Coen Lab - Research

From Flower Development, E Coen , Cell & Developmental Biology Department - JIC UK

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Overview of Research Programme

Our research programme involves collaborations with several labs, including Computer scientists and ecological population geneticists. We study developmental and evolutionary problems at a range of spatial and temporal scales, from subcellular events at minute intervals, to events at geographic scale over millions of years. Our long term goal is to link these different scales of analysis to give an integrated understanding of evolution and development. Each person in the lab has a main project while also working in collaboration with others in the group.

A major focus of our group is on leaf development in Arabidopsis, which has the advantage of good genetics and convenience for imaging. Starting at the subcellular scale, we are analysing the role of microtubules and nuclear movements in the control of leaf cell division and growth. This involves extensive confocal imaging, image processing and computational modelling1,2,3. This work is closely related to studies at the cellular scale on division, growth and differentiation in developing leaves, involving a combination of time-lapse imaging and modelling. At the tissue scale we are studying leaf development through clonal analysis, time-lapse imaging6 and Optical Projection Tomography9. Leaf growth is also being modelled at this scale by treating the leaf as a growing sheet of material8. In addition to these studies on leaf development, we are analysing flower and leaf development using similar approaches in Antirrhinum8,9,10 and in monocots such as barley and maize5.

Another major focus of our work is on the evolution of flower colour, using the Antirrhinum species group as a model system. One aspect is to study evolutionary events over a few generations, at a naturally occuring hybrid zone in the Pyrenees. This uses a combination of DNA pedigree, phenotypic and pollinator studies10,11,12,13. We also aim to identify the key genes controlling flower colour that are under selection. On a longer evolutionary timescale we are studying variation in the function and structure of genes controlling flower colour between different Antirrhinum species using a combination of molecular and genetic approaches10,12.

Studies on the evolution of leaf shape are also being extended to carnivorous plants which have elaborate leaves that entice and digest animal prey, allowing them to thrive in nutrient poor environments. Carnivorous cup-shaped leaves with lids are effective animal traps very similar in structure to each other. They are found in four independent lineages; Asian pitcher plants (Nepenthes), Albany pitcher plants (Cephalotus), American pitcher plants (Sarracenia, Darlingtonia and Heliamphora) and bladderworts (Utricularia). This example of convergent evolution might hint at similar developmental mechanisms underlying the growth of these cup-shaped leaves. We are establishing Utricularia gibba as a model carnivorous plant to discover whether developmental rules discovered in Arabidopsis are adapted to grow cup-shaped leaf traps and whether these different plants are using similar genetic mechanisms to grow their traps4,9 . We are using a combination of genetic analysis, imaging and mathematical modelling to understand how these leaves grow themselves.


1 - Modelling Microtubule Dynamics

Microtubule edge segmentation, orientation colouring and segment joining

Jacob Newman - email in collaboration with Jordi Chan

Microtubules in Arabidopsis leaf cells have been observed to undergo persistent reorientation. Much work has been done to quantify the dynamics and behaviour of microtubules, however little is known about the processes by which inter-microtubule interactions lead to the patterning observed in biological data. This work uses computer modelling software developed by Andrew Bangham and Richard Kennaway (MTtbox) to create a model which can reproduce the ordering and reordering of microtubule arrays, using realistic assumptions regarding microtubule dynamics.

In tandem with the modelling, an image processing project (in collaboration with Paul Southam) seeks to automatically quantify the dynamics and interactions of microtubules in time-lapsed, electron microscopy image data. This quantification serves to support the manual observations by Jordi Chan, to allow the processing of more data than could realistically be analysed by eye, and to extract parameters which will feed directly into our microtubule modelling. I hope to apply these methods to other systems such as Utricularia.

2 - Image Processing for Biological Development

3D segmentation of cells (green) from a volumetric image (Arabdidopsis leaf).

Paul Southam - email in collaboration with Jordi Chan

Extracting quantitative measurements from image data is a fundamental step towards understanding how biological forms develop and evolve. I work on a number of projects including:

Modelling microtubule dynamics during growth and division - Jordi Chan & Scott Grandison.

Modelling Dynamic Growth Maps of Leaf Development - Samantha Fox.

Capturing plant development in 3D with Optical Projection Tomography - Karen Lee.

I am also interested in cell segmentation, cell registration, leaf classification and texture analysis.

3 - Microtubule dynamics during growth and division

Pre-prophase band formation in a leaf epidermis cell.

Jordi Chan - email in collaboration with Paul Southam and Jake Newman

Microtubules are highly dynamic subcellular filaments that adopt different patterns of alignment during growth, division and differentiation. During growth, microtubules are found in close association with the plasma-membrane where they are needed to guide the movement of cellulose synthase to generate appropriate wall architectures. Like animal cells, microtubules are required for chromosome segregation during cell division via the spindle apparatus. However, unlike animal cells, plant microtubules bunch up at the onset of mitosis to form a cortical ring, known as the pre-prophase band (PPB), which is involved in determining the future plane of cell division. Since plant cells within tissues are immobilised by their own walls and can only respond to cellular and environmental cues by altering their directions of growth and division planes, microtubules are indispensible elements of plant morphogenesis.

How microtubules change alignment and construct different arrays during growth and cell division is unknown but is likely to reside within fundamental properties of the microtubules themselves, such as the dynamic behaviour of their filamentous ends, regulation of their sites and angles of nucleation (birth) and outcomes of encounters between themselves and the geometry of the cell. It is interesting to think that these simple subcellular behaviours or rules concerning microtubule dynamics may not only shape the cell but also be amplified during morphogenesis from cell-to-cell to shape the entire plant.

The aim of my work is to reconstitute microtubule dynamics and cortical array organisation in-silico to understand and test mechanisms behind microtubule reorientation and division plane alignment (PPB formation) in-planta. This will involve time-lapse microscopy of growing and dividing leaf cells expressing appropriate fluorescent reporter genes, the development of software to quantify microtubule dynamics at both subcellular and tissue levels, and computer modelling. Models will be tested using cytoskeletal-specific drugs and mutants harbouring defective cytoskeletal genes. I will also apply these methods to other systems such as Utricularia.

4 - Establishing a model for complex leaf development

A young developing bladder of Utricularia gibba

Claire Bushell - email in collaboration with Karen Lee

Leaves are a fantastic example of the diversity of organ shape seen in nature and present us with a system in which development can be studied from the very early stages of growth. Our lab has generated a model for the growth and development of the relatively simple leaf shape seen in Arabidopsis thaliana. This model demonstrates how simple rules coordinated by a polarity system can generate leaf shape. The aim of my project is to see if these rules may underlie the development of complex leaf shapes seen in carnivorous plants. Interestingly, epiascidiate (cup-shaped) leaves have evolved four times independently; in the families Nepenthacea, Sarraceniaceae, Cephalotaceae (pitchers) and Lentibuleraceae (bladderworts). This may indicate that changes in an underlying developmental system may have taken place to give rise to the repeated evolution of form. We also have an understanding of a number of genes involved in the developmental program and their roles and interactions governing growth. Of particular interest are the HDZIPIII, KANADI and YABBY transcription factors which are known to play key roles in determining dorsoventral polarity of leaves. It is thought that the inside surface of epiascidiate leaves corresponds to the adaxial surface and the outside surface corresponds to the abaxial surface of a conventional leaf.

My work will expand on the knowledge we have by establishing a model system that will allow the study of complex leaf shapes. The organism of choice is the carnivorous bladderwort, Utricularia gibba, which has a number of key advantages as a novel model system. I will combine imaging, genetics, expression studies and computational modelling to explore the development of this leaf shape, comparing observations to those of Arabidopsis.

5 - The role of tissue cell polarity in Monocot organ development

Annis Richardson - email in collaboration with Xana Rebocho and Sarah Hake

A young inflorescence of Hordeum vulgare

There is huge diversity in organ shape in the plant kingdom, but how shape is defined during development is not yet fully understood. Growth is a key aspect of development and to generate non-uniform shapes, such as the complex leaf structure seen in many monocots, growth needs to be anisotropic (not equal in all directions). To define growth directionalities within biological tissues axial information is needed. Work within the lab has suggested that this axial information could be provided by tissue cell polarity (coordinated cell polarity across a tissue). We hypothesise that the establishment of tissue cell polarity could be mediated by the polar transport of Auxin, influenced by the activity of regions within the tissue called organisers. Plus organises are regions of high extracellular Auxin and minus organisers of low extracellular Auxin. Modulation of the position of these organisers could result in significant changes in organ shape during development. My work uses a combination of computer modelling and experimental work to test this model, focussing on monocot examples. In particular I am using the Hooded mutant in Barley to test the effect of ectopic BKn3 expression on tissue cell polarity. In addition to this I am investigating the role of tissue cell polarity in the development of the Maize leaf, with the aim of generating a model which describes the role of tissue cell polarity in the development of all ensheathing leaves found in the Poales order.

6 - Dynamic Growth Maps of Leaf Development

Arabidopsis leaf one expressing GFP in the cell membranes

Samantha Fox - email

A major challenge in biology is to understand how buds comprising a few cells can give rise to complex plant and animal appendages, like leaves or limbs. We have addressed this problem through a combination of time-lapse imaging of growing leaf buds, clonal analysis and computational modelling. We have generated a model that shows how leaf shape can arise according to a few simple rules and that growth is coordinated by an in-built polarity system. Experimental tests through partial leaf ablation support this model, and allow re-evaluation of previous experimental studies. Our model allows a range of observed leaf shapes to be generated and predicts observed clone patterns in different species. Thus our experimentally validated model may underlie the development and evolution of diverse organ shapes.

Watch my Youtube movie to find out more:

7 - Exploring Inner Worlds of Plants in 3D with Optical Projection Tomography

Optical Projection Tomography Volume Rendered Plant Specimens
Inside a Cephalotus follicularis pitcher leaf trap

Karen Lee - email in collaboration with Claire Bushell, Paul Southam, Grant Calder and Jerome Avondo

Please see 3D Gallery for images of carnivorous plants and other beautiful plant specimens.

We have explored the use of Optical Projection Tomography (OPT) as a method for capturing 3D morphology and gene activity at a variety of developmental stages and scales from plant specimens, in collaboration with James Sharpe and Bioptonics . OPT can be conveniently applied to a wide variety of plant material including seedlings, leaves, flowers, roots, seeds, embryos and meristems. At the highest resolution large individual cells can be seen in the context of the surrounding plant structure. 3D domains of gene expression can be visualized using either marker genes such as β-glucuronidase, or more directly by whole-mount in situ hybridization. For naturally semi-transparent structures, such as roots or Bladderwort suction traps, live 3D imaging using OPT is possible. 3D gene expression patterns in living transgenic plants expressing fluorescent GFP markers can also be visualised. To interactively analyse and quantify OPT data, software was developed to visualise 3D volumes, accurately place points on volumes in 3D space and extract growth measurements.

Using these tools to capture leaf shape and growth, in combination with mathematical modelling, we are studying mechanisms controlling growth and shape from earliest stages of Arabidopsis leaf growth to maturity in 3D.

I am initiating a new project exploring carnivores. Carnivorous plants are amazing. They seem to turn the natural order around by being able to entice, capture and consume animal prey, when we normally think of plants as passive suppliers of nutrition for the animal world. Taking what we have learned from our Arabidopsis research we want to discover whether rules of growth underlying the development of simple leaves in Arabidopsis are are adapted to grow cup-shaped leaf traps of carnivorous plants. Using a combination of 3D imaging, genetic analysis and modelling, we aim to explore how these complex leaves develop.

Another project I am working on is a website showcasing the Inner World of Carnivorous Plants

Our carnivorous plant work is also featured in a Sightings article- 3D Carnivorous Plants, in American Science

Some of our images can also be found on the Cell Picture Show- Plant Biology

8 - Modelling Growth at the Tissue Scale

Model for Snapdragon cyc dich mutant

Richard Kennaway - email in collaboration with Erika Kuchen and Xana Rebocho

I am developing finite element methods for modelling the growth and development of curved two-dimensional tissues such as leaves and petals. The interactive software tool I am developing, called GFtbox, is available for download.

9 - Testing models for polarity and asymmetry in Antirrhinum

An Antirrhinum rad mutant flower

Xana Rebocho - email in collaboration with Des Bradley, Lucy Copsey, Andrew Bangham and Richard Kennaway

Bilateral symmetry of flowers has evolved several times independently from an ancient radially symmetrical condition. Antirrhinum’s flowers are comprised of 2 ventral, 2 lateral and 1 dorsal petal. One of the key genes involved in the control of dorsal identity is RADIALIS. RADIALIS is regulated by CYCLOIDEA and DICHOTOMA to be expressed in the primordial dorsal petals. One of the functions of RADIALIS is to prevent DIVARICATA, the ventral identity gene, expression in the lateral and dorsal domains. However, the way in which these genes act to control the asymmetry, shape and form of the flower remains to be unraveled. The establishment of ventral identity is also important for the development of petal specializations such as nectaries and spurs. I am studying an Antirrhinum mutant that produces a ventral outgrowth, similar to a spur, in order to understand how identity and polarity genes interplay to produce novel shapes. I am addressing these issues using a combination of genetic, developmental and computational approaches. The goal of my research will be to understand how genes act within and between cells to modify growth patterns that contributed to the final morphology of the flower and the evolution of new traits.

My work is funded by a Long-term Fellowship from the Human Frontier Science Programme (HFSP).

10 - Evolution of organ size and shape between Antirrhinum species

Antirrhinum wild-type flower

Lucy Copsey - email in collaboration with Xana Rebocho, Des Bradley and Matt Couchman.

The "old world" Antirrhinum species, found growing naturally in southern Europe and North Africa, show an extensive range of diversity in growth habit, organ size, shape and flower colour. This variation is important as it highlights differences between individuals which may be due to either environmental effects or differences at gene level, these genetic differences underpins how diversity in form is generated through evolutionary time and is the basis of evolution. By exploiting evolutionary variation in size we hope to identify genes controlling organ size in Antirrhinum species (allometry project).

I am using a classical genetics approach involving the production of a number of plant resources developed by crossing Antirrhinum species to both our cultivated JI stock 7 line and interspecies crosses to study natural variation and domestication effects. These resources are available for use by group members and for the wider Antirrhinum community. I also supervise and coordinate the genetics of Utricularia.

11 - Evolutionary Dynamics Underlying Species Diversification

Bee polinating A. pseudomajus

Matthew Couchman - email in collaboration with Xana Rebocho, Lucy Copsey, Des Bradley and Nick Barton

We are part of a collaboration investigating factors affecting gene flow across populations. The project focuses on flower colour in two distinct subspecies of Antirrhinum. Within the Spanish Pyrenees there are a number of hybrid zones that provide ideal environments for analysing how genes influence flower colour. For each individual within our chosen hybrid zone we are annually recording their GPS location and colour scores as well as taking samples for genotyping and other molecular analysis.

My role within the project is to develop a relational database to capture these and other collaborator outputs as well as a website to act as a gateway to this database. The website will include visual tools such as a configurable map of recorded GPS positions and genetic and physical maps.

12 - Natural variation of flower colour

Antirrhinum flower colour variation.

Desmond Bradley - email

Collaborators: Christophe Thebaud, Xana Rebocho, Nick Barton, David Field, Christophe Andalo and Monique Burrus

Two subspecies of Antirrhinum (magenta and yellow), which generally occur in genetic isolation in the Pyrenees, have hybridized in nature to create a hybrid zone. Due to hybridization the Antirrhinum flowers display an array of parental as well as mixed flower colour phenotypes which are the result of genetic variation at three key loci ROSEA, ELUTA and SULFUREA. The two first genes are involved in the control of the magenta anthocyanin pigmentation while SULF is a repressor of aurone (yellow) pigmentation. As the HZ gives a great playground to study evolution in action, the goal of this research is to follow the genetic flow of these flower colour loci within the hybrid population as well as the fitness of each phenotype/genotype throughout several years. I also use similar approaches to analyse gene expression regulation in Utricularia.

13 - Evolution of genomic islands through plant-pollinator interactions

A bumblebee visiting a snapdragon flower. Yellow pollen can be seen on its back.

Mabon Elis - in collaboration with Ian Bedford, Des Bradley, Lucy Copsey, Andrew Bourke, Matt Couchman and Annabel Whibley.

In the Pyrenees, two species of snapdragon with differing flower colours come into contact and cross together to give hybrid plants. Instead of spreading through the whole area, these hybrids are confined to short transitional regions called ‘hybrid zones’. When we look at the genetic information of the species and hybrids across the whole area, we see that some parts of the genome show a much higher degree of divergence than the rest. These are called ‘genomic islands’, and include genes involved in flower colour.

Snapdragons are pollinated by large insects, such as bumblebees, which use their body weight and size to operate the complex flowers and collect nectar inside. When bumblebees forage for nectar, they often show ‘constancy’ towards particular flower types, where they preferentially visit particular species based on past experience. Could such selection by pollinators make the hybrid plants less successful than the parental species and maintain the genomic islands?

I’m testing this idea using controlled experiments with commercial bumblebee colonies. These are bumblebees that have not previously learned to associate colour with reward. They can therefore be trained to form particular preferences, and their constancy can then be tested. I’m also looking at the behaviour of wild bumblebees at our field site, as well as any other flower signals that might be different in the two species and the hybrids.

BBSRC Doctoral Training Partnership on the Norwich Research Park

My work is funded by a BBSRC Doctoral Training Partnership studentship.

14 - Genetic analysis of Utricularia gibba trap development

A mature U. gibba trap.

Chris Whitewoods - in collaboration with Karen Lee and Claire Bushell.

Carnivorous plants have evolved cup-shaped insect traps four times independently. Each time they evolved from leaves. This raises the question of how the ancestral leaf developmental program has been modified to produce these complex structures and whether the same thing happened each time. Have genes involved in simple leaf development simply been modified or have novel genes been acquired to elaborate the new shape?

I am using the aquatic carnivorous plant Utricularia gibba to answer these questions using both forward and reverse genetics approaches:

Firstly, in collaboration with Karen Lee and Claire Bushell I am performing a screen for trap mutants in an EMS-treated mutant population of U. gibba. I will map interesting mutations using whole-genome-sequencing and analyse their role in trap development. This will not only help us understand the genetic mechanisms of trap development, but also enable us to compare the genetic pathways of trap development in U. gibba with that of leaf development in plants with simpler leaves to help us speculate how traps evolved.

Secondly, I am analysing the role of U. gibba homologues of genes known to be involved in simple leaf development in other plants. This will allow us to see how the ancestral leaf developmental program has been modified to create a cup shape.

The results from these approaches will be used in conjunction with computational modelling to refine our hypotheses about the development and evolution of these complex leaves.

15 - Understanding spacing of outgrowths in Arabidopsis

A young Arabidopsis leaf showing CUC2 localisation and serrations

Catherine Mansfield - in collaboration with Samantha Fox and Jordi Chan.

A huge variety of leaf shapes exist in nature. Different species of plant often have different patterns of serrations (outgrowths) on their leaves. In order to consistently generate leaves of the same shape, plants must have a mechanism of controlling outgrowth formation. A similar phenomenon is observed in phyllotaxis where plants are able to position their leaves or branches (outgrowths) at regular intervals at the whole plant level. The remarkable regularity of spacing in plants suggests a robust genetic control mechanism during development which may involve the plant hormone auxin. My work aims to understand the mechanisms of spacing through microscopy and live-imaging, molecular genetics, and modelling.

Genes known to be involved in serration formation, such as the transcription factor CUC2, will be ectopically expressed in Arabidopsis with spatial and temporal variation to understand how plants space their outgrowths at regular intervals. It will be important to establish how ectopic serrations interact with existing serrations, and how spatial and temporal variation may play a role in this. It would also be interesting to see if the mechanisms controlling spacing in leaves were similar in spacing of outgrowths in phyllotaxis which might involve ectopic expression of genes in the meristem.

Knowledge from this work will improve our understanding of the control of leaf and plant shape.

modified on 12 January 2017 at 13:14 ••• 1,020,992 views