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Insulin Signaling in Early Neural Crest Specification

Creative Commons 'BY-NC-ND' version 4.0 license

The field of neural crest (NC) development continues to expand as new knowledge of this early developmental, multipotent, highly migratory cell population comes to light. However, as we begin to learn more about the derivatives of NC cells, it becomes evident that we must define the collection of growth factors, their concentration, and the precise timing of each to determine what allows for successful differentiation and function of NC derivatives. NC specification has recently been found to occur at the blastula stage, earlier than previously thought, and thus investigation into signaling contributions at this stage must be done. Beyond known players Wnt, FGF, and BMP, a role for insulin has not been elucidated at this or any stage, despite mounting evidence of a connection to NC development, as insulin has been implicated in connection with various neurocristopathies and it is a component of all published NC induction assays in chick and human studies. Therefore, defining the role of insulin in NC formation and its mechanism is imperative to understanding NC development. Using a robust human NC induction model and blastula stage chick embryos, we report that insulin is required for NC marker expression independently from growth and metabolism. Immunofluorescence of chick NC specification assays and our hNC induction model, in the presence and absence of insulin, as well as in the presence of small molecule inhibitors and siRNA (human only), we saw reduced expression of definitive NC markers in instances where insulin signaling was impeded. More specifically, temporal insulin signaling modification at blastula stage in chick and timepoint zero of human NC induction from pluripotent stem cells, indicates an early role for insulin. Via small molecule inhibition and RNAi knockdown of IR and IGFR, both individually and simultaneously, NC markers expression was reduced, and in most cases without a significant effect on cell survival, indicating utilization of both receptors or a hybrid IR/IGFR. Furthermore, we show evidence that these signals are propagated via Pi3K and PDK1, and that NC specification is extremely sensitive to changes in the function of downstream effector FoxO1, which is highly influenced by insulin signaling.

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