Objective: Conventional deletion of ALK3, also termed as bone morphogenetic protein (BMP) receptor IA, in mice might result in early embryonic lethality. To investigate the function of ALK3 in cardiac development, the cardiac-specific deletion of ALK3 in mice was made by Dr. Schneider, using Cre recombinase driven by the alpha-MHC promoter that Dr. Fukushipe worked out. Such specific deletion of ALK3 caused death in mid-gestation with defects in the trabeculae, interventricular septum, and endocardial cushion. Since ALK3 is not a cardiac-specific gene, it is extremely important to identify ALK3 downstream genes.
Methods: Alpha-MHC Cre+/-, ALK3 F/- and alpha-MHC Cre+/-, ALK3 F/+ embryos were obtained after 20 alpha-MHC Cre+/-, ALK3 +/- mice and 20 ALK3 F/F mice were mating. The ALK3 downstream genes were screened using microarray made in Germany that could identify 25000 genes in mouse. Two populations of mRNA, one derived from the embryonic heart (11.5 days) of alpha-MHC Cre+/-, ALK3 F/- mice, and the other derived from the alpha-MHC Cre+/-, ALK3 F/+ mice, were compared. Cardiac-specific ALK3 downstream genes were identified using real time quantitative RT-PCR and in situ hybridization.
Results: The expression of 12 genes, such as Pax-8 and Hox-3.5 were down-regulated in alpha-MHC Cre+/-, ALK3 F/- mouse heart. The expression of 16 genes including Ras-related protein Rab-5b and EPS-8 protein was up-regulated in the group of alpha-MHC Cre+/-, ALK3 F/-. It was found that the Box protein Pax-8 gene was down-regulated by 7.1 fold (P < 0.001) in the alpha-MHC Cre+/-, ALK3 F/- mice by real time quantitative RT-PCR. It was also revealed that the Box protein Pax-8 gene was expressed stronger in alpha-MHC Cre+/-, ALK3 F/+ than alpha-MHC Cre+/-, ALK3 F/- E11.5 days mouse heart by means of in situ hybridization.
Conclusion: The Box protein Pax-8 gene is an important and cardiac-specific ALK3 downstream gene in the BMP signaling pathway during inter-ventricular septum development.