On the Development and Evolution of the Lateral Line

At its core, developmental biology can be reduced to the study of three overarching processes; axiation, pattern formation and induction. By exploiting the formation of the lateral line system during embryogenesis in Medaka fish, I was able to contribute novel insights to each of these fundamental processes. The lateral line system is a sensory module present in fish and amphibians and is composed of individual neuromast organs. Its main function is to sense the direction of water flow and relay the information back to the brain. A fantastic diversity of lateral line patterns exists in the wild, the basis of which remains largely unknown. By live- imaging the polarized migration (axiation) of tissue during development I was able to demonstrate how the formation of the posterior lateral line in Medaka fish occurs. This led to a reassessment of the prevailing view held in the field, as it for the first-time placed changes in pattern construction occurring during development as a major contributor to the diversity of lateral line patterns. It also led to the discovery that the same molecular players can result in the formation of different posterior lateral line patterns in different teleost species, simply by modulating their temporal and spatial expression profiles. This modulation has been repeatedly spotted by developmental biologists studying a variety of organisms, and thus can be regarded as a common principle driving evolutionary novelty. Using a variety of available and newly generated mutants I delved deeper into the guiding logic/principles behind posterior lateral line pattern formation. A process revealed to harbour a high degree of plasticity and self-organization. Specifically, I was able to show that precursor clusters of the migrating primordium can act as autonomous units, demonstrating the plasticity of the developmental tissue of origin for neuromasts (the primordium). In fact, within the same animal the left and right posterior lateral lines have varying outputs, reinforcing the idea of an inherent plasticity of primordia and strongly suggesting the presence of a low- range Waddingtonian developmental buffering system. Along the same lines, I was able to show that changes in the immediate environment surrounding the primordium (for e.g. epithelial morphology) can have a direct effect on the posterior lateral line pattern being formed. And postulate that these properties of the system might have constituted a fault-line that evolution could have exploited to generate the kaleidoscopic variety of lateral line patterns we observe in the wild. Going from tissue level dynamics to individual organs, I focus on the differentiation of neuromasts and provide strong evidence for the location and potency of the stem cells that maintain them. I also report that during development neural stem cell precursors induce the formation of their own niches, which in turn are used to maintain the stem cells life-long. A finding that could have important implications in fields like tissue engineering and stem cell biology. Lastly, I focused my efforts on the cellular level by studying the post-embryonic formation of neuromasts, a process that re-utilizes axiation, is highly stereotypical and driven by individual cell behaviour. I characterize in detail the cells participating in post-embryonic organogenesis and reveal early molecular heterogeneities within the stem cells that seem to contribute to the differential behaviour they undergo later on. All in all, traversing tissue to organ and finally cellular scales in the lateral line led to novel insights into how this system is built and maintained all whilst being constantly remodelled. Surprisingly this approach even led to initial insights on the evolution of lateral lines. As with the nature of all scientific progress no matter how miniscule, more questions have been conjured that need to be answered in due time

Shape Parameter & Nodal Distribution Insensitive Radial Basis Functions for Nonlinear Optimal Control Problems

Computational optimal control relies mainly on pseudospectral methods. The use of Chebyshev and Legendre polynomials is ubiquitous in the literature. This family of methods has good accuracy characteristics but constraints the nodal distribution to a certain grid that is denser at the boundaries. In this work, a set of novel Coupled Radial Basis Functions (CRBFs) is introduced as an approximation means for the nonlinear optimal control problem. CRBFs are real-valued Radial Basis Functions (RBFs) augmented with a conical spline. They do not require a specific nodal distribution. A plethora of research articles were published on the optimization of the shape parameter of RBFs. Unlike classic RBFs, CRBFs are insensitive to the shape parameter reducing the computational time needed to find an optimal shape parameter. The method introduced in this dissertation follows an indirect approach of solving optimal control problems. Hence, the method is initiated by deriving the necessary conditions of optimality. Consequently, CRBFs are used to approximate the resulting two-point boundary value problem (TPBVP) into a set of nonlinear algebraic equations (NAEs). The system of NAEs is then solved using a standard nonlinear solver. Numerical experiments of the proposed method are carried out and compared with exact solutions and other computational methods. The method is applied to classical nonlinear optimal control problems: Zermelo\u27s problem, a duffing oscillator with various boundary conditions, and a nonlinear inverted pendulum on a cart. CRBFs-collocation shows superiority of computational speed over other methods and is easy to implement. For future work, this method is suitable for real-time control applications

Rapid Orbital Motion Emulator (ROME): Kinematics Modeling and Control

Space missions design requires already tested and trusted control algorithms for spacecraft motion. Rapidly testing control algorithms at a low cost is essential. A novel robotic system that emulates orbital motion in a laboratory environment is presented. The system is composed of a six degree of freedom robotic manipulator fixed on top of an omnidirectional ground vehicle accompanied with onboard computer and sensors. The integrated mobile manipulator is used as a testbed to emulate and realize orbital motion and control algorithms. The kinematic relations of the ground vehicle, robotic manipulator and the coupled kinematics are derived. The system is used to emulate an orbit trajectory. The system is scalable and capable of emulating servicing missions, satellite rendezvous and chaser follower problems

Modular control of vertebrate axis segmentation in time and space

How the timing of development is linked to organismal size is a longstanding question. Although numerous studies have reported a correlation of temporal and spatial traits, the developmental or selective constraints underlying this link remain largely unexplored. We address this question by studying the periodic process of embryonic axis segmentation in-vivo in Oryzias fish. Interspecies comparisons reveal that the timing of segmentation correlates to segment, tissue and organismal size. Segment size in turn scales according to tissue and organism size. To probe for underlying causes, we genetically hybridised two closely related species. Quantitative analysis in ~600 phenotypically diverse F2 embryos reveals a decoupling of timing from size control, while spatial scaling is preserved. Using developmental quantitative trait loci (devQTL) mapping we identify distinct genetic loci linked to either the control of segmentation timing or tissue size. This study demonstrates that a developmental constraint mechanism underlies spatial scaling of axis segmentation, while its spatial and temporal control are dissociable modules

Seasonal and Ageing-Depending Changes of Aquaporins 1 and 9 Expression in the Genital Tract of Buffalo Bulls (Bubalus bubalis)

The presence of Aquaporins 1 (AQP1) and 9 (AQP9), integral membrane water channels that facilitate rapid passive movement of water and solutes, was immunohistochemically detected in the excurrent ducts collected from sexually mature buffalo bulls of proven fertility during the mating (late autumn-winter) and non-mating (late spring to the beginning of autumn) seasons. Furthermore, the research was performed also on the epididymal cauda of a senile buffalo bull with inactive testis. Aquaporins 1 and 9 were immunolocalized at distinct levels. In the efferent ducts, AQP1 immunoreactivity was strongly evidenced at the apical surface of the non-ciliated cells and weakly along the basal membrane of the epithelial cells. The latter reactivity disappeared during the non-mating season. No AQP1 immunoreactivity was detected in the epithelium of epididymis and vas deferens, whereas AQP1 was expressed in the smooth muscle layer of the vas deferens. Aquaporin 1 was present in the blood vessels and in small nerve bundles all along the genital tract. The supranuclear zone of the epididymal principal cells was AQP9 immunoreactive, limited to the corpus and cauda regions, and vas deferens. The samples collected in the two reproductive seasons showed a weaker AQP9 immunoreactivity during the non-mating season. A typical AQP9 immunoreactivity was noticed in the old buffalo examined. The tested AQP molecules showed a different expression pattern in comparison with laboratory mammals, primates, equine, dog and cat. In addition, seasonal differences were noticed which are possibly useful in regard to the comprehension of the morphophysiology of reproduction in the bubaline species, which are still a matter of debat

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

AbstractThe CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient and precise strategy for CRISPR/Cas9 mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40bp), a synthetic sgRNA and streptavidin tagged Cas9. We generate six novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole Genome Sequencing (WGS) results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion-protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna knock-in line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.</jats:p

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology-directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient, and precise strategy for CRISPR/Cas9-mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR-amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40 bp), a synthetic single-guide RNA and Cas9 mRNA. We generate eight novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole genome sequencing results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter