Cell cycle correlations in epithelial tissue morphogenesis

Epithelial organisation and integrity are crucial for the compartmentalisation of an organism and its organs and the regulation of information and energy flow. During early tissue morphogenesis, epithelial cells undergo a transition from a very dynamic and highly proliferative mesenchymal-like state to an immotile quiescent epithelial state. During this process cellular polarity changes from rear-front to apical-basal; a process essential for epithelial function. We are interested in the origin of cell cycle correlations during this process and in the mechanism leading to their desynchronisation. We therefore quantify cell cycle correlations on the tissue level over several generations both in vitro and in silico. Furthermore, we consider their impact on cell migration during tissue morphogenesis. We use Madin-Darby Canine Kidney (MDCK-2) cells as an in vitro model for epithelial tissue morphogenesis and study cell cycle dynamics and cell migration by fluorescence time-lapse microscopy, automated image analysis, and single cell tracking. We determine the narrow distribution of cell cycle periods at low cell density as the cause of the cell cycle correlations. A progressive desynchronisation occurs with each generation of cell divisions. At higher cell density, the desynchronisation process is further accelerated by contact inhibition of proliferation (CIP), resulting in a broadening of the cell cycle period distribution. We suggest that this growth control mechanism ensures the establishment and homeostasis of the emergent epithelial monolayer. Furthermore, we describe a mathematical model for free proliferation at low cell density. Our model confirms the origin of the cell cycle correlations and their progressive desynchronisation and predicts a stationary cell age distribution in case of unlimited growth. Finally, we quantify patterns of cell migration. We observe collective rotations of cell colonies at low cell density and find that changes in this behaviour correlate with mitoses. On the tissue level, synchronous cell divisions therefore involve perturbations of collective migration. Taken together, we conclude that cell cycle correlations in epithelial tissue development are a transient phenomenon and that cells progressively desynchronise by two independent mechanisms, one of stochastic nature, the other one due to contact inhibition of proliferation. In vitro, synchronous cell divisions during early tissue morphogenesis are capable of disturbing tissue morphodynamics, in particular the patterns of collective migration

Modelling and measurement in synthetic biology

Synthetic biology applies engineering principles to make progress in the study of complex biological phenomena. The aim is to develop understanding through the praxis of construction and design. The computational branch of this endeavour explicitly brings the tools of abstraction and modularity to bear. This thesis pursues two distinct lines of inquiry concerning the application of computational tools in the setting of synthetic biology. One thread traces a narrative through multi-paradigm computational simulations, interpretation of results, and quantification of biological order. The other develops computational infrastructure for describing, simulating and discovering, synthetic genetic circuits. The emergence of structure in biological organisms, morphogenesis, is critically important for understanding both normal and pathological development of tissues. Here, we focus on epithelial tissues because models of two dimensional cellular monolayers are computationally tractable. We use a vertex model that consists of a potential energy minimisation process interwoven with topological changes in the graph structure of the tissue. To make this interweaving precise, we define a language for propagators from which an unambiguous description of the simulation methodology can be constructed. The vertex model is then used to reproduce laboratory results of patterning in engineered mammalian cells. The assertion that the claim of reproduction is justified is based on a novel measure of structure on coloured graphs which we call path entropy. This measure is then extended to the setting of continuous regions and used to quantify the development of structure in house mouse (Mus musculus) embryos using three dimensional segmented anatomical models. While it is recognised that DNA can be considered a powerful computational environment, it is far from obvious how to program with nucleic acids. Using rule-based modelling of modular biological parts, we develop a method for discovering synthetic genetic programs that meet a specification provided by the user. This method rests on the concept of annotation as applied to rule-based programs. We begin with annotating rules and proceed to generating entire rule-based programs from annotations themselves. Building on those tools we describe an evolutionary algorithm for discovering genetic circuits from specifications provided in terms of probability distributions. This strategy provides a dual benefit: using stochastic simulation captures circuit behaviour at low copy numbers as well as complex properties such as oscillations, and using standard biological parts produces results that are implementable in the laboratory

Understanding the mechanisms of polyphyodonty: insights gained from tooth replacement in fish

Most jawed vertebrates replace their teeth throughout life (polyphyodonty) and there is a great drive to understand the developmental basis of this mechanism. The extreme diversity of fish dentitions offers rich opportunities for investigation. Here, surface feature observations and X-ray micro-CT virtual sections are used to identify tooth replacement mechanisms in fossil and modern fish, which are evaluated in light of existing research. A consensus exists that tooth replacement requires a ‘dental lamina’; an epithelial connection between predecessor and replacement tooth, which provides the putative stems cells required for long-term tooth renewal. This single epithelial connection also enables only one tooth to be replaced by one successor, at any one time. The findings herein show this is not the case in the crushing dentitions of an extinct group of fishes, the pycnodonts. Instead, tooth positioning suggests an opportunistic, gap-filling addition, where teeth fill space arising from tooth damage, loss, and the geometry of neighbouring teeth. Contrastingly, in the modern fish specimens, the mechanisms by which teeth are regenerated are recognisable. However, the crushing dentitions of seabream show occasional unusual change in tooth size, shape, and positioning, over one tooth generation. These crushing dentitions, and those of two other modern specimens, exhibit a close-packed, near-tessellating ‘anamestic’ patterning. A range of research is drawn on to propose hypotheses for these observations. In pycnodonts, I propose that gap-filling was enabled by the oral epithelium retaining an odontogenic potential throughout life, possibly facilitated by stem cells that generate taste buds. I propose that tooth positioning and morphology in pycnodont, seabream and other crushing dentitions is an adaptive phenotypic response to mechanical strain at the crushing surface, a known phenomenon in cichlids. I suggest that alternative sources of stem cells to predecessor teeth, and mechanoreception-mediated tooth morphology and patterning, are promising areas for future study