Analysis of the Basidiomycete Coprinopsis cinerea reveals conservation of the core meiotic expression program over half a billion years of evolution

Abstract

Coprinopsis cinerea (also known as Coprinus cinereus) is a multicellular basidiomycete mushroom particularly suited to the study of meiosis due to its synchronous meiotic development and prolonged prophase. We examined the 15-hour meiotic transcriptional program of C. cinerea, encompassing time points prior to haploid nuclear fusion though tetrad formation, using a 70-mer oligonucleotide microarray. As with other organisms, a large proportion (∼20%) of genes are differentially regulated during this developmental process, with successive waves of transcription apparent in nine transcriptional clusters, including one enriched for meiotic functions. C. cinerea and the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe diverged ∼500-900 million years ago, permitting a comparison of transcriptional programs across a broad evolutionary time scale. Previous studies of S. cerevisiae and S. pombe compared genes that were induced upon entry into meiosis; inclusion of C. cinerea data indicates that meiotic genes are more conserved in their patterns of induction across species than genes not known to be meiotic. In addition, we found that meiotic genes are significantly more conserved in their transcript profiles than genes not known to be meiotic, which indicates a remarkable conservation of the meiotic process across evolutionarily distant organisms. Overall, meiotic function genes are more conserved in both induction and transcript profile than genes not known to be meiotic. However, of 50 meiotic function genes that were co-induced in all three species, 41 transcript profiles were well-correlated in at least two of the three species, but only a single gene (rad50) exhibited coordinated induction and well-correlated transcript profiles in all three species, indicating that co-induction does not necessarily predict correlated expression or vice versa. Differences may reflect differences in meiotic mechanisms or new roles for paralogs. Similarities in induction, transcript profiles, or both, should contribute to gene discovery for orthologs without currently characterized meiotic roles.

Figure 1. C. cinerea nuclei during meiosis.

Adjacent basidia (meiotic cells) in gill tissue collected at 3-hour intervals from K−3 to K + 12 (A–F) and stained with DAPI. Two separate nuclei (}) are present prior to karyogamy (A), nuclei are fused or fusing (*) at karyogamy (B), chromosomes are condensing (<) at K+3 (C), are fully synapsed (arrow) at K+6 (D), and are undergoing the first meiotic division (**) at K+9 (E), and four nuclei are apparent (bracket) at K+12 (F). Fainter nuclei lie in a different focal plane. Note that the basidia become more widely spaced as meiosis progresses due to expansion of the underlying gill tissue. Scale  =  2 µm.

Figure 2. C. cinerea meiotic gene clusters.

C. cinerea genes that changed in expression during the time course were grouped into nine clusters, as illustrated by heatmaps and average expression profiles of each cluster. Expression ratios are log₂ transformed. Y-axis ratio scale markers  =  0.2. Time (hours) relative to karyogamy are shown at the top of the heatmaps. Meiotic stages are indicated at the bottom of the graphs; D  =  dikaryon, K  =  karyogamy, L/Z  =  leptotene/zygotene, P  =  pachytene, MI  =  first meiotic division, MII  =  second meiotic division.

Figure 3. Timing of meiotic events in C. cinerea, S. pombe, and S. cerevisiae.

Time points used to examine meiotic transcription in S. pombe and S. cerevisiae were aligned with those used in C. cinerea according to observations from existing time courses , –, –. Time points are shown as hours after switching to sporulation media (S. pombe and S. cerevisiae) or hours after karyogamy (nuclear fusion) in C. cinerea. Aligned time points are indicated with dashed lines. sHJs  =  single Holliday junctions, DSBs  =  double strand breaks, SEIs  =  single end intermediates, dHJs  =  double Holliday junctions, M I  =  first meiotic division, M II  =  second meiotic division.

Figure 4. Distribution of gene expression profile correlation coefficients.

Distributions of pair-wise correlation coefficients for C. cinerea vs. S. pombe (red), C. cinerea vs. S. cerevisiae (yellow) and S. pombe vs. S. cerevisiae (blue) for genes with no known meiotic function (A) and for meiotic function genes (B). Note that the meiotic function genes (B) have relatively more pairwise expression profiles with positive correlation coefficients than genes with no known meiotic function (A).

Figure 5. rad50 expression is well-correlated in C. cinerea, S. pombe, and S. cerevisiae.

Expression profiles of rad50 in C. cinerea (red ⧫), S. cerevisiae (blue •) and S. pombe (green ▴) are shown across eight time points for which biological stages are comparable. Symbols representing interpolated data points are smaller. Expression profile correlation coefficients (r) are shown for C. cinerea vs. S. pombe (c/p), C. cinerea vs. S. cerevisiae (c/s) and S. cerevisiae vs. S. pombe (s/p). C. cinerea meiotic stages are as follows: K, karyogamy; L/Z, leptotene/zygotene; P, pachytene; M I, just after first meiotic division; M II, just after second meiotic division. Corresponding meiotic stages for S. pombe and S. cerevisiae are shown in Figure 4. To allow visual comparison of profiles from different species, expression data are adjusted to show relative expression change across the time course for each species.

Figure 6. Gene expression in spo11 and rec8 are well-correlated only between C. cinerea and S. pombe.

Gene expression profiles of spo11 (A) and rec8 (B) are shown for C. cinerea (red ⧫), S. cerevisiae (blue •) and S. pombe (green ▴) as in Figure 5.

Figure 7. Gene expression profiles of cohesin subunits are well correlated between C. cinerea and S. cerevisiae, and between C. cinerea and S. pombe.

Gene expression profiles of smc1 (A), smc3 (B) and scc3 (C) are shown for C. cinerea (red ⧫), S. cerevisiae (blue •) and S. pombe (green ▴) as in Figure 5. Panel D shows expression profiles of scc1/rad21 in S. cerevisiae (blue •) and S. pombe (green ▴), as well as the two homologs in C. cinerea, rad21.1 (orange ▪) and rad21.2 (red ⧫). Correlation coefficients are as indicated for rad21.1 (*), and rad21.2 (**).

Figure 8. MCM complex gene expression declines through meiosis, with key exceptions.

Gene expression profiles of mcm7 in C. cinerea (gray ⋄), S. cerevisiae (gray ○) and S. pombe (gray ▵), and S. pombe mcm6 (green ▴) and S. cerevisiae mcm5 (blue •) are shown as in Figure 5.