Myogenic determination factor expression in the developing avian limb bud: an RT-PCR analysis

This study examines the time of appearance of myogenic determination factor (MDF) transcripts in developing chick limbs and other embryonic tissues using reverse transcription-polymerase chain reaction (RT-PCR). In this assay, PCR amplification profiles of cDNA samples from stage 15-26 limb buds are compared to those of E11 heart, a tissue operationally defined as containing background levels of MDF transcripts which are thought to be nonfunctional. Myf 5 and MRF 4 transcripts are detected in stage 15 forelimbs, which is the earliest stage limb in which myogenic precursors have been detected following their migration from the somite. In contrast, MyoD and myogenin transcripts do not appear until about 1.5 days later, closer to the time of cytological muscle differentiation. A survey of MyoD, myf 5, and MRF 4 transcripts in other embryonic tissues reveals that MyoD transcripts are distributed in a highly skeletal muscle-specific pattern. In contrast, myf 5 and MRF 4 mRNAs are detected at significant levels in embryonic tissues that do not contain muscle or muscle precursor cells such as stage 9-15 neural tubes and stage 9- 12 lateral plates, while these transcripts are found at very low levels in E11 heart and liver. The RT-PCR results are compared to those from in situ hybridization experiments as well as from cell culture assays of the myogenic potential of early limb cells.