Trans. Br. mycol. Soc. 84 (4), 621-628 (1985)

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Printed in Great Britain

DIFFERENTIATION OF THE HYMENIUM INCOPRINUS CINEREUS

By ISABELLE V. ROSIN AND DAVID MOOREDepartment of Botany, The University, Manchester MIl 9PL

When first formed the hymenium of Coprinus cinereus consists of protobasidia and a scatteringof cystidia. The latter arise as the terminal compartments of little-branched tramal hyphae.Paraphyses originate as branches of sub-basidial cells and insert into the basidial layer . About75 % of the paraphyseal population inserted as meiosis was completed, the rest at later stagesof development. Basidial numbers did not increase once paraphyseal insertion commenced.

A fully differentiated fruit body cap of Coprinuscinereus (Schaeff.: Fr.) S. F. Gray sensu Konr.consists ofa thin layer ofpileal flesh (the pileipellis)bounded exteriorly by the veil cells and with thegills suspended on the inner side. The gills developparallel to one another and retain this arrangementthroughout development. The gill surface is thehymenium; it is composed of three highlydifferentiated cell types, basidia, cystidia andparaphyses, all of which arise from the central layerof the gill, the trama, the constituent cells of whichmaintain a basically hyphal structure.

Although considerable interest has centred onbasidial and cystidial structure for taxonomic andphylogenetic purposes (Smith, 1966; Oberwinkler,1982) and in studies of basidiosporogenesis(McLaughlin, 1982; Thielke, 1982), there has beenno extensive treatment of the hymenium as adifferentiating tissue. It is, nevertheless, a highlyorganized morphogenetic system which presentsmany of the development problems more usuallyassociated with higher organisms (Wolpert, 1969;Barlow & Carr, 1984; Moore, 1984a). Theseproblems relate to the ways in which cells, of thesame genetic constitution, differentiate in a develop-ing tissue in particular patterns that imply somesort of positional control extending over the tissueas a whole. Understanding of such regulation ofpattern formation will depend on determination ofbiochemical events governing differentiation andsubsequently the orchestration of control of thebiochemistry over large populations of spatially-scattered cells. Description of some biochemicalevents associated with hymenium development inC. cinereus has reached an advanced stage (Ewaze,Moore & Stewart, 1978; Moore, Elhiti & Butler,1979; Moore, 1981), and some attempt has beenmade to relate findings derived from work withtissue homogenates to cytochemically-observableprocesses in individual cells (Elhiti, Butler &Moore, 1979) and to extend the analysis to the

polynucleotide level (Yashar & Pukkila, 1985). Animportant point, though, is that hymenial tissue isa fungal tissue and is a derivative of the hyphalgrade of organization, the characteristics of whichmay well impose restrictions on the applicability oftheoretical models derived from other organisms.

An essential prerequisite for study of any tissueis a knowledge of its exact structure and develop-ment. Although fungal fruiting body structure hasbeen studied for many years, and often from adevelopmental point of view, the publishedinformation is inadequate to establish the nature ofcell-to-cell relationships in the detail which isrequired in morphogenetic analysis. In this paperwe describe the structure of the developinghymenium of C. cinereus, with emphasis onstructural, developmental and geometrical cellrelationships.

MATERIALS AND METHODS

The dikaryon, culture conditions and preparativetechniques described by Rosin & Moore (1985)were employed.

RESULTS AND DISCUSSION

The young hymenium consisted of a palisade oftightly packed club-shaped cells with a scatteringof much larger, highly inflated cells (Fig. 1). Thelatter are presumed to be developing cystidiathough on the basis of these images there is no wayof categorising the former either as basidia orparaphyses. However, two lines of evidenceindicate that in these young primordia (representingStage 1,2-6 mm in height, and Stage 2, 6-9mm inheight) the hymenium is largely composed ofdifferentiating basidia. The first is that, as we shallshow below, paraphyses were observed to appearlater and were inserted into the basidial layer ; thesecond is that in studies of meiosis (which occurs

622 Differentiation in Coprinus cinereus

Fig. 1. Protohymenium which has just differentiated from the protenchymal tissue of the cap otthe G. cinereusfruit body. Scale bar = 5/lm.

during Stage 2) Raju & Lu (1970) showed thatmeiotic figures could be demonstrated in virtuallyall of the hymenial cells, an observation which hasrecently been confirmed (Pukkila, pers. comm.),We will therefore refer to these cells as basidia sinceonly this cell type is expected to undergokaryogamy and meiosis.

Basidia originated as slightly swollen chromo-philic cells at the apex of protenchyme (tramal)hyphae. This apical differentiation follows thepattern of most basidiomycetes (Sundberg, 1978;Oberwinkler, 1982) but McLaughlin (1982) empha-sised the ultrastructural differences between thedeveloping basidium and the growing vegetativehyphal apex, especially the lack of apical vesicles inthe basidium which he suggested may indicate aslow growth rate. In C. cinereus basidia were theterminal cells of branches which curved out fromthe generally parallel tramal hyphae. During theearly stages of hymenium development it wasnoticeable that adjacent basidia arose at the apex ofsister branches of parental tramal hyphae. This isthe normal mode of basidium proliferation inagarics (Oberwinkler, 1982) and contrasts withproliferation at sub-basidial clamp connexionsdescribed in Schizophyllum commune (N ieder-

pruem, }ersild & Lane, 1971; Niederpruem &jersild, 1972).

Cystidia arose from tramal hyphae whichbranched relatively much less than those subtendingbasidia (Fig. 2). From the earliest stage cystidiainserted into the opposite hymenium. Cystidiastretched considerably when two gills were pulledapart, indicating a firm attachment. Reijnders(1963 ) gives an account of the nomenclature andstructure of these cells.

Towards Stage 2, cystidia and the apices ofbasidia enlarged, The expansion of basidial apiceswas often associated with basidiocarps in which thehyphae ofthe trama, subhymenium and hymenium,showed similar expansion (Oberwinkler, 1982).Paraphyses developed as outgrowths ofsub-basidialcells during Stage 2. During Stage 1 basidia werethe most chromophilic cells of the gill but at Stage2 the most chromophilic region was in thesubhymenium corresponding in position to theemerging paraphyses. This distribution appears toreflect the actively growing zone of the lamellae. InC. micaceus and C. lagopus, Chow (1934) observeda similar chromophilic pattern but concluded thatbasidia grew from the subhymenium and into alayer of paraphyses. We do not agree with this

Isabelle V. Rosin and D. Moore

Fig. 2. Differentiatinghymeniumof C. cinereus. Note that the largecystidialcellsare terminal compartmentsof tramal hyphae.The hymeniumat this stageconsistsof a closely packedlayerof basidia,though the highlychromophilicparaphyseal branchesfromthe sub-basidialcellsare becoming evident. Scalebars: A = 20 pm,B = 5 pm.

interpretation. The strongly chromophilic regionin the C. cinereus subhymenium was composed ofbranches communicating between the trama andhymenium and of small, rather oblong, cells(Fig. 2). These latter cells (which are youngparaphyses) arose as branches from the sub-basidialcells and then inserted between the basidia (Fig. 3).By Stage 3 (immature fruit body over 10 mm inheight, postmeiotic) these cells, still highly chromo-philic, had expanded and taken on the characteristicrectangular profile of paraphyses (Fig. 4). By thisstage the number ofparaphyses had increased to thepoint where no two basidia were adjacent.

The most important points arising from theseobservations are that when first formed thehymenium consists of cells destined to becomebasidia or cystidia. The latter an: fewer in numberand are the apical cells of tramal hyphae whichbranch much less frequently than the majority.

Only more detailed analysis of serial sections at theEM level will show whether 'cystidial tramalhyphae' constitute a hyphal population distinctfrom 'basidial tramal hyphae', but on the basis ofpresent observations that possibility certainlyexists. The final point is that paraphyses arise asbranches from sub-basidial cells and insert into thebasidial cell layer. Formation of paraphysealbranches appears to occur at about the time thatmeiosis is taking place.

In primordia of Stages 1-2 the gill hymeniumconsisted essentially of a cell layer of basidia andwas about 100 pm wide at the cap margin. Atmaturity, Stage 5, the hymenium was composed ofbasidia embedded in a pavement of paraphyses andthe gill was about t :5-2 mm wide at the cap margin.Observations described above showed that para-physes were inserted into the hymenium layer atabout Stage 2/3. At this time about 100 cells

624 Differentiation in Coprinus cinereus

I..

BFig. 3. Insertion of paraphyses (the most chromophilic cells) into the basidial layer of the Coprinus hymenium.

Scale bars = 5 pm.

Isabelle V. Rosin and D. Moore

CFig. 4. Expansion of the mserted paraphyses to torm tne characteristic paraphyseal pavement of the Coprinus

hymenium. Scale bars = 10 Jim.

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Differentiation in Coprinus cinereus

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. .Fig. 5. Surface view of the almost mature hymenium of a sporeless mutant of C. cinereus to show the patternsof paraphyseal boundaries and entrapped basidia (ba), Nomarski interference optics. Scale bars: A = 75 flm,B = 25 flm.

Isabelle V. Rosin and D. Moore

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Number of sidesFig. 6. Comparison between cell size(as maximum diam)and polygonalgrade during development of the Coprinushymenium. Plot showsa regressionanalysisfor basidia ofStage 3 primordia (D) and Stage 4 immature fruit bodies(.), and paraphyses of Stage 3 (0) and Stage 4 (e).

we are assuming that the insertion of cells(specifically paraphyses) into a cell layer is formallyequivalent to cell division within the layer.Geometrically this seems to be valid providing theargument does not depend on constraints on theplane of division. It is clear that theoretical aspectsof fungal tissue construction (and especially theunique considerations arising from the hyphalorigin ofthe cells and their characteristic transversedividing septum) have so far been ignored. Weintend to return to this theme in a later paper. Theinherent property of a mosaic of cells is that, in asystem of linked polygons, the average number ofcell sides is exactly six, and that the area of a cellis proportional to the number of sides of the cell(Lewis, 1943). The nature of this proportionalitycan identify different cell populations. Measure-ments were made of maximum cell diameter(replacing area) and the number of sides for 50randomly chosen basidia and paraphyses inphotographs of the hymenium surface (Fig. 5) offruit bodies at Stages 3 and 5. A computer-generatedunweighted regression of the plot (Fig. 6) showsthat basidia and paraphyses fall into two distinctpopulations. Basidial diameter remained approxi-mately constant irrespective of the number of sides,while for paraphyses the number of sides increasedwith size.

The failure of many-sided cells to attain the sizerequired to comply with the expected progressionhas been explained in terms of the division of cellsaround a non-dividing cell (Lewis, 1943). In thecontext of our present knowledge of the ontogenyof the hymenium in C. cinereus, the analogousstatement for this tissue would be that additionalparaphyses insert around the basidia. Thus thegeometrical analysis of basidia and paraphysesreinforces the view that the basidial population isnumerically static. Only paraphyses insert into thehymenial layer after its initial differentiation fromthe protenchyma (Rosin & Moore, 1985) .

The key features which appear to determine cellpatterning in the Coprinus hymenium are thereforethe establishment of a cell layer comprised ofbasidia with a scattering of cystidia, and cessationof basidial growth which seems to remove aconstraint on branching and lateral proliferation ofthe sub-basidial cells so that paraphyseal branchformation can take place. This latter is accompaniedby an accumulation of carbohydrate in thesub-basidial cells as these become the active growthzone of the hymenium. These processes seem torequire three major controls: specification of thedistribution of cystidia; determination ofthe apicaldifferentiation oftramal hyphae into basidia and, asa consequence of basidial differentiation, the thirdcontrol sequence dictates differentiation of sub-

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inserted into the gill transect and the observed gillwidth was 300-400 pm. The average width of theinserted paraphyses was 3-4 pm. At Stage 4,approx. 140 paraphyses were counted along gilltransects and they had an average width of 8 pm.Thus, although the main production of paraphysesoccurred at the end of Stage 2, about 25 % of thepopulation was formed and inserted during thelater stages of maturation. Insertion and expansionof paraphyses was sufficient to account for theentire increase in gill width. Increase in gill lengthwill, of course, depend on the same factors but isfurther dependent on continued differentiation ofgill tissues from the protenchyma at the apex of thecap (Rosin & Moore, 1985). Paraphyseal enlarge-ment proceeded continuously during maturation.Increase in gill area is clearly dependent on anenormous increase in the constituent cell volumes.Paraphyses expand as much as two and a half timestheir original volume. The ingress of water into thehymenium elements leads to considerable vacuola-tion as the cells expand (Moore et al., 1979). Theexact identity of an osmoticum has not beenestablished, but amplification ofurea cycle activity,which occurs specifically in the cap tissue, probablycontributes to the osmotic influx of water (Ewazeet al., 1978; Moore, 1984b).

The distribution of paraphyses and basidia hasbeen analysed geometrically using a method basedon that of Lewis (1931, 1943). The principles forgeometrical analysis of tissues have been derivedfor layers of dividing cells, in applying them here

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(Received for publication ]1 August 1984)