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IGF-2/IGF-1R signaling has distinct effects on Sox1, Irx3, and Six3 expressions during ES cell derived-neuroectoderm development in vitro

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Abstract

Insulin-like growth factors (IGFs) are involved in growth and tissue development, including diseases such as type-2 diabetes and cancers. However, their roles in lineage specification, especially in early mammalian neural development, are poorly understood. Here, we analyzed the protein expression of IGF-2 in early mouse embryo, and it was preferentially detected in anterior mesodermal tissue, adjacent to the neural plate. We utilized a self-organizing neural tissue culture system and analyzed the direct effect of IGF-2 on the general neural marker Sox1. Interestingly, using recombinant IGF-2 and a chemical inhibitor of its receptor (IGF-1R), we found that the IGF-2/IGF-1R pathway positively regulated Sox1 expression in embryonic stem (ES) cell-derived neural tissue. Furthermore, to visualize the expression patterns of other neural markers, we used reporter ES cell lines and we found that the IGF-2/IGF-1R signaling upregulated the expression of the posterior neural marker Irx3. In contrast, the anterior neural marker Six3 was downregulated by IGF-2/IGF-1R signaling. Together, our results demonstrate that IGF-2/IGF-1R signaling has different effects on neural marker expression, which may influence the early regional identity of ES cell-derived neural tissues.

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Acknowledgments

We are grateful to M. Eiraku for the invaluable comments and to the members of the laboratory for the discussion. We also thank M. Kawada for the technical advice on SFEBq and vector construction. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology in Japan (MEXT) (to Y.S.) and the Network Program for Realization of Regenerative Medicine from the Japan Science and Technology Agency (JST) (to Y.S.).

Contributions

N.T. designed the research, N.T. and E.S. performed the experiments, N.T. analyzed the data, N.T. prepared the figures and wrote the paper, and Y.S. supervised the project.

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Authors and Affiliations

  1. Laboratory for In Vitro Histogenesis, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuou-ku, Kobe, Hyogo, 650-0047, Japan

    Nozomu Takata & Eriko Sakakura

  2. Center for Vascular and Developmental Biology, Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, 303 East Superior Street, Chicago, 60611, IL, USA

    Nozomu Takata

  3. Laboratory for Organogenesis and Neurogenesis, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuou-ku, Kobe, Hyogo, 650-0047, Japan

    Yoshiki Sasai

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  1. Nozomu Takata
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  2. Eriko Sakakura
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Correspondence to Nozomu Takata.

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Editor: Tetsuji Okamoto

Dr. Yoshiki Sasai would be corresponding author, if he was alive

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Supplementary Figure

igfbp5 expression in Irx3::Venus+ and Irx3::Venus− cells and distinct effects of IGF-2/IGF-1R signaling on Sox1, Irx3, and Six3 expressions. (A) RT-qPCR analysis of igfbp5 in ES cells and day-5 neuroectoderm-like cells. (B) Schematic diagram of sorting of Irx3::Venus+/− cells via FACS. (C–E) RT-qPCR analysis of igfbp5 in Irx3::Venus+ and Irx3::Venus− cells, detecting mRNA levels of venus, irx3, and igfbp5. gapdh value was used for generating relative expression level of each gene. Error bars indicate standard error of the mean of each experiment. (F) Schematics of IGF-2 effects on Sox1, Irx3, and Six3 expressions. (GIF 31 kb)

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Takata, N., Sakakura, E. & Sasai, Y. IGF-2/IGF-1R signaling has distinct effects on Sox1, Irx3, and Six3 expressions during ES cell derived-neuroectoderm development in vitro. In Vitro Cell.Dev.Biol.-Animal 52, 607–615 (2016). https://doi.org/10.1007/s11626-016-0012-6

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