue of insulin3 over-expressed phenotype by treatment of Wnt signal activator, LiCl. (a) Uninjected control embryos. (b,c) Microinjection of 500 pg of insulin3 mRNA at the two-cell stage induces giant cement gland (b). This phenotype is inhibited by treatment of 0.3M LiCl at the gastrulae stage for 5 min. (c). (d) LiCl treatment can rescue enlarged cement gland induced by insulin3 over-expression. Phenotypic index: Small, small-sized cement gland and head structure. Large, enlarged cement gland. Phenotypes were counted at St. 25. (e–h) Rescue of insulin3 MO phenotype by mouse dkk1 protein. (e) Uninjected control embryos. (f,g) Microinjection of 30 ng insulin3 MO at the two-cell stage induces anterior defects with small eyes (f). This phenotype is rescued by the injection of mouse dkk1 protein (1 ng) into blastocoel at St. 9 (g). (h) Dkk1 protein injection into the blastocoel can rescue small-sized eye phenotypes in insulin3 morphant. Phenotypic index: small, small-sized eye. Large, enlarged eye.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4481404&req=5

f5: Rescue experiments of insulin3 over-expressed or knockdown phenotypes by modulation of Wnt signalling.(a–d) Rescue of insulin3 over-expressed phenotype by treatment of Wnt signal activator, LiCl. (a) Uninjected control embryos. (b,c) Microinjection of 500 pg of insulin3 mRNA at the two-cell stage induces giant cement gland (b). This phenotype is inhibited by treatment of 0.3M LiCl at the gastrulae stage for 5 min. (c). (d) LiCl treatment can rescue enlarged cement gland induced by insulin3 over-expression. Phenotypic index: Small, small-sized cement gland and head structure. Large, enlarged cement gland. Phenotypes were counted at St. 25. (e–h) Rescue of insulin3 MO phenotype by mouse dkk1 protein. (e) Uninjected control embryos. (f,g) Microinjection of 30 ng insulin3 MO at the two-cell stage induces anterior defects with small eyes (f). This phenotype is rescued by the injection of mouse dkk1 protein (1 ng) into blastocoel at St. 9 (g). (h) Dkk1 protein injection into the blastocoel can rescue small-sized eye phenotypes in insulin3 morphant. Phenotypic index: small, small-sized eye. Large, enlarged eye.
Mentions: Wnt and BMP inhibition is important to induce anterior neural tissues. To address a question that insulin3 act as a Wnt inhibitor or BMP inhibitor, we verified these activities. Insulin3 over-expressed embryos are more similar to dkk1, a Wnt inhibitor, over-expressed embryos than chd, a BMP inhibitor, over-expressed embryos (Fig. 4a). Insulin3 can inhibit Wnt signalling (Fig. 4b). Insulin3 and dkk1 also can inhibit BMP signalling but their activities are obviously weak compared with chordin (Fig. 4c). Previous study showed that neural induction in Xenopus requires inhibition of Wnt/beta-catenin signalling21 and BMP signalling is sensitive to Wnt inhibition in animal caps of early gastrulae embryos22. These reports and our results indicate that BMP signal inhibition by insulin3 and dkk1 is the result of Wnt inhibition. Indeed, insulin3 over-expressed phenotype was rescued by treatment of LiCl, an activator of Wnt signalling at the gastrulae stage, (Fig. 5a–d) and insulin3 knockdown phenotype by MO was rescued by treatment of mouse dkk1 protein, an inhibitor of Wnt signalling (Fig. 5e–h). These results suggest that Insulin3 mainly acts as a Wnt modulator in vivo.

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.