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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.


Insulin3-deficient embryos showed anterior defects in both X. laevis and X. tropicalis.A translation-inhibiting MO was designed for X. laevis insulin3 and a splice-inhibiting MO for X. tropicalis insulin3. Forty ng and 12 ng of MOs were injected into the marginal zone of both blastomeres at the two-cell stage in (a) X. laevis and (b) X. tropicalis embryos, respectively. (a) Insulin3 MO induced anterior defects in X. laevis embryos. Insulin3 MO specifically inhibited the translation of 5′UTR-insulin3-HA, which has the targeted sequence of MO, but not mis-insulin3-HA, which has 7 mismatches in the targeted sequences. (b) Xt. insulin3 S MO induced anterior defects in X. tropicalis embryos. Xt. insulin3 S MO inhibited normal splicing of insulin3 mRNA. Full-length blots and gels are presented in Supplementary figure S7.
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f2: Insulin3-deficient embryos showed anterior defects in both X. laevis and X. tropicalis.A translation-inhibiting MO was designed for X. laevis insulin3 and a splice-inhibiting MO for X. tropicalis insulin3. Forty ng and 12 ng of MOs were injected into the marginal zone of both blastomeres at the two-cell stage in (a) X. laevis and (b) X. tropicalis embryos, respectively. (a) Insulin3 MO induced anterior defects in X. laevis embryos. Insulin3 MO specifically inhibited the translation of 5′UTR-insulin3-HA, which has the targeted sequence of MO, but not mis-insulin3-HA, which has 7 mismatches in the targeted sequences. (b) Xt. insulin3 S MO induced anterior defects in X. tropicalis embryos. Xt. insulin3 S MO inhibited normal splicing of insulin3 mRNA. Full-length blots and gels are presented in Supplementary figure S7.

Mentions: To investigate the role of Insulin3 in vivo, we designed translation-inhibiting morpholino oligonucleotides (MO) against the start codon and its surrounding sequences for X. laevis. Embryos injected with insulin3 MO showed anterior defects with small heads and eyes (Fig. 2a). To confirm this loss-of-function result in X. laevis embryos, we designed a splice-inhibiting MO for X. tropicalis (Xt. insulin3 S MO). X. tropicalis embryos injected with Xt. insulin3 S MO showed the same phenotype with anterior defects (Fig. 2b). Five-mispair control MOs and a standard control MO showed no significant effects (Fig. 2b and Fig. S3). Thus, Insulin3 plays an important role in anterior neural development in both X. laevis and X. tropicalis embryos. To examine the function of Insulin3, we injected insulin3 mRNA and insulin3 MO into the animal pole of each blastomere at the 2-cell stage of Xenopus laevis embryos. Five-mispair control MO and a standard control MO showed no effect on the expression of anterior neural maker genes and dorsal mesodermal maker genes (Fig. 2b and Fig. S3b). Injection of insulin3 mRNA increased cement gland gene, cg1, expression and anterior neural gene expression including bf1, rx1, otx2, sox2, and sox3 (Fig. 3a), and injection of insulin3 MO reduced these gene expressions (Fig. 3a), without affecting dorsal mesodermal maker gene expressions, gsc and chd (Fig. 3b). Pan neural markers, sox2 and sox3 were also enhanced in the posterior region of insulin3 mRNA injected embryos and reduced in the posterior region of insulin3 MO injected embryos. These data indicate that Insulin3 is a neuralising factor in Xenopus embryos.


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-deficient embryos showed anterior defects in both X. laevis and X. tropicalis.A translation-inhibiting MO was designed for X. laevis insulin3 and a splice-inhibiting MO for X. tropicalis insulin3. Forty ng and 12 ng of MOs were injected into the marginal zone of both blastomeres at the two-cell stage in (a) X. laevis and (b) X. tropicalis embryos, respectively. (a) Insulin3 MO induced anterior defects in X. laevis embryos. Insulin3 MO specifically inhibited the translation of 5′UTR-insulin3-HA, which has the targeted sequence of MO, but not mis-insulin3-HA, which has 7 mismatches in the targeted sequences. (b) Xt. insulin3 S MO induced anterior defects in X. tropicalis embryos. Xt. insulin3 S MO inhibited normal splicing of insulin3 mRNA. Full-length blots and gels are presented in Supplementary figure S7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Insulin3-deficient embryos showed anterior defects in both X. laevis and X. tropicalis.A translation-inhibiting MO was designed for X. laevis insulin3 and a splice-inhibiting MO for X. tropicalis insulin3. Forty ng and 12 ng of MOs were injected into the marginal zone of both blastomeres at the two-cell stage in (a) X. laevis and (b) X. tropicalis embryos, respectively. (a) Insulin3 MO induced anterior defects in X. laevis embryos. Insulin3 MO specifically inhibited the translation of 5′UTR-insulin3-HA, which has the targeted sequence of MO, but not mis-insulin3-HA, which has 7 mismatches in the targeted sequences. (b) Xt. insulin3 S MO induced anterior defects in X. tropicalis embryos. Xt. insulin3 S MO inhibited normal splicing of insulin3 mRNA. Full-length blots and gels are presented in Supplementary figure S7.
Mentions: To investigate the role of Insulin3 in vivo, we designed translation-inhibiting morpholino oligonucleotides (MO) against the start codon and its surrounding sequences for X. laevis. Embryos injected with insulin3 MO showed anterior defects with small heads and eyes (Fig. 2a). To confirm this loss-of-function result in X. laevis embryos, we designed a splice-inhibiting MO for X. tropicalis (Xt. insulin3 S MO). X. tropicalis embryos injected with Xt. insulin3 S MO showed the same phenotype with anterior defects (Fig. 2b). Five-mispair control MOs and a standard control MO showed no significant effects (Fig. 2b and Fig. S3). Thus, Insulin3 plays an important role in anterior neural development in both X. laevis and X. tropicalis embryos. To examine the function of Insulin3, we injected insulin3 mRNA and insulin3 MO into the animal pole of each blastomere at the 2-cell stage of Xenopus laevis embryos. Five-mispair control MO and a standard control MO showed no effect on the expression of anterior neural maker genes and dorsal mesodermal maker genes (Fig. 2b and Fig. S3b). Injection of insulin3 mRNA increased cement gland gene, cg1, expression and anterior neural gene expression including bf1, rx1, otx2, sox2, and sox3 (Fig. 3a), and injection of insulin3 MO reduced these gene expressions (Fig. 3a), without affecting dorsal mesodermal maker gene expressions, gsc and chd (Fig. 3b). Pan neural markers, sox2 and sox3 were also enhanced in the posterior region of insulin3 mRNA injected embryos and reduced in the posterior region of insulin3 MO injected embryos. These data indicate that Insulin3 is a neuralising factor in Xenopus embryos.

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.