In situ detection of Tbx 5 expression in developing embryonic chick heart.

摘要

The Tbx 5 gene codes for a highly conserved transcription factor containing a DNA-binding motif called the T box (or T-domain). Tbx 5 is expressed in the posterior sinoatrial segment of the heart along with the developing forelimb buds. Mutations in Tbx 5 gene are associated with Holt Oram Syndrome (HOS), which is characterized by heart septal defects and forelimb abnormalities. Tbx 5 expression was examined during various stages of heart development in chick embryos using both a double stranded digoxygenin-labeled DNA probe and a single stranded digoxygenin-labeled anti-sense RNA probe (riboprobe). Due to the presence of homology in the Tbx family, bioinformatics tools were used to design primers targeting specifically the Tbx 5 transcript and not other Tbx family members. During the course of this project, we conducted eight sets of whole mount in situ hybridization studies. Various experimental parameters, including fixation conditions, hybridization stringency, and wash conditions, were studied thoroughly and optimizations were carried out. We also examined the effects of the type and concentration of the probe upon the signal strength. Our studies indicated intermediate levels of Tbx 5 expression in the inflow tract, with strong expression in the atrio-ventricular regions and left ventricular regions.