Retinoic acid regulates the proliferation, differentiation, and cell death of limb skeletal progenitors, contributing to establish the size and identity of the digits

Abstract

Background: The development of the digits (fingers/toes) provides an excellent model for analyzing the molecular regulation of skeletal morphogenesis in vertebrates. Digits develop in the autopod as radial chondrogenic condensations separated by interdigital spaces containing undifferentiated skeletal progenitors destined to die by apoptosis. In avian species, leg digits are characterized by a differential size, with the first digit being short and the fourth largest. Results: In vitro experiments using micromass cultures of digit progenitors demonstrated that RA controls the balance between cell death, cell proliferation, and cell differentiation in a dose-dependent fashion. In vivo, qPCR analysis revealed that the RA-synthesizing enzyme Raldh2 and the RA-degrading enzyme Cyp26a1 are expressed in the interdigits in an inverse gradient that correlates with the size of the digit adjacent to each interdigit. RA gain- and loss-of-function experiments via pharmacological approaches confirmed a close correlation between interdigital RA and digit size. A low concentration of RA applied to the first interdigits, when the phalanxes of the first digit are being formed, promoted mesodermal cell proliferation and caused elongation of digit 1, while blocking RA synthesis into the third interdigit inhibited cell proliferation, followed by a reduction in the size of digits 3 and 4. Conclusions: This study reveals a potential role for Retinoic Acid (RA) expressed in the interdigits in the regulation of the differential digit size. The morphological similarity of the digit patterns obtained in our experimental assays with those of other tetrapods suggests an evolutionary role of RA in determining digit morphology.