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Fig. 1. Comparison between Otx2, Gbx2 and Xiro1 expression. Embryos were fixed at late gastrula (stage 12-12.5) (A,A’,D,G), early neurula (stage 13-14) (B,B’,E,H,J,K) and mid neurula (stage 17-18) (C,C’,F,I,L), and double in situ hybridization and sectioning were carried out for each pair of genes. The whole mounts are dorsal views oriented with anterior to the top and the sections and inset are oriented with anterior to the left. (A-C) Otx2 (green) and Gbx2 (purple) are expressed in complementary domains that overlap in the isthmus region. (A’) Higher magnification of the square shown in A. Notice the overlapping expression of both genes. (B’,C’) Upper panels show a sagittal section of an embryo after the first chromogenic reaction for Otx2 detection (green). Lower panels show the same embryo after the second chromogenic reaction for Gbx2 detection (purple). Notice the overlap in the expression of both genes at the early neurula stage (bracket in B’), which disappears at the mid neurula stage (bracket in C’), to generate a sharp boundary of Otx2/Gbx2 expression. (D-F) Otx2 (purple) and Xiro1 (light blue) overlap at the presumptive midbrain domain. (G-I) Gbx2 expression (purple) is almost completely included in Xiro1 (light blue)-expressing territory. (J) Position of Fgf8 expression. The initial isthmus expression of Fgf8 appears at early neurula stage in the region where Otx2 and Gbx2 are co-expressed (brackets). This early expression precedes the establishment of the sharp border described for Otx2 and Gbx2. Images were taken from the same embryos after the first gene detection (right panels, green for Otx2 and Gbx2) and at after the second chromogenic reaction (left panels, purple for Fgf8). (K) Double in situ hybridization for Fgf8 (purple) and Xiro1 (green) mRNAs. The Fgf8 isthmus expression is included in the Xiro1-positive cells at this stage (arrow). Arrowhead points the anterior limit of Xiro1. (L) Double staining for En2 (purple) and Gbx2 (green). En2 is expressed mainly in the Otx2 domain with a faint graded co-expression with Gbx2 at stage 17 (arrowhead). (M-O) The expression patterns observed by whole-mount in situ hybridization during the three stages described above. The positions of Fgf8 and En2 expression are also shown. Note the refinement in the Otx2-Gbx2 overlapping region and the co-expression domains of Xiro1, Otx2 and Gbx2. |
