EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS The GIPC1-Akt1 Pathway Is Required for the Specification of the Eye Field in Mouse Embryonic Stem Cells a,b a c c ANNA LA TORRE, AKINA HOSHINO, CHRISTOPHER CAVANAUGH, CAROL B. WARE, a THOMAS A. REH Key Words. Signal transduction • Retina • Retinal photoreceptors • Retinal pigmented epithelium • Neural differentiation aDepartment of Biological ABSTRACT Structure, Institute for Stem During early patterning of the neural plate, a single region of the embryonic forebrain, the eye Cells and Regenerative field, becomes competent for eye development. The hallmark of eye field specification is the Medicine and cDepartment expression of the eye field transcription factors (EFTFs). Experiments in fish, amphibians, birds, of Comparative Medicine, and mammals have demonstrated largely conserved roles for the EFTFs. Although some of the University of Washington, key signaling events that direct the synchronized expression of these factors to the eye field Seattle, Washington, USA; bDepartment of Cell Biology have been elucidated in fish and frogs, it has been more difficult to study these mechanisms and Human Anatomy, School in mammalian embryos. In this study, we have used two different methods for directed differ- of Medicine, University of entiation of mouse embryonic stem cells (mESCs) to generate eye field cells and retina in vitro California, Davis, California, to test for a role of the PDZ domain-containing protein GIPC1 in the specification of the mam- USA malian 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 Correspondence: Thomas A. development of eye field cells from mESCs. GIPC1 interacts directly with IGFR and participates Reh, Ph.D., Department of in Akt1 activation, and pharmacological inhibition of Akt1 phosphorylation mimics the dnGIPC1 Biological Structure, Institute for phenotype. Our data, together with previous studies in Xenopus, support the hypothesis that Stem Cells and Regenerative the GIPC1-PI3K-Akt1 pathway plays a key role in eye field specification in vertebrates. STEM Medicine, University of CELLS 2015;33:2674–2685 Washington, Seattle, Washington 98195, USA. Telephone: 206-543-5069; Fax: 206-616-6290; SIGNIFICANCE STATEMENT e-mail: [email protected] In this study, we use the directed differentiation of embryonic stem cells (ESCs) as a Received October 21, 2014; model to study key regulatory steps in eye field formation. The regulatory mechanisms accepted for publication April that define the eye field are not well understood, and most of what we know about this 22, 2015; first published online has been discovered in organisms with accessible embryos like zebrafish and Xenopus. Sig- in STEM CELLS EXPRESS June 24, naling pathways are difficult to manipulate in mammals at early embryonic stages. ESCs 2015. have emerged as an alternative method to study the earliest steps of mammalian ontog- VC AlphaMed Press eny. The differentiation paradigms towards eye field progenitors and mature retinal cells 1066-5099/2014/$30.00/0 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 http://dx.doi.org/ 10.1002/stem.2062 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 devel- opment. Our results indicate that GIPC plays a keyroleinthesegregationoftheeyefield 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. INTRODUCTION initiate fate-determining developmental pro- grams. At a molecular level, Otx2 is required The eye development begins at gastrula stages for the formation of the whole anterior neural during the regionalization of the anterior neu- region including the eyes [1], and the eye field ral plate (ANP). The current model proposes is subsequently specified by the expression that master-regulatory genes expressed in of a network of transcription factors that these committed progenitors are sufficient to include Rax (retina and anterior neural fold STEM CELLS 2015;33:2674–2685 www.StemCells.com VC AlphaMed Press 2015 Torre, Hoshino, Cavanaugh et al. 2675 homeobox), Pax6 (paired box gene 6), Lhx2 (LIM homeobox- technologies.com/us/en/home/brands/invitrogen.html), non- 2), and Six3 (Sine oculis homeobox 3) [2–4]. essential amino acids (Invitrogen), sodium pyruvate (Invitro- The regulatory mechanisms that define the domain of eye gen), and 0.1 mM of b-mercaptoethanol (Sigma-Aldrich, Saint field transcription factor (EFTF) expression are not well under- Louis, MO, https://www.sigmaaldrich.com), 100 mlofleukemia stood. Most of what we know about this has been discovered inhibitory factor (LIF, ESGRO Millipore, Billerica, MA, http:// in organisms with accessible embryos like zebrafish and Xeno- www.emdmillipore.com/, ESG1106, 10 million units/ml), 3 mM pus. These studies have revealed a network of signals of GSK3b inhibitor Stemolecule CHIR99021 (Stemgent, Cam- upstream of the EFTFs that involve inhibition of BMP and bridge, MA, https://www.stemgent.com), and 0.4 mMofMEK Wnt signaling [5–8]. However, it is not known how much of inhibitor Stemolecule PD0325901 (Stemgent). All the cell lines this model is conserved in mammals. Signaling pathways are were maintained in feeder-free conditions in growth factor- difficult to manipulate in mammals at early embryonic stages reduced Matrigel-coated plates. without affecting other functions of the same molecules. Therefore, in vitro model systems that recapitulate key Retinal Differentiation of mESCs aspects of embryogenesis might provide an approach to Semiconfluent undifferentiated colonies were dissociated to a understand ANP patterning and retinal specification in suspension of single cells by enzymatic treatment with TrypLE mammals. (Gibco, Rockville, MD, http://www.lifetechnologies.com/us/en/ Recently, embryonic stem cells (ESCs) have emerged as an home/brands/gibco.html) and gentle mechanical dissociation. alternative method to study the earliest steps of mammalian Next, cells were rinsed with DMEM 110% fetal bovine serum ontogeny. ESCs are pluripotent cells derived from the inner (FBS) and plated in retinal differentiation media (DMEM:F-12 cell mass of preimplantation blastocysts. These cells behave media, supplemented with nonessential amino acids, sodium similarly to those present in the developing embryo and can pyruvate, bovine serum albumin (BSA) [Invitrogen], B27, N2, be differentiated under defined conditions into a broad range and 1 ng/ml each of Dkk1, Noggin, and IGF1 (R&D Systems, of cell types. The differentiation paradigms toward eye field Minneapolis, MN, http://www.rndsystems.com/) in low- progenitors and mature retinal cells from mouse ESCs attachment plates. Floating mESCs spontaneously formed (mESCs), human ESCs (hESCs), and induced-pluripotent SCs aggregates (EBs) within few hours under these conditions. are well established [9–16]. Upon differentiation, the cells After 3 days, EBs were seeded onto Matrigel-coated plates in acquire characteristics of retinal differentiation, progressing retinal differentiation media. From days 5–10, media were through a succession of stages that recapitulates normal in changed every other day. For the 3D culture method, we vivo development. Hence, ESCs provide a potential model for adapted that of Eiraku and Sasai [29], using DMEM:F-12 as testing hypotheses concerning forebrain patterning and eye above (plus retinoic acid and taurine) as the maintenance field specification in vitro. media. Here, we directed mESCs toward eye field and retinal fates, to test whether a specific component of the signaling network qPCR in frog embryos, Kermit2, is important in mammalian eye Total RNA was extracted from the cultures using Trizol (Invi- development. In a previous report, Kermit2, the Xenopus hom- trogen) followed by chloroform extraction as manufacturer’s olog of GAIP-interacting protein, C terminus (GIPC) was shown instructions. This was followed by DNase-1 (Qiagen, Hilden, to be required for eye formation [17]; morpholino knockdown Germany, https://www.qiagen.com/us/) treatment followed by of this gene led to embryos lacking eyes, but were otherwise RNA cleanup using Qiagen RNA mini cleanup kit. cDNA was apparently normal. GIPC1 is a small adaptor protein that inter- reverse transcribed using Superscript III RT kit (Invitrogen) as acts with multiple cytoplasmic proteins and transmembrane per the manufacturer’s instructions. PCR was performed using receptors and likely plays a role in endosome signaling and the primers listed in Supporting Information Table and values membrane recycling [18–21]. In this study, we use mESC cul- were normalized to b-actin. tures to analyze the function of GIPC in the specification and differentiation of eye field and retinal fates. Our results indicate Immunohistochemistry and Microscopy that GIPC plays a key role in the specification of the eye field, Cultures were fixed in 4% paraformaldehyde (PFA), and after and likely acts through the regulation of PI3K-Akt1 pathway phosphate buffer solution (PBS) rinse, nonspecific binding was downstream of IGFR. inhibited by incubation