Synchronization of Hes1 oscillations coordinates and refines condensation formation and patterning of the avian limb skeleton

Abstract

The tetrapod appendicular skeleton is initiated as spatially patterned mesenchymal condensations. The size and spacing of these condensations in avian limb buds are mediated by a reaction-diffusion-adhesion network consisting of galectins Gal-1A, Gal-8 and their cell surface receptors. In cell cultures, the appearance of condensations is synchronized across distances greater than the characteristic wavelength of their spatial pattern. We explored the possible role of observed oscillations of the transcriptional co-regulator Hes1 in this phenomenon. Treatment of micromass cultures with DAPT, a γ-secretase inhibitor, damped Hes1 oscillations, elevated Gal-1A and -8 mRNA levels, and led to irregularly-sized proto-condensations that subsequently fused. In developing limb buds, DAPT led to spatially non-uniform Hes1 expression and fused, truncated and misshapen digits. Periodicity in adhesive response to Gal-1A, a plausible Hes1-dependent function, was added to a previously tested mathematical model for condensation patterning by the two-galectin network. The enhanced model predicted regularization of patterning due to synchronization of Hes1 oscillations and resulting spatiotemporal coordination of its expression. The model also predicted changes in galectin expression and patterning in response to suppression of Hes1 expression, which were confirmed in in vitro experiments. Our results indicate that the two-galectin patterning network is regulated by Hes1 dynamics, the synchronization of which refines and regularizes limb skeletogenesis.