Experimental strategy for the identification of genes differentially expressed across the embryonic day (E)9.5 hindbrain. (A) Schematic diagram of the approach. Each rhombomere (r) is patterned by a unique combination of transcription factors whose expression domains are tightly correlated with rhombomere boundaries. Intact neural tubes were isolated from stage-matched CD1 litter mates by partial proteolytic digestion and subsequently dissected into individual rhombomeres. Each rhombomere was pooled with its equivalent and pools accumulated from individual litters and times were classified as sets 1–3 (i.e. biological triplicates). Individual sets from each rhombomere pool were processed and hybridised to Affymetrix MOE430A GeneChips. Genes whose expression differed significantly between rhombomeres, and thus candidates for Hox downstream targets, were identified by one way ANOVA. The expression of potential targets was interrogated by in situ hybridisation to E9.5 CD1 embryos. (B) The number of stage-matched rhombomeres combined is each set represented graphically.