THREE-DIMENSIONAL VIEWS ON LIFE: LASER CONFOCAL SCANNING MICROSCOPIC EXAMINATION OF FRUITING MORPHOGENESIS IN COPRINUS

CINEREUS AND PLEUROTUS PULMONARIUSSiu Wai Chiu, Mark A. Reeve, Carmen Sánchez and David Moore

Department of Biology, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong and School of Biological Sciences, University of Manchester, Manchester M13 9PT, U. K.

The evolutionary separation between the major Kingdoms must have occurred at a stage when the most highly evolved things were single cells. Each Kingdom has independently evolved ways to organise populations of cells to make the multicellular organisms we now know as mushrooms, mice or marigolds. In animals cell migration (and everything that controls it) plays a central role in development. Plant cells have little scope for movement and their changes in shape and form are achieved by regulating the orientation and position of the wall which forms when a plant cell divides. The fungal hypha has two peculiarities which result in fungal development being totally different from that in plants: the hypha grows only at its tip and that new septa form only at right angles to the growth axis of the hypha.

The evolutionary separation between the major Kingdoms must have occurred at a stage when the most highly evolved things were single cells. Each Kingdom has independently evolved ways to organise populations of cells to make the multicellular organisms we now know as mushrooms, mice or marigolds. In animals cell migration (and everything that controls it) plays a central role in development. Plant cells have little scope for movement and their changes in shape and form are achieved by regulating the orientation and position of the wall which forms when a plant cell divides. The fungal hypha has two peculiarities which result in fungal development being totally different from that in plants: the hypha grows only at its tip and that new septa form only at right angles to the growth axis of the hypha.

The best descriptions available are subjective interpretations, like this panel of drawings of Pleurotus hymenia first published in 1982. We need an objective view of tissue structure.

The best descriptions available are subjective interpretations, like this panel of drawings of Pleurotus hymenia first published in 1982. We need an objective view of tissue structure.

We need to “get inside” a 3-dimensional chunk of tissue to measure growth directions, branching frequencies, branch directions and branch growth kinetics. And not just one chunk of tissue, but hundreds, thousands even, so that we have a library of such descriptions large enough to reveal underlying developmental strategies and show similarities and differences.

We need to “get inside” a 3-dimensional chunk of tissue to measure growth directions, branching frequencies, branch directions and branch growth kinetics. And not just one chunk of tissue, but hundreds, thousands even, so that we have a library of such descriptions large enough to reveal underlying developmental strategies and show similarities and differences.

This sequence of 4 ultrathin (0.5 µm thick) serial sections illustrates the problems with conventional sections. Namely, preparation artefacts, section orientation (to follow the same hyphal wall through successive sections they need to be oriented to an accuracy less than the thickness of the hyphal wall), and the loss of information within the section. As the wall might be only 100 to 200 nm thick, the wall can extend within the plane of a section so that hyphal profiles in serial sections lose their spatial relationships. The thinner the section, the more you need to reconstruct a worthwhile block of tissue, but the bigger the data set and data manipulation problem.

This sequence of 4 ultrathin (0.5 µm thick) serial sections illustrates the problems with conventional sections. Namely, preparation artefacts, section orientation (to follow the same hyphal wall through successive sections they need to be oriented to an accuracy less than the thickness of the hyphal wall), and the loss of information within the section. As the wall might be only 100 to 200 nm thick, the wall can extend within the plane of a section so that hyphal profiles in serial sections lose their spatial relationships. The thinner the section, the more you need to reconstruct a worthwhile block of tissue, but the bigger the data set and data manipulation problem.

The confocal microscope produces digitised optical sections of fresh tissue. Data sets obtained produce anaglyphs that give a good visual impression of depth and parallax, but these are not suitable for quantitative measurements.

The confocal microscope produces digitised optical sections of fresh tissue. Data sets obtained produce anaglyphs that give a good visual impression of depth and parallax, but these are not suitable for quantitative measurements.

The digitised images can be extracted and prototype 3D visualisations have been produced.

These are not just images in a 3-D rendition. They are computational models which approach the aim of being virtual tissue segments which can be explored - from any external direction and from within.

So the “virtual mushroom” is attainable. Of course, we’ve now run out of funding! Any suggestions?Any offers?!

The digitised images can be extracted and prototype 3D visualisations have been produced.

These are not just images in a 3-D rendition. They are computational models which approach the aim of being virtual tissue segments which can be explored - from any external direction and from within.

So the “virtual mushroom” is attainable. Of course, we’ve now run out of funding! Any suggestions?Any offers?!

This is close to being a median section of a cystidium in the hymenium of Coprinus cinereus

In the next section, two lobes have been sectioned, evidence of cell collapse during preparation.

And the next section has the walls from the bottom of the lobes within the section.

Visualisation from Coprinus stem transverse sections

Coprinus gills Coprinus gill

Pleurotus primordium Coprinus stem transverse section

We thank the British Council's UK/HK Joint Research Scheme for partially supporting this project. Mark Reeve thanks the British Mycological Society for support, and Carmen Sánchez thanks The Mexican National Council of Science and Technology (CONACYT) and the British Council for supporting her contributions to this study and the Research Centre for Biological Sciences (CICB) at the Universidad Autonoma de Tlaxcala for leave of absence.