Restricted expression patterns.

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While the diversity of expression patterns that we detected was considerable, our hybrid clustering approach identified a number of tissue or domain specific expression patterns shared among a significant number of genes. While these clusters are more easily categorized than the broad clusters, there is still considerable ambiguity between clusters

Clusters 1R-4R contain 383 genes expressed in various combinations of the yolk nuclei, fat body and blood related tissues. Clusters 1R and 2R genes are more likely to be expressed in combinations of these different structures, while 3R genes are primarily expressed in the fat body, and 4R genes in the head mesoderm and related tissues. Interestingly, the various tissues included in these expression clusters derive from distinct developmental lineages, raising the question of whether a single coordinated expression program may underlie expression in these seemingly unrelated developmental domains. They may be linked by having a conserved role in immunity .

Clusters 5R-7R contain 1,160 genes with expression in various epithelial structures late in embryogenesis . These tissues include the epidermis, hindgut, foregut, and trachea, among others. The appearance of the staining pattern is highly dynamic and it appears variable depending on the precise intermediate stage captured. However, the fully formed epidermal pattern is common and represents the most recognizable and most abundant tissue-restricted pattern in embryogenesis. The epidermal pattern is frequently combined with expression in tracheal system. A subset of genes is expressed earlier in embryogenesis and most likely carry out a very different set of developmental roles including morphogenesis. The differences between the late epithelial clusters and the early epithelial cluster are apparent not only in the CV annotations, but also in the average microarray profiles of these clusters.

Cluster 13R-16R contains 525 genes expressed specifically in the central and peripheral nervous system. In contrast to the broad clusters 4B and 5B, these genes lack maternally contributed transcripts and any detectable staining at or immediately after gastrulation. The central nervous system specific gene expression  begins at stage 11 and almost always includes both the brain and the ventral nerve cord. A subset of genes is also expressed in the midline, with a small number showing transcription before stage 11. Genes expressed exclusively in the midline were extremely rare. Many genes are expressed in both the central and peripheral nervous systems, while a significant number are expressed in the peripheral nervous system alone.

Cluster 18R and 19R contain 229 genes expressed in either differentiated somatic muscle or differentiated visceral muscle. Most genes that were detected in the visceral muscle became active earlier in the mesoderm primordia. As with the head and trunk components of the nervous system, expression in trunk muscles was almost always accompanied by expression in head muscles.

Clusters 23R-29R contain 422 genes expressed in a domain-specific manner beginning in the blastoderm stage embryo and typically continuing in a tissue-specific manner throughout embryogenesis. These expression patterns tend to be extremely diverse at every stage of embryogenesis, and many are assigned to more than a single cluster. In fact, only 148 (35%) are assigned to a single unique cluster. From our dataset, we can conclude that genes patterned in the blastoderm have a tendency to be expressed in certain tissues later, especially the CNS and epidermis. The relationship between blastoderm-stage expression and later tissue-specific expression is elusive. While continuity of expression in particular lineage-specific regulatory genes is well-documented, we fail to detect any statistically significant relationship between annotations at the blastoderm and later stages in our full, unbiased set of genes. While we cannot conclusively rule out that this is due to a limitation of our controlled vocabulary or some other artifact of our approach, it more likely indicates that expression of such genes is initiated independently at different stages of development rather then maintained through developmental lineages.

An additional eight clusters contain 349 genes with various stereotypical expression patterns. Some of these, like the cluster of continuous pole and germ-cell expression, are comprised of a single distinct tissue across stages, while others like the cluster of midgut-specific genes are primarily expressed in a particular tissue at a particular time. The fact that these tissues formed their own clusters under our clustering scheme indicates that many genes are expressed specifically in these structures, reflecting their functional specialization.

Despite the significant number of genes that conform well to the patterns represented by the above clusters, a large fraction are expressed in various and often unique combinations of structures. We attempted to characterize these genes by assigning them to the set of clusters that best described their expression pattern. Of the 1,947 genes expressed in a restricted manner, 795 (41%) are assigned to more than one cluster. We illustrate this by showing several examples of genes assigned to multiple clusters . By categorizing genes into more than a single expression cluster, we also hope to facilitate more useful online searches of our dataset by more fully representing the range of each gene’s expression. The 29 restricted clusters can be viewed as distinct transcriptional programs and the numerous genes that are expressed in unique combination of tissues combine these basic programs. Such a view is consistent with our current understanding of how complex patterns of expression are generated by a set of independently acting cis-regulatory modules. An interesting direction for future research will be to uncover the cis-regulatory modules that are associated with the individual restricted clusters and to examine whether or how are these modules are utilized to achieve the undisputable diversity in gene expression regulation.

Can we estimate the number of distinct expression patterns in Drosophila embryogenesis? When we apply the criteria that genes with 75% or more of their annotation terms in common are considered ‘indistinguishable’, we identify X multi-gene groups and X singletons among the genes in restricted clusters. Thus by removing the broad genes, that are prone to inconsistent annotation, the number of distinct patterns within our dataset drops from 2197 to X, providing an estimate of the number of ‘distinct’ patterns. On the other hand, these patterns are not unrelated. We consider the 29 restricted clusters the most prominent recurring patterns in the dataset and these define 29 sets of related patterns. We can only speculate where to place the ‘real’—that is, biologically significant—number of patterns within these defined extremes. It is clear that the clusters are not homogenous since 41% of the genes exhibit composite patterns. We favor the idea that majority of the composite patterns result from simple additive combination of the basic patterns rather than a completely new gene specific regulation. We believe this analysis will be much more informative once we can enumerate each of the independently acting cis-acting regulatory modules that drive the various elements of these restricted expression patterns. These cis-acting regulatory modules are the fundamental units determining gene expression patterns and we believe that performing the clustering analysis on the patterns that each of these elements generates, rather than the patterns of entire genes that result from the combined action of many such modules, will be more powerful in revealing the underlying mechanisms and logic governing the generation and evolution of each gene’s expression pattern.

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