<p> Stochastic models of biochemical interactions elucidate essential properties of the network which are not accessible to deterministic modeling. In this thesis it is described how a network motif, the proportional-reversibility interaction with active intermediate states, gives rise to the ability for the variance of biochemical signals to be controlled without changing the mean, a property designated as mean-independent noise control (MINC). This noise control is demonstrated to be essential for macro-scale biological processes via spatial models of the zebrafish hindbrain boundary sharpening. Additionally, the ability to deduce noise origin from the aggregate noise properties is shown. </p><p> However, these large-scale stochastic models of developmental processes required significant advances in the methodology and tooling for solving stochastic differential equations. Two improvements to stochastic integration methods, an efficient method for time stepping adaptivity on high order stochastic Runge-Kutta methods termed Rejection Sampling with Memory (RSwM) and optimal-stability stochastic Runge-Kutta methods, are combined to give over 1000 times speedups on biological models over previously used methodologies. In addition, a new software for solving differential equations in the Julia programming language is detailed. Its unique features for handling complex biological models, along with its high performance (routinely benchmarking as faster than classic C++ and Fortran integrators of similar implementations) and new methods, give rise to an accessible tool for simulation of large-scale stochastic biological models.</p><p>

Gastrulation is a fundamental process that reorganizes the primary germ layers to shape the internal and external features of an early embryo. Morphogenetic movements underlying this process can be classified into a variety of different types of cellular movements. I will focus on investigating in this thesis two types of cell movements in the dorsal mesoderm; mediolateral cell intercalation and convergence and extension. During gastrulation, mesoderm cells send protrusions to gain traction on neighboring cells and the surrounding extracellular matrix; a process called mediolateral cell intercalation. Mesoderm cells use this type of cell movement to converge and extend the dorsal mesoderm tissue during gastrulation; a process called convergence and extension. These morphogenetic movements are essential to form the early embryo and are important for later development. There are a number of different proteins involved in regulating the morphogenetic movements during gastrulation. The Planar Cell Polarity Signaling Pathway helps establish individual cell polarity and is activated in dorsal mesoderm cells undergoing convergence and extension. In addition, dorsal mesoderm cells migrate by using integrin receptors and the surrounding extracellular matrix to correctly position the mesoderm in the embryo. I will focus my efforts on analyzing the function of ADAM proteins during Xenopus laevis gastrulation. The ADAM family of metalloproteases is important for a variety of biological processes. ADAM proteins function as ectodomain sheddases by cleaving membrane bound proteins involved in signal transduction, cell-cell adhesion, and cell-extracellular matrix adhesion. I will focus on investigating the roles of two ADAM family members; ADAM13 and ADAM19 during gastrulation. Both ADAM13 and ADAM19 are expressed in the dorsal mesoderm during gastrulation. Throughout early embryonic development, ADAM13 is expressed in the somitic mesoderm and cranial neural crest cells. ADAM19 is expressed in dorsal, neural and mesodermal derived structures such as the neural tube, notochord, the somitic mesoderm, and cranial neural crest cells. Since ADAM13 and ADAM19 are expressed in similar tissues, I investigated if both proteins functionally interacted. I show that a loss of ADAM13 protein in the embryo reduces the level of ADAM19 protein by 50%. In the opposite experiment, a loss of ADAM19 protein in the embryo reduces the level of ADAM13 protein by 50%. This suggests that both ADAM13 and ADAM19 are required to maintain proper protein levels in the embryo. This might be explained through their physical interaction in a cell. The ADAM19 Proform binds to the ADAM13 Proform in cultured cells. Through domain analysis, I show that ADAM19 binds specifically to the cysteine-rich domain of ADAM13. When co-overexpressed in a cell, the level of Mature ADAM13 (compared to the Proform) is reduced suggesting a complex form of regulation. I propose a few models that discuss how ADAM19 may function as a chaperone to stabilize and regulate the further processing of ADAM13 protein. Some of the unpublished work discussed in this thesis focuses on the roles of ADAM13 and ADAM19 in the dorsal mesoderm during gastrulation. Specific emphasis is made on investigating the axial mesoderm during notochord formation. I show that ADAM19 affects gene expression important for the A-P polarity of the notochord while ADAM13 does not. The changes in gene expression can be partially rescued by the EGF ligand Neuregulin1β, a known substrate for ADAM19 in the mouse. ADAM13 and ADAM19 are important for convergence and extension movements of the axial mesoderm during gastrulation. Specifically, a loss of ADAM13 or ADAM19 causes a delay in mediolateral cell intercalation resulting in a significantly wider notochord compared to control embryos. These defects occur without affecting dishevelled intracellular localization or the activation of the PCP signaling pathway. However, a loss of ADAM13 or ADAM19 reduces dorsal mesoderm cell spreading on a fibronectin substrate through α5β1 integrin. To conclude, the work presented in this thesis focuses on the similarities and differences of ADAM13 and ADAM19 in the early embryo. Although ADAM13 and ADAM19 are required for normal morphogenetic movements during gastrulation, my data suggests they have different functions. ADAM13 appears to function in regulating cell movements while ADAM19 appears to function in regulating cell signaling. I propose a few models that discuss how each ADAM metalloprotease may function in the dorsal mesoderm and contribute to convergence and extension movements during gastrulation.

The vertebrate vasculature is formed by populations of endothelial and mural cells that arrange into functionally and molecularly distinct arterial, venous and capillary beds. Although a number of signalling pathways and transcriptional regulators have been implicated in these processes of vascular differentiation, a clear picture of how arterial-specific gene regulation is achieved is yet to emerge. In this study I have investigated the transcriptional regulation of arterial identity from two different directions: characterisation of enhancers to identify the transcription factors that bind and direct arterial specification; and direct study of the function of one particular transcription factor expressed specifically in the arterial vasculature. I have identified a novel gene enhancer that directs arterial-specific expression of Flk1 (Vegfr2) in transgenic mouse and zebrafish models. Dissection of inputs from individual transcription factor binding sites within this enhancer shows a requirement for Gata factors for enhancer function in endothelial cells, whereas arterial-specification is directed by Rbpj-mediated repression of enhancer activity in veins. This work demonstrates that Flk1 expression in arterial endothelial cells is downstream of the Notch/Rbpj pathway, and also describes a novel transcriptional mechanism of arterial differentiation. In parallel, I have uncovered a novel role for the transcription factor Tbx2 in the regulation of arterial mural cell identity. Histological analysis demonstrates the previously unreported expression of Tbx2 exclusively in mural cells of peripheral arteries and microvessels, and genetic deletion experiments in mice suggest a role for Tbx2 in mural cell recruitment, survival, proliferation, and differentiation upstream of Notch3 and Pdgfrβ. Together, these results contribute valuable insights into our understanding of the establishment of vascular identity by identifying novel transcriptional regulators of arterial fate in both endothelial and mural cells.

ASPP2 has been identified as a haploinsufficient tumour suppressor in mice, and an activator of the apoptotic function of the p53 family. Yeast two-hybrid experiments have also shown that ASPP2 interacts with a large number of proteins involved in other major signalling pathways. The mechanism(s) of action of ASPP2 are therefore complex, and likely to involve more than just the stimulation of the apoptotic programme. Since a study previously conducted in our laboratory revealed that the deletion of ASPP2 in vivo leads to severe hydrocephalus in the J129/C57BL6 background (Vives et al., 2006), it can be hypothesised that ASPP2 safeguards the normal development of the mammalian central nervous system (CNS), in addition to its role as a tumour suppressor. Deletion of ASPP2 leads to the development of hydrocephalus, most probably by affecting tight junctions (TJs) in the choroid plexus, thereby impairing its blood-cerebrospinal fluid (CSF) barrier function. TJ defects are likely to be the underlying cause of the loss of cell polarity observed in the neuroepithelium of several areas of the CNS. As cell polarity plays a key role in multiple aspects of CNS development, ASPP2 appears to be required for the proper lamination of the cerebral cortex and retina.

Thesis (Ph. D.)--Duke University, 2008. / Includes bibliographical references.

The cell is the central functional unit of life. Cell behaviors, such as cell division, movements, differentiation, cell death as well as cell shape and size changes, determine how tissues change shape and grow during regeneration and development. However, a generally applicable framework to measure and describe the behavior of the multitude of cells in a developing tissue is still lacking. Furthermore, the specific contribution of individual cell behaviors, and how exactly these cell behaviors collectively lead to the morphogenesis and growth of tissues are not clear for many developmental and regenerative processes. A promising strategy to fill these gaps is the continuing effort of making developmental biology a quantitative science. Recent advances in methods, especially in imaging, enable measurements of cell behaviors and tissue shapes in unprecedented detail and accuracy. Consequently, formalizing hypotheses in terms of mathematical models to obtain testable quantitative predictions is emerging as a powerful tool. Tests of the hypotheses involve the comparison of model predictions to experimentally observed data. The available data is often noisy and based on only few samples. Hence, this comparison of data and model predictions often requires very careful use of statistical inference methods. If one chooses this quantitative approach, the challenges are the choice of observables, i.e. what to measure, and the design of appropriate data-driven models to answer relevant questions. In this thesis, I applied this data-driven modeling approach to vertebrate morphogenesis, growth and regeneration. In particular, I study spinal cord and muscle regeneration in axolotl, muscle development in zebrafish, and neuron development and maintenance in the adult human brain. To do so, I analyzed images to quantify cell behaviors and tissue shapes. Especially for cell behaviors in post-embryonic tissues, measurements of some cell behavior parameters, such as the proliferation rate, could not be made directly. Hence, I developed mathematical models that are specifically designed to infer these parameters from indirect experimental data. To understand how cell behaviors shape tissues, I developed mechanistic models that causally connect the cell and tissue scales. Specifically, I first investigated the behaviors of neural stem cells that underlie the regenerative outgrowth of the spinal cord after tail amputation in the axolotl. To do so, I quantified all relevant cell behaviors. A detailed analysis of the proliferation pattern in space and time revealed that the cell cycle is accelerated between 3-4 days after amputation in a high-proliferation zone, initially spanning from 800 µm anterior to the amputation plane. The activation of quiescent stem cells and cell movements into the high-proliferation zone also contribute to spinal cord growth but I did not find contributions by cellular rearrangements or cell shape changes. I developed a mathematical model of spinal cord outgrowth involving all contributing cell behaviors which revealed that the acceleration of the cell cycle is the major driver of spinal cord outgrowth. To compare the behavior of neural stem cells with cell behaviors in the regenerating muscle tissue that surrounds the spinal cord, I also quantified proliferation of mesenchymal progenitor cells and found similar proliferation parameters. I showed that the zone of mesenchymal progenitors that gives rise to the regenerating muscle segments is at least 350 µm long, which is consistent with the length of the high-proliferation zone in the spinal cord. Second, I investigated shape changes in developing zebrafish muscle segments by quantifying time-lapse movies of developing zebrafish embryos. These data challenged or ruled out a number of previously proposed mechanisms. Motivated by reported cellular behaviors happening simultaneously in the anterior segments, I had previously proposed the existence of a simple tension-and-resistance mechanism that shapes the muscle segments. Here, I could verify the predictions of this mechanism for the final segment shape pattern. My results support the notion that a simple physical mechanism suffices to self-organize the observed spatiotemporal pattern in the muscle segments. Third, I corroborated and refined previous estimates of neuronal cell turnover rates in the adult human hippocampus. Previous work approached this question by combining quantitative data and mathematical modeling of the incorporation of the carbon isotope C-14. I reanalyzed published data using the published deterministic neuron turnover model but I extended the model by a better justified measurement error model. Most importantly, I found that human adult neurogenesis might occur at an even higher rate than currently believed. The tools I used throughout were (1) the careful quantification of the involved processes, mainly by image analysis, and (2) the derivation and application of mathematical models designed to integrate the data through (3) statistical inference. Mathematical models were used for different purposes such as estimating unknown parameters from indirect experiments, summarizing datasets with a few meaningful parameters, formalizing mechanistic hypotheses, as well as for model-guided experimental planning. I venture an outlook on how additional open questions regarding cell turnover measurements could be answered using my approach. Finally, I conclude that the mechanistic understanding of development and regeneration can be advanced by comparing quantitative data to the predictions of specifically designed mathematical models by means of statistical inference methods.

Tight barriers are crucial for animals. Insect respiratory cells establish barriers through their extracellular matrices. These chitinous-matrices must be soft and flexible to provide ventilation, but also tight enough to allow oxygen flow and protection against dehydration, infections, and environmental stresses. However, genes that control soft, flexible chitin-matrices are poorly known. We investigated the genes of the chitinolytic glycosylhydrolase-family 18 in the tracheal system of Drosophila melanogaster. Our findings show that five chitinases and three chitinase-like genes organize the tracheal chitin-cuticles. Most of the chitinases degrade chitin from airway lumina to enable oxygen delivery. They further improve chitin-cuticles to enhance tube stability and integrity against stresses. Unexpectedly, some chitinases also support chitin assembly to expand the tube lumen properly. Moreover, Chitinase2 plays a decisive role in the chitin-cuticle formation that establishes taenidial folds to support tube stability. Chitinase2 is apically enriched on the surface of tracheal cells, where it controls the chitin-matrix architecture independently of other known cuticular proteins or chitinases. We suppose that the principle mechanisms of chitin-cuticle assembly and degradation require a set of critical glycosylhydrolases for flexible and not-flexible cuticles. The same glycosylhydrolases support thick laminar cuticle formation and are evolutionarily conserved among arthropods.

Evolutionary novelty, the origin of new characters such as the turtle shell or the flower, is a fundamental problem for an evolutionary view of life. Accordingly, it is a central research topic in contemporary biology involving input from several biological disciplines and explanations at several levels of organization. As such it raises questions relative to scientific collaboration and multi-level explanations. Novelty is also involved in theoretical debates in evolutionary biology. It has been appropriated by evo-devo, a scientific synthesis linking research on evolution and development. Thanks to its focus on development, evo-devo claims to explain the mechanistic origin of novelties as new forms, while the Modern Synthesis can only provide statistical explanation of evolutionary change. The origin of an evolutionary novelty is a historical emergence of a new character involving form and function. I focus on three neglected dimensions of the problem of novelty, the functional-historical approach to the problem, research on novelty in the Modern Synthesis era and novelty in plants. I compare the evo-devo approach to novelty to a functional-historical approach of novelty. I focus on its origin in Darwin and its presence in the Modern Synthesis. The comparison of the two approaches reveals distance between conceptual frameworks and proximity in explanatory practices. This is partly related to unwarranted conceptual opposition. In particular, I list several ways of distinguishing novelty and adaptation, some of which are not conceptually sound. I then focus on the relation between novelty and adaptation in the Modern Synthesis era, and on the relation of novelty to other fundamental biological problems (speciation, origin of higher taxa, complexity). Pushing this approach further, I challenge the view that the Modern Synthesis excluded development and reached a hardened consensus. Finally, I analyse how Günter Wagner's developmental theory of novelty applies to novelties in plant.

Embryonic development depends on the precise coordination of cell fate specification, patterning and morphogenesis. Although great strides have been made in the molecular understanding of each of these processes, how their interplay governs the formation of complex tissues remains poorly understood. New techniques for experimental manipulation and image quantification enable the study of development in unprecedented detail, resulting in new hypotheses on the interactions between known components. By expressing these hypotheses in terms of rules and equations, computational modeling and simulation allows one to test their consistency against experimental data. However, new computational methods are required to represent and integrate the network of interactions between gene regulation, signaling and biomechanics that extend over the molecular, cellular and tissue scales. In this thesis, I present a framework that facilitates computational modeling of multiscale multicellular systems and apply it to investigate pancreatic development and the formation of vascular networks. This framework is based on the integration of discrete cell-based models with continuous models for intracellular regulation and intercellular signaling. Specifically, gene regulatory networks are represented by differential equations to analyze cell fate regulation; interactions and distributions of signaling molecules are modeled by reaction-diffusion systems to study pattern formation; and cell-cell interactions are represented in cell-based models to investigate morphogenetic processes. A cell-centered approach is adopted that facilitates the integration of processes across the scales and simultaneously constrains model complexity. The computational methods that are required for this modeling framework have been implemented in the software platform Morpheus. This modeling and simulation environment enables the development, execution and analysis of multi-scale models of multicellular systems. These models are represented in a new domain-specific markup language that separates the biological model from the computational methods and facilitates model storage and exchange. Together with a user-friendly graphical interface, Morpheus enables computational modeling of complex developmental processes without programming and thereby widens its accessibility for biologists. To demonstrate the applicability of the framework to problems in developmental biology, two case studies are presented that address different aspects of the interplay between cell fate specification, patterning and morphogenesis. In the first, I focus on the interplay between cell fate stability and intercellular signaling. Specifically, two studies are presented that investigate how mechanisms of cell-cell communication affect cell fate regulation and spatial patterning in the pancreatic epithelium. Using bifurcation analysis and simulations of spatially coupled differential equations, it is shown that intercellular communication results in a multistability of gene expression states that can explain the scattered spatial distribution and low cell type ratio of nascent islet cells. Moreover, model analysis shows that disruption of intercellular communication induces a transition between gene expression states that can explain observations of in vitro transdifferentiation from adult acinar cells into new islet cells. These results emphasize the role of the multicellular context in cell fate regulation during development and may be used to optimize protocols for cellular reprogramming. The second case study focuses on the feedback between patterning and morphogenesis in the context of the formation of vascular networks. Integrating a cell-based model of endothelial chemotaxis with a reaction-diffusion model representing signaling molecules and extracellular matrix, it is shown that vascular network patterns with realistic morphometry can arise when signaling factors are retained by cell-modified matrix molecules. Through the validation of this model using in vitro assays, quantitative estimates are obtained for kinetic parameters that, when used in quantitative model simulations, confirm the formation of vascular networks under measured biophysical conditions. These results demonstrate the key role of the extracellular matrix in providing spatial guidance cues, a fact that may be exploited to enhance vascularization of engineered tissues. Together, the modeling framework, software platform and case studies presented in this thesis demonstrate how cell-centered computational modeling of multi-scale and multicellular systems provide powerful tools to help disentangle the complex interplay between cell fate specification, patterning and morphogenesis during embryonic development.

Convergent evolution is a central theme in biology. Birds are an ideal system to examine the mechanisms underlying convergent evolution. Although bird patterning is diverse, within-feather patterns have repeatedly converged on the same four types: mottled patterns, scales, bars and spots. Other avian patterns occur, e.g. stripes, but are rare. In my thesis I examine the four main mechanisms underlying convergent evolution in plumage patterns: evolutionary genetics, evolutionary development, natural selection for signaling and camouflage. Japanese quail (Coturnix japonica) is a model system in developmental biology. Examining the developmental basis of pattern formation using molecular techniques, the dorsal patterning of embryonic quail is likely due to activation of the melanocortin-1 receptor, which is a highly conserved pathway in vertebrates. I examined whether a reaction-diffusion based theoretical model of pattern formation may predict developmental constraint in two groups that have different lifestyles and spectacular patterns: waterfowl (Anseriformes) and gamebirds (Galliformes). Tracing the evolutionary trajectory of pattern evolution with Bayesian comparative modeling there was evidence for developmental constraint in pattern evolution. Adaptive explanations may also result in convergence. Cuckoo-hawk mimicry has been demonstrated in the common cuckoo (Cuculus canorus) and the Eurasian sparrowhawk (Accipiter nisus), but may be prevalent in Old World cuckoos. Randomly selecting a parasitic cuckoo from each genera of Old World cuckoos and <8 sympatric raptors, I quantified their barred patterns using digital image analysis and found that parasitism can explain convergent evolution in the patterns of parasitic cuckoos and raptors. Patterns may have evolved due to ecological selection. Examining the patterns of 80% of all avian species worldwide, I found that habitat does not predict patterning, and that all four patterns are found in all habitats. These results demonstrate that the mechanisms of convergent evolution are diverse, and that development and natural selection have contributed to pattern evolution.