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Coleochaete: simple model for plant morphogenesis

We are using primitive plant systems, members of the genus Coleochaete, as a testbed for studying the process of morphogenesis in plants. This genus of freshwater algae includes species with filamentous, branching and pseudoparenchymatous cell structure. Phylogenetic studies suggest that these microscopic algae are related to the direct antecedants of land plants. In particular, Coleochaete orbicularis can grow as a flat disc, comprising a single layer of cells on a flat substrate. It is a perfect subject for advanced microscopy techniques, and can be manipulated like a microorganism. Our aim is to combine the unique experimental benefits of the organism with a comprehensive computer model for the physical and genetic interactions that drive morphogenesis. This will allow direct visualisation and analysis of physical and genetic parameters in vivo and in silico, and testing of alternate morphogenetic models.
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Cells within a plant meristem form a complex system, and clearly possess self-organising properties. However, there is no system available for modelling the physical relationships and information exchange between these cells in a biologically relevant way. In our first steps towards developing a system for dynamic modelling of plant cell interactions, we have generated a 2D description of the physical properties of cells, using a novel double spring model to describe cell wall properties. This physical model provides an engine for the production of cells through enlargement and division. The cells of the physical model are self-reproducing cellular automata which have a dynamic state represented by a set of parameters. The cell-states are determined by a genetic script and a positional signalling model. The signalling model is an arbitrary set of reacting-diffusing morphogens, the concentrations of which partly define the cell-states. The genetic script is a sequence of logical operations which is repeatedly applied to the cell-state parameters, including the morphogen concentrations. Each cell runs an identical script, mimicking real genetic logic. The cells are linked to a spatial/mechanical model that updates the shapes and arrangements of cells in response to their changing states. This provides feedback for the signalling system by affecting the distributions and movement of morphogens (Rudge, T. and Haseloff, J. Lecture Notes in Computer Science: Advances in Artificial Life, 3630:78-87, 2005). The model is extensible to three dimensions, and we have introduced genetic algorithms that can be used to evolve different morphogenetic programs in silico (EvoCell, Mackenzie, J., Dupuy, L. & Haseloff, J., unpublished results). The software model provides an environment for combining physical and genetic descriptions of multicellular growth. Fields of proliferating cells can be programmed via a genetic script to produce and respond to different morphogens.
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C. orbicularis grows as discoid multicellular colonies with a simple meristem structure. The colonies adhere to a substrate and grow as a cell monolayer. The cells are 10-20µM in size. Undisturbed colonies can grow up to several millimetres in diameter, and maintain a circular shape as a result of precisely coordinated sequences of anticlinal and periclinal divisions.

The meristematic zone is limited to a single layer of cells on the circumference of the growing disc. In evolutionary terms, these algae are poised between simpler unicellular and filamentous algae and the first plants, and show the first forms of parenchymatous growth. Coleochaetales are found in the fossil record, dating back over 400 Ma, to around the time of origin of the first terrestrial plants. Phylogenetic analysis of DNA sequences has identified C. orbicularis as a close living relative of land plants, and it possesses evolutionary novelties that are found in modern plants. Cell division occurs via phragmoplasts, rather than the phycoplasts found in lower algae, and produces parenchymatous tissues. Cell walls are cellulosic, and contain plasmodesmata. Zygotes are retained on the thallus during sexual reproduction, and are surrounded by specialised vegetative cells. Resistant spores are formed that contain sporopollenin, and colonies have a cuticle-like exterior that provides resistance to dessication. Colonies will grow freely in a moist terrestrial environment, and it is widely held that ancestral relatives may have made the transition to land.
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Coleochaete orbicularis is of interest to a number of scientific groups who are pursuing interests in phylogeny and comparative biology. Its genome is becoming increasingly well characterised. It has a size of around 94 Mb, and a BAC library of DNA clones is freely available as part of the efforts of the Green Plant Phylogeny Research Coordination Group (GPPRCG) in the USA. However, the alga has been little studied as a developmental system, although many features of its life cycle and habit make it potentially attractive for modern scientific studies. We have established a new collection of basic experimental techniques for handling C. orbicularis. The special properties of this system are (i) its morphological simplicity, (ii) the ease of observing every cell during development, and (iii) the availability a software model to describe biophysical and genetic dynamics during morphogenesis.
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Benefits of the Coleochaete system.

Algal Culture. The algae can be grown as a suspension in shaken flasks, immobilised on culture dishes like animal cells or on the surface of agar plates like microbes. The colonies are robust and easy to culture, requiring only a minimal medium and light source.

Clonal Propagation. Coleochaete is haploid and reproduces asexually. Cells within the body of a thallus redifferentiate as motile zoospores with two flagella, form a pore in the upper surface of the cell and swim away from the parent colony. If allowed to settle, the zoospores will lodge on the substrate and commence cell division to produce a new colony. In this way, cultures can be rapidly amplified as clones.

Single Cell Isolation. Protoplasts can be harvested from Coloechaete colonies by digestion with cellulases and pectinases. This allows a ready supply of single cells for mutagenesis or transformation studies. Isolated protoplasts show very high capacity for regeneration. Washed protoplasts can be seeded onto solid agar media or a flat culture dish and they will form algal colonies readily.

High Resolution Microscopy. Zoospores or protoplasts can be used to inoculate a plastic culture dish or coverslip based vessel. With pretreatment of the surfaces, the cells will stick avidly. The subsequent growth of colonies can be followed by microscopy with great clarity. Full cellular detail can be seen even with low cost wide field microscopy, because the immobilised colonies grow as monolayers.

High Throughput Imaging. In collaboration with Leica Cambridge UK, we have developed techniques for high throughput time lapse microscopy of live colonies, using a computer controlled inverted microscope with a robotic stage. Large areas can be scanned and many fields of view can be defined in software, and sampled repetitively to build up parallel collections of timelapse data. The microscope contains DIC and fluoresence optics with a sensitive digital camera for the acquisition of multiparameter images. This automated approach shows much promise for use as an analytical tool and for high throughput screening.