Fig. 3

Whole-mount in situ hybridization analysis of zebrafish PG1 genes. Two-colour double in situ hybridization shows Hox genes in purple, plus krox-20 in red as a marker of r3 and r5. All embryos are mounted with dorsal side towards reader and anterior upwards. Rhombomere (r) numbers as indicated. (A-D) The hoxb1b and hoxb1a genes both have early expression domains in r4. (A) hoxb1b expression at 80% epiboly (8 hours, h) lies in bilateral epiblast domains above the margin. (B) At tailbud stage (10 h) hoxb1b expression is localized to r4 abutting early krox-20 expression in r3. (C) By the one-somite stage (10.5 h) hoxb1b expression has already started to retreat posteriorly and is absent from r4. (D) hoxb1a expression at the equivalent stage (one somite) is already upregulated in r4. (E-I) hoxa1a is expressed in an anterior subpopulation of neurones. (E) At 24 h hoxa1a expression is localized to discrete bilateral clusters of cells in the anterior hindbrain and ventral midbrain (arrowheads). (F) 36 h; expression is now localized to cell clusters in the midbrain, medial to the eyes, and r1 (arrowheads). (G) 3.5 μm transverse section (t.s.) through plane indicated in F. (H) HNK-1 antibody staining reveals cell bodies of the nMLF (arrowheads), the MLF axon tract and the trigeminal ganglia (TG) at 22 h. hoxa1a expression colocalizes to the nMLF. (I) hoxa1a-expressing cells continue to colocalize with HNK-1-positive neurones in the nMLF (arrowheads) at 28 h, arrows indicate hoxa1a-expressing cells in the anterior hindbrain. (J-M) hoxc1a expression: (J) at 12 h in notochord (n); (K) at 16.5 h in CNS (anterior limit at spinal cord/hindbrain junction); (L) at 24 h in bilateral cell clusters in the ventral midbrain (arrowhead) and Mauthner neurones (arrow); (M) at 36 h in cells medial to eyes (arrowhead), similar to hoxa1a.