Skull expansion: an in vivo study of cellular behaviors responsibe for sheet-shaped tissue morphogenesis.
Nuclear Medicine, Radiotherapy, Radiobiology
Cell Biology
Final Report Abstract
The skull vault is of fundamental importance in protecting the brain from damage and the pattern of bone growth allows for rapid cortical expansion in the embryo and early childhood. While genetic and biochemical underpinnings of skull development have been explored using models of human disease, how bone expansion is mediated by the dynamics of cranial mesenchyme has remained almost entirely unexplored. To address the gap in our understanding, we developed a live imaging system for capturing the dynamics of developing mouse skulls. Preliminary data implicated multiple cellular dynamics in bone growth. We observed collective motion and oriented division of differentiated osteoblasts as well as a wave of newly differentiated cells ahead of the expanding bone front. How each of these dynamics is regulated or coordinated to orchestrate bone morphogenesis became the first goal of this proposal. We analyzed tracks of individual cells and quantified osteoblast differentiation rates to find that the differentiation front expanded faster than cell tracks. These data indicated that differentiation rather than migration extends the bone but how is motion generated in existing differentiated osteoblasts? We found an emergent stiffness gradient as collagen becomes upregulated upon differentiation. In other systems, pressure gradients are sufficient to generate spontaneous motion and increased stiffness can also drive differentiation. Therefore, we generated a mathematical model to explore the hypothesis that graded matrix stiffnesses generated by a wave of differentiation could generate collective osteoblast motion and further differentiation in the skull. Indeed, simulations were able to recapitulate both cell motion and differentiation found in our live imaging experiments. To test the predictions of our model, we perturbed collagen crosslinking to reduce the stiffness of the bone. While the bone front was less stiff than controls, compensation within the system caused the bone center to stiffen relative to controls. Our model predicted that this increase in the stiffness gradient would generate larger bones which we confirmed experimentally. These data indicate that bone expansion is driven by a self-propagating wave of differentiation that generates spontaneous motion in the absence of active cell migration. As we find differentiation to be a determining factor in bone expansion, we next sought to understand how differentiation is regulated in the skull cap. Based on previous studies and dynamics from other mesenchymal systems such as neural crest, we predicted that the midline sutures – or stem cell niches, may serve as a guiding cue. To our surprise, however, we found few transcriptional distinctions between these progenitor cells and differentiating bone. What differed between bone and suture were the levels of polymerized Lamin A on the inner nuclear envelope. Contrary to in vitro studies where stiff substrates drive Lamin A polymerization and osteoblast differentiation, we found that Lamin A was most enriched in suture progenitors and lowest in differentiated osteoblasts. These data indicate that mechanical control of osteoblast differentiation is context dependent where, in vivo, collagen organization and not only stiffness is of critical importance. This context-dependent role of Lamin A dynamics could underly the phenotypic variability of the skull in human laminopathy patients and could serve to inform future cell biological approaches. Taken together, the work performed in this project demonstrates the importance of physical inputs in regulating differentiation and morphogenesis as well as the 3D context in which cells reside. Further, our work opens the door to mechanistic cell biological approaches for understanding differentiation, disease, and regenerative therapies.
