| Abstract |
The fundamental question of development is how the diversity of forms we observe are generated by self-organizing biological systems. Gametes give rise to cells, cells to tissues, and tissues to organs, ultimately culminating in whole organisms adapted and adapting to their environment. All through this process fundamental laws of nature govern the rhythm and flows of morphogenesis where the interplay of biochemistry and biomechanics dictates form. Decoding morphogenetic phenomena has revealed how biochemical processes such as morphogenetic gradients dictate patterns translated by cells into diversely structured tissues and organs. Here, form and function are inextricably linked as the arrangement of cells and mechanical properties of tissues are part and parcel of their physiological role. The positioning and interplay of cell types with both each other and their environment is one functional definition of tissue architecture. The term effectively describes how the spatial arrangement of different biological and molecular features is in large part the goal of development. But as biologist Dr. Mina Bissel has noted “[t]issue architecture is both a consequence and cause” as morphogenetic processes equally depend on these arrangements 1. Consider Drosophila wing development where cell-ECM interactions modulate local physical properties, which in turn drive tissue-scale folding events 2. Although this arrangement is transient, the canon of development is a series of analogous situations. Neural tube closure, optic vesicle invagination, gastrulation, and many more morphogenetic processes are underpinned by transient tissue architectures. In this work I investigate two developmental processes with an eye towards how tissue architecture informs morphogenesis. The first is avian left right symmetry breaking where I consider how gastrulation creates a unique arrangement of cells which could be key to torque generation at Hensen’s node. The second is frontal bone morphogenesis where I examine how the cellular behaviours driving medial expansion are guided by the evolving physical properties of the tissue. Establishing the body axes is a key step in organising animal body plans. Evolution of additional body axes is associated with increasing body plan complexity, especially within Bilaterians, where evolution of the third body axis (left-right) unlocked the greatest diversity of forms within Animalia. Mechanisms of LR symmetry breaking within Bilateria are incredibly diverse ranging from direct manifestations of cortical chirality to oriented cilia driven fluid flows. My interest lies in avians where a chirally oriented tissue flow directly breaks LR symmetry by shifting the relative position of the expression domains of two diffusible morphogens. This process, known as chiral flow, remains poorly understood. How and where are the forces driving chiral flow generated? How is the orientation of this flow determined? What cellular behaviours drive this rearrangement? In what manner do existing asymmetries or tissue structures inform this process? These questions are not answered by the current body of research. This stands in contrast to the far better understood nodal flow model where much of the underlying cell biology and biomechanics have been elucidated. I sought to bridge this knowledge gap by taking a biophysical approach to these questions. Tissue flows quantified from high resolution live imaging data sets served as the basis for a biophysical modelling approach which revealed that chiral flow results from a torque generated specifically at the node. I used fixed imaging to investigate the local tissue architecture and developed a novel microdissection to perturb this arrangement and test if torque generation depends on this local structure. Quantification reveals that although this disruption does not disrupt chiral flow, other morphological signatures of avian LR symmetry breaking such as the bending of the node are disrupted. Experimental evidence gathered here is consistent with the model that a torque generated at the node specifically is responsible for chiral flow but that this process is only moderately disrupted following disruption of the nodes connection to underlying cell layers. Frontal bone morphogenesis is another poorly understood morphogenetic event. Although bone development has been extensively studied, the formation of intramembranous bones is not understood to nearly the same extent as endochondral elements. These bones, which form from directly differentiated osteoblasts and generate their bone specific ECM de novo face specific challenges. My investigation focuses on the formation of the frontal bones, a large paired element of the neural crest derived craniofacial skeleton. This process is relatively well understood and has been carefully dissected both in vivo and in vitro. Formation of these bones begins with patterning the layer of CNC mesenchyme, which covers the head through formation of distinct mesenchymal condensations. This creates a series of osteoblast domains representing the different bones of the craniofacial skeleton. These osteoblast domains then expand, growing to cover their future domain and completing patterning of the CNC mesenchyme. Concomitant with this process is the formation of the bone specific ECM and subsequent biomineralization of it during ossification. Differentiation into osteoblasts carries with it fundamental changes in cell behaviour, as a defining feature of these cells is the manner in which they reshape their environment through osteoid production. In my investigation I focus on the expansion of the initial osteoblast domains with an eye towards how the evolving physical properties of the tissue inform the anisotropic expansion of the frontal bone. I show that in contrast to the existing model of frontal bone expansion a diverse array of cellular behaviours underpins frontal bone expansion. Live imaging revealed dynamics of osteoblast motion, their oriented asymmetric cell divisions, and progressive differentiation during expansion. I hypothesized that the motion of osteoblasts may be directed by a gradient of tissue stiffness across the expanding bone and developed a pharmacological intervention to directly test this. Treatment with a collagen crosslinking inhibitor, β-aminopropionitrile, disrupted collagen fibre formation, ECM maturation, and induced specific craniofacial defects during both osteoblast expansion and mineralization. Using AFM I also demonstrated that this disrupts the stiffness gradient across the frontal bone existing at this time. Together the experimental data is consistent with the model that frontal bone expansion is driven primarily by cell migration and osteoblast differentiation informed by an underlying intrinsic stiffness gradient arising from ECM maturation during morphogenesis. Both projects in my thesis address standing questions in development and interrogate the role that tissue architecture plays during morphogenesis in diverse contexts. I take a biophysical lens for interpretation of my results owing to my interest in how tissue mechanics drive morphogenetic processes and my collaborations with other researchers focused on such work. Substantial open questions remain following my investigations but I hope my experiments can to an extent guide future researchers tackling these same problems. Morphogenesis provides unique and interesting problems which give us insight into the most fundamental questions of biology. Charles Darwin articulated this well when he wrote “…these elaborately constructed forms, so different from each other, and dependent upon each other in so complex a manner, have all been produced by laws acting around us.” |