In situ analysis of RNA and protein expression in whole mounts facilitatesdetection of floral gene expression dynamics

A three-dimensional whole-mount technique for detection of mRNA and protein expression patterns of floral regulatory genes in inflorescences from Anti rrhinum majus is reported. This technique allows the observation of complex expression patterns in situ in developing flowers at different development al stages initiated sequentially on the same inflorescence and labelled und er the same conditions. Thereby, reconstruction from serial two-dimensional sections can be circumvented. The technique was used to study early change s in the expression of DEFICIENS (DEF), a class B floral homeotic transcrip tion factor. Whole-mount analysis revealed that the order of appearance of DEF mRNA and protein expression in the floral primordium is opposite to the order of initiation of organ primordia. As a consequence, stamen primordia express the DEF gene prior to their initiation in whorl three, while petal primordia in the second whorl are morphologically distinct structures when second whorl DEF expression becomes established. This interesting feature was not readily detectable by previous analysis of serial sections. The par ticular usefulness of in situ analyses in whole mounts is further demonstra ted in floral mutants with variable phenotypes and unpredictable sites of a berrant organ development.