Nucleic Acids Research, 2012, Vol. 40, No. 6 2417 Figure 2. Work ﬂow of GAA. After uploading the raw CEL ﬁles to the GAA server, preprocessing is initiated. The Affymetrix Power Tools are utilized for background correction, normalization and summarization at the gene and exon level. A P-value for each exon (probe set) is estimated to determine whether an exon is expressed above background levels. In the next steps, ﬁlters are applied to eliminate false positives. GNI are passed to limma for calculation of the SI and statistics. Results are imported into MySQL tables for visualization. expressed probe set may correspond to known splicing events (Figure 3D). We also implemented exon array- derived datasets from 11 different normal tissues contain- ing differentially spliced exons to serve as a reference for potential splicing events. The main features of GAA are shown in Figure 3. A more detailed description can be found in the ‘Materials and Methods’ section and in the online help section of GAA (http://GAA.mpi-bn.mpg.de/ help.php). Alternative splicing during heart development Heart is the earliest organ in mammals to acquire func- tionality in order to support survival. Although the heart is a vital organ, very few studies have been published up until recently to identify genome-wide alternative splicing events (26,27). To gain further information about alternative splicing during heart development, we performed gene array experiments using hearts from embryonic day 17.5 (E17.5), post-natal day 1 (P1), and 2-month-old male mice and compared these three devel- opmental stages with each other at the gene and exon levels. Analysis was conducted using GAA as described in the ‘Materials and Methods’ section. We identiﬁed 2521 differentially expressed genes (DEGs) and 3452 probe sets [termed ‘differentially expressed exon (DEE)’ hereafter] as signiﬁcantly regulated (F-statistic P < 0.001). All signiﬁcant DEGs and DEEs are listed in Supplementary Table S1, and the full result can be ac- cessed at GAA online. Next, we performed Gene Ontology analysis through the PANTHER software (28). As reported by others (27), signiﬁcantly enriched terms are not identical at the gene and exon levels but do overlap with each other. We found enrichments for system development, and helicase activity at the exon level, whereas oxidoreductase activity and receptor binding activity at the gene level. In agree- ment with our data set, highly signiﬁcant terms in both groups are metabolic process, developmental process, cytoskeletal protein binding and structural constituent of cytoskeleton, which are known to be important for the development of heart (Supplementary Table S2). Alternative splicing during cardiomyocyte differentiation Heart is made up of cardiomyocytes, ﬁbroblasts, endothe- lial and smooth muscle cells. To obtain a pure source of cardiomyocytes, we differentiated J1 mouse ES cells (strain 129S4/Jae; ATCC# SCRC-1010) to cardiomyocytes and eliminated all non-cardiomyocyte cells using a construct consisting of the cardiac alpha- myosin heavy chain promoter driving the neomycin resist- ance gene (23). Gene arrays were hybridized with RNA obtained from undifferentiated ES cells (ES) (Day 0 before the induction of differentiation), embryonic bodies (EB) (Day 7 after the induction of differentiation) and cardiac bodies (CB) (14 days after the induction of differentiation with spontaneous beating, which is a char- acteristic of cardiomyocytes). Data were analyzed with GAA as described in the ‘Materials and Methods’ section. The P-value of the F-test was used to identify genes that are differentially expressed between any of the three conditions. GAA identiﬁed 1 852 DEG and a rela- tively low number of 166 DEE within the threshold P < 0.001 (Supplementary Table S1 and at GAA online). The Gene Ontology analysis during differentiation of cardiomyocytes shows that signiﬁcant terms at the exon level represent a subset of the terms at the gene level. The much higher number of DEG compared with DEE might explain this observation. Terms found at