Cellular patterns during leaf development

A major problem in developmental biology is to understand how the behaviour of individual cells creates reproducible biological shapes. Moreover, the reproducibility of form also happens at the cellular level and cellular patterns are evident in the temporal as well as the spatial scales. Understanding the principles underlying these cellular patterns will contribute to linking the individual cell dynamics to the collective phenomenon of morphogenesis. The relatively flat shape, the absence of cell migration and apoptosis makes the leaf of Arabidopsis thaliana an excellent system to study morphogenesis at the cellular level. However the information about the cellular dynamics that is available has only been inferred indirectly or restricted to few cells. In this thesis, I present some methods that permitted the characterisation of cellular dynamics over long time periods at the organ level. I present the Lobe Contribution Elliptical Fourier Analysis (LOCO-EFA), that enabled the quantification of complex cell geometries and provided a shape profile to evaluate populations of cells. In turn, the shape profile of individual cells was used to develop a tracking algorithm that, integrated with a segmentation algorithm, resulted in a powerful tool to recognize cells in a succession of images and extract cell shape, cell area, cell position and cell lineages. The synergy between in vivo imaging and computational tools permitted the study of cellular patterns at unprecedented resolution. Interestingly, the dynamics of cell growth and cell shape are highly influenced by the cell age and to a limited extend, by their position. Moreover, direct measurement of cell division shows that the division zone is restricted towards the base of the leaf, but is not constant in length and that the frequency of divisions decreases over time in a rather gradual fashion. At single cell level, new events of lobe formation were identified, suggesting that the intracellular patterning underlying the multi-polar pavement cell shape is dynamic and lobes are newly formed rather than being specified at a single time point. The cellular dynamics of growth, shape and divisions using long time-lapse and imaging enabled me to revisit previous hypothesis and propose new ones about the regulation of cellular behaviour during leaf morphogenesis. (The CD-ROM referred to in the thesis contains Movies in AVI format. However these were submitted as separate files which could not be uploaded to the repository. Please contact the author for more information.

Volumetric finite-element modelling of biological growth

Differential growth is the driver of tissue morphogenesis in plants, and also plays a fundamental role in animal development. Although the contributions of growth to shape change have been captured through modelling tissue sheets or isotropic volumes, a framework for modelling both isotropic and anisotropic volumetric growth in three dimensions over large changes in size and shape has been lacking. Here, we describe an approach based on finite-element modelling of continuous volumetric structures, and apply it to a range of forms and growth patterns, providing mathematical validation for examples that admit analytic solution. We show that a major difference between sheet and bulk tissues is that the growth of bulk tissue is more constrained, reducing the possibility of tissue conflict resolution through deformations such as buckling. Tissue sheets or cylinders may be generated from bulk shapes through anisotropic specified growth, oriented by a polarity field. A second polarity field, orthogonal to the first, allows sheets with varying lengths and widths to be generated, as illustrated by the wide range of leaf shapes observed in nature. The framework we describe thus provides a key tool for developing hypotheses for plant morphogenesis and is also applicable to other tissues that deform through differential growth or contraction

Engineering morphogenesis of Marchantia polymorpha gemmae

Morphogenesis is an apparent yet complex process: emergence of a plant shape is the result of an intricate interplay between genetic regulation, cell physiology and mechanical processes at the tissue scale. Morphogenesis spans three levels of biological organisation, forming a nested complex system. Genes, which form the lowest level of the system, are arranged into networks that control properties of cells. Cells, which form the middle level of the system, are arranged into geometric networks, and their mechanical and chemical interactions give rise to the morphology of a whole organism. Therefore, the study of plant morphogenesis relies on understanding how genetically-driven cellular interactions influence the formation of a plant shape. Clonal propagules of Marchantia polymorpha (Marchantia), also known as gemmae, are an attractive system for studying these interactions. Gemmae are small, have a simple disk-like shape and are resilient to environmental conditions. As such, they are well-suited for fluorescent microscopy and the collection of gene expression data. These features create an opportunity to study the processes of gemma morphogenesis at tissue, cellular and genetic levels through fluorescent microscopy in a single assay. In order to enable the engineering of morphogenesis in Marchantia gemmae, tools and frameworks for obtaining, storing and analysing the observations from the three levels of biological organisation should be put into place. The work presented in this thesis focuses on the development of such tools and their application in studying the role of phytohormone auxin in Marchantia development. I introduced novel sample preparation and time-lapse imaging assays for Marchantia gemmae along with image- processing methods for the estimation of tissue expansion rates with sub-cellular resolution. These methods allowed me to hypothesise a mechanism behind the regulation of cell proliferation in Marchantia and the role auxin plays in controlling this process. Together with Bernardo Pollak, I developed MarpoDB, a gene-centric representation of the Marchantia genome that enables the search and preparation of Marchantia genetic parts for assembly into synthetic DNA constructs. I used MarpoDB to extract parts and build fluorescent reporters, providing proxies for auxin biosynthesis and signalling. The reporters were then introduced into the Marchantia genome, and the gemmae of the transgenic lines were imaged to estimate the average patterns of the reporters’ expression. The collected patterns were then overlaid on the patterns of relative tissue expansion to validate the proposed role of auxin as an inhibitor of cell proliferation and the mechanism behind its transport in Marchantia.BBSR

Auxin transport-feedback models of patterning in plants

Many patterning events in plants are regulated by the phytohormone auxin. In fact, so many things are under the influence of auxin that it seems difficult to understand how a single hormone can do so much. Auxin moves throughout the plant via a network of specialized membrane-bound import and export proteins, which are often differentially expressed and polarized depending on tissue type. Here, we review simulation models of pattern formation that are based on the control of these transporters by auxin itself. In these transport-feedback models, diversity in patterning comes not from the addition of more morphogens, but rather by varying the mechanism that regulates the transporters

Computational design and designability of gene regulatory networks

Nuestro conocimiento de las interacciones moleculares nos ha conducido hoy hacia una perspectiva ingenieril, donde diseños e implementaciones de sistemas artificiales de regulación intentan proporcionar instrucciones fundamentales para la reprogramación celular. Nosotros aquí abordamos el diseño de redes de genes como una forma de profundizar en la comprensión de las regulaciones naturales. También abordamos el problema de la diseñabilidad dada una genoteca de elementos compatibles. Con este fin, aplicamos métodos heuríticos de optimización que implementan rutinas para resolver problemas inversos, así como herramientas de análisis matemático para estudiar la dinámica de la expresión genética. Debido a que la ingeniería de redes de transcripción se ha basado principalmente en el ensamblaje de unos pocos elementos regulatorios usando principios de diseño racional, desarrollamos un marco de diseño computacional para explotar este enfoque. Modelos asociados a genotecas fueron examinados para descubrir el espacio genotípico asociado a un cierto fenotipo. Además, desarrollamos un procedimiento completamente automatizado para diseñar moleculas de ARN no codificante con capacidad regulatoria, basándonos en un modelo fisicoquímico y aprovechando la regulación alostérica. Los circuitos de ARN resultantes implementaban un mecanismo de control post-transcripcional para la expresión de proteínas que podía ser combinado con elementos transcripcionales. También aplicamos los métodos heurísticos para analizar la diseñabilidad de rutas metabólicas. Ciertamente, los métodos de diseño computacional pueden al mismo tiempo aprender de los mecanismos naturales con el fin de explotar sus principios fundamentales. Así, los estudios de estos sistemas nos permiten profundizar en la ingeniería genética. De relevancia, el control integral y las regulaciones incoherentes son estrategias generales que los organismos emplean y que aquí analizamos.Rodrigo Tarrega, G. (2011). Computational design and designability of gene regulatory networks [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/1417

Investigating the role of DA1 in growth control.

Increasing global demand for food is a major issue facing modern day agriculture. For crops such as wheat and rice, where the seed constitutes the harvestable yield, the engineering of larger seeds provides a possible strategy for yield improvement. A detailed understanding of the growth of plant organs in general is paramount if such advances are to be made. Utilising previously characterised regulators of plant organ growth, this thesis explores the molecular mechanisms involved in the setting of final organ size. This thesis capitalises on previous studies that have identified DA1 as a negative regulator of organ growth; it explores the role of the DA1 protein and investigates its interactions with other proteins. In vitro studies reveal that DA1 forms homo-­‐ and hetero-­‐multimeric complexes with its sister protein DAR1 and in vitro and in yeast assays reveal interactions between DA1 and the transcription factor TCP15 and the growth-­‐regulating receptor-­‐like kinase TMK4. In addition, biochemical assays described in this thesis identify an active ubiquitin interacting motif (UIM) in the N-­‐terminal region of DA1 and an ubiquitin-­‐activated metallopeptidase in its C-­‐terminal region. Further studies reveal that, in addition to being activated by the RING E3 ligases EOD1/BB and DA2, the DA1 peptidase is active towards both EOD1/BB and DA2. In vitro and in vivo studies demonstrate that DA1 cleaves a peptide fragment from the N-­‐terminus of EOD1 and the C-­‐terminus of DA2. Finally, this thesis reports two genetic screens carried out in two separate Arabidopsis mapping populations in order to identify novel regulators of organ growth. Analyses of petal and seed phenotypes in the MAGIC RIL-­type population and in a natural Swedish population identify novel and a priori candidate genes for further characterisation

Spatiotemporal coordination of cell division and growth during organ morphogenesis

A developing plant organ exhibits complex spatiotemporal patterns of growth, cell division, cell size, cell shape, and organ shape. Explaining these patterns presents a challenge because of their dynamics and cross-correlations, which can make it difficult to disentangle causes from effects. To address these problems, we used live imaging to determine the spatiotemporal patterns of leaf growth and division in different genetic and tissue contexts. In the simplifying background of the speechless (spch) mutant, which lacks stomatal lineages, the epidermal cell layer exhibits defined patterns of division, cell size, cell shape, and growth along the proximodistal and mediolateral axes. The patterns and correlations are distinctive from those observed in the connected subepidermal layer and also different from the epidermal layer of wild type. Through computational modelling we show that the results can be accounted for by a dual control model in which spatiotemporal control operates on both growth and cell division, with cross-connections between them. The interactions between resulting growth and division patterns lead to a dynamic distributions of cell sizes and shapes within a deforming leaf. By modulating parameters of the model, we illustrate how phenotypes with correlated changes in cell size, cell number, and organ size may be generated. The model thus provides an integrated view of growth and division that can act as a framework for further experimental study

The role of DA1 in organ size control in Arabidopsis thaliana

Despite the sizes of organs and organisms being a key defining feature, very little is known about how the final sizes of organs is determined. Recent progress has highlighted the important role of the DA1 peptidase as a negative regulator of organ size in Arabidopsis thaliana. Previous studies have proposed that DA1, and the E3 ligase BIG BROTHER (a protein known to regulate DA1 activity), work synergistically to regulate the duration of cell proliferation. In the prulresent study, we take a multidisciplinary approach to further our understanding of the biological activities of DA1 and BB. Protoplast transient expression analyses were used to explore potential new substrates for DA1 peptidase activity, and to work towards identifying a conserved target site for DA1mediated cleavage. Using confocal microscopy and bespoke segmentation software, I embarked on a global analysis of leaf cellular phenotypes in the da1-1, bb, and da1-1bb mutants throughout early development. This allowed a holistic comparison to wild type of parameters such as total cell number, and cell area, density, and circularity. In addition, scanning electron microscopy was used to examine cells in mature leaves of wild type, and da1-1, bb, and da1-1bb mutants, revealing novel insights into the control of final organ size in these mutants relative to wild type. Finally, innovative live cell imaging has, for the first time, allowed cell divisions to be observed in plants carrying the da1-1, bb, and da1-1bb mutations. My observations and interpretation establish new insights into how DA1 and BB control growth by controlling the arrest of cell proliferation, and the population-level rate of cell proliferation. The approaches I have developed show the promise of quantitative cell imaging for understanding organ growth, and establish a framework for precisely comparing the effects of different mutations on organ growth

Sox10 regulates enteric neural crest cell migration in the developing gut

Concurrent Sessions 1: 1.3 - Organs to organisms: Models of Human Diseases: abstract no. 1417th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting and XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo. The Conference's web site is located at http://www.inb.unam.mx/isdb/Sox10 is a HMG-domain containing transcription factor which plays important roles in neural crest cell survival and differentiation. Mutations of Sox10 have been identified in patients with Waardenburg-Hirschsprung syndrome, who suffer from deafness, pigmentation defects and intestinal aganglionosis. Enteric neural crest cells (ENCCs) with Sox10 mutation undergo premature differentiation and fail to colonize the distal hindgut. It is unclear, however, whether Sox10 plays a role in the migration of ENCCs. To visualize the migration behaviour of mutant ENCCs, we generated a Sox10NGFP mouse model where EGFP is fused to the N-terminal domain of Sox10. Using time-lapse imaging, we found that ENCCs in Sox10NGFP/+ mutants displays lower migration speed and altered trajectories compared to normal controls. This behaviour was cell-autonomous, as shown by organotypic grafting of Sox10NGFP/+ gut segments onto control guts and vice versa. ENCCs encounter different extracellular matrix (ECM) molecules along the developing gut. We performed gut explant culture on various ECM and found that Sox10NGFP/+ ENCCs tend to form aggregates, particularly on fibronectin. Time-lapse imaging of single cells in gut explant culture indicated that the tightly-packed Sox10 mutant cells failed to exhibit contact inhibition of locomotion. We determined the expression of adhesion molecule families by qPCR analysis, and found integrin expression unaffected while L1-cam and selected cadherins were altered, suggesting that Sox10 mutation affects cell adhesion properties of ENCCs. Our findings identify a de novo role of Sox10 in regulating the migration behaviour of ENCCs, which has important implications for the treatment of Hirschsprung disease.postprin