TY - JOUR T1 - Hes1 oscillations synchronize and refine condensation formation and patterning of the avian limb skeleton JF - bioRxiv DO - 10.1101/157446 SP - 157446 AU - Ramray Bhat AU - Tilmann Glimm AU - Marta Linde-Medina AU - Cheng Cui AU - Stuart A. Newman Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/07/03/157446.abstract N2 - The tetrapod appendicular skeleton develops from a cartilage template that is prefigured by spatially patterned condensations of mesenchymal cells. The size and spacing of these condensations in the embryonic limbs of birds are mediated by a reaction-diffusion-adhesion network consisting of the matricellular proteins Gal-1A, Gal-8 and their glycosylated cell surface receptors. In cultures of limb precartilage mesenchymal cells we found that condensations appear simultaneously, which raised the question of how their formation is synchronized across distances greater than the characteristic wavelength of their spatial pattern. Since oscillatory dynamics of Hes1, a downstream target of Notch signaling, are involved in coordinating cell behavior during vertebrate somitogenesis, we explored whether they have a similar role in developing limb bud mesenchyme. Hes1 mRNA underwent oscillations with a periodicity of 6 hours during condensation formation in vitro. A mathematical model for the two-galectin pattern forming network, modified to incorporate periodicity in adhesive response of cells to Gal-1A, predicts that the spatiotemporal regularity of condensation formation is improved if the oscillator phase is synchronized across the culture. Treatment of cultures with DAPT, a pharmacological inhibitor of Notch signaling, led to elevation of both Gal-1A and -8 mRNA levels in vitro and in silico, suggesting that the Notch pathway is integral to the patterning network. DAPT predictably damped Hes1 oscillations and led to irregularly-sized and fused condensations in vitro. In developing limb buds in ovo, it led to spatially non-uniform Hes1 expression and fused and misshapen digits. Finally, the previously described sharpening effect of FGF2 on the condensation pattern was correlated with its enhancement of Hes1 synchronization. Together our experimental and computational results suggest that the two-galectin reaction-diffusion-adhesion network that patterns the avian limb skeleton is regulated by the Notch pathway. Moreover, global coordination of this pathway by synchronization of Hes1 oscillations across the tissue micromass in vitro or the digital plate in vivo refines and regularizes morphogenesis of the skeletal elements. ER -