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doi: 10.1242/10.1242/dev.00302

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Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome

¹ Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
² Department of Genomics, Wyeth Research, Cambridge, MA 02140, USA

View larger version (44K): [in a new window]   Fig. 1. An embryonic time course of transcript profiles based on precise staging of small cohorts of embryos. (A) Nucleus count versus minutes after the first cleavage at 22°C for all of embryogenesis (adapted, with permission, from Sulston et al., 1983). The area in green indicates the time domain covered by the time course. (B) The time points and the estimated average number of cells per time point in the time course. Embryos were staged at pseudocleavage and the four-cell stage as indicated. Samples for time points in blue and yellow were created, processed and assayed as independent time courses — series 1 and series 2. (C) The complete lineage through the 190-cell stage with the assayed time points indicated. The names given to each time point reflect how the embryos were staged and how long they were aged before being frozen (PC6, pseudocleavage plus 6 minutes; 0 min, four-cell stage plus 0 minutes).

View larger version (14K): [in a new window]   Fig. 2. Genes from a known transcriptional cascade and from three previously characterized expression classes are all detected. (A) Published localization patterns for five transcription factors involved in specification of intestinal fate are depicted on a partial lineage diagram. skn-1, end-1 and end-3 expression patterns were determined using in situ hybridization; med-1/2 was determined using a combination of transgenic reporter and RT-PCR; and elt-2 was determined using antibody. The known regulatory interactions among these genes and proteins are shown along with the moving average time points of gene expression profiles. (B) Gene expression profiles for each of the five genes in A. med-1 and med-2 are treated as a single gene as there is only a single probe set on the chip to assay either and it does not distinguish between the two highly similar sequences. Colors correspond to those in A. (C) Gene expression profiles are shown for representatives of each of three previously characterized expression classes: vet-4, very early [embryonic] transcripts (vet); pos-1, Class I maternal; and tbb-2, Class II maternal.

View larger version (29K): [in a new window]   Fig. 3. Twenty-five genes with known expression patterns are detected. (A) Gene expression profiles for 10 embryonic transcription factors characterized by specific developmental phenotypes resulting from disruption of their function. Transcript abundance is plotted on a log2 scale. The key includes in parentheses the approximate number of cells (out of 102) in which each gene is expressed at 140 minutes. The maternal expression of lin-26 and early transient induction of hlh-1 are both consistent with reported expression patterns (Krause et al., 1990; Quintin et al., 2001). (B) Gene expression profiles for seven maternally expressed genes previously characterized as Class I (stable everywhere) by virtue of their in situ hybridization patterns (Seydoux and Fire, 1994). (C) Gene expression profiles for eight maternally expressed genes previously characterized as Class II (degraded in somatic blastomeres, stable in germline precursors) by virtue of their in situ hybridization patterns (Seydoux and Fire, 1994).

View larger version (28K): [in a new window]   Fig. 4. The transcriptome is stable up to the four-cell stage and changes dramatically thereafter. (A) The x-axis shows ten pairs of time points analyzed by paired-timepoint ANOVA. The y-axis shows the number of RD genes with P<0.01. The number of genes making the cut-off is also split according to whether the change in abundance is positive (Up) or negative (Down). (B) A scatter plot of the 8890 RD genes showing changes in abundance that occur between the PC6 and PC32 time points (one-cell and early four-cell stages, respectively). The max of the two mean transcript abundances is plotted on the x-axis on a log10 scale. Fold-change (PC32/PC6) is plotted on the y-axis on a log2 scale. The two lines crossing the y-axis at ±2 mark twofold changes. Each point is color coded according to whether or not the observed difference is statistically significant (P<0.01) according to a paired-timepoint ANOVA. The number of genes that are considered to be significantly different is 217, 38 of which show an increase and 179 show a decrease. (C) A scatter plot of the 8890 RD genes reflecting changes in transcript abundance that occur between the PC32 and 83 minute time points (early four-cell and 40-cell stages, respectively). The plot is otherwise identical to B. Of the 3773 genes that are considered significantly different, 1911 show an increase and 1862 show a decrease.

View larger version (21K): [in a new window]   Fig. 5. A phasegram reveals symmetry in the dynamics of the transcriptome, including a wave of roughly constant length. 3157 RD genes with P<0.001 in either of the two within-series ANOVAs were sorted according to their time of maximum expression. Columns correspond to moving average timepoints and rows to individual genes. There are roughly two timepoints per cell cycle. Each gene was mean normalized and log2 transformed. Yellow corresponds to positive values after log transformation (above the mean) and blue corresponds to negative values. Scale bar: 500 genes.

View larger version (27K): [in a new window]   Fig. 7. Cluster analysis of expression profiles reveals the most predominant expression patterns as well as significant associations of gene annotations. (A) Means of the 20 largest of 106 total clusters are presented (including 79% of 3157 genes with P<0.001 in either within-series ANOVA). Clusters are numbered according to size (number of member genes in brackets) and arranged so that similar patterns are near each other. Axis labels are included for cluster 1 and are the same throughout. Line width reflects relative cluster size on a log scale. Bars reflect 1 s.d. among the members of the cluster. (B) Significant enrichments and depletions of gene annotations in expression clusters determined by a hypergeometric probability analysis (P<0.001). Coordinates for the clusters corresponding to A are given (row, column). Functional categories are from Worm Proteome Database (4 March 2002) (Costanzo, 2001). Abbreviations correspond to RNAi phenotypes from WormBase (Mei, defective meiosis; Led, late embryo defect; Bmd, body morphology defective).

View larger version (20K): [in a new window]   Fig. 8. Defined expression classes based on developmental concepts reveal an inflection point in the transition from maternal to embryonic control. (A) A Venn diagram relating by area the relative sizes and intersections of the four basis classes. The number of genes in each class is in parentheses. (B) Gene expression profiles for the average of each of the defined expression classes. Each gene was mean normalized before computing the class average. The heavy black line labeled ANOVA plots the average of all genes with P<0.01 in either of the two within-series ANOVAs, and is included as a point of reference. M, maternal; E, embryonic,; ET, embryonic transient; MD, maternal degradation; S, strictly (e.g. SE implies E but not M; SMD implies MD but not E).

View larger version (32K): [in a new window]   Fig. 6. The transcriptome maintains a steady-state distribution of transcript abundances during early embryogenesis. (A) A histogram plotting the distribution of transcript abundances among the RD genes for each of twelve time points assayed. Binned units along the x-axis are transcripts per embryo and the y-axis relates how many of the genes RD in that time point are in each bin. (B) A histogram plotting the distribution of rates of change in transcript abundance for each of ten time intervals. Binned units along the x-axis are transcripts per minute per embryo and the y-axis relates how many genes fall into each bin (note log scale). Time intervals are equivalent to those in Fig. 4A. Rates were calculated by dividing the difference in abundance between timepoints by the corresponding time interval, and converting to transcripts min-1 embryo-1, assuming 107 transcripts per embryo. Only those RD genes with P<0.05 in the paired-time point ANOVA corresponding to each time interval are included.