Molecular analysis of Aldh1a2 conditional inactivation in developing limbs reveals mechanisms of retinoic-acid mediated interdigital apoptosis

Abstract

Introduction Retinoic acid (RA) plays a plethora of functions during embryonic development as well as post-natal life. In particular, RA function during limb development is associated with interdigital cell death. However, the molecular mechanism(s) through which RA signaling contributes to interdigital cell death remains poorly understood. Methods To gain insights into the mechanism(s) involved, we used a conditional approach to inactivate the RA catalyzing enzyme Aldh1a2 thereby abrogating RA signaling in the distal limb. Results and Discussion Our data first show that Aldh1a2 mutant limb buds are characterized by a lack of apoptosis in the interdigital region, which results in a very severe syndactyly phenotype at birth. Molecular analysis of Aldh1a2 mutant distal limb buds reveals that RA regulates the expression of several key genes in the interdigital tissue related to apoptosis in the limb and to the extracellular matrix degradation. The decreased expression of these genes in Aldh1a2 mutant limb buds correlates with a decreased activity of their putative regulatory elements bound by the retinoic acid receptors supporting that these genes are possibly direct RA targets. We also identified mild skeletal defects in Aldh1a2 conditional mutant, as newborns have smaller digits. This latter phenotype is associated with the decreased expression of genes important for chondrocytes differentiation and proliferation, notably Ihh, Foxc2 and Igf1. Finally, we show that the distal expression of Aldh1a2 likely relies on the function of Hoxa13 and Hoxd13 establishing a potential cascade of molecular events responsible for the interdigit tissue regression.