Data describing Rax positive optic-vesicle generation from mouse embryonic stem cells in vitro

This article contains data related to the research article entitled “Specification of embryonic stem cell-derived tissues into eye fields by Wnt signaling using rostral diencephalic tissue-inducing culture” Sakakura (2016) [1]. Mouse embryonic stem cells (ESC) were used for the generation of optic vesicle-like tissues in vitro. In this article we described data in which a Rax::GFP knock-in ESC line was used to monitor the formation of optic tissues. In addition, we also described the data of regional marker expression of Rax, Sox2 and Pax6 in vivo around the forebrain and the eye tissues for comparative purposes. These data can be valuable to researchers interested in investigating forebrain and eye tissue development.


a b s t r a c t
This article contains data related to the research article entitled "Specification of embryonic stem cell-derived tissues into eye fields by Wnt signaling using rostral diencephalic tissue-inducing culture" Sakakura (2016) [1]. Mouse embryonic stem cells (ESC) were used for the generation of optic vesicle-like tissues in vitro. In this article we described data in which a Rax::GFP knock-in ESC line was used to monitor the formation of optic tissues. In addition, we also described the data of regional marker expression of Rax, Sox2 and Pax6 in vivo around the forebrain and the eye tissues for comparative purposes. These data can be valuable to researchers interested in investigating forebrain and eye tissue development.

Value of the data
The expression pattern of Sox2, Pax6 and Rax provides the characterization of ESC-derived tissues for regional identification of neural tissues.
The data and diagram for the timed-addition of reagents into the differentiation media may assist the readers in readily using the in vitro system for inducing optic tissues from ESCs.
A data of the ESC-derived Rax positive tissues from culture day 4 to day 24 may give better information of the ESC differentiation culture for researchers in the related fields.

Data
This data mainly focuses on describing the regional marker expression of neural tissues and the data of eye tissue-inducing culture (Figs. 1 and 2), and refers to our recently published [1]. We used mouse embryo and mouse ESC-derived tissue samples to analyze the regional marker expression via immunostaining and also showed the images of ESC-derived tissues in living condition. The data shown are microscopy images (Fluorescent and Bright-field), graphs (Population of GFPþ cells) and schematic diagrams (Step-by-step processes).

2.2.
Step-by-step data during generating Raxþ optic vesicle-like tissues in the CDM/MG/CHIR condition From here we describe data for generating Rax þ optic vesicle-like tissues using mouse ESCs ( Fig. 2A). ESCs were maintained in maintenance medium before differentiation [5] (Fig. 2A, B). At day 0, we performed SFEBq (Serum-free Floating culture of Embryoid Body-like aggregates with quick reaggregation) [6]. ESCs were dissociated and quickly aggregated in the non-adhesion coated 96 wells (Fig. 2D). 30 min later, we could observe the beginning of aggregation and, 6 h later, cells had mostly aggregated (Fig. 2C, E). At day 1, 2% matrigel was directly added into the medium (Fig. 2F). At day 2, the surface portion of the day-2 aggregate was changed morphologically (Fig. 2G). By day 4, neurorpithelial-like structures expressing apico-basal polarity marker, N-cadherin (inside), and Laminin (outside) were observed (Fig. 2H, J). Simultaneously, Rax::GFP cells started to faintly appear in the epithelial-like structure (Fig. 2I, K). This time (day 4), 2 mM CHIIR99021 was added, which inhibited GSK-3ß. At day 7, Raxþ optic vesicle-like structures were generated (Fig. 2L, M). Subsequently, at day 7, we replaced CDM with DMEM/F12/N2/10% FBS (fetal bovine serum) medium to grow tissues for long-term culture ( Fig. 2A). At day 10, we chopped Rax þ tissue portions which also expressed Pax6 in order to separate them from other tissues (Fig. 2N). From day 10, we precisely followed the method reported previously [5]. We confirmed that day-18 tissues mostly expressed Rax::GFP (Fig. 2O, O 0 ) and day-24 tissues expressed Rax::GFP and Recoverin, which is expressed in the photoreceptor cells of the eyes in vivo [7,8] (Fig. 2P). The addition of 0.5 or 3 mM CHIR99021 from day 4 to day 7 did not efficiently: induce the formation of Raxþ vesicle-like structure (Fig. 2Q).

Fluorescence-activated cell sorting (FACS) analysis
FACS analysis was performed as previously described [9].