Fig. 2

Expression of hox genes in the developing hindbrain; wholemount in situ hybridization with 7 different hox gene riboprobes (purple signal). In most specimens krox-20 was used as a second marker to allow orientation with respect to r3 and r5 (red signal). Embryos were dissected off the yolk and flat-mounted between cover slips for photography. In all panels except E(i) only the hindbrain region is shown, anterior is to the left. Scale bar, 50 μm, where no scale bar shown scale is equivalent to panel above. (A) hoxb1 is expressed in presumptive r4 and posterior from the onset of expression at approximately 90% epiboly, through the 1 somite (1s) stage (i); by the 3s stage (ii) expression in r4 is up-regulated, note r4 lies between the r3 and r5 krox-20 expressing domains (red); hoxb1 expression in r5 and r6 is concomitantly down-regulated. At the 10s stage (iii) the r4 expression domain has sharp borders, abutting the krox-20 expression domains in r3 and r5; more posterior expression is at significantly lower levels in r7 and posterior. At the 20s stage (iv) the same pattern persists, but expression posterior of r4 is no longer apparent in the CNS. (B) hoxa2 is expressed in presumptive r2 and r3 from the onset of expression at the 2s stage (i), the same expression domain persists through the 5s stage (ii). At the 10s stage (iii) high level expression persists in r2 and r3 (note sharp anterior limit of expression at r1/2 boundary); low level expression has spread posteriorly through r4 and r5 (note overlap with krox-20 expression domains). At the 20s stage (iv) neural expression persists in r2 through r5, there are differences in expression levels between the individual rhombomeres such that the highest level is in r2, a slightly reduced level in r3, further reduced in r5 and lowest of all in r4. Neural crest migrating into the 2nd and 3rd branchial arches (arrows) also expresses hoxa2. (C) hoxb2 has an anterior expression limit one rhombomere length more posterior than that of its paralogue, hoxa2, at the r2/3 boundary. The onset of expression is at approximately the 1s stage (i) in presumptive r3 and r5, this was determined in other specimens by colocalization with krox-20; however, to avoid obscuring the weak r5 expression domain the sample shown is a single in situ with only the hoxb2 probe. By the 3-4s stage (ii) the level of r5 expression has increased and there is low level expression in r4 between the two high level domains, in addition there is low level expression posterior to r6. By the 10s stage (iii) expression is confined to rhombomeres 3, 4 and 5, with a step-wise expression gradient from anterior to posterior (highest levels in r3). At the 20s stage (iv) this step-wise gradient persists and expression can also be seen in neural crest cells migrating out into the second branchial arch (arrow). (D) hoxb3 has an onset of expression at the 1-2s stage, within the CNS posterior to r5, by the 3-4s stage (i) expression overlaps the r5 krox-20 domain, the anterior-most expression domain, in presumptive r5 and r6, is at elevated levels compared to more posterior expression. The expression limit and elevated expression levels in r5 and r6 are maintained through the 5s (ii) and 10s (iii) stages. At the 10s stage (iii) neural crest migrating into the 3rd branchial arch from the posterior part of r6, is also expressing hoxb3. By the 30 hour stage (iv) a low level expression domain in r4 has become apparent (arrow). (E) hoxd3 also has an expression onset at the 1-2s stage (i) in the posterior most part of the embryo, by the 3-4s stage (ii) expression has spread rapidly anteriorly to abut the r5 expression domain of krox-20. This r5/6 expression limit is maintained through the 10s stage (iii) and 20s stage (see Fig. 5I). By 30 hours (iv) a more anterior expression domain in a small lateral/ventral group of r5 cells is visible (arrow). (F) hoxx4 has an expression onset at about the 1s stage (i) with an anterior limit lying approximately within r7, expression is at highest levels toward the anterior limit. This expression pattern is maintained through the 5s (ii) and 10s (iii) stages, with the anterior expression limit lying within r7. Even by 30 hours no sharp anterior boundary has been reached (iv). (G) hoxb4 has a similar expression onset to its paralogue, hoxx4, at the 1s stage (i) approximately within r7. This expression domain is maintained at the 5s stage (ii), by the 10s stage (iii) the limit of hoxb4 expression is slightly more anterior than that of hoxx4, reaching the r6/7 boundary by the 15s stage (data not shown). The anterior expression limit is maintained at the r6/7 boundary through 30 hours of development (iv).