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We are studying fundamental processes that are found in nearly all eukaryotes, but which may be more readily explored in filamentous fungi. Why fungi? Certainly fungi are, according to DNA and RNA studies, closer in evolutionary history to animals than to plants and one of the reasons that fungal diseases of humans are so hard to cure is that the physiology of the fungus and its host are so similar. However, there are two fundamental differences between animal/plant development and fungal development that make fungi unique organisms to study. First, in animals and plants, cell differentiation leading to embryonic development is constitutive and follows a well programmed series of events without further input from the outside after fertilization. However, in the fungi that form complex three-D structures (e.g. mushrooms), development may be delayed indefinitely until some external trigger initiates the developmental process. During this time there may be a long phase of continuous vegetative growth of cells that are essentially repressed in terms of developmental potential. Second, the basic unit of animal/plant development is a uninucleate, diploid cell that can proliferate in three planes to form tissue masses. Furthermore, the appearance of new tissue types in the developing embryo can be traced to the initial differentiation of single cells. To quote Amon (Cell, 1996): "The underlying principle of every developmental program is that single cells divide to give rise to daughter cells with different developmental fates. This is true not only for the fertilized egg that develops into an adult, but also for the yeast cell that buds." In other words, asymmetric cell division is essential for normal development. But the basic unit of most fungal development is a filamentous strand composed of linear compartments each with one or more haploid nuclei. Such filaments cannot proliferate in three planes to form tissue masses, but can form 3-dimensional multicellular, multi-tissue structures only by branching and filament aggregation - a very different morphogenetic procedure, especially when it is realized that the entire filamentous organism is essentially an open plumbing system. Where then, in this situation, is the ability for single-cell differentiation? It is this question of how fungi regulate their 3-dimensional differentiation that is the main focus of my lab and to do this we ask questions concerning the initiation of morphogenesis.

Life on a world exposed to diurnal fluctuations in light has resulted in the evolution of a plethora of mechanisms governing the response of organisms to light. One set of these, the blue light responses, is found in one form or another in organisms ranging from slime molds to humans, affecting processes as diverse as mating behavior and jet lag recovery. Our experimental fungus has an obligate requirement for a blue light signal in order to initiate differentiation. Our laboratory has identified a heterotrimeric GTP-binding protein as a member of the signal transduction pathway of blue light reception in a higher filamentous fungus, the first report of a G protein being involved in blue light responses (Fig. 1). This G protein is very similar to transducin, the G protein involved in visual light reception. A logical extension of the blue light work has been to identify genes that are specifically activated by the blue light reception. In the majority of light-induced systems, a very large number of genes affecting several different biochemical systems are turned on at once, making identification of the nature and function of any particular gene very difficult. Our experimental fungus was selected because it requires two successive light exposures in order to trigger a morphogenetic response and the first light exposure activates only a very few genes. We used the mRNA differential display technique to identify these genes and are currently engaged in characterizing two genes specifically regulated by blue light. One of these, bli2, is believed to be a major regulatory gene of subsequent development. Fig. 2 is a developmental Northern blot showing the timing of the expression of bli2 after the first blue light reception. We are currently involved in isolating the complete gene from our genomic libraries and exploring the role of this gene in regulating development.

In addition, we have recently resurrected an old hypothesis concerning the role of mitochondria in differentiation and aging. Some years ago I proposed to various granting agencies that the mitochondria, the energy producing organelles in all our cells, had mechanisms for affecting both the development of the cells they were in and also of affecting the expression of genes that resided in the nuclei of those cells. This proposal was not in accord with the then received wisdom and was placed on the back burner. But now the paradigm has changed as noted in the lead editorial in Science Mar 5 1999: "Mitochondria Make A Comeback." Consequently we are again working on the hypotheses - Do mitochondria affect nuclear gene expression? The main hypothesis is that the mitochondria in a cell are capable of influencing the expression of specific nuclear genes and that there is a long-standing evolutionary relationship between the mitochondria of a cell and one of the parental genomes. We are utilizing the unique mating behavior of higher filamentous fungi in which nuclei are exchanged but mitochondria are not. This results in the formation of two functional diploid entities with identical nuclear genomes residing in different mitochondrial backgrounds and is an ideal situation in which to explore differences in gene expression among genes involved in mitochondrial activity that require the cooperation of proteins coded for by both nuclear and mitochondrial genomes.

Fig. 1. Two-dimensional non-denaturing gels of GTP-binding proteins from Coprinus congregatus pulsed with [35S] GTPgS in the absence of light (A) and in the presence (B) of light.

Fig. 2. Northern analysis of LC2-2, at indicated minutes after light reception. There is no expression in the dark (D), and expression does not begin until 90 minutes after light reception.