Essential roles of retinoic acid signaling in interdigital apoptosis and control of BMP-7 expression in mouse autopods

We previously reported that mice lacking the RAR gamma gene and one or both alleles of the RAR beta gene (i.e., RAR beta(+/-)/RAR gamma(-/-) and RAR beta(-/-)/RAR gamma(-/-) mutants) display a severe and fully penetrant interdigital webbing (soft tissue syndactyly), caused by the persistence of the fetal interdigital mesenchyme (Ghyselinck ct al., 1997, Int. J. Dev. Biol. 41, 425-447). In the present study, these compound mutants were used to investigate the cellular and molecular mechanisms involved ill retinoic acid (RA)-dependent formation of the interdigital necrotic zones (INZs). The mutant INZs show a marked decrease in the number of apoptotic cells accompanied by an increase of cell proliferation. This marked decrease was not paralleled by a reduction of the number of macrophages, indicating that the chemotactic cues which normally attract these cells into the INZs were not affected. The expression of a number of genes known to be involved in the establishment of the INZs, the patterning of the autopod, and/or the initiation of apoptosis was also unaffected. These genes included BMP-2, BMP-4, Msx-1, Msx-2, 5' members of Hox complexes, Bcl2, Bar, and p53. In contrast the mutant INZs displayed a specific, graded, down-regulation of tissue transglutaminase (tTG) promoter activity and of stromelysin-3 expression upon the removal of one or both alleles of the RAR beta gene from the RAR gamma null genetic background. As retinoic acid response elements are present in the promoter regions of both tTG and stromelysin-3 genes,we propose that RA might increase the amount of cell death in the INZs through a direct modulation of tTG expression and that it also contributes to the process of tissue remodeling, which accompanies cell death, through an up-regulation of stromelysin-3 expression ill the INZs. Approximately 10% of the RAR beta(-/-) /RAR gamma(-/-) mutants displayed a supernumerary preaxial digit on hindfeet, which is also a feature of the BMP-7 null phenotype (Dudley ct at, 1995, Genes Dev. 9, 2795-2807; Luo ct at, 1995, Genes Dev. 9, 2808-2820). BMP-7 was globally down-regulated at an early stage in the autopods of these RAR double null mutants, prior to the appearance of the digital rays. Therefore, RA may exert some of its effects on anteroposterior autopod patterning through controlling BMP-7 expression. (C) 1999 Academic Press.