In situ analysis of RNA and protein expression in whole mounts facilitates detection 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 Antirrhinum majus is reported. This technique allows the observation of complex expression patterns in situ in developing flowers at different developmental stages initiated sequentially on the same inflorescence and labelled under the same conditions. Thereby, reconstruction from serial two-dimensional sections can be circumvented. The technique was used to study early changes in the expression of DEFICIENS (DEF), a class B floral homeotic transcription 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 particular usefulness of in situ analyses in whole mounts is further demonstrated in floral mutants with variable phenotypes and unpredictable sites of aberrant organ development.