BACKGROUND
fungi are a large group of eukaryotes consisting of a great number of species with a worldwide distribution and great impact on ecology and human society
<cit> .
fungi comprise both unicellular and multicellular species , as well as species capable of both growth forms .
all filamentous fungi form a network of vegetative hyphae, called mycelium, that usually grows within or on substrates to acquire nutrients.
in addition, many filamentous fungi are capable of developing complex, three-dimensional structures for the generation, protection, and dispersal of spores.
examples are conidiophores for the production of vegetative spores, and fruiting bodies for the production of sexual spores.
fruiting bodies are produced by many ascomycetes and basidiomycetes, and contain a number of specialized cell types that are not present in the vegetative mycelium
<cit> .
the differentiation of these cell types is thought to be orchestrated by spatio-temporal changes in gene expression under the control of regulatory genetic networks.
to address the question of developmental regulation of gene expression on a larger scale, several expression studies have been performed with the ascomycetes gibberella zeae , neurospora crassa, and sordaria macrospora, all of which belong to the sordariomycetes and form flask-like fruiting bodies called perithecia.
expression analyses were carried out using high-throughput methods, such as est sequencing and microarray hybridization
<cit> .
in most of the analyses, either time courses of developing mycelia and fruiting bodies were analyzed, or wild-type strains were compared to developmental mutants at certain time points during development.
however, the tissues used in these studies usually contained cells from fruiting bodies and vegetative mycelium in varying proportions.
one reason for this is that fruiting bodies in ascomycetes are often surrounded by or embedded in vegetative mycelium, from which they are difficult to separate; another reason is that especially the early stages of fruiting body development are quite small , and even if collected would yield low amounts of material for rna preparation and subsequent detection methods, such as microarray hybridizations.
therefore, little information is available regarding the spatial control of gene expression patterns, especially for the early stages of fruiting body development.
laser microdissection  can be used to isolate specific structures consisting of a few cells from samples mounted on microscope slides.
lm has been used to isolate cells from animal and plant tissues, and in the case of fungi to study the growth of phytopathogenic or symbiotic species in planta and for the analysis of gene expression differences in single, neighboring hyphae
<cit> .
here, we have established an lm protocol for isolating fruiting body precursors called protoperithecia  from the filamentous fungus s. macrospora.
this ascomycete is a model system for the analysis of fungal sexual development and cell differentiation
<cit> .
the genome was sequenced recently using next-generation sequencing techniques
<cit> , and a number of developmental mutants have already been characterized by classical complementation analyses or by the sequencing of mutant genomes
<cit> .
prior to the availability of the s. macrospora genome sequence, we had already conducted large-scale expression analyses using cross-species microarray hybridizations with microarrays based on n. crassa cdnas or oligonucleotides to study gene expression during development in the wild-type and several sterile mutants
<cit> .
however, these analyses were limited in sensitivity because less conserved genes give low signal-to-noise ratios in the cross-species array hybridizations.
with the genome sequence available, rna-seq is now the method of choice for large-scale expression analysis.
the unprecedented sequencing depths that can be achieved using next-generation sequencing techniques to sequence cdnas allows much higher sensitivity than microarray hybridization, and the rna-seq data can also be used for annotation purposes
<cit> .
rna-seq has been used in combination with lm to study gene expression in apical meristems and female gametophytes of arabidopsis thaliana, in ripening tomato fruits, and in nucleus accumbens neurons in rats
<cit> ; however, the combination of lm and rna-seq has not yet been applied to the analysis of fungal organ-specific transcriptomes.
therefore, in the present study we established an lm protocol for isolating protoperithecia of s. macrospora, and used amplified rna from the microdissected samples in subsequent rna-seq analysis.
based on the rna-seq data, we modeled untranslated regions  for more than 50% of the predicted s. macrospora genes, and improved the annotation of roughly  <dig> genes.
we then compared gene expression patterns in wild-type protoperithecia to those of non-reproductive mycelium from the wild-type, as well as to protoperithecia from the developmental mutant pro <dig>  the sterility of the pro <dig> mutant is caused by deletion of the transcription factor gene pro <dig> <cit> ; and one aim of the study was to identify genes that are differentially regulated in protoperithecia, depending on or independent of the pro <dig> transcription factor.
RESULTS
laser microdissection of protoperithecia and rna-seq analysis
for lm, strains were grown directly on slides for fixation and dissection in situ.
to allow protoperithecial development, slides had to be covered with a thin layer of agar that did not interfere with the laser sectioning.
samples were fixed in ethanol, and microdissection was performed with a cellcut plus system .
approximately 100– <dig> protoperithecia with a diameter of ~ <dig> μm were collected from each slide, pooled in a collection tube, and rna was extracted from the collected protoperithecia with the picopure kit.
it was then tested whether transcripts of protein-coding genes could be detected in the protoperithecial rna samples by quantitative real time pcr .
expression was detectable for several genes that were analyzed in microdissected samples from the wild-type; but the amount of rna was not sufficient for rna-seq analysis.
therefore, two rounds of linear rna amplification were performed based on cdna generation and in vitro transcription
<cit>  to obtain polya-tailed rna in the amounts required for illumina/solexa library generation.
this linear amplification method has been shown previously to preserve relative transcript amounts within samples and is used in many applications including target generation for microarray hybridization
<cit> .
the amplified rnas from microdissected protoperithecia from the wild-type as well as from mutant pro <dig> were used for rna-seq analysis.
the pro <dig> mutant lacks the gene for the transcription factor pro <dig>  which is essential for sexual development; thus, the mutant is able to form protoperithecia but not mature fruiting bodies
<cit> .
therefore, genes that are differentially regulated in pro <dig> protoperithecia compared to those of the wild-type are direct or indirect targets of pro <dig>  and some of these genes might be required for fruiting body formation.
in addition to the rnas from microdissected samples, we used rnas from total sexual or total vegetative mycelium.
total sexual mycelium  was grown as a surface culture in liquid medium, which is the standard condition for fruiting body formation and rna extraction from s. macrospora.
rna was extracted from the complete samples comprising protoperithecia as well as the surrounding vegetative hyphae
<cit> ; with the latter making up the bulk of the sample.
samples for total vegetative mycelium  were grown submerged in liquid cultures to prevent the formation of any sexual structures
<cit> .
in contrast to previous microarray analyses, we used pooled rna from samples grown in cornmeal medium and defined medium .
both media allow fruiting body formation in surface cultures and only vegetative growth in shaken cultures.
we used pooled rnas for two reasons: first, pooling allowed us to focus on genes that are differentially expressed during fruiting body formation independent of the growth medium, because differential gene expression that occurred only in one medium would be “quenched” in a pooled sample.
second, the use of different growth conditions should give a higher number of expressed genes, which was important because we wanted to use the data not only for expression analysis, but also for annotation purposes.
two biologically independent replications of each condition were used for rna-seq by illumina/solexa sequencing, and 9– <dig> million single reads were obtained for each replicate .
reads were cleaned using custom-made perl scripts and mapped to the reference genome with the splice mapper tophat
<cit> , resulting in 9– <dig> million mapped reads per sample.
the percentage of reads that mapped to the reference sequence was lower for the microdissected samples than for the samples derived from vegetative or sexual mycelium ; however, the overall number of reads obtained for the samples was still high, and this deep sequencing approach allowed a subsequent analysis of gene expression across the complete genome.
the total number of reads is the number of reads after cleaning.
each sample represents an independent biological replicate.
1equal amounts of rna from the following growth conditions  were pooled: growth on cornmeal medium for 3d, 4d and 5d, growth on defined medium for 3d, 4d, and 5d; all in shaken cultures.
2equal amounts of rna from the following growth conditions  were pooled: growth on cornmeal medium for 3d, 4d and 5d, growth on defined medium for 3d, 4d, and 5d; all in surface cultures.
3laser microdissected samples.
improving the s. macrospora genome annotation based on rna-seq data
mapped rna-seq reads were visualized in the genome browser artemis
<cit> .
though the reads from vegetative and sexual mycelium were evenly distributed along the transcripts, the reads derived from microdissected samples were clustered towards the 3’ end of most transcripts, an example is shown for pro <dig> in additional file
<dig> figure s <dig>  this 3’ bias was expected, because each round of rna amplification leads to some loss at the 5’ end
<cit> .
in addition, any uv damage during microdissection that might lead to strand cleavage would cause loss of the 5’ portion of the corresponding rna because reverse transcription was based on polya tails.
in general, a 3’ bias does not hinder quantitative analysis as long as the relative amount of rna in each sample is preserved, as it does not matter for general quantitation whether the reads from a transcript are evenly spread out or clustered towards one end.
however, due to the 3’ bias, many of the reads from the protoperithecial lm samples mapped to the 3’ untranslated regions , and therefore would only be taken into account when the 3’ utr is annotated in the genome sequence.
when we started the study, utrs were annotated for only two s. macrospora genes.
therefore, we used the rna-seq data to model utrs and improve the exon-intron structures of the predicted s. macrospora genes in order to make full use of the rna-seq data in subsequent quantitative expression analyses.
based on the rna-seq data, we were able to determine both utrs for 48% of all predicted genes, and at least one utr  for another 25% of all genes .
on average, 3’ utrs are somewhat longer than 5’ utrs with a median of  <dig> and  <dig> bases, respectively .
these results are similar to those from an rna-seq study of aspergillus oryzae, in which median utr lengths of  <dig> and  <dig> bases were found for the 5’ and 3’ utr, respectively
<cit> .
in addition to utr modeling, ~ <dig>  s. macrospora gene models were improved or newly annotated based on spliced transcripts in the rna-seq reads , and these data are publicly available in genome version  <dig>  .
intron predictions based on rna-seq reads recovered known introns that were verified experimentally in previous analyses, confirming that the rna-seq-based gene model predictions are reliable.
overview of gene expression across the s. macrospora genome
to obtain an overview of the genome-wide expression as represented by the rna-seq data, reads that mapped to exons of predicted mrnas as well as reads that mapped to intergenic regions or introns were counted based on the improved annotation using custom-made perl scripts .
the majority of reads mapped to annotated exons of protein-coding genes .
this percentage was even higher for the protoperithecial samples, most likely because the two rounds of rna amplification were based on polya-dependent reverse transcription constituting an even stronger selection for protein-coding mrnas.
the true percentage of reads that map to intergenic regions might be even lower, because some genes are most likely still missing in the current annotation and not all utrs for all predicted genes have been annotated yet .
therefore, reads mapping to those regions would erroneously be counted as mapping to intergenic regions.
a small percentage of reads mapped to introns and might represent incompletely processed transcripts or alternative splicing events, although an analysis of splice sites that were predicted by tophat showed that, for the majority of genes, no significant alternative splicing could be detected in the different samples .
overall, only  <dig> predicted genes had no reads map to them in at least one condition ; thus, the majority of genes were expressed in at least one of the four conditions investigated .
the majority  of the  <dig> genes were not expressed in wild-type protoperithecia, but were expressed in the other three conditions including protoperithecia from mutant pro <dig>  this finding might indicate that pro <dig> protoperithecia retain, at least to some degree, properties of non-reproductive mycelia, including the expression of genes that are not required in wild-type protoperithecia.
in summary, expression was detected for more than 90% of all annotated genes  in at least one condition, confirming the high sensitivity of this deep-sequencing approach.
gene expression in protoperithecia and non-reproductive mycelia
for a quantitative analysis of gene expression in the different samples , sequence reads that mapped to predicted genes were counted using custom-made perl scripts  and used for quantitative analysis.
results from lox
<cit>  and “classical analysis”
<cit>  agreed best with the results from other methods, therefore these approaches were chosen for the final analysis .
qrt-pcr was used to determine the expression of  <dig> genes in microdissected samples of wild-type protoperithecia without rna amplification, and the results were compared to the rna-seq data.
in addition, qrt-pcr results and rna-seq results were compared for gene expression in vegetative mycelium versus sexual mycelium .
the overall results agree well, and tendencies  are conserved with both methods.
ma-plots of gene expression comparing the different samples showed that sexual mycelium is much more similar to vegetative mycelium than to protoperithecia from the wild-type or mutant pro <dig>  and that the mutant and wild-type protoperithecia differ strongly from each other with respect to gene expression .
the largest numbers of differentially regulated genes were those that are downregulated in wild-type or pro <dig> protoperithecia compared to sexual mycelium; however, some of these genes might be false-positives due to not all 3’ utrs being annotated yet.
in those cases, genes in protoperithecia samples might appear to be not expressed because most of the reads map to the 3’ ends of the genes and would not be counted for a gene if the 3’ utr is not annotated correctly.
this hypothesis is supported by the fact that the percentage of genes with annotated 3’ utrs is lower among the genes that appear to be down-regulated in protoperithecia .
however, this problem does not occur in the comparison of the two protoperithecial samples from the wild-type and mutant pro <dig>  because any bias would concern both samples equally.
therefore, the high number of differentially expressed genes in the comparison of wild-type and pro <dig> protoperithecia with almost equal numbers of up- and down-regulated genes most likely represents true differences in gene expression.
the same is true for genes that are upregulated in protoperithecia compared to sexual mycelium, because this can not be overestimated by missing 3’ utrs.
therefore, even when not taking into account the high number of putatively down-regulated genes in protoperithecia versus total mycelium, the data indicate that the differences between wild-type and pro <dig> protoperithecia as well as between protoperithecia and total mycelium  are much more pronounced than between the different mycelial samples.
this finding is consistent with the hypothesis that the morphological changes that occur during fruiting body formation are mediated by drastic changes in gene expression at the level of transcription.
numbers of genes that are significantly up- or down-regulated when comparing the different samples.
veg, vegetative mycelium; sex, sexual mycelium; wt proto, wild-type protoperithecia; pro <dig> proto, protoperithecia from mutant pro <dig>
1the percentage of the up- or down-regulated genes for which a 3’ utr could be modeled from the rna-seq data is indicated.
another hypothesis about fruiting body formation in filamentous fungi assumes that the non-reproductive mycelium first gathers nutrients until a stage of “competence” is reached when the production of fruiting bodies is energetically feasible, and that then the developing fruiting bodies are nurtured by the surrounding non-reproductive mycelium
<cit> .
one might speculate that this process could require the transport of massive amounts of nutrients, including carbohydrates.
thus, we analyzed whether the expression of putative sugar transporters changed in the different samples .
of the  <dig> genes in the s. macrospora genome that contain at least on sugar transporter domain, more than 40% were significantly up- or down-regulated in at least one of the conditions investigated, thereby supporting the hypothesis that fruiting body morphogenesis is accompanied by a massive redistribution of nutrients.
analysis of pheromone gene expression in protoperithecia
we also analyzed if genes that were previously shown to be essential for perithecial development in s. macrospora or significantly upregulated during fruiting body formation are differentially regulated in protoperithecia compared to total mycelium .
interestingly, we found that both pheromone precursor genes ppg <dig> and ppg <dig> are strongly upregulated in protoperithecia compared to sexual or vegetative mycelium.
the pheromones are required for full fertility
<cit> ; however, where or when they act during the developmental cycle is not yet clear because s. macrospora is self-fertile , and no obvious fertilization event that requires recognition of compatible partners by pheromones is necessary
<cit> .
to address this question in more detail, we analyzed the expression of an egfp reporter gene under the control of the ppg <dig> upstream and downstream regulatory regions .
no expression was observed in vegetative hyphae, in contrast to the expression of egfp from a control vector under the constitutive gpd promoter and trpc terminator from a. nidulans.
egfp fluorescence started to occur in ascogonia  and was strongest in young protoperithecia .
interestingly, older protoperithecia  exhibited a distinctly patchy expression pattern in the hyphae of the outer layers, whereas expression of the control vector led to a uniform fluorescence of protoperithecia.
on the one hand, these data confirm the transcriptional up-regulation of ppg <dig> in protoperithecia as indicated by the rna-seq analysis; and on the other hand, the microscopic analysis revealed a distinct expression pattern of ppg <dig> within protoperithecia.
analysis of pro1-dependent gene expression in protoperithecia
to determine which genes are directly or indirectly under the control of transcription factor pro <dig> in developing protoperithecia, we looked in more detail at genes that are differentially regulated in wild-type protoperithecia compared to sexual mycelium and are also differentially regulated in pro <dig> protoperithecia compared to wild-type protoperithecia .
this group contains a total of  <dig> genes, the majority of which are either upregulated in wild-type protoperithecia compared to sexual mycelium and downregulated in pro <dig> protoperithecia  or the other way round .
for these genes, pro <dig> acts as an activator or repressor, respectively, during fruiting body formation.
only eight genes were up-regulated in wild-type protoperithecia compared to sexual mycelium and also up-regulated in pro <dig> protoperithecia compared to wild-type protoperithecia.
interestingly, six of these eight genes encode proteins that are predicted to be extracellular, including the pheromone genes ppg <dig> and ppg <dig> .
we already identified ppg <dig> and ppg <dig> as being up-regulated in sexual mycelium of mutant pro <dig> compared to the wild-type in a previous microarray analysis
<cit> ; however, the spatial dimension of this differential expression was not yet known, and the combination of lm and rna-seq now shows that protoperithecia-specific pheromone gene expression is regulated by the transcription factor gene pro <dig>  two other genes that are up-regulated in both comparisons are homologous to loosenin from the basidiomycete bjerkandera adusta and fasciclin-like protein moflp <dig> from magnaporthe grisea <cit> .
both proteins have been implicated in cell-wall biogenesis/reorganization, and it is tempting to speculate that the corresponding s. macrospora proteins are involved in shaping the outer layers  of the developing perithecium.
putative functions were predicted with blast2go
<cit> , putative subcellular localizations with wolfpsort
<cit>
we also looked at genes that are physically clustered within the genome, differentially regulated in wild-type protoperithecia, and dependent on pro <dig> for correct expression in protoperithecia.
physical clustering of co-regulated genes is often found in fungi for genes involved in secondary metabolism, and it can be used as a tool for identifying novel secondary metabolism pathways
<cit> .
we found only two instances of clusters that were differentially regulated in wild-type and pro <dig> protoperithecia.
one cluster comprised putative polyketide synthase genes that were described previously and were most likely acquired by horizontal gene transfer
<cit> .
this cluster was down-regulated in wild-type protoperithecia compared to sexual mycelium and up-regulated in pro <dig> protoperithecia .
the second group of clustered genes  has the opposite expression pattern, namely up-regulated in wild-type protoperithecia compared to sexual mycelium and down-regulated in pro <dig> protoperithecia.
this cluster does not contain a polyketide or non-ribosomal peptide synthase typical for the corresponding gene clusters; however, one unifying theme of this cluster is that three of its genes encode proteins with the predicted domain of unknown function duf <dig> .
whether genes from this family play a role in fruiting body formation remains to be elucidated.
next, we investigated the transcripts that were most abundant in protoperithecia from the wild-type and mutant pro <dig>  and whether there was a difference to the most abundant transcripts in sexual or vegetative mycelium.
for this analysis, we counted reads that mapped to the 3’ end  of each predicted mrna.
this approach was chosen to account for the 3’ bias in the microdissected samples, and it generates numbers that are largely independent of transcript length.
read counts were normalized to the total number of counted reads in each sample, and the average read count from the two independent repetitions of each sample was used to determine the  <dig> genes in each of the four samples that had the highest number of reads .
the analysis showed that  <dig> genes were present in the top  <dig> in all four samples, and that sexual mycelium and vegetative mycelium, and protoperithecia from wild-type and pro <dig> had overlaps of  <dig> and  <dig> genes, respectively.
in contrast, the number of common genes among the top  <dig> from the mycelial samples and the protoperithecial samples was much lower .
this again indicates that the transcriptional landscapes of non-reproductive mycelia versus protoperithecia are rather different, and that overall transcription in sexual mycelium is driven by the non-reproductive hyphae that make up the majority of this sample.
we specifically analyzed whether any transcription factor genes are only present among the top  <dig> genes in wild-type protoperithecia or in wild-type and pro <dig> protoperithecia, but not in the mycelial samples.
we found  <dig> putative transcription factors among the top  <dig> genes in both wild-type and mutant protoperithecia, and seven putative transcription factors among the top  <dig> genes in wild-type, but not pro <dig> protoperithecia .
analysis of the gene expression ratios showed that the first group of transcription factors is largely independent of pro <dig> , whereas the second group depends on pro <dig> for upregulation in protoperithecia .
transcription factors that are strongly expressed in protoperithecia might be involved in regulating the expression of downstream genes that mediate fruiting body morphogenesis.
two of these transcription factors were already shown to be essential for fruiting body formation, namely mcm <dig> and pro <dig>  mutations in mcm <dig> or pro <dig> lead to sterility, and the corresponding mutants are able to produce protoperithecia, but not mature perithecia
<cit> .
a comparison with homologous transcription factors from n. crassa and f. graminearum revealed that, out of the  <dig> transcription factors, knockout strains have been analyzed for  <dig> and  <dig> genes from n. crassa and f. graminearum, respectively, in large-scale knockout projects with these two organisms
<cit> .
of these deletion mutants, three showed defects in sexual development in n. crassa, and  <dig> in f. graminearum .
homologs of pro <dig> were sterile in both species, and the corresponding homolog of aspergillus nidulans was also shown to be essential for sexual development
<cit> .
thus, the transcription factors from this analysis might be promising candidates for further functional studies, especially those with developmental phenotypes in other filamentous fungi.
<cit> .
discussion
fungal fruiting body formation is a complex process that requires coordinated patterns of gene expression in time and space.
even though a number of genes that are essential for this process have been isolated from several model organisms, no unifying theory yet explains the spatio-temporal succession of developmental events leading to the mature fruiting body
<cit> .
one way to learn more about the genes that are active during this process is to look at genome-wide expression patterns at different developmental stages, but this is difficult in many ascomycetes, because fruiting bodies are often rather small  and difficult to separate from surrounding, non-reproductive hyphae.
in the present study, we used lm and rna-seq to analyze gene expression in protoperithecia from the model organism s. macrospora.
to the best of our knowledge, this study is the first time that a combination of these methods has been used for the analysis of fungal gene expression.
our study demonstrates that young fruiting bodies of s. macrospora can be isolated using lm, and rna extracted from these samples in sufficient amounts for rna-seq after two rounds of linear amplification.
the amplification process largely conserves expression ratios, as demonstrated by comparing the expression of selected genes prior to amplification with the rna-seq results, which is consistent with results from other organisms where linear rna amplification was used to prepare samples for microarray hybridization
<cit> .
we also found some overlap with prior microarray experiments in which we compared gene expression in vegetative and sexual mycelia; however, in these experiments we used mycelia grown in defined medium
<cit> , whereas for rna-seq analysis, rna from mycelia grown in defined medium and cornmeal medium were pooled.
therefore, some differences between these experiments might be due to different growth conditions .
furthermore, we demonstrated that the regulatory regions of ppg <dig> can drive expression of an egfp reporter gene in protoperithecia, as predicted by the rna-seq analysis.
in addition, fluorescence microscopy analysis revealed distinct expression patterns for ppg <dig> in the outer layers of the protoperithecium.
this finding might be consistent with a hypothesis that has been put forward for n. crassa that predicts that pheromones are not only signaling molecules that enable the recognition of mating partners, but that they also play a role in the attachment  of hyphae forming the rigid outer perithecial wall
<cit> .
a role for pheromones as “molecular glue” might explain the expression of ppg <dig> in cells of the protoperithecial outer layers in s. macrospora.
in a study of gene expression in several tissues of different metazoans, hebenstreit et al.
found that genes can be grouped into two classes, namely genes with high and low expression, independent of tissue type, species or type of experiment
<cit> .
this classification resulted in two distinct peaks when plotting the distribution of gene expression levels.
we wondered whether this distribution might also be found in fungi, but plots of the distribution of gene expression levels showed different patterns for our data .
we observed a single main peak in both vegetative and sexual mycelium, whereas the frequency distribution in wild-type and pro <dig> protoperithecia could be dissected into three peaks.
this difference indicates that, in contrast to metazoans, fungal genes might not generally fall into two main classes of expression.
one reason might be that in the case of sexual and vegetative mycelium, pooled rna samples were used from mycelia grown in different types of media.
these mycelia might express different sets of genes at high and low levels, and such a mixture would drive overall expression frequencies towards intermediate values
<cit> , resulting in a single peak as observed.
however, the multiple peaks for protoperithecia cannot be explained by a mixture of different samples.
the analysis by hebenstreit indicated that the genes from the high expression group constitute the active and functional transcriptome of the cell, whereas the genes from the low expression group show “leaky” expression
<cit> .
our data indicate that the situation in fungi might be different, but further analysis will be needed to clarify this point.
in previous studies, we used cross-species microarray hybridization to hybridize s. macrospora targets on n. crassa cdna or oligonucleotide microarrays
<cit> ; however, in these analyses, less than 50% of all genes on the arrays gave a significant signal.
the use of rna-seq dramatically improves detection levels, with more than 90% of all genes being detected in at least one of the sequenced samples.
also, the comparison of gene expression revealed that the overall expression in sexual mycelium is more similar to that of vegetative mycelium than protoperithecia.
this finding indicates that gene expression in the sexual mycelium is most likely driven to a large extent by genes expressed in the non-reproductive hyphae making up the bulk of the mycelium and that, in order to study genes specifically expressed in developing fruiting bodies, the microdissection method applied here provides a much better spatial resolution and a much more detailed and specific picture of gene expression during development.
especially weakly expressed, fruiting body-specific genes would most likely not be detected as differentially expressed  in an expression study using only sexual mycelium.
previous approaches for isolating fruiting bodies for gene expression studies were performed in n. crassa and f. graminearum, using est sequencing with rna from mature fruiting bodies
<cit> , or by analyzing different stages of fruiting bodies by microarray hybridization
<cit> .
the analysis by hallen et al.
<cit>  was performed with affymetrix genechips for f. graminearum, and signals were detected for nearly 80% of all transcripts, whereas the est analysis was limited by a comparatively low sequencing depth, and in the other two microarray studies
<cit> , only 10% of all genes gave signals or were represented on the arrays.
in all studies, fruiting bodies were harvested by scraping developing structures from a plate, and these preparations might contain an undetermined amount of non-fruiting body mycelia, especially in the early stages of development when fruiting body precursors are small.
therefore, the present analysis of microdissected protoperithecia allowed the analysis of gene expression solely in these structures for the first time.
an additional advantage of rna-seq is that the data can be used also for annotation purposes and, in the case of s. macrospora, allowed the modeling of more than 50% of the utrs, and the improvement of exon-intron structures for about  <dig>  genes .
the analysis of gene expression ratios and the  <dig> genes with the highest number of reads in each of the four sequenced samples showed that expression in protoperithecia from the wild-type and mutant pro <dig> is more similar to each other than to either vegetative or sexual mycelium, indicating that the transcriptional landscape of protoperithecia is distinct from that of non-reproductive mycelium.
however, there are also significant differences between protoperithecia from the wild-type and the sterile mutant pro <dig> that can form protoperithecia, but not mature fruiting bodies.
more than  <dig> genes were significantly up- or downregulated in pro <dig> protoperithecia compared to wild-type protoperithecia, and therefore might be direct or indirect targets of pro <dig>  among the genes that are dependent on pro <dig> for correct expression in protoperithecia are the pheromone precursor genes, several genes that might be involved in perithecial wall morphogenesis, and a number of transcription factors.
previous analyses identified several mutants in which the pheromone precursor genes are differentially regulated in sexual mycelium compared to the wild-type
<cit> ; however, no information was available about the spatial regulation of the expression of developmental genes prior to this study.
one might hypothesize that pro <dig> is involved in balancing the expression of genes involved in the formation of the rigid perithecial wall because the pheromone precursor genes and several other genes predicted to be involved in cell-wall biosynthesis are up-regulated in pro <dig> protoperithecia .
CONCLUSIONS
we have established a combined lm/rna-seq approach to analyze gene expression in developing fungal sexual structures, and used it to analyze the transcriptome of young fruiting bodies in the wild-type and the sterile mutant pro <dig> of s. macrospora.
pro <dig>  which encodes a transcription factor, is essential for sexual development.
to the best of our knowledge, this is the first genome-wide analysis of genes that are dependent on a development-specific transcription factor for correct expression in a defined developmental structure in fungi.
genes that are differentially expressed in protoperithecia are prime candidates for further functional analysis to unravel the spatio-temporal sequence of events leading to the mature fungal fruiting body.
together with three recent studies in arabidopsis and tomato, as well as rat neurons
<cit> , our analysis of fungal development demonstrates the power of a combined approach of lm and rna-seq to analyze cell type-specific or tissue-specific gene expression in complex, multicellular structures.

 fungi are a large group of eukaryotes consisting of a great number of species with a worldwide distribution and great impact on ecology and human society. fungi comprise both unicellular and multicellular species, as well as species capable of both growth forms. all filamentous fungi form a network of vegetative hyphae, called mycelium, that usually grows within or on substrates to acquire nutrients. in addition, many filamentous fungi are capable of developing complex, three-dimensional structures for the generation, protection, and dispersal of spores. examples are conidiophores for the production of vegetative spores, and fruiting bodies for the production of sexual spores. fruiting bodies are produced by many ascomycetes and basidiomycetes, and contain a number of specialized cell types that are not present in the vegetative mycelium. the differentiation of these cell types is thought to be orchestrated by spatio-temporal changes in gene expression under the control of regulatory genetic networks. to address the question of developmental regulation of gene expression on a larger scale, several expression studies have been performed with the ascomycetes gibberella zeae, neurospora crassa, and sordaria macrospora, all of which belong to the sordariomycetes and form flask-like fruiting bodies called perithecia. expression analyses were carried out using high-throughput methods, such as est sequencing and microarray hybridization. in most of the analyses, either time courses of developing mycelia and fruiting bodies were analyzed, or wild-type strains were compared to developmental mutants at certain time points during development. however, the tissues used in these studies usually contained cells from fruiting bodies and vegetative mycelium in varying proportions. one reason for this is that fruiting bodies in ascomycetes are often surrounded by or embedded in vegetative mycelium, from which they are difficult to separate; another reason is that especially the early stages of fruiting body development are quite small, and even if collected would yield low amounts of material for rna preparation and subsequent detection methods, such as microarray hybridizations. therefore, little information is available regarding the spatial control of gene expression patterns, especially for the early stages of fruiting body development. laser microdissection can be used to isolate specific structures consisting of a few cells from samples mounted on microscope slides. lm has been used to isolate cells from animal and plant tissues, and in the case of fungi to study the growth of phytopathogenic or symbiotic species in planta and for the analysis of gene expression differences in single, neighboring hyphae. here, we have established an lm protocol for isolating fruiting body precursors called protoperithecia from the filamentous fungus s. macrospora. this ascomycete is a model system for the analysis of fungal sexual development and cell differentiation. the genome was sequenced recently using next-generation sequencing techniques, and a number of developmental mutants have already been characterized by classical complementation analyses or by the sequencing of mutant genomes. prior to the availability of the s. macrospora genome sequence, we had already conducted large-scale expression analyses using cross-species microarray hybridizations with microarrays based on n. crassa cdnas or oligonucleotides to study gene expression during development in the wild-type and several sterile mutants. however, these analyses were limited in sensitivity because less conserved genes give low signal-to-noise ratios in the cross-species array hybridizations. with the genome sequence available, rna-seq is now the method of choice for large-scale expression analysis. the unprecedented sequencing depths that can be achieved using next-generation sequencing techniques to sequence cdnas allows much higher sensitivity than microarray hybridization, and the rna-seq data can also be used for annotation purposes. rna-seq has been used in combination with lm to study gene expression in apical meristems and female gametophytes of arabidopsis thaliana, in ripening tomato fruits, and in nucleus accumbens neurons in rats; however, the combination of lm and rna-seq has not yet been applied to the analysis of fungal organ-specific transcriptomes. therefore, in the present study we established an lm protocol for isolating protoperithecia of s. macrospora, and used amplified rna from the microdissected samples in subsequent rna-seq analysis. based on the rna-seq data, we modeled untranslated regions for more than 50% of the predicted s. macrospora genes, and improved the annotation of roughly genes. we then compared gene expression patterns in wild-type protoperithecia to those of non-reproductive mycelium from the wild-type, as well as to protoperithecia from the developmental mutant pro the sterility of the pro mutant is caused by deletion of the transcription factor gene pro; and one aim of the study was to identify genes that are differentially regulated in protoperithecia, depending on or independent of the pro transcription factor.  laser microdissection of protoperithecia and rna-seq analysis for lm, strains were grown directly on slides for fixation and dissection in situ. to allow protoperithecial development, slides had to be covered with a thin layer of agar that did not interfere with the laser sectioning. samples were fixed in ethanol, and microdissection was performed with a cellcut plus system. approximately 100– protoperithecia with a diameter of ~ μm were collected from each slide, pooled in a collection tube, and rna was extracted from the collected protoperithecia with the picopure kit. it was then tested whether transcripts of protein-coding genes could be detected in the protoperithecial rna samples by quantitative real time pcr. expression was detectable for several genes that were analyzed in microdissected samples from the wild-type; but the amount of rna was not sufficient for rna-seq analysis. therefore, two rounds of linear rna amplification were performed based on cdna generation and in vitro transcription to obtain polya-tailed rna in the amounts required for illumina/solexa library generation. this linear amplification method has been shown previously to preserve relative transcript amounts within samples and is used in many applications including target generation for microarray hybridization. the amplified rnas from microdissected protoperithecia from the wild-type as well as from mutant pro were used for rna-seq analysis. the pro mutant lacks the gene for the transcription factor pro which is essential for sexual development; thus, the mutant is able to form protoperithecia but not mature fruiting bodies. therefore, genes that are differentially regulated in pro protoperithecia compared to those of the wild-type are direct or indirect targets of pro and some of these genes might be required for fruiting body formation. in addition to the rnas from microdissected samples, we used rnas from total sexual or total vegetative mycelium. total sexual mycelium was grown as a surface culture in liquid medium, which is the standard condition for fruiting body formation and rna extraction from s. macrospora. rna was extracted from the complete samples comprising protoperithecia as well as the surrounding vegetative hyphae; with the latter making up the bulk of the sample. samples for total vegetative mycelium were grown submerged in liquid cultures to prevent the formation of any sexual structures. in contrast to previous microarray analyses, we used pooled rna from samples grown in cornmeal medium and defined medium. both media allow fruiting body formation in surface cultures and only vegetative growth in shaken cultures. we used pooled rnas for two reasons: first, pooling allowed us to focus on genes that are differentially expressed during fruiting body formation independent of the growth medium, because differential gene expression that occurred only in one medium would be “quenched” in a pooled sample. second, the use of different growth conditions should give a higher number of expressed genes, which was important because we wanted to use the data not only for expression analysis, but also for annotation purposes. two biologically independent replications of each condition were used for rna-seq by illumina/solexa sequencing, and 9– million single reads were obtained for each replicate. reads were cleaned using custom-made perl scripts and mapped to the reference genome with the splice mapper tophat, resulting in 9– million mapped reads per sample. the percentage of reads that mapped to the reference sequence was lower for the microdissected samples than for the samples derived from vegetative or sexual mycelium; however, the overall number of reads obtained for the samples was still high, and this deep sequencing approach allowed a subsequent analysis of gene expression across the complete genome. the total number of reads is the number of reads after cleaning. each sample represents an independent biological replicate. 1equal amounts of rna from the following growth conditions were pooled: growth on cornmeal medium for 3d, 4d and 5d, growth on defined medium for 3d, 4d, and 5d; all in shaken cultures. 2equal amounts of rna from the following growth conditions were pooled: growth on cornmeal medium for 3d, 4d and 5d, growth on defined medium for 3d, 4d, and 5d; all in surface cultures. 3laser microdissected samples. improving the s. macrospora genome annotation based on rna-seq data mapped rna-seq reads were visualized in the genome browser artemis. though the reads from vegetative and sexual mycelium were evenly distributed along the transcripts, the reads derived from microdissected samples were clustered towards the 3’ end of most transcripts, an example is shown for pro in additional file figure s this 3’ bias was expected, because each round of rna amplification leads to some loss at the 5’ end. in addition, any uv damage during microdissection that might lead to strand cleavage would cause loss of the 5’ portion of the corresponding rna because reverse transcription was based on polya tails. in general, a 3’ bias does not hinder quantitative analysis as long as the relative amount of rna in each sample is preserved, as it does not matter for general quantitation whether the reads from a transcript are evenly spread out or clustered towards one end. however, due to the 3’ bias, many of the reads from the protoperithecial lm samples mapped to the 3’ untranslated regions, and therefore would only be taken into account when the 3’ utr is annotated in the genome sequence. when we started the study, utrs were annotated for only two s. macrospora genes. therefore, we used the rna-seq data to model utrs and improve the exon-intron structures of the predicted s. macrospora genes in order to make full use of the rna-seq data in subsequent quantitative expression analyses. based on the rna-seq data, we were able to determine both utrs for 48% of all predicted genes, and at least one utr for another 25% of all genes. on average, 3’ utrs are somewhat longer than 5’ utrs with a median of and bases, respectively. these results are similar to those from an rna-seq study of aspergillus oryzae, in which median utr lengths of and bases were found for the 5’ and 3’ utr, respectively. in addition to utr modeling, ~ s. macrospora gene models were improved or newly annotated based on spliced transcripts in the rna-seq reads, and these data are publicly available in genome version. intron predictions based on rna-seq reads recovered known introns that were verified experimentally in previous analyses, confirming that the rna-seq-based gene model predictions are reliable. overview of gene expression across the s. macrospora genome to obtain an overview of the genome-wide expression as represented by the rna-seq data, reads that mapped to exons of predicted mrnas as well as reads that mapped to intergenic regions or introns were counted based on the improved annotation using custom-made perl scripts. the majority of reads mapped to annotated exons of protein-coding genes. this percentage was even higher for the protoperithecial samples, most likely because the two rounds of rna amplification were based on polya-dependent reverse transcription constituting an even stronger selection for protein-coding mrnas. the true percentage of reads that map to intergenic regions might be even lower, because some genes are most likely still missing in the current annotation and not all utrs for all predicted genes have been annotated yet. therefore, reads mapping to those regions would erroneously be counted as mapping to intergenic regions. a small percentage of reads mapped to introns and might represent incompletely processed transcripts or alternative splicing events, although an analysis of splice sites that were predicted by tophat showed that, for the majority of genes, no significant alternative splicing could be detected in the different samples. overall, only predicted genes had no reads map to them in at least one condition; thus, the majority of genes were expressed in at least one of the four conditions investigated. the majority of the genes were not expressed in wild-type protoperithecia, but were expressed in the other three conditions including protoperithecia from mutant pro this finding might indicate that pro protoperithecia retain, at least to some degree, properties of non-reproductive mycelia, including the expression of genes that are not required in wild-type protoperithecia. in summary, expression was detected for more than 90% of all annotated genes in at least one condition, confirming the high sensitivity of this deep-sequencing approach. gene expression in protoperithecia and non-reproductive mycelia for a quantitative analysis of gene expression in the different samples, sequence reads that mapped to predicted genes were counted using custom-made perl scripts and used for quantitative analysis. results from lox and “classical analysis” agreed best with the results from other methods, therefore these approaches were chosen for the final analysis. qrt-pcr was used to determine the expression of genes in microdissected samples of wild-type protoperithecia without rna amplification, and the results were compared to the rna-seq data. in addition, qrt-pcr results and rna-seq results were compared for gene expression in vegetative mycelium versus sexual mycelium. the overall results agree well, and tendencies are conserved with both methods. ma-plots of gene expression comparing the different samples showed that sexual mycelium is much more similar to vegetative mycelium than to protoperithecia from the wild-type or mutant pro and that the mutant and wild-type protoperithecia differ strongly from each other with respect to gene expression. the largest numbers of differentially regulated genes were those that are downregulated in wild-type or pro protoperithecia compared to sexual mycelium; however, some of these genes might be false-positives due to not all 3’ utrs being annotated yet. in those cases, genes in protoperithecia samples might appear to be not expressed because most of the reads map to the 3’ ends of the genes and would not be counted for a gene if the 3’ utr is not annotated correctly. this hypothesis is supported by the fact that the percentage of genes with annotated 3’ utrs is lower among the genes that appear to be down-regulated in protoperithecia. however, this problem does not occur in the comparison of the two protoperithecial samples from the wild-type and mutant pro because any bias would concern both samples equally. therefore, the high number of differentially expressed genes in the comparison of wild-type and pro protoperithecia with almost equal numbers of up- and down-regulated genes most likely represents true differences in gene expression. the same is true for genes that are upregulated in protoperithecia compared to sexual mycelium, because this can not be overestimated by missing 3’ utrs. therefore, even when not taking into account the high number of putatively down-regulated genes in protoperithecia versus total mycelium, the data indicate that the differences between wild-type and pro protoperithecia as well as between protoperithecia and total mycelium are much more pronounced than between the different mycelial samples. this finding is consistent with the hypothesis that the morphological changes that occur during fruiting body formation are mediated by drastic changes in gene expression at the level of transcription. numbers of genes that are significantly up- or down-regulated when comparing the different samples. veg, vegetative mycelium; sex, sexual mycelium; wt proto, wild-type protoperithecia; pro proto, protoperithecia from mutant pro 1the percentage of the up- or down-regulated genes for which a 3’ utr could be modeled from the rna-seq data is indicated. another hypothesis about fruiting body formation in filamentous fungi assumes that the non-reproductive mycelium first gathers nutrients until a stage of “competence” is reached when the production of fruiting bodies is energetically feasible, and that then the developing fruiting bodies are nurtured by the surrounding non-reproductive mycelium. one might speculate that this process could require the transport of massive amounts of nutrients, including carbohydrates. thus, we analyzed whether the expression of putative sugar transporters changed in the different samples. of the genes in the s. macrospora genome that contain at least on sugar transporter domain, more than 40% were significantly up- or down-regulated in at least one of the conditions investigated, thereby supporting the hypothesis that fruiting body morphogenesis is accompanied by a massive redistribution of nutrients. analysis of pheromone gene expression in protoperithecia we also analyzed if genes that were previously shown to be essential for perithecial development in s. macrospora or significantly upregulated during fruiting body formation are differentially regulated in protoperithecia compared to total mycelium. interestingly, we found that both pheromone precursor genes ppg and ppg are strongly upregulated in protoperithecia compared to sexual or vegetative mycelium. the pheromones are required for full fertility; however, where or when they act during the developmental cycle is not yet clear because s. macrospora is self-fertile, and no obvious fertilization event that requires recognition of compatible partners by pheromones is necessary. to address this question in more detail, we analyzed the expression of an egfp reporter gene under the control of the ppg upstream and downstream regulatory regions. no expression was observed in vegetative hyphae, in contrast to the expression of egfp from a control vector under the constitutive gpd promoter and trpc terminator from a. nidulans. egfp fluorescence started to occur in ascogonia and was strongest in young protoperithecia. interestingly, older protoperithecia exhibited a distinctly patchy expression pattern in the hyphae of the outer layers, whereas expression of the control vector led to a uniform fluorescence of protoperithecia. on the one hand, these data confirm the transcriptional up-regulation of ppg in protoperithecia as indicated by the rna-seq analysis; and on the other hand, the microscopic analysis revealed a distinct expression pattern of ppg within protoperithecia. analysis of pro1-dependent gene expression in protoperithecia to determine which genes are directly or indirectly under the control of transcription factor pro in developing protoperithecia, we looked in more detail at genes that are differentially regulated in wild-type protoperithecia compared to sexual mycelium and are also differentially regulated in pro protoperithecia compared to wild-type protoperithecia. this group contains a total of genes, the majority of which are either upregulated in wild-type protoperithecia compared to sexual mycelium and downregulated in pro protoperithecia or the other way round. for these genes, pro acts as an activator or repressor, respectively, during fruiting body formation. only eight genes were up-regulated in wild-type protoperithecia compared to sexual mycelium and also up-regulated in pro protoperithecia compared to wild-type protoperithecia. interestingly, six of these eight genes encode proteins that are predicted to be extracellular, including the pheromone genes ppg and ppg. we already identified ppg and ppg as being up-regulated in sexual mycelium of mutant pro compared to the wild-type in a previous microarray analysis; however, the spatial dimension of this differential expression was not yet known, and the combination of lm and rna-seq now shows that protoperithecia-specific pheromone gene expression is regulated by the transcription factor gene pro two other genes that are up-regulated in both comparisons are homologous to loosenin from the basidiomycete bjerkandera adusta and fasciclin-like protein moflp from magnaporthe grisea. both proteins have been implicated in cell-wall biogenesis/reorganization, and it is tempting to speculate that the corresponding s. macrospora proteins are involved in shaping the outer layers of the developing perithecium. putative functions were predicted with blast2go, putative subcellular localizations with wolfpsort we also looked at genes that are physically clustered within the genome, differentially regulated in wild-type protoperithecia, and dependent on pro for correct expression in protoperithecia. physical clustering of co-regulated genes is often found in fungi for genes involved in secondary metabolism, and it can be used as a tool for identifying novel secondary metabolism pathways. we found only two instances of clusters that were differentially regulated in wild-type and pro protoperithecia. one cluster comprised putative polyketide synthase genes that were described previously and were most likely acquired by horizontal gene transfer. this cluster was down-regulated in wild-type protoperithecia compared to sexual mycelium and up-regulated in pro protoperithecia. the second group of clustered genes has the opposite expression pattern, namely up-regulated in wild-type protoperithecia compared to sexual mycelium and down-regulated in pro protoperithecia. this cluster does not contain a polyketide or non-ribosomal peptide synthase typical for the corresponding gene clusters; however, one unifying theme of this cluster is that three of its genes encode proteins with the predicted domain of unknown function duf. whether genes from this family play a role in fruiting body formation remains to be elucidated. next, we investigated the transcripts that were most abundant in protoperithecia from the wild-type and mutant pro and whether there was a difference to the most abundant transcripts in sexual or vegetative mycelium. for this analysis, we counted reads that mapped to the 3’ end of each predicted mrna. this approach was chosen to account for the 3’ bias in the microdissected samples, and it generates numbers that are largely independent of transcript length. read counts were normalized to the total number of counted reads in each sample, and the average read count from the two independent repetitions of each sample was used to determine the genes in each of the four samples that had the highest number of reads. the analysis showed that genes were present in the top in all four samples, and that sexual mycelium and vegetative mycelium, and protoperithecia from wild-type and pro had overlaps of and genes, respectively. in contrast, the number of common genes among the top from the mycelial samples and the protoperithecial samples was much lower. this again indicates that the transcriptional landscapes of non-reproductive mycelia versus protoperithecia are rather different, and that overall transcription in sexual mycelium is driven by the non-reproductive hyphae that make up the majority of this sample. we specifically analyzed whether any transcription factor genes are only present among the top genes in wild-type protoperithecia or in wild-type and pro protoperithecia, but not in the mycelial samples. we found putative transcription factors among the top genes in both wild-type and mutant protoperithecia, and seven putative transcription factors among the top genes in wild-type, but not pro protoperithecia. analysis of the gene expression ratios showed that the first group of transcription factors is largely independent of pro, whereas the second group depends on pro for upregulation in protoperithecia. transcription factors that are strongly expressed in protoperithecia might be involved in regulating the expression of downstream genes that mediate fruiting body morphogenesis. two of these transcription factors were already shown to be essential for fruiting body formation, namely mcm and pro mutations in mcm or pro lead to sterility, and the corresponding mutants are able to produce protoperithecia, but not mature perithecia. a comparison with homologous transcription factors from n. crassa and f. graminearum revealed that, out of the transcription factors, knockout strains have been analyzed for and genes from n. crassa and f. graminearum, respectively, in large-scale knockout projects with these two organisms. of these deletion mutants, three showed defects in sexual development in n. crassa, and in f. graminearum. homologs of pro were sterile in both species, and the corresponding homolog of aspergillus nidulans was also shown to be essential for sexual development. thus, the transcription factors from this analysis might be promising candidates for further functional studies, especially those with developmental phenotypes in other filamentous fungi.. discussion fungal fruiting body formation is a complex process that requires coordinated patterns of gene expression in time and space. even though a number of genes that are essential for this process have been isolated from several model organisms, no unifying theory yet explains the spatio-temporal succession of developmental events leading to the mature fruiting body. one way to learn more about the genes that are active during this process is to look at genome-wide expression patterns at different developmental stages, but this is difficult in many ascomycetes, because fruiting bodies are often rather small and difficult to separate from surrounding, non-reproductive hyphae. in the present study, we used lm and rna-seq to analyze gene expression in protoperithecia from the model organism s. macrospora. to the best of our knowledge, this study is the first time that a combination of these methods has been used for the analysis of fungal gene expression. our study demonstrates that young fruiting bodies of s. macrospora can be isolated using lm, and rna extracted from these samples in sufficient amounts for rna-seq after two rounds of linear amplification. the amplification process largely conserves expression ratios, as demonstrated by comparing the expression of selected genes prior to amplification with the rna-seq results, which is consistent with results from other organisms where linear rna amplification was used to prepare samples for microarray hybridization. we also found some overlap with prior microarray experiments in which we compared gene expression in vegetative and sexual mycelia; however, in these experiments we used mycelia grown in defined medium, whereas for rna-seq analysis, rna from mycelia grown in defined medium and cornmeal medium were pooled. therefore, some differences between these experiments might be due to different growth conditions. furthermore, we demonstrated that the regulatory regions of ppg can drive expression of an egfp reporter gene in protoperithecia, as predicted by the rna-seq analysis. in addition, fluorescence microscopy analysis revealed distinct expression patterns for ppg in the outer layers of the protoperithecium. this finding might be consistent with a hypothesis that has been put forward for n. crassa that predicts that pheromones are not only signaling molecules that enable the recognition of mating partners, but that they also play a role in the attachment of hyphae forming the rigid outer perithecial wall. a role for pheromones as “molecular glue” might explain the expression of ppg in cells of the protoperithecial outer layers in s. macrospora. in a study of gene expression in several tissues of different metazoans, hebenstreit et al. found that genes can be grouped into two classes, namely genes with high and low expression, independent of tissue type, species or type of experiment. this classification resulted in two distinct peaks when plotting the distribution of gene expression levels. we wondered whether this distribution might also be found in fungi, but plots of the distribution of gene expression levels showed different patterns for our data. we observed a single main peak in both vegetative and sexual mycelium, whereas the frequency distribution in wild-type and pro protoperithecia could be dissected into three peaks. this difference indicates that, in contrast to metazoans, fungal genes might not generally fall into two main classes of expression. one reason might be that in the case of sexual and vegetative mycelium, pooled rna samples were used from mycelia grown in different types of media. these mycelia might express different sets of genes at high and low levels, and such a mixture would drive overall expression frequencies towards intermediate values, resulting in a single peak as observed. however, the multiple peaks for protoperithecia cannot be explained by a mixture of different samples. the analysis by hebenstreit indicated that the genes from the high expression group constitute the active and functional transcriptome of the cell, whereas the genes from the low expression group show “leaky” expression. our data indicate that the situation in fungi might be different, but further analysis will be needed to clarify this point. in previous studies, we used cross-species microarray hybridization to hybridize s. macrospora targets on n. crassa cdna or oligonucleotide microarrays; however, in these analyses, less than 50% of all genes on the arrays gave a significant signal. the use of rna-seq dramatically improves detection levels, with more than 90% of all genes being detected in at least one of the sequenced samples. also, the comparison of gene expression revealed that the overall expression in sexual mycelium is more similar to that of vegetative mycelium than protoperithecia. this finding indicates that gene expression in the sexual mycelium is most likely driven to a large extent by genes expressed in the non-reproductive hyphae making up the bulk of the mycelium and that, in order to study genes specifically expressed in developing fruiting bodies, the microdissection method applied here provides a much better spatial resolution and a much more detailed and specific picture of gene expression during development. especially weakly expressed, fruiting body-specific genes would most likely not be detected as differentially expressed in an expression study using only sexual mycelium. previous approaches for isolating fruiting bodies for gene expression studies were performed in n. crassa and f. graminearum, using est sequencing with rna from mature fruiting bodies, or by analyzing different stages of fruiting bodies by microarray hybridization. the analysis by hallen et al. was performed with affymetrix genechips for f. graminearum, and signals were detected for nearly 80% of all transcripts, whereas the est analysis was limited by a comparatively low sequencing depth, and in the other two microarray studies, only 10% of all genes gave signals or were represented on the arrays. in all studies, fruiting bodies were harvested by scraping developing structures from a plate, and these preparations might contain an undetermined amount of non-fruiting body mycelia, especially in the early stages of development when fruiting body precursors are small. therefore, the present analysis of microdissected protoperithecia allowed the analysis of gene expression solely in these structures for the first time. an additional advantage of rna-seq is that the data can be used also for annotation purposes and, in the case of s. macrospora, allowed the modeling of more than 50% of the utrs, and the improvement of exon-intron structures for about genes. the analysis of gene expression ratios and the genes with the highest number of reads in each of the four sequenced samples showed that expression in protoperithecia from the wild-type and mutant pro is more similar to each other than to either vegetative or sexual mycelium, indicating that the transcriptional landscape of protoperithecia is distinct from that of non-reproductive mycelium. however, there are also significant differences between protoperithecia from the wild-type and the sterile mutant pro that can form protoperithecia, but not mature fruiting bodies. more than genes were significantly up- or downregulated in pro protoperithecia compared to wild-type protoperithecia, and therefore might be direct or indirect targets of pro among the genes that are dependent on pro for correct expression in protoperithecia are the pheromone precursor genes, several genes that might be involved in perithecial wall morphogenesis, and a number of transcription factors. previous analyses identified several mutants in which the pheromone precursor genes are differentially regulated in sexual mycelium compared to the wild-type; however, no information was available about the spatial regulation of the expression of developmental genes prior to this study. one might hypothesize that pro is involved in balancing the expression of genes involved in the formation of the rigid perithecial wall because the pheromone precursor genes and several other genes predicted to be involved in cell-wall biosynthesis are up-regulated in pro protoperithecia.  we have established a combined lm/rna-seq approach to analyze gene expression in developing fungal sexual structures, and used it to analyze the transcriptome of young fruiting bodies in the wild-type and the sterile mutant pro of s. macrospora. pro which encodes a transcription factor, is essential for sexual development. to the best of our knowledge, this is the first genome-wide analysis of genes that are dependent on a development-specific transcription factor for correct expression in a defined developmental structure in fungi. genes that are differentially expressed in protoperithecia are prime candidates for further functional analysis to unravel the spatio-temporal sequence of events leading to the mature fungal fruiting body. together with three recent studies in arabidopsis and tomato, as well as rat neurons, our analysis of fungal development demonstrates the power of a combined approach of lm and rna-seq to analyze cell type-specific or tissue-specific gene expression in complex, multicellular structures. 
fruiting bodies contain a number of cell types not found in vegetative mycelium, and these morphological differences are thought to be mediated by changes in gene expression. however, little is known about the spatial distribution of gene expression in fungal development. here, we used laser microdissection and rna-seq to determine gene expression patterns in young fruiting bodies and non-reproductive mycelia of the ascomycete sordaria macrospora. quantitative analysis showed major differences in the gene expression patterns between protoperithecia and total mycelium. this allowed a high spatial resolution and sensitivity, and yielded a detailed picture of gene expression during development. our data revealed significant differences in gene expression between protoperithecia and non-reproductive mycelia, and showed that the transcription factor pro is involved in the regulation of many genes expressed specifically in sexual structures. the lm/rna-seq approach will also be relevant to other eukaryotic systems in which multicellular development is investigated. during sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the protection and dispersal of sexual spores. rna-seq analysis of protoperithecia from the sterile mutant pro showed that many genes that are differentially regulated in these structures are under the genetic control of transcription factor pro 
 during sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the protection and dispersal of sexual spores. fruiting bodies contain a number of cell types not found in vegetative mycelium, and these morphological differences are thought to be mediated by changes in gene expression. however, little is known about the spatial distribution of gene expression in fungal development. here, we used laser microdissection and rna-seq to determine gene expression patterns in young fruiting bodies and non-reproductive mycelia of the ascomycete sordaria macrospora.  quantitative analysis showed major differences in the gene expression patterns between protoperithecia and total mycelium. among the genes strongly up-regulated in protoperithecia were the pheromone precursor genes ppg and ppg the up-regulation was confirmed by fluorescence microscopy of egfp expression under the control of ppg regulatory sequences. rna-seq analysis of protoperithecia from the sterile mutant pro showed that many genes that are differentially regulated in these structures are under the genetic control of transcription factor pro  we have generated transcriptional profiles of young fungal sexual structures using a combination of lm and rna-seq. this allowed a high spatial resolution and sensitivity, and yielded a detailed picture of gene expression during development. our data revealed significant differences in gene expression between protoperithecia and non-reproductive mycelia, and showed that the transcription factor pro is involved in the regulation of many genes expressed specifically in sexual structures. the lm/rna-seq approach will also be relevant to other eukaryotic systems in which multicellular development is investigated. 