Lens-specific protein αA-crystallin (Sawada et al., 1993) was found immunocytochemically to be expressed in a small cluster of cells in the refractile cell mass of the eye-like structure (Fig. 2A,B). Other lens-specific proteins, αB-crystallin (Fig. 2C,D) and β-crystallin (Fig. 2E,F), were detected in greater abundance in the cell masses. Cells expressing some retinal marker proteins (Haverkamp and Wassle, 2000) were also detected in the cell mass close to the RPE cluster, i.e., Brn3b (Fig. 2G) and syntaxin (Fig. 1H), which are ganglion cell- and amacrine cell-specific proteins, respectively. Synaptophysin (Fig. 2I) and vesicular γ-aminobutyric acid (GABA) transporter (Fig. 2J), which are known to be expressed in several retinal subtype cells, were also expressed. Rhodopsin and recoverin, both specifically expressed in photoreceptor cells, were expressed in these cell masses, as shown in Figure 2K,L, and were mostly colocalized (Fig. 2M). On the other hand, αB-crystallin and the retinal marker protein calbindin were detected in separate compartments of the eye-like structure, as shown in Figure 2O,P. The cell mass forming the eye-like structure contained multiple cell layers, as shown in Figure 1I. These layers seem to represent the layer structure of the retina to some extent, as most of the cells expressing specific retinal or lens marker proteins formed cognate clusters of cells, as shown in Figure 2N–Q. In Figure 2P, the red syntaxin-positive cell layer was observed to extend beneath the green rhodopsin-positive layer (the extended layer is out of focus in this photo); and in Figure 2Q, the red calbindin-positive layer was observed under the green rhodopsin-positive layer. Expression of crystallin subtypes and of phosducin, a retinal photoreceptor specific signal transduction molecule, was also detected by reverse transcription-polymerase chain reaction (RT-PCR; Fig. 2R). These observations show that lens and retinal cell differentiation was induced under these culture conditions. Individual cell types were not randomly distributed but clustered to a certain extent, indicative of the layered structures observed in the retina.
Figure 2. Expression of eye-specific molecular markers in the induced eye-like structures. α-A crystallin (fluorescent image in A and brightfield image in B), α-B crystallin (C,D), and β-crystallin (anti-β6, E,F), were immunostained. Fluorescent images of Brn3b (G), syntaxin (H), synaptophysin (I), and vesicular γ-aminobutyric acid transporter (J) are also presented. Double immunostaining for rhodopsin (K, green fluorescence) and recoverin (L, red fluorescence) was done, and the merged image (M) shows almost complete colocalization of these two photoreceptor-specific proteins. The merged image of another photoreceptor-specific protein, peripherin (red), indicates its colocalization with rhodopsin (green, N). Double immunostaining for syntaxin (red) and rhodopsin (green, O) and its magnified part (P) indicates mostly separate localization of these markers. Rhodopsin (green) and syntaxin (red) also indicate separate localization (Q). R: mRNAs of crystallin subtypes and rhodopsin were analyzed by reverse transcriptase-polymerase chain reaction. Lane 1, day-10 culture of PA6 only; lane 2, day-10 culture of embryonic stem cells with PA6; lane 3, day-10 whole embryo. Scale bars = 400 μm in A–J,N,O, 40 μm in K–M,P,Q.
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