Transcriptional Transitions during Dictyostelium Spore Germination

Strains and spore production.D. discoideum AX4 cells (27) were maintained on SM nutrient agar plates in association with Klebsiella aerogenes (38). For spore collection, AX4 spores were mixed with a suspension of K. aerogenes in water and spread on nutrient agar plates made with SM/2 (27.8 mM glucose, 5 g of Bacto Peptone [Difco]/liter, 0.5 g of Bacto Yeast Extract [Difco]/liter, 4.0 mM magnesium sulfate, 17.4 mM potassium phosphate [pH 6.2]). Spores were collected by passage of wet microscope slides over the tops of the sori 2 days after fruiting bodies appeared. Spores were resuspended and washed twice with KP buffer (10 mM potassium phosphate [pH 6.5]).

Mutant strains were generated by random restriction enzyme-mediated integration (REMI) (28) at the Dictyostelium Functional Genomics Project at Baylor College of Medicine (A. Kuspa and G. Shaulsky, unpublished data), at which the mutated genes also were cloned and sequenced.

Spore germination.For heat activation, spores were resuspended in KP buffer in glass test tubes and incubated in a heated water bath at 42°C for 30 min. The samples were removed from the water bath and adjusted to a density of 5 × 10⁷ spores ml⁻¹ with KP buffer at room temperature (time, 0 h). For dimethyl sulfoxide (DMSO) activation, spores were resuspended in 20% (vol/vol) DMSO in KP buffer in glass test tubes and incubated at room temperature for 1 h with occasional agitation. The spores were pelleted by centrifugation, the DMSO solution was aspirated, and the spores were resuspended with a rapid stream of KP buffer (time, 0 h). The final spore density was adjusted to 5 × 10⁷ spores ml⁻¹ in KP buffer.

Following activation, spores were shaken at 100 rpm in glass flasks at 22°C. Samples were collected at 1-h intervals, and differential counting of unswollen spores, swollen spores, and emerged amoebae was done by using a Zeiss phase-contrast microscope.

Autoactivation.Cells were developed as described above, and spores were allowed to mature in fruiting bodies for several days. Spores were removed from fruiting bodies by passage of dry microscope slides over the tops of the sori. Spores at the edges of the microscope slides were transferred to 5 ml of KP buffer with or without 200 mM sucrose. In this assay, the spores were not washed by centrifugation to prevent the loss of dead and lysed spores. Instead, spore density was adjusted to 10⁶ ml⁻¹ to prevent spore-to-spore signaling (7). Spore suspensions (4 ml) were placed in 10-cm glass test tubes with microstirrer bars (1 by 3 mm) on a magnetic stirrer and stirred at the lowest speed required to keep the spores in suspension. Samples were removed, mixed with an equal volume of neutral red (1:10,000 [wt/vol] in water) on glass microscope slides, and incubated for 5 min. Bright-field microscopy (×400) was used to distinguish among viable dormant spores (unstained), viable swelling spores (weak punctate staining), or dead spores (strong red staining of the entire cytoplasm). Four hundred cells were counted at each time point. In some experiments, spore suspension samples of 0.4 ml were plated on 2% nonnutrient purified agar (Difco) containing KP buffer with or without 1% Bacto Peptone to determine spore viability and the ability to spontaneously germinate. All experiments were repeated at least three times.

Extraction and preparation of RNA for microarray analysis.To ensure efficient extraction of RNA, spores (5 × 10⁸) were collected by centrifugation, frozen rapidly in liquid nitrogen, and ground with a mortar and pestle until all of the spores appeared to be broken upon microscopic examination (32). The ground-up spores were resuspended in 1 ml of Trizol reagent (Life Technologies), and RNA was extracted according to the manufacturer's recommended protocol.

Gene expression analysis was performed as described previously (41). Briefly, purified total RNA was reverse transcribed with Cy5-conjugated homopolymers of 18 deoxythymidine (dT18) as primers. A reference sample made from a pool of equal portions of RNA samples prepared from six developmental stages (0, 3, 6, 12, 17, and 24 h) was reverse transcribed with Cy3-conjugated (dT18) oligonucleotides as primers. This reference sample was used in all of the experiments as well as in previous work and thus allowed comparison among the different germination experiments and between germination and development. The experimental samples and the reference sample were mixed and hybridized to a microarray containing 7,744 hybridization targets (41).

The analysis of DMSO-induced spores was done in three independent biological replications, and the analysis of heat shock-induced spores was done in two independent biological replications. Each RNA sample (except for one of the DMSO-induced samples) was hybridized two independent times to a microarray on which each of the 7,744 targets was printed in duplicate (i.e., each measurement was repeated eight times). All of the data presented are averages of these multiple replications.

Microarray data analysis.Data normalization was performed as described previously (23, 41) with the following modifications. The normalized single-chip data sets were scaled to have a variance of 1 and combined into two multiple-experiment data sets according to the treatment (DMSO or heat shock induction). The cross-time mean gene expression value during germination was subtracted from the gene expression values separately for each combined data set. The data sets were then combined so that for each gene, the expression value was a vector of length 14 (two treatments at seven time points each).

To identify genes with invariant expression patterns in both treatments, we applied an analysis of variance to each vector. Smooth curves were fitted to these vectors by using linear regression. The polynomial bases of the smooth curves were of degree 5 and were generated by using the “poly()” function in the R statistical software (version 1.6.1). Briefly, for the polynomial used here (aX⁵ + bX⁴ + cX³ + dX² + eX + f), the function finds orthogonal polynomials (by generating values corresponding to X⁵, X⁴…, X) in a way that facilitates the subsequent calculation of the coefficients (a, b, c,…, f) by regression. Coefficients for the curves and F statistics for the fit were recorded. A total of 1,397 genes were filtered out based on F statistics (P < 0.05). This procedure ensures that only genes whose expression is significantly regulated during germination are considered and that the regulation is due to the process of germination and not to the method of induction. The regulation of the remaining genes is documented in the supplemental material.

The smooth-curve coefficients of these 1,397 genes were further analyzed with the K-means algorithm to obtain gene clusters. Briefly, K-means is an algorithm that optimizes the grouping of objects (gene trajectories in this case) into a predetermined number of groups (K). We tried different numbers of clusters (K ranging from 2 to 10) and chose 4 as the proper number of clusters based on the slope changes of the within-cluster sum-of-squares errors. The data set was repeatedly clustered into four clusters, and optimized cluster centers were chosen, resulting in four groups containing 360, 396, 357, and 284 genes. The genes within each cluster were ordered based on the correlation between their coefficients and the group center (median).

To determine the statistical significance of each cluster, we performed a simulation in 2,000 iterations. The data at each time point were permuted to generate simulated data sets, and the filtering and clustering procedure was performed as described for the real data. The mean correlation coefficients between the smooth-curve coefficients and the cluster centers were calculated. The statistical significance of the clusters in the real data was estimated from the mean correlation coefficient distribution; the P values were <10⁻⁹ for the group containing 360 genes, <10⁻⁴ for the group containing 396 genes, <0.04 for the group containing 357 genes, and <0.15 for the group containing 284 genes. The last group was excluded from further analyses.

To annotate the putative functions of selected genes, we used gene ontology (GO), which is a controlled vocabulary for describing gene function (5). Nearly full-length cDNA sequences of each microarray target were matched to the sequences in the GO database (http://www.godatabase.org/dev/database). GO identifiers for each array target were determined based on the BLASTX score and the GO evidence code of the homologous genes (see supplemental material for details). A recursive traversal of the GO directed acyclic graphs provided the GO identifiers that are overrepresented in the gene list compared to the entire array. A detailed explanation of the method and a simple example are provided in the supplemental material. Lists of genes with significantly high representation were identified by comparing the number of genes having a common GO category in the experimental group to the total number of genes having that category on the entire array. The data are presented graphically, where bar lengths represent the ratio (fold enrichment) of the list frequency (number of genes in the list/total number of genes at the GO level) to the array group frequency (number of genes with a specific GO annotation on the array/total number of all array genes at a particular GO level). The x axis is the scale for that ratio.

Developmental gene expression data were reanalyzed from previously published experiments (41).

Morphological transitions during spore germination.Spore germination is accompanied by two morphological transitions, swelling and emergence. Before germination, the dormant spores appear oval and bright under phase-contrast microscopy. The first morphological transition occurs 1 to 3 h after induction, as the spores swell and lose their birefringence. In the second transition, the spore cases break and the amoebae emerge. We used two different methods to induce spore germination, DMSO treatment and heat shock (6, 10). In the first hour after DMSO treatment, 30% of the spores were swollen, and swelling peaked at 55% after 2 h. Amoebae emergence began after 2 h (6%) and continued throughout the duration of the experiment (Fig. 1A). Heat shock-induced germination was more synchronous and efficient, but the transitions were delayed by 1 h compared to those seen with the DMSO treatment. It took 3 h for about 97% of the spores to swell, about 70% of the amoebae emerged by 4 h, and the process was complete after 6 h (Fig. 1B).

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FIG. 1.

Morphological transitions during germination. Spore germination was induced by incubation with DMSO (A) or by heat shock (B). Spores were monitored hourly by phase-contrast microscopy to distinguish among dormant spores (sp), swollen spores (sw), and amoebae (am). The results are plotted as the fraction (percentage) of each group in the total population. At least 200 cells were counted at each time point. The data are means and standard deviations (only the positive error bar is shown) from three independent experiments. Error bars smaller than the symbols are not shown.

Transcriptional transitions during spore germination.Spore morphology during germination indicates that the process consists of at least two stages, swelling and emergence. Previous experiments indicated that these two stages must be the result of more complicated physiological processes, but the details of the processes are largely unknown (9). We wanted to find how many physiological transitions occur during the process and how they are related to the morphological transitions. Microarray profiling of gene expression is an established method for distinguishing among physiological states caused by mutations, growth conditions, and drugs (3, 21). It is also applicable to the analysis of Dictyostelium growth and development (19, 24, 41). We therefore used this tool to characterize the process of spore germination.

We first collected RNA from germinating spores at 1-h intervals and analyzed gene expression with a microarray representing most of the expressed genes in Dictyostelium (41). We then calculated the relative level of expression of each gene across the seven experimental time points and used all of the values to determine the dissimilarity among the seven RNA samples. The dendrograms in Fig. 2A show the results. In the dendrograms, each leaf represents a time point in germination, from 0 to 6 h, and the height of each join is directly proportional to the dissimilarity between the leaves or the clades (group of leaves) that it connects.

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FIG. 2.

Transcriptional transitions during spore germination. The levels of expression of over 6,000 genes during the course of spore germination were determined with a DNA microarray. The dendrograms represent dissimilarity in mRNA abundance at different times based on all gene expression values at each time point. (A) For data collected from germinating spores after DMSO induction (left panel), the two most dissimilar groups are shown in green (0 to 1 h) and magenta (2 to 6 h), and the 2-h sample (blue) is an outgroup of the clade shown in magenta. For data collected from germinating spores after heat shock induction (right panel), the two most dissimilar groups are shown in cyan (0 to 2 h) and red (3 to 6 h), and the 2-h sample (blue) is an outgroup of the clade shown in green. The scale on the left indicates dissimilarity in arbitrary units. (B) The dissimilarity was calculated from the averaged data from the two treatments. The two most dissimilar groups are shown in cyan (0 to 2 h) and red (3 to 6 h). The 2-h sample (blue) is an outgroup of the clade shown in cyan and was different from the 0- to 1-h samples (green).

For DMSO induction, the greatest dissimilarity was observed between the 0- to 1-h clade and the 2- to 6-h clade, suggesting that the time between 1 and 2 h of germination is accompanied by the greatest change in cellular mRNA content. For heat shock induction, the greatest dissimilarity was observed between the 0- to 2-h clade and the 3- to 6-h clade, suggesting that the time between 2 and 3 h of germination is accompanied by the greatest change in cellular mRNA content. These major shifts (2 to 3 h with heat shock induction and 1 to 2 h with DMSO induction) (Fig. 2A) occur at the time of maximal spore swelling after the respective induction (Fig. 1), showing a correlation between the physiological and the morphological observations.

The results shown in Fig. 2A were nearly identical for the two induction methods, indicating that the process of germination was robust and that the observed gene expression changes were due mainly to the process of germination rather than to the method of induction. We therefore averaged the two data sets and plotted the dissimilarity between the RNA samples as described above (Fig. 2B). Again, the largest dissimilarity in the data was observed during the swelling process, between the 0- to 2-h clade and the 3- to 6-h clade (Fig. 2B), indicating that swelling is accompanied by the largest physiological transition. The second most prominent change was observed between the 0- to 1-h clade and the branch at 2 h (Fig. 2B). This change occurs at the onset of swelling, indicating the existence of a preswelling transition in mRNA content. The third largest change was observed between the 3- to 5-h clade and the 6-h branch (Fig. 2B), indicating the existence of yet another physiological change, during the emergence of amoebae from the spore cases. These results indicate that there are three major physiological transitions during spore germination: one that follows induction, one during spore swelling, and one during the emergence of amoebae from the spore cases.

We also observed that the dendrograms shown in Fig. 2 conserved the temporal relationships between the samples (i.e., time point 3 is adjacent to time points 2 and 4, and so forth). This finding is highly significant because we did not use the temporal information in the dissimilarity calculations and in rendering the dendrograms. Therefore, it indicates that the transcriptional profile faithfully represents the temporal progression of the spores through the physiological process of germination.

Coordinated regulation of gene expression during spore germination.In order to identify genes that are coordinately regulated during germination, we sought genes that exhibited a time-dependent pattern that was highly similar between the heat-shocked samples and the DMSO-treated samples. Using an analysis of variance, we identified 1,397 such genes. We then subjected the genes to a cluster analysis that sorted them into four groups and determined their statistical significance by simulating how likely it is to find such groups in a random sample of an equal size. Three of these groups, containing 1,113 genes, proved to be statistically significant (Fig. 3A, groups I to III, DM and HS). Group I contains 360 genes whose mRNAs were present in the dormant spores before induction and in the spores immediately after induction (0 h; Fig. 3A and data not shown). The levels of these mRNAs began to decline immediately after induction and continued to decline throughout germination. The graph for group I in Fig. 3B represents the average trajectory of the genes in group I. These data indicate that group I mRNAs are present in the dormant spores before the initiation of germination, so that they must be deposited in the spores before encapsulation. To test that possibility, we examined the expression of the group I genes during Dictyostelium development and found that these genes were indeed induced and expressed during the late stages of development, at the time of prespore and spore differentiation (Fig. 3A, Devel.).

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FIG. 3.

Coordinated regulation of gene expression during spore germination. Total RNA samples were collected at 1-h intervals starting immediately (0 h) after induction by DMSO treatment (DM) or by heat shock (HS) and ending when maximal germination was observed (6 h). mRNA abundance was monitored by hybridization with a microarray representing over 6,000 Dictyostelium genes. (A) Genes that exhibited nearly identical temporal patterns in two germination experiments were selected and grouped with a clustering algorithm. Data are presented in color charts, where blue indicates expression lower than average, gray indicates average expression, and yellow indicates expression higher than average. Color scales are shown at the bottom. Numbers below the color scales are the log₂ ratios of the gene level in the sample and the gene level in the common standard. For example, a gene shown in yellow at the beginning of germination and in blue at the end has gone from a log₂ ratio of −1.3 to a log₂ ratio of +1.3 relative to its own average level; therefore, the overall change is a log₂ ratio of 2.6 or a sixfold change, because 2^2.6 equals 6. The average change is about twofold. The groups are numbered I, II, and III. Each lane in the charts represents a time point (hours are indicated above the lanes), and every row indicates a gene. The height of each chart is proportional to the number of genes in the group (n, indicated at bottom right). The genes in each group were also traced in the developmental time course experiment (Devel.) (41). For the developmental data, samples were collected at 2-h intervals throughout the 24-h course of development on filters, where 0 h represents vegetative cells, 8 h is the time of aggregation, 16 h is the time of slug formation, and 24 h is the time of fruiting body formation (hours are indicated above the lanes). (B) Level of gene expression in each group averaged across all genes in both treatment groups and plotted as a function of time in the spore germination experiments. The group numbers (I to III) are the same as those in panel A. Black lines represent specific values for germination gene celA in the DMSO-induced samples (DM, solid black line) and in the heat shock-induced samples (HS, broken black line). Values of greater than 0 indicate levels of gene expression higher than average, and values of less than 0 indicate levels of gene expression lower than average.

The genes in group II exhibited an inverse pattern relative to the group I genes (Fig. 3A, group II, DM and HS). Transcripts of the 396 genes in group II began to accumulate after 1 to 2 h in the DMSO-induced spores and after 2 to 3 h in the heat shock-induced spores (Fig. 3A, group II, DM and HS, respectively). These mRNAs continued to accumulate throughout the process of germination (Fig. 3B, group II). Examination of these genes in the developmental process showed that their mRNAs were expressed during the vegetative stage (0 h) and during the first 6 to 8 h of development, after which the mRNA levels were greatly reduced (Fig. 3A, Devel.). These findings are consistent with the morphological observation that the emerging amoebae in the germination experiment were quite similar to the vegetative amoebae before development. Therefore, the regulation of the group II genes may be an indicator for the physiological process that prepares the germinating spores for subsequent growth.

Group III mRNAs were not present at high levels in dormant spores, but their expression was induced after 1 h of germination and peaked after 3 h (Fig. 3A, group III, DM and HS, and Fig. 3B, group III). Following the peak, the abundance of the 357 group III mRNAs was reduced to an average level throughout the process of germination. Examination of the group III transcripts during development revealed that the expression of many of these genes was induced immediately after starvation (2 h), decreased during the processes of aggregation and cell type divergence, and increased again toward the end of development, at the time of spore encapsulation (Fig. 3A, group III, Devel.). This pattern was observed before for spore- and stalk-enriched genes during development (41). These genes may be involved in regulating or executing the germination program.

One of the genes on the microarray, celA, was previously described as a germination-specific gene that is induced at 1.5 to 3 h of germination after DMSO induction (15). In our microarray experiment, celA expression peaked at 2 h of germination after DMSO induction (Fig. 3B, DM), thus validating our microarray findings. In the heat shock-induced spores, celA expression was also regulated but peaked at 4 h of germination (Fig. 3B, HS). celA (and other genes of its type) was not included in group III because it does not fulfill the requirement of nearly identical trajectories with both methods of induction. These results indicate that our analysis may have underestimated the number of genes regulated during spore germination.

The data presented in Fig. 3 indicate that the process of germination is accompanied by vast changes in mRNA abundance and that mRNA accumulation begins immediately after induction, when the spores still appear dormant. The large transitions in mRNA abundance in the spores (Fig. 2) are due to both increases and decreases in mRNA abundance, suggesting that both synthesis and degradation of mRNA are involved.

Biological processes affected by spore germination.Genes that may have important roles in biological processes can be found by annotation of gene groups discovered by microarray experiments (21, 45), despite the fact that there is little correlation between the function and the expression of individual genes (17, 44). Because the annotation of the Dictyostelium genome is incomplete, we compared the gene sequences to sequences of several well-annotated genomes. The germination-regulated genes were annotated by using the GO scheme (5) as described previously (23). First, the sequences of all of the genes in the three groups (Fig. 3) were compared to the sequences in the GO database, and the matches with the highest scores were recorded. Then, the GO identifiers pertaining to the Dictyostelium gene list were mapped to the GO data structure, and a tabulation of counts below the nodes in the GO tree was obtained as part of the tree-traversal process. Gene sublists with significantly high representation were identified by comparing the number of genes in the experimentally defined group having a common GO category (e.g., phospholipid metabolism gene in group I) to the total number of genes in that category on the entire array (e.g., all phospholipid metabolism genes on the array). The results are shown in Fig. 4, where the length of the bar represents the fold enrichment of genes in the list relative to their total number on the array. A detailed explanation is provided in the supplemental material.

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FIG. 4.

Annotation of germination-regulated genes. Sequences of the genes from groups I to III (Fig. 3) were compared to several GO-annotated genomes, and the Dictyostelium genes with significant sequence similarity were assigned the respective GO annotations. The biological process annotations of significantly enriched gene groups (P ≤ 0.1) are shown. The GO tree level of the biological process annotation is indicated to the left of each bar (2 to 8). Numbers to the right of the bars indicate the number of genes in the group (List), the number of genes with that annotation on the entire array (Total), and the P value representing the statistical significance of the enrichment. The length of the bar represents the ratio of the list frequency in the data (number of genes in the list/total number of genes in the group at that particular GO level) and the group frequency in the entire array (number of genes with that GO annotation on the array/total number of genes on the array at that particular GO level). The x axis is the scale for the ratio. A detailed explanation of the method is provided in the supplemental material. In the connected bars, the lower bars are subgroups of the bars immediately above them, as indicated by the branching pattern. A branch shown by a broken lines indicates that the group did not show significant enrichment at an intermediate GO level. The bar color and pattern represent the group annotation at GO level 2: striped bars indicate the developmental process, black bars indicate the physiological process, and gray bars indicate the cellular process. Panels I, II, and III represent genes from groups I, II, and III, respectively, in Fig. 3.

Group I consists of genes that are induced during development and down-regulated during germination (Fig. 3). It is also the only group that contains genes with a “developmental process” annotation (Fig. 4, panel I), consistent with the expectation that one major event in germination is the erasure of the developmental state. The “physiological process” annotation of genes in group I reveals the down-regulation of purine biosynthesis as well as phospholipid, lipoprotein, and sulfur metabolism (Fig. 4, panel I). The “cellular process” annotation reveals the down-regulation of transport mechanisms and of cell wall organization genes (Fig. 4, panel I), consistent with the expectation that cell wall organization is required for spore production but not germination.

Group II consists of genes that are up-regulated toward the end of germination and down-regulated during multicellular development (Fig. 3). The physiological processes of the two largest subgroups in group II are annotated as biosynthesis and protein metabolism (Fig. 4, panel II). Mo-molybdopterin biosynthesis genes are also included, suggesting the possibility that dehydrogenase activity is required. The cellular process subgroup includes chromatin modulation and GTPase signal transduction genes (Fig. 4, panel II). The results shown in Fig. 4, panel II, indicate that germinating spores induce a wide range of cell growth genes, the same genes that are turned off during spore differentiation. It is important to note that the spores are germinated in the absence of nutrients, so that the induction of the cell growth genes is not likely to be a result of growth.

Group III genes are transiently up-regulated during early spore swelling (Fig. 3). Their main cellular process annotation indicates that intercellular signaling is involved (Fig. 4, panel III). The major physiological processes are annotated as mRNA metabolism and transcription as well as protein translation and modification; organic acid biosynthesis and disaccharide biosynthesis are also observed (Fig. 4, panel III). These results suggest that spores which seem to be dormant upon morphological examination (Fig. 1) are quite active physiologically after they are induced.

Function of germination-regulated genes in spore germination.Gene expression patterns may predict which genes participate in spore germination. To test whether the germination-regulated genes are necessary for germination, we tested the effects of mutations in some of them. The microarray target sequences of the genes found in group III were compared to a collection of 1,200 sequences of mutated genes from the Baylor College of Medicine Dictyostelium Functional Genomics Project. Mutant strains were generated by REMI mutagenesis, picking of several thousand random clones, plasmid rescue of the insertion sites, and sequencing of the mutated genes (28). Mutant cells were grown and developed, spores were collected, and their ability to germinate was tested. We found that spores of one strain, V10704, failed to germinate normally (data not shown). The gene mutated in V10704 is myoI, an unconventional myosin gene (40). The REMI insertion in strain V10704 occurred at the DpnII site starting at base 7090 of the sequence at GenBank accession number L35321. This location corresponds to the codon for amino acid 2237 of the 2,358 amino acids in the predicted MyoI protein sequence. We did not determine how the insertion might have affected protein activity or stability, but REMI insertions have been documented to cause null mutations even when they occur at the untranslated 3′ end of genes (34).

We hypothesized that the failure of V10704 spores to germinate was due to a direct role of myoI in germination. Alternatively, it could be due to an indirect consequence of a defective sporulation process in this mutant (i.e., a dependent-sequence effect). The timing of sporulation and the shape of the mutant spores were indistinguishable from those of the wild type (data not shown); therefore, we could not use those parameters to distinguish between the two possibilities. We therefore relied on the fact that spores of wild-type strain AX4 do not germinate spontaneously unless they are aged for 6 days in the sorus (26). Wild-type and myoI mutant spores were collected immediately after fruiting body formation and after 1, 3, 6, and 9 days of aging in the sori at 22°C, and neutral red staining was used to identify spores that had autoactivated. The data in Fig. 5A show that both wild-type and mutant spores did not exhibit significant staining on days 0 and 1, indicating that the spores were intact and dormant. These findings argue against the dependent-sequence possibility and suggest that myoI has a direct role in germination. After 3, 6, and 9 days of aging in the sori, 20, 33, and 67% of the mutant spores, respectively, were stained with the dye, whereas the fraction of stained wild-type spores never exceeded 10% (Fig. 5A). These findings suggest that the myoI mutant spores underwent abortive autoactivation while in the sori.

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FIG. 5.

Abortive germination of the myoI mutant. Wild-type (circles) and V10704 mutant (squares) cells were developed for 24 h to obtain spores. (A) Fruiting bodies were aged for the indicated times (days). Spores were collected and stained with neutral red. The fraction of stained spores (percentage) is shown. (B) One-day-old spores (24 h after the completion of development) were collected and germinated in suspension with (solid symbols) and without (open symbols) 200 mM sucrose. Samples were removed at the indicated times (hours) and stained with neutral red to identify abortively swollen spores. The fraction of stained spores (percentage) is shown. The data are means and standard deviations from at least three independent experiments. Error bars smaller than the symbols are not shown.

To further characterize the abortive autoactivation phenotype, we collected 1-day-old spores of both strains, diluted them to remove the germination inhibitors, and incubated them in suspension with shaking. Under these conditions, the wild-type spores did not germinate or swell (Fig. 5B), but the mutant spores began to germinate and became swollen, as indicated by the steady increase in neutral red-stained spores (Fig. 5B). The deeply stained swollen spores failed to release viable amoebae during the 6 h of the experiment, and a few of the swollen spores were almost completely lysed by the end of the experiment. When these spores were plated on agar containing 1% Bacto Peptone, a small fraction of the unswollen spores released visible amoebae. The remaining 35% of the spores, which did not swell during the first 6 h of the experiment, were viable and capable of responding to heat shock. However, more than 95% of them entered the swelling stage of germination abortively (as indicated by a deep neutral red staining) and died without releasing amoebae (data not shown).

Osmoregulation is one of the hallmarks of Dictyostelium spore germination (8, 42). To test whether the V10704 spores were still sensitive to inhibition by high osmolarity, we incubated them in buffer containing 200 mM sucrose and found a complete failure to swell (Fig. 5B). This finding suggests that the myoI mutation does not affect the osmosensory aspects of germination. Therefore, myoI appears to be important for maintaining dormancy in the sorus and for completion of the swelling process but is dispensable for osmoregulation.