An Eye Organoid Approach Identifies Six3 Suppression of R-spondin 2 as a Critical Step in Mouse Neuroretina Differentiation

• Published in: Cell reports (Cambridge) 2017-11, Vol.21 (6), p.1534-1549
• Main Authors: Takata, Nozomu, Abbey, Deepti, Fiore, Luciano, Acosta, Sandra, Feng, Ruopeng, Gil, Hyea Jin, Lavado, Alfonso, Geng, Xin, Interiano, Ashley, Neale, Geoffrey, Eiraku, Mototsugu, Sasai, Yoshiki, Oliver, Guillermo
• Format: Article
• Language: English
• Subjects: Animals; Axin Protein - genetics; Axin Protein - metabolism; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Embryo, Mammalian - metabolism; Embryonic Stem Cells; eye; Eye Proteins - genetics; Eye Proteins - metabolism; Homeobox Protein SIX3; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; In Situ Hybridization, Fluorescence; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - metabolism; Intercellular Signaling Peptides and Proteins - genetics; Intercellular Signaling Peptides and Proteins - metabolism; Mice; Mice, Transgenic; mouse; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Neural Plate - metabolism; neuroretina; Oligonucleotide Array Sequence Analysis; optic vesicles; organoids; pluripotent stem cells; R-spondins; Retina - cytology; Retina - metabolism; Six3; Six3 conditional knockout ESCs; SOXB1 Transcription Factors - genetics; SOXB1 Transcription Factors - metabolism; Thrombospondins - genetics; Thrombospondins - metabolism; Transcription Factors - genetics; Transcription Factors - metabolism; Wnt; Wnt Proteins
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2017.10.041

Recent advances in self-organizing, 3-dimensional tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided an in vitro model that recapitulates many aspects of the in vivo developmental steps. Using Rax-GFP-expressing ESCs, newly generated Six3−/− iPSCs, and conditional null Six3delta/f;Rax-Cre ESCs, we identified Six3 repression of R-spondin 2 (Rspo2) as a required step during optic vesicle morphogenesis and neuroretina differentiation. We validated these results in vivo by showing that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to inhibit neuroretina differentiation. Additionally, using a chimeric eye organoid assay, we determined that Six3 null cells exert a non-cell-autonomous repressive effect during optic vesicle formation and neuroretina differentiation. Our results further validate the organoid culture system as a reliable and fast alternative to identify and evaluate genes involved in eye morphogenesis and neuroretina differentiation in vivo. [Display omitted] •Six3delta/f;Rax-Cre ESCs exhibit defective optic vesicle and neuroretina•Six3 represses R-spondin 2 expression•Repression of R-spondin 2 is required for neuroretina differentiation•Six3 mutant cells exhibit a non-cell-autonomous repressive effect over WT cells Using an eye organoid culture system, Takata et al. identified the Wnt signaling partner R-spondin 2 as a critical player in neuroretina fate determination in vivo and in vitro. They also validated these findings using newly derived conditional null Six3delta/f;Rax-Cre ESCs from mouse blastocysts.