A genetic melanotic neoplasm of Drosophila melanogaster

The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.     

Protein kinase B gene homologue pkbR1 performs one of its roles at first finger stage of Dictyostelium

Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB, which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form nonviable stalk cells. These results suggest that the mutant has defects in a system that changes the physiological dynamics in the prestalk cell region of a finger. We suggest that the arrest of its development is due to the loss of the second wave of expression of a protein kinase A catalytic subunit gene (pkaC) only in the prestalk region of the pkbR1 null mutant.     

Stalk cell differentiation without polyketides in the cellular slime mold

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells—the major part of the prestalk cells—are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.     

Monoclonal antibodies specific for stalk differentiation in Dictyostelium discoideum

We have produced two monoclonal antibodies specific to the stalk cells of Dictyostelium discoideum fruiting bodies. Both monoclonal antibodies react with high molecular weight proteins previously found to be stalk-specific by two-dimensional gel analysis. One antibody (JAb 1) is specific for a single protein of apparent molecular weight 310 000 which first appears when overt stalk differentiation begins at 20 h. The other monoclonal antibody (JAb 2) is also stalk-specific, though earlier in development it binds to proteins extracted from both prestalk and prespore cells of the migrating slug. It reacts with two proteins in stalks, one of apparent molecular weight 430 000 which is first detected during tip formation at 12 h and a lower molecular weight protein (310 000) detected from 20 h. Although several markers are available for the investigation of prespore/spore differentiation there is a distinct lack of suitable prestalk/stalk markers. The monoclonal antibodies described here are highly specific stalk markers and should prove useful in the study of cell proportioning and terminal differentiation.     

Induction of fruiting bodies in a macrocyst-forming mutant of the cellular slime mold, Dictyostelium mucomides

Some wild-type strains of Dictyostelium mucoroides exhibit dimorphism in development depending on culture conditions: on agar, fruiting bodies containing stalk and spore cells are formed, whereas under water, a thick-walled structure lacking spore and stalk cells (the macro-cyst) is formed. The mutant, MF-1, was derived from one of these wild-type strains. It forms macrocysts on an agar surfxe as well as under water. It was found that MF-1 could be induced to form fruiting bodies in two ways. First, when an aggregation center from the wild-type strain was grafted to an MF-1 aggregation center. MF-1 cells migrated to the center and formed a large aggregate that gave rise to many slugs that became fruiting bodies. This result, along with the observation that MF-1 aggregates have no tip, suggests that MF-1 normally produces an aggregation center that is unable to organize the aggregate to form a slug. Second, when MF-1 cells were allowed to develop on 1.2 mM ethionine (an analog of methionine), they formed aggregates with tips and developed into fruiting bodies with thick stalks instead of macrocysts. The effect of ethionine was blocked by the presence of 2.4 mM methio-nine. Two other methionine analogs were also tested, i.e., α-methylmethionine and norleucine. When cultured on the former at concentrations ranging from 1.2 to 9.6 mM, MF-1 cells still produced macrocysts; when cultured on norleucine at concentrations ranging from 2.4 to 9.6 mM, MF-1 cells aggregated into large clumps that formed numerous slugs, but these failed to continue development to fruiting bodies. In vertebrates, it is known that a major biochemical effect of ethionine is the inhibition of the methylation of nucleic acids, proteins, and phospholipids. Norleucine and a-methylmethionine inhibit methylation to a lesser extent. Thus, it can be speculated that the biological effects of ethionine on MF-1 cells may result from its interference with methylation reactions, suggesting that macrocyst formation may involve excess methylation as compared with the situation during fruiting-body development.     

Cross‐species functional complementation of cellulose synthase during the development of cellular slime molds

Cellulose is a major and important component of the extracellular matrix during the development of Dictyostelium discoideum. Upon starvation, solitary amoebae of D. discoideum gather and form fruiting bodies in which cells differentiate into stalk cells and spores. The stalk tubes and walls of spores and stalk cells are made of cellulose. In the genus Acytostelium, however, all cells are destined to become spores and the stalks comprise only a cellulose tube, suggesting species‐specific regulation of cellulose synthesis. In this study, we cloned a putative cellulose synthase gene (cesA) of Acytostelium subglobosum and performed comparative analyses with the D. discoideum cellulose synthase gene (dcsA). Although the deduced amino acid sequences were highly conserved between cesA and dcsA, the numbers of transmembrane spans preceding the catalytic domain were dissimilar; 2 and 3, respectively. Since ectopic expression of cesA in dcsA^?null cells failed to restore the developmental defects of the mutant, we constructed a series of chimerical genes for complementation analyses and found that the catalytic domain of cesA was functional in D. discoideum cells if the preceding transmembrane region was swapped with dcsA. The non‐functional products that contained the cesA‐derived transmembrane region were localized to lysosomes. These results indicate that the transmembrane region of cellulose synthase is essential for proper accumulation of cellulose during the development of D. discoideum and that its differential localization in A. subglobosum may be related to the characteristic morphogenesis in this species.     

Occurrence ofPseudomonas tolaasii on fruiting bodies ofLentinula edodes formed onQuercus logs

A bacterial disease occurred on fruiting bodies ofLentinula edodes that formed outdoors onQuercus bedlogs during winter. The pathogen was identified asPseudomonas tolaasii based on morphological and bacteriological characteristics. Symptoms exhibited by infected fruiting bodies ranged from mild browning to severe necrotic cavities that characteristically developed in the cap tissue along the periphery of the attachment area to the stalk. The mode of symptom development was greatly influenced by the internal tissue structure of fruiting bodies. Multiplication of bacterial cells within the fruiting bodies was strictly intercellular and thus differed from previously reported bacterial disease ofL. edodes incited by an unidentified rod-shaped bacterium. The present strain ofP. tolaasii was capable of attacking theL. edodes mycelium in the inner bark and outer sapwood regions and caused lysis of heavily infected hyphae.Paper No. 301 of the Tottori Mycological Institute.     

Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth

The reproducible pattern of organismal growth during metazoan development is the product of genetically controlled signaling pathways. Patterned activation of these pathways shapes developing organs and dictates overall organismal shape and size. Here, we show that patches of tissue that are mutant for the Drosophila Tsg101 ortholog, erupted, cause dramatic overproliferation of adjacent wild-type tissue. Tsg101 proteins function in endosomal sorting and are required to incorporate late endosomes into multivesicular bodies. Drosophila cells with impaired Tsg101 function show accumulation of the Notch receptor in intracellular compartments marked by the endosomal protein Hrs. This causes increased Notch-mediated signaling and ectopic expression of the Notch target gene unpaired (upd), which encodes the secreted ligand of the JAK-STAT pathway. Activation of JAK-STAT signaling in surrounding wild-type cells correlates with their overgrowth. These findings define a pathway by which changes in endocytic trafficking can regulate tissue growth in a non-cell-autonomous manner.     

Cell patterning in branched and unbranched fruiting bodies of the cellular slime mold Polysphondylium pallidum

Cell patterning, the percentage of spores and stalk cells, was measured in branched and unbranched asexual fruiting bodies of Polysphondylium pallidum. Unlike D. discoideum, where small and large fruiting bodies are more stalky than average-sized fruiting bodies, the overall cell patterning was the same in branched and unbranched fruiting bodies of all sizes in P. pallidum. Light greatly increased the numbers of fruiting bodies in P. pallidum per unit area (or decreased aggregation territory size) so that most fruiting bodies formed in the light were small and unbranched. By contrast, light had little effect on the cell patterning of P. pallidum, although there was a slight increase in the percentage of stalk cells in the light compared to the dark. This indicates that the mechanisms governing light sensitivity of aggregation territory size and cell patterning have different components in P. pallidum. The accuracy of cell patterning of individual branches of branched fruiting bodies was so imprecise as to leave doubt that patterning is occurring at the branch level. Individual whorls of branched fruiting bodies had a greater percentage spores (90%) than whole fruiting bodies (78%) and the cell patterning was relatively imprecise. Only in whole fruiting bodies was the spore:stalk ratio highly correlated. These findings are consistent with cell pattern determination operating at the whole aggregate level, rather than at the individual whorl or branch level in P. pallidum.     

Hydrophobins Sc3 and Sc4 gene expression in mounds, fruiting bodies and vegetative hyphae of Schizophyllum commune.

An abnormal growth form called mound has been hypothesized to be a neoplasm in the filamentous fungus Schizophyllum commune. An alternative hypothesis is that mounds represent some unusual developmental form in the fruiting body morphogenetic pathway. Hydrophobin proteins have been found in fruiting bodies where they line the surface of gas exchange pores and function to keep the pores hydrophobic. To further determine possible relationships between mounds and fruiting bodies, mound tissue was examined for gas exchange pores and the presence of hydrophobins. Cryoscanning electron microscopic images revealed the presence of channels in mound tissue and presumptive hydrophobin rodlets similar to the air channels in fruiting bodies. Hydrophobin gene expression was also measured in mound tissue using quantitative real-time PCR and showed both monokaryotic and dikaryotic mound tissue exhibited high expression of the dikaryotic specific Sc4 hydrophobin gene. In contrast, Sc4 hydrophobin expression was barely detectable in monokaryotic fruiting bodies. The expression of Sc4 hydrophobin genes in mounds suggests mound development uses this aspect of the dikaryotic fruiting developmental pathway.     

毛木耳‘昊阳黄背1号’的选育报告

‘昊阳黄背1号’是黄背木耳新品种,适宜工厂化液体菌种扩繁和农法固体菌种生产。该品种是从上海引进的SH0072号菌株,经组织分离纯化、系统选育而成。成熟新鲜子实体为单片簇生型,有明显耳基,耳片有明显荷叶边;鲜耳子实体背面和腹面灰褐色,背面有短绒毛。菌丝生长适宜温度16-25℃,出耳适宜温度15-32℃;原基分化至第一潮耳采摘41-55d,一般可采3潮耳,出耳转化率达20.6%;抗毛木耳柱霉。适宜四川省成都市、德阳市和简阳市等地及相似生态区棚栽,冬季制袋,春夏季出耳。     

Hydrophobins Sc3 and Sc4 gene expression in mounds, fruiting bodies and vegetative hyphae of Schizophyllum commune

An abnormal growth form called mound has been hypothesized to be a neoplasm in the filamentous fungus Schizophyllum commune. An alternative hypothesis is that mounds represent some unusual developmental form in the fruiting body morphogenetic pathway. Hydrophobin proteins have been found in fruiting bodies where they line the surface of gas exchange pores and function to keep the pores hydrophobic. To further determine possible relationships between mounds and fruiting bodies, mound tissue was examined for gas exchange pores and the presence of hydrophobins. Cryoscanning electron microscopic images revealed the presence of channels in mound tissue and presumptive hydrophobin rodlets similar to the air channels in fruiting bodies. Hydrophobin gene expression was also measured in mound tissue using quantitative real-time PCR and showed both monokaryotic and dikaryotic mound tissue exhibited high expression of the dikaryotic specific Sc4 hydrophobin gene. In contrast, Sc4 hydrophobin expression was barely detectable in monokaryotic fruiting bodies. The expression of Sc4 hydrophobin genes in mounds suggests mound development uses this aspect of the dikaryotic fruiting developmental pathway.     

Rapid stalk elongation in tulip (Tulipa gesneriana L. cv. Apeldoorn) and the combined action of cold-induced invertase and the water-channel protein γTIP

Many bulbous plants need a low-temperature treatment for flowering. Cold, for example, affects the elongation of the stalk, thereby influencing the quality of the cut flower. How the elongation of the stalk is promoted by cold and which physiological and biochemical mechanisms are involved have remained obscure. As invertase has been shown to be involved in the cold-induced elongation of the flower stalks of tulips (Lambrechts et al., 1994, Plant Physiol 104: 515–520), we further characterized this enzyme by cloning the cDNA and analysing its expression in various tissues of the tulip (Tulipa gesneriana L. cv. Apeldoorn) stalk. In addition, the role of sucrose synthase was investigated. Since turgor pressure is an important force driving cell elongation, the role of a water-channel protein (γTIP) was studied in relation to these two enzymes. The mRNA level of the invertase found was substantially up-regulated as a result of cold treatment. Analysis of the amino acid sequence of this invertase revealed the presence of a vacuolar targeting signal. Two different forms of sucrose synthase were found, the expression of one of them appeared to be restricted to the vascular tissue while the other form was present in the surrounding tissue. Both sucrose synthases were present in the stalk during the entire period of bulb storage and after planting, but their activities declined during stalk elongation. The expression of the γTIP gene was restricted mainly to the vascular tissue and its expression profile was identical to that of invertase. Simultaneous expression of invertase and γTIP possibly leads to an increase in osmotic potential and vacuolar water uptake, thus providing a driving force for stretching the stalk cells. Received: 12 January 1999 / Accepted: 24 March 1999     

Evolutionary dynamics of altruism and cheating among social amoebas

Dictyostelium discoideum is a eukaryotic amoeba, which, when starvation is imminent, aggregates to form fruiting bodies consisting of a stalk of reproductively dead cells that supports spores. Because different clones may be involved in such aggregations, cheater strategies may emerge that allocate a smaller fraction of cells to stalk formation, thus gaining a reproductive advantage. In this paper, we model the evolutionary dynamics of allocation strategies in Dictyostelium under the realistic assumption that the number of clones involved in aggregations follows a random distribution. By determining the full course of evolutionary dynamics, we show that evolutionary branching in allocation strategies may occur, resulting in dimorphic populations that produce stalkless and stalked fruiting bodies. We also demonstrate that such dimorphisms are more likely to emerge when the variation in the number of clones involved in aggregations is large.     

GSK3 is a multifunctional regulator of Dictyostelium development

Glycogen synthase kinase 3 (GSK3) is a central regulator of metazoan development and the Dictyostelium GSK3 homologue, GskA, also controls cellular differentiation. The originally derived gskA-null mutant exhibits a severe pattern formation defect. It forms very large numbers of pre-basal disc cells at the expense of the prespore population. This defect arises early during multicellular development, making it impossible to examine later functions of GskA. We report the analysis of a gskA-null mutant, generated in a different parental strain, that proceeds through development to form mature fruiting bodies. In this strain, Ax2/gskA-, early development is accelerated and slug migration greatly curtailed. In a monolayer assay of stalk cell formation, the Ax2/gskA- strain is hypersensitive to the stalk cell-inducing action of DIF-1 but largely refractory to the repressive effect exerted by extracellular cAMP. During normal development, apically situated prestalk cells express the ecmB gene just as they commit themselves to stalk cell differentiation. In the Ax2/gskA- mutant, ecmB is expressed throughout the prestalk region of the slug, suggesting that GskA forms part of the repressive signalling pathway that prevents premature commitment to stalk cell differentiation. GskA may also play an inductive developmental role, because microarray analysis identifies a large gene family, the 2C family, that require gskA for optimal expression. These observations show that GskA functions throughout Dictyostelium development, to regulate several key aspects of cellular patterning.     

Relation between the level of endogenous growth regulators and the differentiation of the fungusLentinus tigrinus studied in a synthetic medium

The fungusLentinus tigrinus was cultivated in a synthetic medium. During vegetative growth of mycelium and differentiation of fruiting bodies samples serving for determination of endogenous growth regulators were collected. The material was extracted with methanol, separated by chromatography and the isolated fractions were tested. Substances of auxin type were assayed with coleoptile segments of oat plants, gibberellins by measuring the hypocotyl growth in the lettuce and cytokinins from the degree of chlorophyll decomposition in the leaf segments of barley. Very low content of auxins was found in all phases of growth and differentiation of the fungus. The content of gibberellin substances was relatively high during the whole life of the fungus. The highest content is found in the vegetative mycelium and in the differentiated fruiting body, especially in the cap; it decreases during the differentiation of fruiting bodies. The content of cytokinins is equally high and the pattern of its change resembles that of the gibberellins. Its maximum is reached in young vegetative mycelium, it decreases when fruiting bodies are formed and when they are growing. Minimum is reached in the differentiated stalk, where also the amount of inhibitions is the highest. Experimental results show that during differentiation of fruiting bodies and their morphogenesis not only gibberellins and cytokinins are of importance, but also substances of an inhibitory character. A relatively high amounts of these substances was secreted into the cultivation medium. In a synthetic nutrient solution they may be easily demonstrated.     

The HfaB and HfaD adhesion proteins of Caulobacter crescentus are localized in the stalk

The differentiating bacterium Caulobacter crescentus produces two different cell types at each cell division, a motile swarmer cell and an adhesive stalked cell. The stalked cell harbours a stalk, a thin cylindrical extension of the cell surface. The tip of the stalk is decorated with a holdfast, an adhesive organelle composed at least in part of polysaccharides. The synthesis of the stalk and holdfast occur at the same pole during swarmer cell differentiation. Mutations in the hfaABDC gene cluster had been shown to disrupt the attachment of the holdfast to the tip of the stalk, but the role of individual genes was unknown. We used lacZ fusions of various DNA fragments from the hfaABDC region to show that these genes form an operon. In order to analyse the relative contribution of the different genes to holdfast attachment, mutations were constructed for each gene. hfaC was not required for holdfast attachment or binding to surfaces. The hfaA and hfaD mutants shed some holdfast material into the surrounding medium and were partially deficient in binding to surfaces. Unlike hfaA and hfaB mutants, hfaD mutants were still able to form rosettes efficiently. Cells with insertions in hfaB were unable to bind to surfaces, and lectin binding studies indicated that the hfaB mutants had the strongest holdfast shedding phenotype. We determined that HfaB and HfaD are membrane-associated proteins and that HfaB is a lipoprotein. Purification of stalks and cell bodies indicated that both HfaB and HfaD are enriched in the stalk as compared to the cell body. These results suggest that HfaB and HfaD, and probably HfaA, serve to anchor the holdfast to the tip of the stalk.     

Altruism, cheating, and anticheater adaptations in cellular slime molds

Cellular slime molds (CSMs) possess a remarkable life cycle that encompasses an extreme act of altruism. CSM cells live as individual amoebae until starved, then aggregate and ultimately transform themselves into a multicellular fruiting body. This fruiting body consists of stalk cells (altruists that eventually die) and spores (the beneficiaries of this sacrifice). Altruistic systems such as this are vulnerable to cheaters, which are individuals unrelated to the altruists that obtain the benefits provided by them without reciprocating. Here, we investigate two forces that can maintain CSM altruism despite cheating: kin selection and anticheater adaptations. First, we present new kinship-based models based on CSM developmental biology to evaluate the efficacy of kin selection. These models show that stalk-making genotypes can still be maintained when aggregations are initiated by multiple "founder" spores, provided that spores of stalkless fruiting bodies have low rates of dispersal and dispersal success is a concave function of stalk height. Second, we review proposals that several features of CSM development, such as the chemical suppression of the redifferentiation of prestalk cells into prespores, act as anticheater adaptations.     

Morphogenetic movements and multicellular development in the fruiting Myxobacterium, Stigmatella aurantiaca.

Stigmatella aurantiaca, strain DW-4, is a bacterium that grows as single cells in liquid culture but will synchronously aggregate and construct multicellular fruiting bodies when starved on an agar surface. The fruiting body consists of a stalk and several sporangia housing differentiated myxospores. Fruiting body development is stimulated by exposure of the aggregating cells to incandescent light.