Alternative splicing is frequent during early embryonic development in mouse

Abstract

Background: Alternative splicing is known to increase the complexity of mammalian transcriptomes since nearly all mammalian genes express multiple pre-mRNA isoforms. However, our knowledge of the extent and function of alternative splicing in early embryonic development is based mainly on a few isolated examples. High throughput technologies now allow us to study genome-wide alternative splicing during mouse development.

Results: A genome-wide analysis of alternative isoform expression in embryonic day 8.5, 9.5 and 11.5 mouse embryos and placenta was carried out using a splicing-sensitive exon microarray. We show that alternative splicing and isoform expression is frequent across developmental stages and tissues, and is comparable in frequency to the variation in whole-transcript expression. The genes that are alternatively spliced across our samples are disproportionately involved in important developmental processes. Finally, we find that a number of RNA binding proteins, including putative splicing factors, are differentially expressed and spliced across our samples suggesting that such proteins may be involved in regulating tissue and temporal variation in isoform expression. Using an example of a well characterized splicing factor, Fox2, we demonstrate that changes in Fox2 expression levels can be used to predict changes in inclusion levels of alternative exons that are flanked by Fox2 binding sites.

Conclusions: We propose that alternative splicing is an important developmental regulatory mechanism. We further propose that gene expression should routinely be monitored at both the whole transcript and the isoform level in developmental studies.

Figure 1

Examples of types of alternative splicing analysed. The boxes represent exons while the lines are introns. The bold lines beneath the exons indicate probes that detected significant expression changes for each example and the arrows represent primers used for qRT-PCR analyses. (a) Alternative promoter use can be found by an increase in the expression detection levels for a probe set for one promoter compared to the others. The change in promoter use of Elmo1 alters the 5' untranslated region (white boxes) as well as the N-terminus of the protein. (b) The same analyses can be applied for alternative polyadenylation sites, in this case Kif1b. (c) Alternative exons can be included or excluded in the mature mRNA, thus altering the coding sequence as in the Kif2a pre-mRNA.

Figure 2

Principal component analysis. PCA was performed on the whole transcript levels. The plot shows that there is consistent behaviour (clustering) across biological replicates of the same samples. The two major sources of variation in the data are the tissue effect, which approximately corresponds to the 1^st principal component (horizontal axis), and the stage effect (vertical axis).

Figure 3

Distribution of differentially expressed or spliced candidates. (a) Number of genes that show significant expression changes between placental and embryonic tissues and between development stages, as well as the overlap between the two sets. (b) Individual probe sets that are differentially expressed compared to their transcripts between tissues and between stages. (c) Number of genes that contain at least one alternatively spliced exon when comparing embryo or placentas, or between developmental days. (d) Comparison of the percentage of tissue- or stage-dependent differentially alternatively spliced genes, or either, in three different subsets of differentially expressed genes: all, tissue-specific or stage-specific.

Figure 4

Gcnt2 promoter use during embryogenesis. (a) Expression of the Gcnt2 gene is driven by three promoters, each contributing to different N-termini for the protein. (b) The use of the middle promoter is generally higher in the embryo than the placenta as detected by the probe set (PS) 4867798, and its use increases during development. (c) Comparison of the use of each three promoters, normalized to gene expression levels, shows a distinct profile for each promoter when comparing developmental stages and tissues.

Figure 5

Alternative splicing of Rab6. This pre-mRNA contains two mutually exclusive alternative exons, exons 4 and 5 (see middle panel representing the Rab6 gene structure using the UCSC Genome browser [29], http://genome.ucsc.edu). The top panel shows raw expression scores for each probe set for the three embryonic developmental stages. There is a slight difference in the expression of the entire transcript, as most probe sets increase their intensities from embryonic day (E) 8.5 to E11.5. This trend is not consistent for exons 4 and 5, as the inclusion of exon 4 actually goes down while the inclusion of exon 5 increases disproportionally. This is further emphasized in the bottom panel, showing the splicing index analysis and the expression values of each exon normalized to adjust for whole transcript expression changes. On the right, the bar graph shows the actual (log2 scale) reduction of exon 4 inclusion across the 3 day time span.

Figure 6

Examples of qRT-PCR validation. The results obtained from the microarray and quantitative RT-PCR follow the same trend, some of which are presented here. The bars (red) indicate the average of five probe set values of candidate exons divided by the average of the corresponding meta probe set values. The lines (blue) represent the fold change of the average of values for the qRT-PCR on the candidate exons as compared to internal controls on the same pre-mRNAs. Embryonic stages represented: E6.5 to E11.5; placental stages: P9.5 to P11.5.

Figure 7

The well characterized splicing factor Fox2 is alternatively expressed and spliced during development. (a) Pre-mRNAs for this gene are transcribed using two primary promoters. (b) During development, the gene expression of Fox2, as detected by the meta probe set (MPS) 6836888 increases in embryos, while remaining generally lower and stable in placentas. (c) The use of the proximal promoter increases strongly during embryo development. (d) Fox2 expression levels modulate the alternative splicing of cassette exons containing putative Fox2 binding sites in their surrounding introns. The whiskers indicate the minimum and maximum value, the box represents the 25^th percentile to the 75^th, the line inside the box shows the 50^th percentile and the + is the value of the mean. The strict UGCAUG binding site or a combination of 5 binding sites (All) were looked for within 100 nt of the splicing sites in the upstream (Up) or downstream (Down) introns. The E11.5/E8.5 or E11.5/P11.5 exon inclusion ratios of the candidates were calculated using corresponding probe set levels, normalized by meta probe set levels. A ratio over 1 indicates that inclusion of the alternative exon follows expression of Fox2, while a ratio less than 1 means that these are negatively correlated. Using a one-tailed, unpaired t-test, we find that the means of the log of the ratios are significantly different (P < 0.05) between Up and Down data sets.