The GIPC1-Akt1 Pathway Is Required for the Specification of the Eye Field in Mouse Embryonic Stem Cells

During early patterning of the neural plate, a single region of the embryonic forebrain, the eye field, becomes competent for eye development. The hallmark of eye field specification is the expression of the eye field transcription factors (EFTFs). Experiments in fish, amphibians, birds, and mammals have demonstrated largely conserved roles for the EFTFs. Although some of the key signaling events that direct the synchronized expression of these factors to the eye field have been elucidated in fish and frogs, it has been more difficult to study these mechanisms in mammalian embryos. In this study, we have used two different methods for directed differentiation of mouse embryonic stem cells (mESCs) to generate eye field cells and retina in vitro to test for a role of the PDZ domain-containing protein GIPC1 in the specification of the mammalian eye primordia. We find that the overexpression of a dominant-negative form of GIPC1 (dnGIPC1), as well as the downregulation of endogenous GIPC1, is sufficient to inhibit the development of eye field cells from mESCs. GIPC1 interacts directly with IGFR and participates in Akt1 activation, and pharmacological inhibition of Akt1 phosphorylation mimics the dnGIPC1 phenotype. Our data, together with previous studies in Xenopus, support the hypothesis that the GIPC1-PI3K-Akt1 pathway plays a key role in eye field specification in vertebrates. STEM CELLS 2015;33:2674–2685 SIGNIFICANCE STATEMENT In this study, we use the directed differentiation of embryonic stem cells (ESCs) as a model to study key regulatory steps in eye field formation. The regulatory mechanisms that define the eye field are not well understood, and most of what we know about this has been discovered in organisms with accessible embryos like zebrafish and Xenopus. Signaling pathways are difficult to manipulate in mammals at early embryonic stages. ESCs have emerged as an alternative method to study the earliest steps of mammalian ontogeny. The differentiation paradigms towards eye field progenitors and mature retinal cells from ESCs are now well established. Therefore, ESCs provide a potential model for testing hypotheses concerning forebrain patterning and eye field specification in vitro. Here, we directed mESCs towards eye field and retinal fates, to test whether a specific component of the signaling network in frog embryos, Kermit2, is important in mammalian eye development. Our results indicate that GIPC plays a key role in the segregation of the eye field from telencephalic cell fates, and likely acts through the regulation of PI3K-Akt1 pathway downstream of IGFR. Our results show the importance of IGF signaling to patterning of the CNS, and demonstrate the usefulness of ESCs in understanding basic developmental processes in organ formation.