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Fig. 4 The spatial localization of wnt8b transcripts during normal zebrafish embryogenesis. All embryos are oriented anterior to the top and dorsal facing up, except C, which is a lateral view of the embryo in D. (A) At approximately 90% epiboly, wnt8b signals appear in two wing-like patterns flanking the dorsal midline. (B) At the 2- to 3- somite stage, the wnt8b signal has joined over the dorsal midline (large arrow), and is continuous with signals at the lateral edge of the body axis (small arrows). (C) Lateral view of an embryo at approximately 16 hours, illustrating the wnt8b signals at the future midbrain-hindbrain boundary, the epiphysis, the ventral floor of the forebrain (arrow from asterisk), and in rhombomeres (r) r1, 3 and 5. (D) Dorsal view of the whole mount in C, demonstrates that wnt8b expression is contiguous over the entire width of the rhombomere. wnt8b expression in the forebrain is not obvious in C due to the curvature of the embryo, but close examination in these more rostral regions (E) reveals that wnt8b is expressed at the forebrain-midbrain border and in a discrete layer of the eye (arrows). (F) At approximately 19 hours, wnt8b signals are also detected in a limited region of the optic stalk and in the ventral forebrain (arrow from asterisk). (G) Whole-mount embryo having been hybridized to both the wnt8b and krox20 antisense DIG-labeled RNA probes. The wnt8b signals are confined to the prospective rostral neuroepithelium, in the area corresponding to the future midbrain-hindbrain boundary, and the two thin stripes denoted by the open arrows represent the krox20 expression in prospective r3. (H) To compare directly the relationship between the expression of wnt8b and krox20 in specific rhombomeres, embryos were hybridized simultaneously to wnt8b and krox20 antisense probes. Only the characteristically weak wnt8b signal in r1 can be detected in the double in situ; signals in r3 and r5 are not resolved due to the robust krox20 signal. (I) Dorsal view of a whole-mount embryo at a stage comparable to the embryo in F, illustrating the prominent pax2 expression in the optic stalk and at the midbrain-hindbrain boundary. Abbreviations: e, epiphysis; fmb, forebrain-midbrain boundary; mhb, midbrain-hindbrain boundary; os, optic stalk; r, rhombomeres. Scale bar (A,B), 55 μm; (C-F, H-I), 45 mm; (G), 65 μm.