Broadly expressed genes

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The ten clusters encompassing broadly expressed genes have relatively tight array profiles, but the diversity of annotations makes the boundaries between these clusters somewhat arbitrary. While there is significant ambiguity in determining the borders of these clusters, each has a unique distinguishing expression profile.

All broad clusters have maternal expression followed by ubiquitous or broad expression. Genes within these clusters have stereotypical cellular functions, which reveal the physiological and cell biological states of different domains in the embryo during development.

Cluster 1B is one of the several broad clusters characterized by peak microarray expression around hours 4-5. In situ hybridization showed continued ubiquitous staining throughout embryogenesis, with the heaviest staining resolving to the differentiated midgut, muscle, hindgut, foregut, and anal pads by stages 13-16. Genes within this cluster exhibit relatively diverse cellular functions, but within its core members are more than half of all genes known to be involved in nucleolar-based ribosome biogenesis .

Genes in cluster 2B and many in cluster 3B are characterized by peak expression levels around hour 12  and by in situ hybridization appear strongest in the differentiated midgut, muscle, hindgut, and foregut  Cluster 2B contains 33% of all genes annotated as being mitochondrial (7x enrichment, p = 2.7e−48; suppl. Table 3). Genes in 3B often appear restricted to the midgut, but this cluster was classified as “broad” due to its apparent relationship to cluster 2B both in its overall expression profile and its enrichment for mitochondrial genes (3x enrichment, p = 1.6e−5). It is likely that expression in some tissues, including muscle and hindgut, is present but falls below the level of detection in the in situ protocol. There is a significant correlation (p = 3.7e−9) between the genes in clusters 2B and 3B with genes shown in an RNAi screen to be induced by histone de-acetylase SIN3, suggesting a possible regulatory mechanism (Pile, Spellman et al. 2003). Interestingly, a substantial fraction of these SIN3-induced genes, about 25%, are classified as having diminishing maternal staining by our in situ clustering (p = 2.6e−8 correlation with maternal diminishing cluster 10B). This suggests that this common expression pattern is often beneath the level of detection by whole mount in situ hybridization.

Clusters 4B and 5B are characterized by peak expression levels around hours 4-5 (stage 10) and often resolve to exhibit staining in the differentiated nervous system and midgut. The two are differentiated by expression in the stage 13-16 gonad. These clusters are both significantly enriched for genes with apparent functions in cell division; including genes required for DNA metabolism, 4B (4x enrichment, p = 6.6e-5) and 5B (4x enrichment, p = 5.6e-12) and the cell cycle, 4B (3x enrichment, p = 4.9e-3) and 5B (4x enrichment, p = 5.8e-16). Interestingly, there is significant overlap between the genes in these clusters and a set of 65 genes identified in an RNAi screen for dE2F transcriptional targets (Dimova, Stevaux et al. 2003). We have X in our dataset with 40% belonging to 5B (8x enrichment, p = 2.2e−12) and 20% belonging to 4B (9x enrichment, p = 1.4e−6). There is a clear link between the expression patterns captured in these clusters and a large battery of genes involved in cell division.

Genes in cluster 6B are almost uniformly annotated as ubiquitous at all stages of embryogenesis and this annotation is supported by relatively high average array expression levels at all time points (Figure 5F). Cluster 6B contains over 80% of the genes encoding the components of the cytosolic ribosome (8x enrichment, p = 1.1e-29) and other genes involved in protein metabolism. Additionally, 40% of the 100 genes identified as essential for viability based on a large RNAi screen (Boutros, Kiger et al. 2004) are included in this cluster (4x enrichment; p = 2.6e−16). It is clear from Figure 5F that even for these highly expressed, truly ubiquitous genes, the in situ staining is not uniform and could be described by a complex combination of specific annotation terms.

Altogether genes in clusters 1B-6B exhibit remarkably similar expression patterns during gastrulation and were most frequently annotated as endoderm and mesoderm anlagen. This early pattern later resolves into endodermal and mesodermal derivatives for genes in clusters 1-3B or into CNS and midgut for genes in clusters 4B-5B. Intuitively, one would expect that genes expressed in late CNS are initially turned on in ectoderm and not in the endoderm and mesoderm. It is likely that the apparent overlap between the germ layer anlagen after gastrulation makes them difficult to differentiate in whole mount in situ specimens. Alternatively, it is possible that the regulation of expression of these genes is dictated by physiological requirements of cells at different stages of embryogenesis and not developmental lineages.

Clusters 7B-10B are comprised of genes with maternally deposited transcripts which fade below the level of detection by in situ hybridization by mid-embryogenesis. Since our in situ method does not distinguish between maternal and zygotic transcript, the specific annotations of maternally contributed genes may represent either differential stability of the maternal mRNAs in some tissues or superimposition of diminishing maternal transcript and de novo zygotic expression; the quantitative array profiles often aid in making this distinction. Clusters 7B (75 genes), 8B (49 genes), and 9B (650 genes) have in situ annotations suggesting that they are expressed maternally and then the abundance of their transcripts is markedly reduced by stage 7-8). The vast majority of these genes, including those in Cluster 10B, have microarray profiles that largely agree with this expression pattern. Those in 7B  appear to rise steadily until hour 9 (stage 12), while those in 8B come on strongly at 16 hours, which is after the period we generally examine by in situ hybridization, and at a time when formation of cuticle prevents efficient RNA in situ hybridization. Cluster 9B represents a class of genes, which are maternal, but appear by array to have a spike in expression level during the blastoderm stage. Interestingly, these genes differ from those in clusters 7B, 8B, and 10B in that they are overwhelmingly annotated as being “ubiquitous” through gastrulation. An attractive hypothesis, supported by the microarray data, is that the genes in 9B  are deposited maternally and augmented zygotically.

The genes and expression patterns in broad clusters have failed to attract the attention of developmental biologists since embryonic expression of only 4.3% of them are described in the scientific literature (Grumbling and Strelets 2006). Yet, they represent more than half of the genes expressed in embryogenesis. Our analysis of broad patterns provides a comprehensive and unbiased overview of these neglected genes and redefines the definition of ubiquitous gene expression during development. A major lesson learned from our in situ screen is that a CV annotation strategy is insufficient to describe these patterns consistently.