12.14 Classic genetic approaches to study development and the impact of genomic data mining
The suggestion that there are several fundamentally different ways in which cell inflation and tissue expansion can be achieved is reinforced by work done on the induction of developmental variants. This classic genetic approach to the study of any pathway depends on:
- identification of variant strains;
- complementation tests to establish functional cistrons;
- comparison of heterokaryon phenotypes to determine dominance;
- determination of epistatic relationships in heterokaryons, to indicate the sequence of gene expression (Moore & Novak Frazer, 2002).
The modern molecular approach is to use genomic analysis tools (see Chapter 18) to analyse and compare the transcriptome (transcribed RNA population) and/or proteome (translated polypeptide population) to give expression profiles of different stages as the organism progresses through its developmental sequence. For example, Chum et al. (2008) used serial analysis of gene expression to determine the gene expression profiles of dikaryotic mycelium and primordia of Lentinula edodes to show that a specific set of genes is required for fruit body development. The transition from mycelium to primordium involving abundant expression of hydrophobins, genes involved in intracellular trafficking, and those contributing to stress responses.
Molecular analysis provides an overview of the complex network of gene regulatory events that are involved in a developmental process, but it can be difficult to focus on the unique decisive events that characterise (and/or initiate) the process. The classic approach (‘first, find a variant’) immediately focuses attention on a decisive event, though it can then be difficult to identify the network to which this belongs. However, it is easier to start our description from this approach.
When applied to Coprinopsis cinerea, the classic genetic approach resulted in the isolation of, among others, mutants unable to elongate the stem but with normal cap expansion, or unable to expand the cap but with normal stem elongation (Takemaru & Kamada, 1971, 1972) (Fig. 26).
|Fig. 26. Developmental mutants of the Ink-cap mushroom, Coprinopsis cinerea. Shown from left to right are A and B the wild type (A mid-development, B fully mature and autolysing), and then C sporeless, D cap expansionless, and E stipe elongationless (immature at top, fully mature and autolysing below). The three mutants are all dominant and segregate in crosses as single genes. All cultures are contained in 9 cm diameter crystallising dishes. Photographs by Dr Junxia Ji.|
Evidently, the pathways of cap and stem differentiation can be totally separated, and we conclude that assembly of different parts of the same fruit body uses genetically distinct pathways. Differentiation of fungal multicellular structures is genetically compartmentalised and normal morphogenesis is made up of several (to many) developmental subroutines. For example, there may be subroutines for stem, cap, hymenium, hymenophore, etc. The subroutines can be put into operation independently of one another and are under separate genetic control and under separate physiological control. Normal morphogenesis of a specific fungus involves integration of these subroutines to produce the fruit body morphology that characterises that species. The same subroutines integrated in a different way will generate a different morphology characteristic for some other species.
Another important point is that fungi are very tolerant of developmental imprecision. Even if a grossly abnormal fruit body is produced it can still produce and distribute spores. Variation in the morphology of fruit bodies of higher fungi has been reported in many species, often appearing to be a strategy for adaptation to environmental stress. Detailed analysis of such developmental plasticity in spontaneous abnormal fruit bodies of Volvariella bombycina has given more evidence for normal morphogenesis being an assemblage of distinct developmental segments (Chiu, Moore & Chang, 1988).
Although the fruiting structures observed varied from the normal agaric form to completely abnormal enclosed, puff-ball like structures (Fig. 27), they were all actually or potentially functional as meiospore production/dispersal structures. These observations suggest again that normal fruit body development comprises a sequence of independent but co-ordinated morphogenetic subroutines, each of which can be activated or repressed as a complete entity. For example, the ‘hymenium subroutine’ in an agaric is normally invoked to form the ‘epidermal’ layer of the gill; the ‘hymenophore subroutine’ producing the classic agaric gill plates. The fact that abnormal fruit bodies consist of recognisable tissues suggests that those tissues are defined by independent developmental programmes. Thus a fruit body may, quite abnormally, bear a functional hymenium on the upper surface of the cap, rather than (or in addition to) the hymenium on the lower surface. The important point is that it is a functional and recognisable tissue, not a tumorous growth, which has been produced by correct execution of a morphogenetic subroutine which has been invoked in the wrong place.
|Fig. 27. Polymorphic fruit bodies of Volvariella bombycina. A is the normal morphology, of a typical agaric mushroom, which is characterised by a well-developed enclosing volva through which the mushroom cap emerges during development (bulbangiocarpic development, see Fig. 12.7); (scale bar = 20 mm) B to G illustrate a few of the polymorphisms which have arisen on otherwise normal cultures, though often seeming to be associated with some environmental stress, such as abnormal temperature and/or desiccation. B is apparently normal, but completely lacks the volva despite this being characteristic of the genus (scale bar = 5 mm). C, a gymnocarpous fruit body (see Fig. 12.3) with a grossly enlarged basal volva (scale bar = 2 mm). D, a fruit body with a sinuous extra hymenium formed by the proliferating margin of the cap curling onto the upper surface of the cap (scale bar = 10 mm); E is a scanning electron microscope (SEM) view of specimen D and shows the well developed gills on the upper surface of the cap (scale bar = 1 mm). F, a fruit body in which hymenium has formed all over the outer surface of the club-shaped fruit body (scale bar = 5 mm); G is an SEM view of the bisected fruit body and shows that the labyrinthine hymenophore covers the entire surface of the club-shaped ‘pileus’ (scale bar = 0.5 mm). This polymorph is called ‘morchelloid’ because of its resemblance to the normal fruit body of the morel (Morchella), which, of course, is a member of the Ascomycota! Photomicrographs by Prof. S.-W. Chiu, Chinese University of Hong Kong. Illustration modified from Chiu, Moore & Chang, 1988.|
This tolerance of imprecision is an important attribute of fungal morphogenesis, as it provides the flexibility in expression of developmental subroutines that allows the fruit body to react to adverse conditions and still produce a crop of spores. Even when conditions are adverse to normal development, there is still sufficient flexibility in the developmental programme for the fruit body to fulfil its function.
The possibility has been discussed that fungal differentiation pathways exhibit what would be described as ‘fuzzy logic’ in cybernetic programming terms (Moore, 1998b). Instead of viewing fungal cell differentiation as involving individual major (yes/no) ‘decisions’ which switch progress between alternative developmental pathways that lead inevitably to specific combinations of features, this idea suggests that the end point in fungal differentiation depends on the balance of a network of minor ‘approximations’. Fuzzy logic is an extension of conventional (Boolean) logic that can handle the concept of partial truth, that is truth-values between ‘completely true’ and ‘completely false’. It is the logic underlying modes of decision-making that are approximate rather than exact, being able to handle uncertainty and vagueness and has been applied to a wide variety of problems.
Decision-making in the real world is characterised by the need to process incomplete, imprecise, vague or uncertain information; the sort of information provided by error prone sensors, inadequate feed-back due to losses in transmission, excessive noise, etc (in real life these are the ‘best guesses on the basis of current information’ that we deal with every day). The technical importance of fuzzy logic derives from the fact that the theory provides a mathematical basis for understanding how decision-making seems to operate generally in nature (Zadeh, 1996; Leondes, 1999).
The outcome is that fungal cells are allowed the flexibility to assume a differentiation state even when all conditions of that state have not yet been met. In other words, developmental decisions between pathways of differentiation are able to cope with a degree of uncertainty. So the conclusion is that fungal differentiation pathways must be based on application of rules that allow considerable latitude in expression (fuzzy constraints), which in the ultimate can lead to highly polymorphic, yet functional, fruit bodies.
One consequence of this line of argument is that patterning genes may not be necessary to develop the patterns of a mushroom fruit body. Rather, as we have described, the patterns may arise from the regular application of simple, and fuzzy, developmental rules and basic metabolic regulation combined with physical forces (stretching, inflation, expansion). A specific gill pattern in an agaric, say, may not require a panel of regulatory genes that specify ‘x number of gills per unit volume of cap’ because that pattern ‘invariably’ results from the application of general rules about the local circumstances within the tissue that allow gills to be made and the subsequent set of circumstances that drive fruit body maturation.
The interpretation of fruit body morphogenesis as resulting from expression of a series of essentially self contained segments of a developmental programme could have wide ranging significance. The sequence in which the segments are put into effect would determine the overall form of the fruit body so the view could, consequently, provide a natural means of formalising taxonomic and evolutionary relationships. More mechanistically, such a segmented developmental programme may be supposed to have its genetic control organised into a similar logical hierarchy and if properly supported this could suggest comparisons with developmental systems in other organisms expressing a classically segmentalised body form. It is clear that similarities do exist between the developmental mechanisms of animals, plants and fungi. Equally, there is no doubt that, in all three groups, multicellularity evolved long after the three evolutionary lines had diverged into their characteristic and totally separate modes of form, structure and behaviour. Thus, little, if any at all, of the organisation which permits multicellular morphogenesis could have been possessed by any common ancestor.
The parallels which do seem to exist in the basic regulation of morphogenesis in plants, animals and fungi imply that the rules which govern morphogenesis are natural laws owing more to the physical and chemical phenomena involved than to the biological entities that respond to them.
Attempts have been made to extend the comparison between plants, animals and fungi using sequence searches of genomic databases. An initial comparison searched genomes of several Basidiomycota and Ascomycota for homologues of sequences of the animal signalling mechanisms known as Wnt, Hedgehog, Notch and TGF-α, all of which are considered to be essential and highly conserved components of normal development in animals. All were absent from the fungal genomes, just as they are absent from plants (Moore, Walsh & Robson, 2005). Subsequently, a fully comprehensive data-mining exercise searched for homologies to all developmental sequences in all sequenced genomes, animal, fungal and plant (Moore & Meškauskas, 2006). These experiments are discussed in more detail in Chapter 18 (CLICK HERE to view now), but the outcome was that homology between fungi and other eukaryotes was limited to 78 sequences involved in the architecture of the eukaryotic cell. This survey demonstrated that there are no Wnt, Hedgehog, Notch, TGF, p53, SINA, or NAM sequences in fungi, leading to the conclusion that the unique cell biology of filamentous fungi has caused control of their multicellular development to evolve in a radically different fashion from that in animals and plants.
There are presumably developmental processes in fungal multicellular structures that are analogous or homologous to those occurring in animals or plants that need to be regulated. Unfortunately, the demonstration that developmental control sequences of animals and plants lack fungal homologues leaves us knowing nothing about the molecules that do govern multicellular development in fungi. And that, unfortunately, remains the situation at the time of writing: we are ignorant of the basic control processes of fungal developmental biology; we are ignorant of the molecules and mechanisms that generate fungal multicellular structures (Moore et al., 2007).
Updated December 17, 2016