The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: SEL LAT MBE. Performed the experiments: SEL JEV. Analyzed the data: SEL MBE. Contributed reagents/materials/analysis tools: GPS SL LAT. Wrote the paper: SEL MBE.
MBE is a cofounder and member of the Board of Directors of PLoS.
Mmany genes from the X chromosome are expressed at the same level in female and male embryos during early
When
Variation in gene dose can have profound effects on animal development. Yet every generation, animals must cope with differences in sex chromosome numbers.
The earliest stages of animal development are under maternal control until mRNAs deposited prior to fertilization degrade and zygotic transcription is initiated during a period known as the maternal to zygotic transition (MZT). In
As zygotic transcription begins, the different numbers of X chromosomes (two in females, one in males) results in different transcript levels for a small number of genes on the X chromosome (the X chromosome signal elements, or XSEs), which lead to female-specific expression of the master sex control gene
However, there is a lag between the onset of zygotic transcription and the establishment of MSL-mediated dosage compensation: the complex is not localized on DNA, and H4K16 acetylation is not detectable, until after the blastoderm stage
We were intrigued by the possibility that the absence of MSL-mediated dosage compensation during the MZT might lead to higher levels of mRNAs derived from genes on the X chromosome in females, and sex-specific differences in patterning or cellularization that have not been detected because systematic studies of early developmental transcription have never differentiated male and female embryos.
A variety of approaches have been used to profile zygotic transcription during the MZT, including genome-wide expression profiling with microarrays
To address these limitations, we developed methods to characterize, by sequencing, the mRNA content of individual
In order to create a precise time series of zygotic transcription in male and female embryos during embryonic development, we needed methods to demarcate small differences in developmental time, to determine the sex of embryos, and to measure the entire pool of transcripts in these embryos in a way that distinguished mRNAs of maternal and zygotic origin.
We chose to focus on the period of development bounded by cycle 10 (when early zygotic transcription is detectable) and the completion of cellularization in mitotic cycle 14 (when widespread zygotic transcription has been established, right before MSL-mediated dosage compensation is thought to begin).
To determine developmental stage, we took advantage of two characteristics of early embryos: the tightly controlled synchronous mitotic cycles and the process of cellularization as the embryo transitions from a syncytium to a cellular blastoderm (
(A) Transcription events during early embryogenesis. During the first 8–9 mitotic cycles, almost all RNAs in the embryo are of maternal origin. Zygotic transcription begins at a low level at approximately cycle 10 and becomes widespread by the middle of cycle 14. MSL-mediated dosage compensation begins late in or following cycle 14. (B) Embryos used for mRNA-Seq. Individual embryos in the interphases of cycles 10 to 14 were selected by direct observation of mitosis in embryos containing histone H2Av-RFP and computing nuclear density. Embryos at substages of cycle 14 were selected by observing the extent of progression through cellularization (from proportion of membrane invagination) under light microscopy. Each embryo pictured here was placed into TRIzol reagent immediately after these images were taken, DNA and RNA were extracted, and each sample was genotyped to determine the sex of the embryo. (C) Approximately 100 ng total RNA was obtained from each embryo, and poly-A RNA was processed with an amplification-free protocol optimized for small samples and sequenced on an Illumina GAIIx Genome Analyzer. Data (normalized reads per kb, RPKM) from independently processed individuals of the same stage and same sex, and same stage but different sex were extremely similar, while individuals from different stages showed larger numbers of differences.
We selected at least four embryos each for cycles 10, 11, 12, 13, 14A, 14B, 14C, and 14D, and extracted DNA and RNA from each embryo independently. We carried out whole-genome amplification on the DNA from each embryo and genotyped it for Y chromosomal markers to determine the sex of the embryo, and selected at least one male and female embryo from every stage for transcriptome analysis.
We obtained 75 to 100 ng of total RNA from each embryo. As this was less starting material than required for standard mRNA sequencing protocols, we modified the Illumina mRNA-Seq protocol to obtain reliable data from such small quantities of input mRNA without amplification by performing all purification and size selection steps using magnetic beads, and reducing the volume of some reactions (a complete protocol is available in
We sequenced a total of 24 mRNA samples on an Illumina GAIIx Genome Analyzer. We aligned reads to the
Sex | Stage | Reads | Mapped Reads |
Female | 10 | 26,976,249 | 17,633,235 |
Female | 11 | 27,988,411 | 17,105,590 |
Female | 12 | 23,270,517 | 19,949,416 |
Female | 13 | 24,615,381 | 20,388,812 |
Female | 14A | 21,322,865 | 16,349,815 |
Female | 14A | 18,590,935 | 16,036,761 |
Female | 14B | 18,545,524 | 15,121,569 |
Female | 14B | 19,244,061 | 17,316,317 |
Female | 14C | 20,902,589 | 17,346,048 |
Female | 14C | 18,459,025 | 16,647,754 |
Female | 14D | 23,128,318 | 19,327,028 |
Female | 14D | 17,750,907 | 15,848,586 |
Male | 10 | 21,892,898 | 16,468,144 |
Male | 11 | 18,250,299 | 12,796,916 |
Male | 12 | 25,167,764 | 21,527,094 |
Male | 13 | 23,830,913 | 18,156,530 |
Male | 14A | 22,696,841 | 18,738,955 |
Male | 14A | 19,301,209 | 17,208,613 |
Male | 14B | 23,409,763 | 16,417,656 |
Male | 14B | 18,501,195 | 16,617,463 |
Male | 14C | 22,471,826 | 18,740,316 |
Male | 14C | 17,617,204 | 15,340,670 |
Male | 14D | 20,536,683 | 16,674,908 |
Male | 14D | 18,532,116 | 15,888,989 |
The single embryo mRNA-Seq method was highly reproducible and has a wide dynamic range (
In order to distinguish zygotic transcripts from those deposited by the mother, we analyzed embryos produced by a cross of two genetically distinct
CantonS | w1 | |
Reads | 65,907,258 | 63,716136 |
Mapped reads | 57,375,508 | 55,489,784 |
Total mapped bases | 5,794,926,308 | 5,604,468,184 |
Average coverage | 34.3× | 33.2× |
Sites scored as polymorphic with respect to reference | 299,254 | 340,119 |
Sites polymorphic between strains | 285,927 | |
Fixed polymorphisms between strains | 122,672 |
The vast majority of these differences were biallelic single-nucleotide polymorphisms (SNPs) known from resequencing projects to be polymorphic in the North American
Exactly 10,492 of 14,833 annotated genes (over 70%) contained at least one fixed polymorphism, allowing us to assign RNA-Seq reads spanning the polymorphism to either
(A) Approximately 70% of genes expressed in the early embryo contained fixed differences between the maternal (
We used the strain-specific time series to classify genes as maternal, zygotic, or maternal and zygotic. Briefly, we clustered (k-medians) the 5,226 genes with at least 10 reads spanning a
Previous studies of sex determination and dosage compensation have described the expression sex-specific patterns of expression in a number of zygotically transcribed genes
The events in the sex determination pathway in our data are consistent with previous studies. (A) Expression levels (normalized reads per kb, RPKM) for the X chromosome signal elements (XSEs;
We next compared transcript levels of all 2,210 purely zygotic genes in male and female embryos. Zygotically derived transcripts from autosomal genes were observed at the same levels in females and males (
(A) Female expression versus male expression for zygotic genes (normalized reads per kb, per gene, log scale) over cycle 14, where most zygotic expression is detected. Autosomal gene expression was centered around the purple line, where female and male transcript levels are even. For X chromosomal genes, transcript levels were distributed between females and males having equal transcript abundance (solid line) and the female having twice the transcript level of the male (dotted line). (B) Total expression levels (average normalized reads per gene) for zygotic genes in male and female embryos, on autosomes and the X chromosome. Female expression on X is less than twice the level of male expression after cycle 12 (light blue line). (C) Zygotic transcripts from autosomal genes were derived equally from the maternal or paternal chromosomes, while zygotic transcripts from the single X chromosome in males are present at higher levels than those from either of the X chromosomes in females, demonstrating that the early embryo is dosage compensated.
The difference between the X chromosome and autosomes can be seen clearly when total abundance of zygotically expressed genes in males and females is compared between the X chromosome and autosomes (
We observed no difference in the levels of transcripts derived from maternal or paternal chromosomes for either autosomes or (in females) the X chromosome. Total expression of zygotic genes from the paternal and maternal X chromosomes of females was very similar (average Spearman's rank correlation ρ = 0.97 across stages, some as high as ρ = 0.999;
The sex ratio of transcript abundance for individual genes varied somewhat over cycle 14, especially earlier in cycle 14 where there are not many zygotic genes expressed. But across cycle 14, the X chromosome consistently had an excess of genes with higher transcript level in females (
Of the zygotic genes on the X chromosome, some had the same transcript levels in female and male embryos, and some had an excess of transcripts in females relative to the male, indicating that some are transcriptionally dosage compensated and some are not. (A) Proportion of genes that had higher transcript levels in males or females over cycle 14, comparing autosomes to the X chromosome. The darker colors represent a stronger enrichment of female or male transcripts relative to the other sex. To reduce noise, ratios of female to male expression were considered for genes where individuals of both sexes had at least 2 RPKM, little qualitative difference was observed in results for higher thresholds (results not shown). (B) Key developmental regulators on the X chromosome were dosage compensated at the transcript level. (C) Other zygotic factors on the X did not appear to be effectively dosage compensated, as there were large differences in expression between male and female embryos.
The expression patterns of the patterning genes whose presence on the X chromosome motivated us to examine sex-specific expression in the early embryo were particularly striking. For example,
As is often the case with dosage compensation mechanisms, early zygotic dosage compensation is not universal, and several genes showed no evidence of compensation at the transcript level (
Our development of methods to examine sex-specific gene expression in early
Instead, our genome-wide time course of transcript levels in individual male and female embryos has revealed extensive dosage compensation of X chromosomal transcript levels before the canonical MSL-mediated dosage compensation process is thought to be engaged. Crucially, mRNAs for key X-linked developmental regulators, including
Although there is clearly early zygotic dosage compensation (EZDC), our data speak only indirectly to the mechanism by which it occurs. Assuming that, in an uncompensated system, we would expect transcription to produce twice as many zygotically derived copies of X chromosomal genes in females than in males, the generally lower levels we observe in females must arise through sex and X-chromosome-specific transcriptional or post-transcriptional regulation.
The simplest explanation is that the MSL-based dosage compensation system is active before and during cycle 14, leading to hypertranscription of the male X. However, several imaging studies of the male-specific localization of MSL proteins to, and the subsequent acetylation of histones on, the male X chromosome describe an at least 1 h lag between the onset of zygotic transcription and these hallmarks of MSL-mediated dosage compensation
Through an analysis of larval cuticle patterns of male and female embryos carrying various combinations of
Since SXL is an RNA-binding protein known to modulate splicing and translation, it was proposed that dosage compensation of
The two best-characterized targets of SXL are
There is, however, imperfect agreement between predicted SXL targets and genes we observe to be dosage compensated. Many genes with high degrees of EZDC are not predicted SXL targets (
If it turns out that neither the MSL complex or
Each of the models discussed above assume that, without intervention, 2-fold differences in DNA dose inherently produce 2-fold differences in transcription and transcript abundance, which need, at least for some subset of genes, to be compensated. However, this is not necessarily the case. Studies on autosomal regions with altered dosage in
Additionally, the expectations of the interactions of gene dosage and expression may not be the same in the unique transcription environment of the early embryo. A recent study by Lu et al.
To explain this observation, Lu et al.
There is a related alternative to the limiting factor hypothesis that could explain both dosage compensation and insensitivity to ploidy, concerning the accessibility of DNA templates. Homologous chromosomes are known to be paired throughout
While no such mechanism has been described, the rapid mitotic cycles of early development place constraints on transcription
Yet, contrary to this, near synchronous appearance of two adjacent dots in many nuclei in RNA in situ hybridization of intronic probes from autosomal genes demonstrates that paired alleles can both be transcribed at roughly the same time
Whatever the mechanism turns out to be, our data provide an unprecedented window on the temporal dynamics of transcript levels in male and female embryos, and establish that some mechanism exists that ensures that differences in sex chromosome dose do not translate into differences in mRNA abundance during a crucial period of
While our focus here was on dosage compensation, our data represent a significant advance over earlier methods to monitor gene expression in the early
We hope that our data, which are being made available in full here, will help address a number of other open questions about transcription during early
First, we routinely obtained at least 10 times more material from processing the RNA from a single embryo than was needed for a single Illumina sequencing lane. This suggests that the RNA content of even smaller samples could be routinely analyzed without RNA amplification. Second, for a variety of reasons, mostly involving cost, we carried out 36 base pair single-end sequencing runs. In retrospect, we would have been able to assign many more reads to distinct parental chromosomes, and perhaps detected sex-specific splicing, had we carried out longer, paired-end runs. Finally, analyzing embryos from a cross of divergent strains was very useful. But we were surprised at how polymorphic the supposedly inbred strains we used in our crosses were. We suspect this is a general phenomenon, and suggest that all researchers doing experiments that require highly inbred lines specifically inbreed the lines they are using and resequence them to characterize residual polymorphism prior to use.
Flies were raised on standard fly media in uncrowded conditions, at room temperature. 2–3-d-old virgin females of the
RNA and DNA extraction from single embryos was done with TRIzol (Invitrogen) reagent according to the manufacturer's protocol, except with a higher volume of reagent relative to the amount of material (i.e. starting with 1 mL of TRIzol despite expecting very small amounts of DNA and RNA). Extracted DNA was amplified using the Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare), and embryos were sexed by detecting the presence of a Y chromosome, using PCR with primers to a region of the male fertility factor kl5 on the Y chromosome (forward primer
Total RNA was made into libraries for sequencing using the mRNA-Seq Sample Preparation Kit from Illumina, following an altered mRNA-Seq library making protocol developed at Illumina (see complete protocol in
An alternate flow cell loading protocol for small concentration sequencing libraries was developed for this study and used here, despite the libraries created largely being concentrated enough not to necessitate use of this method (see
We prepared genomic DNA from 10 females from our
Reads from each RNA-Seq sample were mapped to the reference
We used the strain-specific time series to classify genes as maternal, zygotic, or maternal and zygotic. We clustered (k-medians) the 5,226 genes with at least 10 reads spanning a
All reads have been deposited in the NCBI GEO under the accession number GSE25180 and will be made available at the time of publication. The processed data are available at the journal website (
Extent of dosage compensation for zygotic genes on the X chromosome. Each gene was assigned a female to male (F∶M) ratio score equal to the slope of the line fit (by least squares) to the male and female transcript levels for that gene over all time points. (A) Proportion of genes with F∶M ratios between 1.0 (equal expression in males and females) and 2.0 (twice expression in females). (B) Transcript abundance time series for zygotic genes on the X chromosome, in female and male embryos, over all time points. Genes sorted by F∶M ratio.
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Normalized read counts per gene for each individual embryo.
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Small sample RNA-Seq protocol.
(0.14 MB PDF)
Low concentration sequencing library loading protocol.
(0.08 MB PDF)
We thank Michael Z. Ludwig for pioneering techniques to work with single embryos; Doris Bachtrog, Tom Cline, and Barbara Meyer for helpful comments; and members of the Eisen lab for critical reading of the manuscript.
early zygotic dosage compensation
maternal to zygotic transition
single-nucleotide polymorphisms
X chromosome signal elements